WO2001096571A2

WO2001096571A2 - Human protein phosphatase iic abi2

Info

Publication number: WO2001096571A2
Application number: PCT/EP2001/006666
Authority: WO
Inventors: Yonghong Xiao
Original assignee: Bayer Aktiengesellschaft
Priority date: 2000-06-16
Filing date: 2001-06-13
Publication date: 2001-12-20
Also published as: WO2001096571A3; AU2001285738A1

Abstract

Reagents which regulate human protein phosphatase IIC ABI2 activity and reagents which bind to human protein phosphatase IIC ABI2 gene products can be used, inter alia, to treat Asthma, COPD and disorders associated with an increase in apoptosis, including AIDS and other infectious or genetic immunodeficiencies, peripheral or central nervous system disease including neurodenerative disease, myelodysplasia, ischemic injuries, toxin-induced diseases, wasting diseases, viral infections, and osteoporosis. Regulation of human protein phosphatase IIC ABI2 also can be used to treat disorders associated with a decrease in apoptosis, including cancer. Inflammatory disorders also can be treated.

Description

REGULATION OF HUMAN PROTEIN PHOSPHATASE IIC ABI2

TECHNICAL FIELD OF THE INVENTION

The invention relates to the regulation of human protein phosphatase IIC ABI2 activity for therapeutic effects.

BACKGROUND OF THE INVENTION

The protein phosphorylation/dephosphorylation cycle is one of the major regulatory mechanisms employed by eukaryotic cells to control cellular activities. See U.S. Patent 5,853,997. It is estimated that more than 10% of the active proteins in a typical mammalian cell are phosphorylated. During protein phosphorylation/- dephosphorylation, phosphate groups are transferred from adenosine triphosphate molecules to a protein by protein kinases and are removed from the protein by protein phosphatases.

Protein phosphatases function in cellular signaling events that regulate cell growth and differentiation, cell-to-cell contacts, the cell cycle, and oncogenesis. Three protein phosphatase families have been identified as evolutionarily distinct. These include the serine/threonine phosphatases, the protein tyrosine phosphatases, and the acid/alkaline phosphatases (Carbonneau & Tonks, Ann. Rev. Cell Biol 8, 463-93, 1992).

The serine/threonine phosphatases are either cytosolic or associated with a receptor. On the basis of their sensitivity to two thermostable proteins, inhibitors 1 and 2, and their divalent cation requirements, the serine/threonine phosphatases can be separated into four distinct groups: PP-I, PP-IIA, PP-IIB, and PP-IIC. PP-I dephosphorylates many of the proteins phosphorylated by cyclic AMP-dependent protein kinase and is therefore an important regulator of many cyclic AMP mediated, hormone responses in cells. PP-IIA has broad specificity for control of cell cycle, growth and proliferation, and DNA replication and is the main phosphatase responsible for reversing the phosphorylations of serine/threonine kinases. PP-IIB, or calcineurin (Cn), is a Ca⁺²-activated phosphatase; it is involved in the regulation of such diverse cellular functions as ion channel regulation, neuronal transmission, gene transcription, muscle glycogen metabolism, and lymphocyte activation. PP-IIC is a Mg⁺²-clependent phosphatase which participates in a wide variety of functions, including regulating cyclic AMP-activated protern-kinase activity, Ca⁺²-dependent signal transduction, tRNA splicing, and signal transmission related to heat shock responses. PP-IIC is a monomeric protein with a molecular mass of about 40-45 kD.

One α and several β isoforms of PP-IIC have been identified (Wenk et al., FEBS Lett. 297, 135-38, 1992; Terasawa et al, Arch. Biochem. Biophys. 307, 342-49, 1993; and Kato et al, Arch. Biochem. Biophys. 375,387-93, 1995).

Because of the importance of protein phosphatases in a variety of biological functions, there is a need in the art to identify additional protein phosphatases which can be regulated to provide therapeutic effects.

SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods of regulating a human protein phosphatase IIC ABI2. These and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a protein phosphatase IIC ABI2 polypeptide comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 2;

the amino acid sequence shown in SEQ ID NO. 2; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 16;

the amino acid sequence shown in SEQ ID NO. 16;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 18;

the amino acid sequence shown in SEQ ID NO. 18;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 19;

the amino acid sequence shown in SEQ ID NO. 19.

Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a protein phosphatase IIC ABI2 polypeptide comprising an amino acid sequence selected from the group consisting of:

the amino acid sequence shown in SEQ ID NO. 2;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 4;

the amino acid sequence shown in SEQ ID NO. 4; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 6;

the amino acid sequence shown in SEQ ID NO. 6;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 8;

the amino acid sequence shown in SEQ ID NO. 8;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 10;

the amino acid sequence shown in SEQ ID NO. 10;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 12;

the amino acid sequence shown in SEQ ID NO. 12;

amino acid sequences wliich are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 14;

the amino acid sequence shown in SEQ ED NO. 14;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 15;

the amino acid sequence shown in SEQ ID NO.15; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 16;

the amino acid sequence shown in SEQ ID NO. 16;

the amino acid sequence shown in SEQ ID NO.18;

the amino acid sequence shown in SEQ ID NO. 19.

Binding between the test compound and the protein phosphatase IIC ABI2 polypeptide is detected. A test compound which binds to the protein phosphatase IIC ABI2 polypeptide is thereby identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the activity of the protein phosphatase IIC ABI2.

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 1 ;

the nucleotide sequence shown in SEQ ID NO. 1 ; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 3;

the nucleotide sequence shown in SEQ ID NO. 3;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 5;

the nucleotide sequence shown in SEQ ID NO.5;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 7;

the nucleotide sequence shown in SEQ ID NO. 7;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 9;

the nucleotide sequence shown in SEQ ID NO. 9;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 11 ;

the nucleotide sequence shown in SEQ ID NO.11 ;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 13;

the nucleotide sequence shown in SEQ ID NO. 13; nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 17;

the nucleotide sequence shown in SEQ ID NO. 17.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the protein phosphatase IIC ABI2 through interacting with the protein phosphatase IIC ABI2 mRNA.

Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a protein phosphatase IIC ABI2 polypeptide comprising an amino acid sequence selected from the group consisting of:

the amino acid sequence shown in SEQ ID NO. 2;

the amino acid sequence shown in SEQ ID NO.4;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 6;

the amino acid sequence shown in SEQ ID NO. 6; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 8;

the amino acid sequence shown in SEQ ID NO. 8;

the amino acid sequence shown in SEQ ID NO.10;

the amino acid sequence shown in SEQ ID NO. 12;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 14;

the amino acid sequence shown in SEQ ID NO. 14;

amino acid sequences which are at least about 50%ι identical to the amino acid sequence shown in SEQ ID NO. 15;

the amino acid sequence shown in SEQ ED NO.15;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 16;

the amino acid sequence shown in SEQ ID NO. 16; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ D NO. 18;

the amino acid sequence shown in SEQ ED NO.18;

the amino acid sequence shown in SEQ ID NO. 19.

A protein phosphatase IIC ABI2 activity of the polypeptide is detected. A test compound which increases protein phosphatase IIC ABI2 activity of the polypeptide relative to protein phosphatase IIC ABI2 activity in the absence of the test compound is thereby identified as a potential agent for increasing extracellular matrix de- gradation. A test compound which decreases protein phosphatase IIC ABI2 activity of the polypeptide relative to protein phosphatase IIC ABI2 activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a protein phosphatase IIC ABI2 product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:

the nucleotide sequence shown in SEQ ID NO. 1;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 3; the nucleotide sequence shown in SEQ ED NO. 3;

the nucleotide sequence shown in SEQ ID NO.5;

the nucleotide sequence shown in SEQ ID NO. 7;

the nucleotide sequence shown in SEQ ED NO. 9;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 11;

the nucleotide sequence shown in SEQ ID NO.11 ;

the nucleotide sequence shown in SEQ ID NO. 13;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 17; - li ¬

the nucleotide sequence shown in SEQ ID NO. 17.

Binding of the test compound to the protein phosphatase IIC ABI2 product is detected. A test compound which binds to the protein phosphatase IIC ABI2 product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 1;

the nucleotide sequence shown in SEQ ID NO. 1;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 3;

the nucleotide sequence shown in SEQ ID NO. 3;

the nucleotide sequence shown in SEQ ID NO.5;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 7; the nucleotide sequence shown in SEQ ED NO. 7;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ED NO. 9;

the nucleotide sequence shown in SEQ ID NO. 9;

the nucleotide sequence shown in SEQ ED NO.11 ;

the nucleotide sequence shown in SEQ ID NO. 13;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO. 17;

the nucleotide sequence shown in SEQ ID NO. 17.

Protein phosphatase IIC ABI2 activity in the cell is thereby decreased.

The invention thus provides reagents and methods for regulating human protein phosphatase IIC ABI2 which can be used inter alia, to treat disorders associated with an increase in apoptosis, including AIDS and other infectious or genetic immunodeficiencies, neurodegenerative diseases, myelodysplasia, ischemic injuries, toxin-induced diseases, wasting diseases, viral infections, and osteoporosis; disorders associated with a decrease in apoptosis, including cancer; and inflammatory disorders. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 2 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 3 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 4 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 5 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 6 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 7 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 8 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 9 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 10 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 11 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 12 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 13 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide.

Fig. 14 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide. Fig. 15 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide. Fig. 16 shows the amino acid seuqence of the protein identified with Swiss

Prot Accession No. 004719. Fig. 17 shows the DNA-sequence encoding a protein phosphatase IIC ABI2 polypeptide. Fig. 18 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide. Fig. 19 shows the amino acid sequence of a protein phosphatase IIC ABI2 polypeptide.

Fig. 20 shows the BLASTP alignment of the polypeptide with the amino acid sequence of Fig. 4 with the protein having Swiss Prot Accession No.

O04719. Fig. 21 shows the BLOCKS search results. Fig. 22 shows the relative expression of human phosphatase IIC ABI2 in respiratory cells and tissues. Fig. 23 shows the relative expression of human phosphatase IIC ABI2 in various human tissues and the neutrophil-like cell line HL60. Fig. 24 shows a BLASTP alignment Fig. 25 shows a HMMPFAM - alignment.

Fig. 26 shows the relative expression of human phosphatase IIC ABI2 in human tissues.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide and being selected from the group consisting of:

a) a polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide comprising an amino acid sequence selected from the group consisting of: amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 2; the amino acid sequence shown in SEQ ED NO. 2; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 18; the amino acid sequence shown in SEQ ED NO.18; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 19; the amino acid sequence shown in SEQ ID NO. 19;

b) a polynucleotide comprising the sequence of SEQ ID NOS. 1 ;

c) a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b);

d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d).

Furthermore, it has been discovered by the present applicant that a novel protein phosphatase IIC ABI2, particularly a human protein phosphatase IIC ABI2, is a discovery of the present invention. Human protein phosphatase IIC ABI2 is 41% identical over 131 amino acids to the protein identified with SwissProt Accession

No. O04719 and annotated as "protein phosphatase 2C ABI2 (PP2C)" (FIG. 1). A BLOCKS search indicates that human protein phosphatase IIC ABI2 contains multiple protein phosphatase domains (FIG. 2). The extended genomic sequence encoding human protein phosphatase IIC ABI2 (SEQ ID NO.l) contains multiple ESTs, which are shown in SEQ ID NOS.3, 5, 7, 9, 11, 13, and 15, indicating that this coding sequence is expressed. Polypeptides

Protein phosphatase IIC ABI2 polypeptides according to the invention comprise at least 75, 100, 125, 150, 175, 200, 250, 300, or 350 contiguous amino acids of SEQ

ED NO.2, 18 or 19 or a biologically active variant thereof, as defined below. A protein phosphatase IIC ABI2 polypeptide of the invention therefore can be a portion of a protein phosphatase IIC ABI2 molecule, a full-length protein phosphatase IIC ABI2 molecule, or a fusion protein comprising all or a portion of a protein phosphatase IIC ABI2 molecule.

Biologically Active Variants

Protein phosphatase IIC ABI2 variants which are biologically active, i.e., retain a protein phosphatase IIC ABI2 activity, also are protein phosphatase IIC ABI2 - polypeptides. Preferably, naturally or non-naturally occurring protein phosphatase IIC ABI2 variants have amino acid sequences which are at least about 50, preferably about 55, 60, 70, more preferably about 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to an amino acid sequence shown in SEQ ID NO.2, 18 or 19. Percent identity between a putative protein phosphatase IIC ABI2 variant and an amino acid sequence of SEQ ID NO.2, 18 or 19 is determined with the Needleman/Wunsch algorithm (Needleman and Wunsch, J.Mol. Biol. 48; 443-453, 1970) using a Blosum62 matrix with a gap creation penalty of 8 and a gap extension penalty of 2 (S. Henikoff and J.G. Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992).

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine. Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, such as DNASTAR software. Whether an amino acid change results in a biologically active protein phosphatase IIC ABI2 polypeptide can readily be determined by assaying for protein phosphatase TLC ABI2 activity, as is known in the art and described, for example, in the specific examples below.

Fusion Proteins

Fusion proteins are useful for generating antibodies against protein phosphatase IIC ABI2 amino acid sequences and for use in various assay systems. For example, fusion proteins can be used to identify proteins which interact with portions of a protein phosphatase IIC ABI2 polypeptide, including its active site and phosphatase domains. Methods such as protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two-hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A protein phosphatase IIC ABI2 fusion protein comprises two protein segments fused together by means of a peptide bond. Contiguous amino acids for use in a fusion protein can be selected from the amino acid sequences shown in SEQ ID NO.2, 18 or 19 or from a biologically active variant thereof, such as those described above. For example, the first protein segment can comprise at least 75, 100, 125, 150, 175, 200, 250, 300, or 350 contiguous amino acids of SEQ ID NO.2, 18 or 19 or a biologically active variant thereof. Preferably, a fusion protein comprises the active site of the protein phosphatase IIC ABI2 or the functional domains shown in FIG. 2. The first protein segment also can comprise full-length protein phosphatase

IIC ABI2. The second protein segment can be a full-length protein or a protein fragment or polypeptide. Proteins commonly used in fusion protein construction include β- galactosidase, β-glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chloramphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VS V- G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DNA binding domain (DBD) fusions, GAL4

DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the protein phosphatase IIC ABI2 polypeptide-encoding sequence and the heterologous protein sequence, so that the protein phosphatase IIC ABI2 polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two protein segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises protein phosphatase IIC ABI2 coding sequences disclosed herein in proper reading frame with nucleotides encoding the second protein segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA),

Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA- KITS). Identification of Species Homologs

Species homologs of human protein phosphatase IIC ABI2 can be obtained using protein phosphatase IIC ABI2 polynucleotides (described below) to make suitable probes or primers to screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of protein phosphatase IIC ABI2, and expressing the cDNAs as is known in the art.

Polynucleotides

A protein phosphatase IIC ABI2 polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a protein phosphatase IIC ABI2 polypeptide. A nucleotide sequence encoding the human protein phosphatase IIC ABI2 polypeptide shown in SEQ ID NO.2 is shown in SEQ ID NO.l. A full-length coding sequence is provided in nucleotides 215-1270 of SEQ ID NO.17. These nucleotides encode the amino acid sequence shown in SEQ ID NO.18.

Degenerate nucleotide sequences encoding human protein phosphatase IIC ABI2 polypeptides, as well as homologous nucleotide sequences which are at least about

50, preferably about 55, 60, 65, 70, more preferably about 75, 80, 85, 90, 95, 96, 97,

98, or 99% identical to the protein phosphatase IIC ABI2 coding sequences shown in

SEQ ID NO.l and 17 also are protein phosphatase IIC ABI2 polynucleotides.

Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of protein phosphatase IIC ABI2 polynucleotides which encode biologically active protein phosphatase IIC ABI2 polypeptides also are protein phosphatase IIC ABI2 polynucleotides. Identification of Variants and Homologs

Variants and homologs of the protein phosphatase IIC ABI2 polynucleotides disclosed above also are protein phosphatase IIC ABI2 polynucleotides. Typically, homologous protein phosphatase IIC ABI2 polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known protein phosphatase IIC ABI2 polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each—homologous sequences can be identified which contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches.

Species homologs of the protein phosphatase IIC ABI2 polynucleotides disclosed herein can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of protein phosphatase IIC ABI2 polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al, J. Mol. Biol. 81, 123 (1973). Variants of human protein phosphatase IIC ABI2 polynucleotides or protein phosphatase IIC ABI2 polynucleotides of other species can therefore be identified, for example, by hybridizing a putative homologous protein phosphatase IIC ABI2 polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO.l, 3, 5, 7, 9, 11, 13 or 17 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising protein phosphatase IIC ABI2 polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to protein phosphatase IIC ABI2 polynucleotides or their complements following stringent hybridization and/or wash conditions are also protein phosphatase IIC ABI2 polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20°C below the calculated T_m of the hybrid under study. The T_m of a hybrid between a protein phosphatase IIC ABI2 polynucleotide having a coding sequence disclosed herein and a polynucleotide sequence which is at least about 50, 55, 60, 65, 70, preferably about 75, 90, 96, or 98% identical to that nucleotide sequence can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m = 81.5°C - 16.6(log₁₀ [Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 60011), where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65°C, or 50% formamide, 4X SSC at 42°C, or 0.5X SSC, 0.1% SDS at 65°C. Highly stringent wash conditions include, for example, 0.2X SSC at 65°C.

Preparation of Polynucleotides

A naturally occurring protein phosphatase IIC ABI2 polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or synthesized using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated protein phosphatase IIC ABI2 polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments which comprise protein phosphatase IIC ABI2 nucleotide sequences. Isolated polynucleotides are in preparations which are free or at least 70, 80, or 90%o free of other molecules.

Protein phosphatase IIC ABI2 cDNA molecules can be made with standard molecular biology techniques, using protein phosphatase IIC ABI2 mRNA as a template. Protein phosphatase IIC ABI2 cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of protein phosphatase IIC ABI2 polynucleotides, using either human genomic DNA or cDNA as a template.

Alternatively, synthetic chemistry techniques can be used to synthesize protein phosphatase IIC ABI2 polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a protein phosphatase IIC ABI2 polypeptide having, for example, the amino acid sequence shown in SEQ ID NO.2, 18 or 19 or a biologically active variant thereof.

Obtaining Full-Length Polynucleotides

The partial sequences of SEQ ID NOS.l, 3, 5, 7, 9, 11, and 13 and the coding sequence shown in nucleotides 215-1270 of SEQ ID NO.17 can be used to identify regulatory elements of the full-length gene from which they were derived. The partial sequences can be nick-translated or end-labeled with ³²P using polynucleotide kinase using labeling methods known to those with skill in the art (BASIC METHODS

IN MOLECULAR BIOLOGY, Davis et al, eds., Elsevier Press, N.Y., 1986). A lambda library prepared from human tissue can be directly screened with the labeled sequences of interest or the library can be converted en masse to pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al., 1989, pg. 1.20).

Both methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured, and the DNA is fixed to the filters. The filters are hybridized with the labeled probe using hybridization conditions described by Davis et al, 1986. The partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification. The resulting autoradio- gra s are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque. The colonies or plaques are selected and expanded, and the DNA is isolated from the colonies for further analysis and sequencing.

Positive cDNA clones are analyzed to determine the amount of additional sequence they contain using PCR with one primer from the partial sequence and the other primer from the vector. Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot Analysis.

Once one or more overlapping cDNA clones are identified, the complete sequence of the clones can be determined, for example after exonuclease III digestion (McCombie et al, Methods 3, 33-40, 1991). A series of deletion clones are generated, each of which is sequenced. The resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence. Various PCR-based methods can be used to extend the nucleic acid sequences encoding the disclosed portions of human protein phosphatase IIC ABI2 to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DNA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first ^""one. Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer

Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68 - 72°C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations are used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al, Nucleic Acids Res. 19, 3055-3060, 1991. Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA. This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Also, random-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.

Obtaining Polypeptides

Protein phosphatase IIC ABI2 polypeptides can be obtained, for example, by purification from cells, by expression of protein phosphatase IIC ABI2 polynucleotides, or by direct chemical synthesis. Protein Purification

Protein phosphatase IIC ABI2 polypeptides can be purified from cells, including cells which have been transfected with protein phosphatase IIC ABI2 expression constructs. Human germinal B cells and normal prostate epithelial cells are especially useful sources of protein phosphatase IIC ABI2 polypeptides. A purified protein phosphatase IIC ABI2 polypeptide is separated from other compounds which normally associate with the protein phosphatase IIC ABI2 polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified protein phosphatase IIC ABI2 polypeptides is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. Enzymatic activity of the purified preparations can be assayed, for example, as described in the specific examples, below.

Expression of Polynucleotides

To express a protein phosphatase IIC ABI2 polypeptide, a protein phosphatase IIC ABI2 polynucleotide can be inserted into an expression vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding protein phosphatase IIC

ABI2 polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y, 1989. A variety of expression vector/host systems can be utilized to contain and express sequences encoding a protein phosphatase IIC ABI2 polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors

(e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector ~ enhancers, promoters, 5' and 3' untranslated regions — which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses

(e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a protein phosphatase EC ABI2 polypeptide, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the protein phosphatase IIC ABI2 polypeptide. For example, when a large quantity of a protein phosphatase EC ABI2 polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the sequence encoding the protein phosphatase

IIC ABI2 polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264, 5503-5509, 1989 or pGEX vectors (Promega, Madison, Wis.) can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or Factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al., Methods Enzymol 153, 516-544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding protein phosphatase C ABI2 polypeptides can be driven by any of a number of promoters.

For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu EMBOJ. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al, EMBO J. 3, 1671-1680, 1984; Broglie et al, Science 224, 838-843, 1984; Winter et al, Results

Probl Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express a protein phosphatase EC ABI2 polypeptide. For example, in one such system Autographa californica nuclear poly- hedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding protein phosphatase IIC ABI2 polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of protein phosphatase IIC ABI2 polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which protein phosphatase EC ABI2 polypeptides can be expressed (Engelhard etal, Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be utilized in mammalian host cells.

For example, if an adenovirus is used as an expression vector, sequences encoding protein phosphatase IIC ABI2 polypeptides can be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus which is capable of expressing a protein phosphatase IIC

ABI2 polypeptide in infected host cells (Logan & Shenk, Proc. Natl Acad. Sci. 81, 3655-3659, 1984). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DNA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding protein phosphatase IIC ABI2 polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a protein phosphatase EC ABI2 polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding, sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl Cell Differ. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process an expressed protein phosphatase IIC ABI2 polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HE 293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, VA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein. Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express protein phosphatase IIC ABI2 polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced protein phosphatase EC ABI2 sequences.

Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.

Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase

(Wigler et al., Cell 11, 223-32, 1977) and adenine phosphoribosylrransferase (Lowy et al, Cell 22, 817-23, 1980). Genes which can be employed in tk^" or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al, Proc. Natl. Acad. Sci. 77, 3567-70, 1980); npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et al., J. Mol Biol 150, 1-14, 1981); and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992 supra). Additional selectable genes have been described, for example trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine

(Hartman & Mulligan, Proc. Natl Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol Biol 55, 121-131, 1995). Detecting Expression of Polypeptides

Although the presence of marker gene expression suggests that the protein phosphatase EC ABI2 polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a protein phosphatase IIC ABI2 polypeptide is inserted within a marker gene sequence, transformed cells containing sequences which encode a protein phosphatase EC ABI2 polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a protein phosphatase IIC ABI2 polypeptide under the control of a single promoter.

Expression of the marker gene in response to induction or selection usually indicates expression of the protein phosphatase IIC ABI2 polynucleotide.

Alternatively, host cells which contain a protein phosphatase IIC ABI2 poly- nucleotide and which express a protein phosphatase IIC ABI2 polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.

The presence of a polynucleotide sequence encoding a protein phosphatase EC ABI2 polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a protein phosphatase IIC ABI2 polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a protein phosphatase IIC ABI2 polypeptide to detect transformants which contain a protein phosphatase IIC ABI2 polynucleotide.

A variety of protocols for detecting and measuring the expression of a protein phosphatase IIC ABI2 polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a protein phosphatase EC ABI2 polypeptide can be used, or a competitive binding assay can be employed.

These and other assays are described in Hampton et al, SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 158, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding protein phosphatase IIC ABI2 polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a protein phosphatase IIC ABI2 polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase, such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a protein phosphatase IIC

ABI2 polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode protein phosphatase EC ABI2 polypeptides can be designed to contain signal sequences which direct secretion of protein phosphatase IIC ABI2 polypeptides through a prokaryotic or eukaryotic cell membrane.

Other constructions can be used to join a sequence encoding a protein phosphatase IIC ABI2 polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, Wash.). The inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the protein phosphatase IIC ABI2 polypeptide can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a protein phosphatase IIC ABI2 polypeptide and 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site. The histidine residues facilitate purification on EVIAC (immobilized metal ion affinity chromatography as described in Porath et al., Prot. Exp. Purif 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the protein phosphatase IIC ABI2 polypeptide from the fusion protein. Vectors which contain fusion proteins are disclosed in Kroll et al, DNA Cell Biol. 12, 441-453, 1993).

Chemical Synthesis

Sequences encoding a protein phosphatase IIC ABI2 polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers et al, Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp.

Ser. 225-232, 1980). Alternatively, a protein phosphatase IIC ABI2 polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence. For example, protein phosphatase EC ABI2 polypeptides can be produced by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269, 202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of protein phosphatase EC ABI2 polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic protein phosphatase IIC ABI2 polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the protein phosphatase IIC ABI2 polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce protein phosphatase IIC ABI2 polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence. The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter protein phosphatase IIC ABI2 polypeptide- encoding sequences for a variety of reasons, including modification of the cloning, processing, and or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a protein phosphatase IIC ABI2 polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable of binding an epitope of a protein phosphatase IIC

ABI2 polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a protein phosphatase EC

ABI2 polypeptide can be used therapeutically, as well as in immunochemical assays, including but not limited to Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immuno- radiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody which specifically binds to the immunogen.

Typically, an antibody which specifically binds to a protein phosphatase IIC ABI2 polypeptide provides a detection signal at least 5-, 10-, or 20- fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to protein phosphatase EC ABI2 polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a protein phosphatase EC ABI2 polypeptide from solution.

Protein phosphatase IIC ABI2 polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a protein phosphatase IIC ABI2 polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueriή) and Corynebacterium parvum axe especially useful.

Monoclonal antibodies which specifically bind to a protein phosphatase IIC ABI2 polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et αl, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote βt al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol Cell Biol. 62, 109-120, 1984).

In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl Acad. Sci. 81, 6851-6855. 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, one can produce humanized antibodies using recombinant methods, as described in GB2188638B. Antibodies which specifically bind to a protein phosphatase EC ABI2 polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies which specifically bind to protein phosphatase IIC ABI2 polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J.

Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in Mallender & Voss, 1994, J. Biol. Chem. 269, 199-206.

A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Altematively, single-chain antibodies can be produced directly using, for example, filamentous phage technology. Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J. Immunol Meth. 165, 81- 91.

Antibodies which specifically bind to protein phosphatase IIC ABI2 polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies of the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a protein phosphatase IIC ABI2 polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences which are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of protein phosphatase IIC ABI2 gene products in the cell. Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkylphosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol 20, 1-8, 1994; Sonveaux, Meth. Mol Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of protein phosphatase EC ABI2 gene expression can be obtained by designing antisense oligonucleotides which will form duplexes to the control, 5', or regulatory regions of the protein phosphatase IIC ABI2 gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al., in Huber & Carr,

MOLECULAR AND IMMUNOLOGIC APPROACHES, Furura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful duplex formation between an antisense oligonucleotide and the complementary sequence of a protein phosphatase IIC ABI2 polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a protein phosphatase IIC ABI2 polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent protein phosphatase IIC ABI2 nucleotides, can provide targeting specificity for protein phosphatase IIC ABI2 mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non- complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular protein phosphatase IIC ABI2 polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a protein phospr ase IIC ABI2 polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, internucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol 10, 152-158, 1992;

Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin.

Struct. Biol 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.

Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a protein phosphatase EC ABI2 polynucleotide can be used to generate ribozymes which will specifically bind to mRNA transcribed from the protein phosphatase EC ABI2 polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within a protein phosphatase IIC ABI2 RNA target are initially identified by scanning the RNA molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the protein phosphatase IIC ABI2 target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. The suitability of candidate targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribo- nuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related; thus, upon hybridizing to the protein phosphatase IIC ABI2 target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, liposome-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease protein phosphatase EC ABI2 expression. Alternatively, if it is desired that the cells stably retain the DNA construct, it can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. The DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors which induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of protein phosphatase EC ABI2 mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whose products interact with human phosphatase IIC ABI2. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, COPD. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human phosphatase IIC ABI2 gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al, Nature 308, 149-53; Lee et al, Proc. Natl Acad. Sci. U.S.A. 88, 2825, 1984), and differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311), and microarrays.

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human phosphatase IIC ABI2. For example, treatment may include a modulation of expression of the differentially expressed genes and/or the gene encoding the human phosphatase IIC ABI2. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human phosphatase IIC ABI2 gene or gene product are up-regulated or down-regulated. Screening Methods

The invention provides methods for identifying modulators, i.e., candidate or test compounds which bind to protein phosphatase IIC ABI2 polypeptides or polynucleotides and or have a stimulatory or inhibitory effect on, for example, expression or activity of the protein phosphatase EC ABI2 polypeptide or polynucleotide, so as to regulate degradation of the extracellular matrix. Decreased extracellular matrix degradation is useful for preventing or suppressing malignant cells from metastasizing. Increased extracellular matrix degradation may be desired, for example, in developmental disorders characterized by inappropriately low levels of extracellular matrix degradation or in regeneration.

The invention provides assays for screening test compounds which bind to or modulate the activity of a protein phosphatase IIC ABI2 polypeptide or a protein phosphatase IIC ABI2 polynucleotide. A test compound preferably binds to a protein phosphatase EC ABI2 polypeptide or polynucleotide. More preferably, a test compound decreases a protein phosphatase IIC ABI2 activity of a protein phosphatase IIC ABI2 polypeptide or expression of a protein phosphatase IIC ABI2 polynucleotide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recombinantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997.

Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl

33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A.

89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl Acad. Sci. 97, 6378-6382, 1990; Felici, J. Mol Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to protein phosphatase IIC ABI2 polypeptides or polynucleotides or to affect protein phosphatase IIC ABI2 activity or protein phosphatase IIC ABI2 gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format. Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UN-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent

5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together. Binding Assays

For binding assays, the test compound is preferably a small molecule which binds to and occupies the active site or the fad-like domain of the protein phosphatase EC ABI2 polypeptide, thereby making the active site or phosphatase domains inaccessible to substrate such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules. In binding assays, either the test compound or the protein phosphatase IIC ABI2 polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound which is bound to the protein phosphatase EC ABI2 polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a protein phosphatase IIC ABI2 polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a target polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a protein phosphatase IIC ABI2 polypeptide. (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a protein phosphatase IIC ABI2 polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA). Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol 5, 699-705, 1995. BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In yet another aspect of the invention, a protein phosphatase IIC ABI2 polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g.,

U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the protein phosphatase IIC ABI2 polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one constmct a poly- nucleotide encoding a protein phosphatase IIC ABI2 polypeptide is fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence that encodes an unidentified protein ("prey" or "sample") is fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form a protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with the protein phosphatase IIC ABI2 polypeptide.

It may be desirable to immobilize either the protein phosphatase IIC ABI2 poly- peptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the protein phosphatase EC ABI2 polypeptide (or polynucleotide) or the test compound can be bound to a solid support. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the protein phosphatase IIC ABI2 polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a protein phosphatase IIC ABI2 polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, a protein phosphatase EC ABI2 polypeptide is a fusion protein comprising a domain that allows the protein phosphatase IIC ABI2 polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed protein phosphatase EC ABI2 polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing polypeptides or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a protein phosphatase IIC ABI2 polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated protein phosphatase IIC ABI2 polypeptides or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a protein phosphatase IIC ABI2 polypeptide polynucleotides, or a test compound, but which do not interfere with a desired binding site, such as the active site or a phosphatase domain of the protein phosphatase IIC ABI2 polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the protein phosphatase IIC ABI2 polypeptide (or polynucleotides) or test compound, enzyme-linked assays which rely on detecting a protein phosphatase IIC ABI2 activity of the protein phosphatase EC ABI2 polypeptide, and SDS gel electrophoresis under non-reducing conditions.

Screening for test compounds which bind to a protein phosphatase EC ABI2 polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a protein phosphatase IIC ABI2 polynucleotide or polypeptide can be used in a cell-based assay system. A protein phosphatase IIC ABI2 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, including neoplastic cell lines such as the colon cancer cell lines HCT116, DLD1,

HT29, Caco2, SW837, SW480, and RKO, breast cancer cell lines 21-PT, 21-MT, MDA-468, SK-BR3, and BT-474, the A549 lung cancer cell line, and the H392 glioblastoma cell line, can be used. An intact cell is contacted with a test compound. Binding of the test compound to a protein phosphatase IIC ABI2 polypeptide or polynucleotide is determined as described above, after lysing the cell to release the protein phosphatase IIC ABI2 polypeptide- or polynucleotide-test compound complex.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease a protein phosphatase IIC ABI2 activity of a protein phosphatase IIC ABI2 polypeptide. Protein phosphatase IIC ABI2 activity can be measured, for example, using the methods described in the specific examples, below. Protein phosphatase IIC ABI2 activity can be measured after contacting either a purified protein phosphatase IIC

ABI2 polypeptide, a cell extract, or an intact cell with a test compound. A test compound which decreases protein phosphatase EC ABI2 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing human protein phosphatase IIC ABI2 activity. A test compound which increases protein phosphatase IIC ABI2 activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human protein phosphatase EC ABI2 activity.

Gene Expression

In another embodiment, test compounds which increase or decrease protein phosphatase IIC ABI2 gene expression are identified. A protein phosphatase IIC ABI2 polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the protein phosphatase IIC ABI2 polynucleotide is determined. The level of expression of protein phosphatase IIC ABI2 mRNA or polypeptide in the presence of the test compound is compared to the level of expression of protein phosphatase IIC ABI2 mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of protein phosphatase IIC ABI2 mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of protein phosphatase EC ABI2 mRNA or polypeptide is less expression. Alternatively, when expression of the mRNA or protein is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of protein phosphatase IIC ABI2 mRNA or polypeptide expression.

The level of protein phosphatase EC ABI2 mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or protein. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a protein phosphatase IIC ABI2 polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a protein phosphatase IIC ABI2 polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell which expresses a protein phosphatase IIC ABI2 polynucleotide can be used in a cell-based assay system. The protein phosphatase IIC ABI2 polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, including neoplastic cell lines such as the colon cancer cell lines HCT116, DLD1, HT29, Caco2, SW837, SW480, and RKO, breast cancer cell lines 21-PT, 21- MT, MDA-468, SK-BR3, and BT-474, the A549 lung cancer cell line, and the H392 glioblastoma cell line, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions which can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise a protein phosphatase IIC ABI2 polypeptide, protein phosphatase IIC ABI2 polynucleotide, antibodies which specifically bind to a protein phosphatase IIC ABI2 polypeptide, or mimetics, agonists, antagonists, or inhibitors of a protein phosphatase IIC ABI2 polypeptide. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as

Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.

Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of admini- stration.

Diagnostic Methods

The human protein phosphatase IIC ABI2 and polynucleotides encoding it can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences which encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding human protein phosphatase IIC ABI2 in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease. Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,

4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of human protein phosphatase IIC ABI2 also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays,

Western blot analysis, and ELISA assays. Therapeutic Indications and Methods

Human phosphatase IIC ABI2 can be regulated to treat COPD. Chronic obstructive pulmonary (or airways) disease (COPD) is a condition defined physiologically as airflow obstraction that generally results from a mixture of emphysema and peripheral airway obstraction due to chronic bronchitis (Senior & Shapiro, Pulmonary Diseases and Disorders, 3d ed., New York, McGraw-Hill, 1998, pp. 659- 681, 1998; Barnes, Chest 117, 10S-14S, 2000). Emphysema is characterized by destmction of alveolar walls leading to abnormal enlargement of the air spaces of the lung. Chronic bronchitis is defined clinically as the presence of chronic productive cough for three months in each of two successive years. In COPD, airflow obstraction is usually progressive and is only partially reversible. By far the most important risk factor for development of COPD is cigarette smoking, although the disease does occur in non-smokers.

Chronic inflammation of the airways is a key pathological feature of COPD (Senior & Shapiro, 1998). The inflammatory cell population comprises increased numbers of macrophages, neutrophils, and CD8⁺ lymphocyes. Inhaled irritants, such as cigarette smoke, activate macrophages which are resident in the respiratory tract, as well as epithelial cells leading to release of chemokines (e.g., interleukin-8) and other chemotactic factors. These chemotactic factors act to increase the neutrophil - monocyte trafficking from the blood into the lung tissue and airways. Neutrophils and monocytes recruited into the airways can release a variety of potentially damaging mediators such as proteolytic enzymes and reactive oxygen species.

Matrix degradation and emphysema, along with airway wall thickening, surfactant dysfunction, and mucus hypersecretion, all are potential sequelae of this inflammatory response that lead to impaired airflow and gas exchange.

Human phosphatase IIC ABI2 can be regulated to treat Asthma. There is evidence suggesting that human lung mast cells and basophils contain low levels of the Mg2⁺- dependent protein phosphatase activity characteristic of protein phosphatase-EC (Peirce MJ, Munday MR, Peachell PT. Role of protein phosphatases in the regulation of human mast cell and basophil function. Am J Physiol. 1999 Dec;277(6 Pt l):C1021-8). Human protein phosphatase IIC ABI2 contributes to this phosphatase activity and the activity is important in both the signaling from membrane receptors in these cells, such as the FceRl high-affinity IgE receptor, and the consequent release of inflammatory mediators, such as histamine, leukotrienes, prostaglandins, and cytokines. Our own expression studies showing relatively high expression of human protein phosphatase IIC ABI2 in the lung and in immune tissues such as thymus and spleen (FIGS 22 and 23) are consistent with this molecule playing an important role in inflammatory or allergic responses in the lung.

Human phosphatase EC ABI2 can be regulated to treat peripheral or central nervous system disease. It shows a high expression in the nervous system (FIG. 26), suggesting a role in the control of neural function. A number of different protein phosphatases and protein tyrosine phosphatases have well described roles in neural function (Harrison S, Page CP, Spina D. Airway nerves and protein phosphatases. Gen Pharmacol. 1999 Mar;32(3):287-98). Human protein phosphatase EC ABI2 has similar functions. Importantly, phosphatases are thought to have modulatory effects in neurons, serving to dampen neuronal activites such as the release of neuropeptides.

In diseases where neuronal responses are pathologically heightened or lowered, for example in the exaggerated responses to allergens and other triggers in the lungs of asthmatics, regulating the activity of human protein phosphatase IIC ABI2 has therapeutic effects.

During fetal development, decreased expression of human protein phosphatase IIC ABI2 may cause an increase in apoptosis with no adverse effects to the subject. However, in other situations and in adults, decreased expression of human protein phosphatase IIC ABI2 may cause an increase in apoptosis which is detrimental to the subject. Therefore, in one embodiment, human protein phosphatase IIC ABI2 or a portion or biologically active variant thereof may be administered to a subject to prevent or treat a disorder associated with an increase in apoptosis. Such disorders include, but are not limited to, AEDS and other infectious or genetic immunodeficiencies, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebellar degeneration, myelodysplasia syndromes such as aplastic anemia, ischemic injuries such as myocardial infarction, stroke, and reperfusion injury, toxin-induced diseases such as alcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseases such as cachexia, viral infections such as those caused by hepatitis B and C, and osteoporosis. In another embodiment, an agonist which is specific for human protein phosphatase IIC ABI2 may be used to prevent or treat a disorder associated with increased apoptosis including, but not limited to, those listed above. In still another embodiment, a vector capable of expressing human protein phosphatase IIC ABI2, or a fragment or a derivative thereof, may be used to prevent or treat a disorder associated with increased apoptosis including, but not limited to, those listed above.

Human protein phosphatase IIC ABI2 agonists and antagonists may be used to mimic, augment or inhibit the action of the enzyme, wliich may be useful to treat osteoporosis, Paget's disease, degradation of bone implants particularly dental implants. Osteoporosis is a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk . It is the most common human metabolic bone disorder. Established osteoporosis includes the presence of fractures.

Bone turnover occurs by the action of two major effector cell types within bone: the osteoclast, which is responsible for bone resorption, and the osteoblast, which synthesizes and mineralizes bone matrix. The actions of osteoclasts and osteoblasts are highly coordinated. Osteoclast precursors are recmited to the site of turnover; they differentiate and fuse to form mature osteoclasts which then resorb bone.

Attached to the bone surface, osteoclasts produce an acidic microenvironment in a tightly defined junction between the specialized osteoclast border membrane and the bone matrix, thus allowing the localized solubilization of bone matrix. This in turn facilitate the proteolysis of demineralized bone collagen. Matrix degradation is thought to release matrix-associated growth factor and cytokines, which recrait osteoblasts in a temporally and spatially controlled fashion. Osteoblasts synthesize and secrete new bone matrix proteins, and subsequently mineralize this new matrix. In the normal skeleton this is a physiological process which does not result in a net change in bone mass. In pathological states, such as osteoporosis, the balance between resorption and formation is altered such that bone loss occurs. See WO 99/45923.

The osteoclast itself is the direct or indirect target of all currently available osteoporosis agents with the possible exception of fluoride. Antiresorptive therapy prevents further bone loss in treated individuals. Osteoblasts are derived from multipotent stem cells which reside in bone marrow and also gives rise to adipocytes, chondrocytes, fibroblasts and muscle cells. Selective enhancement of osteoblast activity is a highly desirable goal for osteoporosis therapy since it would result in an increase in bone mass, rather than a prevention of further bone loss. An effective anabolic therapy would be expected to lead to a significantly greater reduction in fracture risk than currently available treatments.

The agonists or antagonists to the newly discovered polypeptides may act as antiresorptive by directly altering the osteoclast differentiation, osteoclast adhesion to the bone matrix or osteoclast function of degrading the bone matrix. The agonists or antagonists could indirectly alter the osteoclast function by interfering in the synthesis and/or modification of effector molecules of osteoclast differentiation or function such as cytokines, peptide or steroid hormones, proteases, etc.

The agonists or antagonists to the newly discovered polypeptides may act as anabolics by directly enhancing the osteoblast differentiation and /or its bone matrix forming function. The agonists or antagonists could also indirectly alter the osteoblast function by enhancing the synthesis of growth factors, peptide or steroid hormones or decreasing the synthesis of inhibitory molecules. In a further embodiment, human protein phosphatase EC ABI2 or a fragment or derivative thereof may be added to cells to stimulate cell proliferation. In particular, human protein phosphatase IIC ABI2 may be added to a cell or cells in vivo using delivery mechanisms such as liposomes, viral based vectors, or electroinjection for the purpose of promoting regeneration or cell differentiation of the cell or cells. In addition, human protein phosphatase EC ABI2 may be added to a cell, cell line, tissue, or organ culture in vitro or ex vivo to stimulate cell proliferation for use in heterologous or autologous transplantation. In some cases, the cell will have been selected for its ability to fight an infection or a cancer or to correct a genetic defect in a disease such as sickle cell anemia, β thalassemia, cystic fibrosis, or Huntington's chorea.

In another further embodiment, an agonist which is specific for human protein phosphatase IIC ABI2 may be administered to a cell to stimulate cell proliferation, as described above.

In another further embodiment, a vector capable of expressing human protein phosphatase EC ABI2 or a portion or a biologically active variant thereof, may be administered to a cell or cells in vivo using delivery mechanisms, or to a cell to stimulate cell proliferation, as described above.

Increased expression of human protein phosphatase IIC ABI2 may be associated with increased cell proliferation. Therefore, in one embodiment, an antagonist of human protein phosphatase IIC ABI2 or a portion or a biologically active variant thereof may be administered to a subject to prevent or treat cancer. Cancer is a disease fundamentally caused by oncogenic cellular transformation. There are several hallmarks of transformed cells that distinguish them from their normal counterparts and underlie the pathophysiology of cancer. These include uncontrolled cellular proliferation, unresponsiveness to normal death-inducing signals (immortalization), increased cellular motility and invasiveness, increased ability to recrait blood supply through induction of new blood vessel formation (angiogenesis), genetic instability, and dysregulated gene expression. Narious combinations of these aberrant physiologies, along with the acquisition of drag-resistance frequently lead to an intractable disease state in which organ failure and patient death ultimately ensue.

Most standard cancer therapies target cellular proliferation and rely on the differential proliferative capacities between transformed and normal cells for their efficacy. This approach is hindered by the facts that several important normal cell types are also highly proliferative and that cancer cells frequently become resistant to ^"these agents. Thus, the therapeutic indices for tradϊtfonal anti-cancer therapies rarely exceed 2.0.

The advent of genomics-driven molecular target identification has opened up the possibility of identifying new cancer-specific targets for therapeutic intervention that will provide safer, more effective treatments for cancer patients. Thus, newly discovered tumor-associated genes and their products can be tested for their role(s) in disease and used as tools to discover and develop innovative therapies. Genes playing important roles in any of the physiological processes outlined above can be characterized as cancer targets.

Genes or gene fragments identified through genomics can readily be expressed in one or more heterologous expression systems to produce functional recombinant proteins. These proteins are characterized in vitro for their biochemical properties and then used as tools in high-throughput molecular screening programs to identify chemical modulators of their biochemical activities. Agonists and/or antagonists of target protein activity can be identified in this manner and subsequently tested in cellular and in vivo disease models for anti-cancer activity. Optimization of lead compounds with iterative testing in biological models and detailed pharmacokinetic and toxicological analyses form the basis for drag development and subsequent testing in humans. Cancers which can be treated according to the invention include, but are not limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, and particularly, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and utems. In one aspect, an antibody specific for human protein phosphatase IIC ABI2 may be used directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express human protein phosphatase IIC ABI2.

In still another embodiment, a vector expressing the complementary sequence or antisense of the polynucleotide encoding human protein phosphatase IIC ABI2 or a portion or biologically active variant thereof may be administered to a subject to prevent or treat a disorder associated with cell proliferation including, but not limited to, the types of cancer listed above.

In a further embodiment, an antagonist of human protein phosphatase EC ABI2 or a portion or a biologically active variant thereof may be administered to a subject to prevent or treat inflammation of any type and, in particular, that which results from a particular disorder or conditions. Such disorders and conditions associated with inflammation include, but are not limited to, Addison's disease, adult respiratory distress syndrome, allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, atrophic gastritis, glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritable bowel syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, and autoimmune thyroiditis; complications of cancer, hemodialysis, extracorporeal circulation; viral, bacterial, fungal, parasitic, protozoal, and helminthic infections and trauma. In one aspect, an antibody specific for human protein phosphatase EC ABI2 may be used directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express human protein phosphatase IIC ABI2.

In another further embodiment, a vector expressing the complementary sequence or antisense of the polynucleotide encoding human protein phosphatase IIC ABI2 or a portion or a biologically active variant thereof may be administered to a subject to prevent or treat inflammation of any type including, but not limited to, those listed above.

In other embodiments, any of the therapeutic proteins, antagonists, antibodies, agonists, complementary sequences or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

The invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an anti- sense nucleic acid molecule, a specific antibody, ribozyme, or a polypeptide-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein. A reagent which affects protein phosphatase IIC ABI2 activity can be administered to a human cell, either in vitro or in vivo, to reduce protein phosphatase IIC ABI2 activity. The reagent preferably binds to an expression product of a human protein phosphatase EC ABI2 gene. If the expression product is a polypeptide, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells which have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using -a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about 30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung or liver.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about

0.5 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and

400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid composition and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a tumor cell, such as a tumor cell ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods which are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al Trends in Biotechnol 11, 202-05 (1993); Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE

TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wu et al, J. Biol. Chem. 266, 338-42 (1991).

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases extracellular matrix degradation relative to that which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅o (the dose therapeutically effective in

50% of the population) and LD₅o (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect.

Factors which can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half- life and clearance rate of the particular formulation. Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about

50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about

100 μg of DNA.

If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides which express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a protein phosphatase IIC ABI2 polynucleotide or activity of a protein phosphatase IIC ABI2 polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a protein phosphatase IIC ABI2 polynucleotide or the activity of a protein phosphatase IIC ABI2 polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to protein phosphatase IIC ABI2-specific mRNA, quantitative RT-PCR, immunologic detection of a protein phosphatase EC ABI2 polypeptide, or measurement of protein phosphatase IIC ABI2 activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergis- tically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

The above disclosure generally describes the present invention, and all patents and patent applications cited in this disclosure are expressly incorporated herein. A more complete understanding can be obtained by reference to the following specific examples which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

EXAMPLE 1

Detection of protein phosphatase IICABI2 activity

The polynucleotide of SEQ ID NO. 1 is inserted into pGEX vector and expressed as a fusion protein with glutathione S-transferase. The fusion protein is purified from lysed cells by adsorption by glutathion-agarose-beads followed by elution in the presence of free glutathione. The activity of the fusion protein (protein phosphatase IIC ABI2 polypeptide of SEQ ) NO. 18) is assessed according to the following procedures: ^"~~

Phosphorylase kinase (EC 2.7.1.38), protein kinase A (3':5'-cyclic AMP dependent) phosphorylase b (EC 2.4.1.1), and crade histone (type 2AS) are obtained from Sigma Chemical Co. Okadaic acid can be obtained from a variety of commercial sources. Phosphohistone with a specific activity >4.5 x 10⁶ dpm/nmol incorporated phosphate is prepared by the phosphorylation of bovine brain histone (type 2AS from Sigma Chem. Co) with 3':5'-cAMP-dependent protein kinase (from rabbit muscle) in the presence of γ³²P-ATP essentially as described by Honkanen et al. (J. Biol. Chem. 265, 19401-04 (1990) and Mol. Pharmacol. 40, 577-83 (1991). The reaction is started by the addition of protein kinase A (1 mg) to a 20 mM Tris-buffer (pH 6.2) containing 20 mg of histone, 1 mCi γ³² P-ATP (150 μM ATP), 100 μM cAMP, 5 mM DTT, and 5 mM MgCl₂. The final volume is 4 ml, and the phosphorylation reaction is allowed to continue for 3.5 hours at 30°C.

The reaction is terminated by the addition of 1.3 ml of ice cold 100% trichloroacetic acid. After placing the tube in ice for 10 minutes, the precipitated phosphohistone is collected by centrifugation at 3000 x g for 5 minutes. The supernatant is discarded, and the pellet is redissolved in 4 ml of 0.8 M Tris-Cl (pH 8.5). Trichloroacetic acid (1.3 ml of 100%) w/v) is added to precipitate the phosphohistone a second time, and the precipitation-resuspension washing procedure is repeated 5 times. The pellet produced after the final trichloroacetic acid precipitation is washed 2 times with 4 ml of ethanokethyl ether (1:4; v/v) and then 2 additional times with 4 ml acidified ethanokethyl ether (1:4; 0.1 N HCl). The washed phoshohistone pellet is allowed to air dry and resuspended in 5 mM Tris HCl (pH 7.4).

Phosphorylase α is prepared essentially according to the methods described in Honkanen et al., Mol. Pharmacol. 40, 577-83 (1991). Briefly, ³² P-phosphorylase α is prepared by the phosphorylation of phosphorylase β with phosphorylase kinase using 30 mg of phosphorylase b, 1.4 mCi of γ³² P-ATP (to give 1 x 10⁴ cpm pmole^"1) and 100 U of phosphorylase kinase. The phosphorylation reaction is carried out for

1.5 hour at pH 8.2 and 30°C. After termination of the reaction, phosphorylase α is crystallized by adjustment of the pH to 6.8 and placing the mixture on ice. The crystals are collected by centrifugation and washed extensively with 20 mM Tris-HCL, 50 mM 2-mercaptoethanol, pH 6.8.

After washing, the crystals are dissolved by the addition of NaCl to achieve a final concentration of 100 mM. The solution is dialyzed overnight at 4 °C against 20 mM Tris-HCL, 50 mM 2-mercaptoethanol, pH 6.8. (2 x 4 liters). The phosphorylase α, which recrystalizes during dialysis, is redissolved in assay buffer containing 100 mM NaCl for immediate use or 100% glycerol for short term storage. This results in phosphorylase α with a specific activity of approximately 6 x 10 cpm/nmol of incorporated phosphate.

Determination of protein phosphatase activity. Protein phosphatase activity against phosphohistone is determined by the quantification of liberated P from phosphohistone according to previously established methods (see Honkanen et al., J. Biol. Chem. 265, 19401-04, 1990; Honkanen et al, Mol Pharmacol. 40, 577-83, 1991; Critz & Honkanen, Neuroprotocols 6, 78-83, 1995). Assays (80 μl final volume) are conducted in 50 mM Tris-buffer (pH 7.4) containing 0.5 mM DTT, 4 mM EDTA, and 2 μg fusion protein. The assay is initiated by the addition of substrate (30 μl) to a

1.5 ml microfuge tube containing 50 μl of dilute homogenate. Assays are conducted at 30°C for 10 minutes and are stopped by the addition of 100 μl of IN H₂SO₄ containing 1 mM K₂HPO₄.

³² P-Phosphate liberated by the enzyme is then extracted as a phosphomolybdate complex and measured according to the methods of Killilea et al, Arch. Biochem.

Biophys. 191, 638-46, 1978). Briefly, free phosphate is extracted by adding 20 μl of ammonium molybdate (7.5% w/v in 1.4 N H₂SO₄) and 250 μl of isobutanokbenzene (1:1, v/v) to each tube. The tubes are mixed vigorously for approximately 10 seconds followed by centrifugation at 14,000 x g for 2 minutes. Aliquots of the upper phase (100 μl) are removed for counting, and radioactivity is quantified with a scintillation counter. It is shown that the fusion protein (polypeptide of SEQ ED NO. 18) has protein phosphatase IIC ABI2 activity.

EXAMPLE 2

Expression of recombinant human protein phosphatase IICABI2

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of a recombinant human protein phosphatase EC ABI2 in yeast. The encoding DNA sequence is derived from the coding sequence shown in nucleotides 215-1270 of SEQ ID NO.17. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its 5 '-end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag, and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added.

After digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes, the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoris, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast. The yeast is cultivated under usual conditions in 5 liter shake flasks, and the recombinantly produced protein isolated from the culture by affinity chromatography (Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human protein phosphatase IIC ABI2 is obtained.

EXAMPLE 3

Identification of a test compound which binds to a protein phosphatase IIC ABI2 polypeptide

Purified protein phosphatase IIC ABI2 polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Protein phosphatase IIC ABI2 polypeptides comprise the amino acid sequence shown in SEQ ED NO.2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells. Binding of a test compound to a protein phosphatase IIC ABI2 polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound which increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound was not incubated is identified as a compound which binds to a protein phosphatase IIC ABI2 polypeptide. EXAMPLE 4

Measurement of Protein Phosphatase Activity and the Preparations of Phos- phoprotein Substrates

Phosphorylase kinase (EC 2.7.1.38), protein kinase A (3 ':5 '-cyclic AMP dependent) phosphorylase b (EC 2.4.1.1), and crade histone (type 2AS) are obtained from Sigma Chemical Co. Okadaic acid can be obtained from a variety of commercial sources. Phosphohistone with a specific activity >4.5 x 10⁶ dpm/nmol incorporated phosphate is prepared by the phosphorylation of bovine brain histone (type 2 AS from Sigma

Chem. Co) with 3':5'-cAMP-dependent protein kinase (from rabbit muscle) in the presence of γ³²P-ATP essentially as described by Honkanen et al. (J. Biol. Chem. 265, 19401-04 (1990) and Mol Pharmacol. 40, 577-83 (1991). The reaction is started by the addition of protein kinase A (1 mg) to a 20 mM Tris-buffer (pH 6.2) containing 20 mg of histone, 1 mCi γ³² P-ATP (150 μM ATP), 100 μM cAMP, 5 mM DTT, and 5 mM MgCl₂. The final volume is 4 ml, and the phosphorylation reaction is allowed to continue for 3.5 hours at 30°C.

The reaction is terminated by the addition of 1.3 ml of ice cold 100% trichloroacetic acid. After placing the tube in ice for 10 minutes, the precipitated phosphohistone is collected by centrifugation at 3000 x g for 5 minutes. The supernatant is discarded, and the pellet is redissolved in 4 ml of 0.8 M Tris-Cl (pH 8.5). Trichloroacetic acid (1.3 ml of 100% w/v) is added to precipitate the phosphohistone a second time, and the precipitation-resuspension washing procedure is repeated 5 times.

The pellet produced after the final trichloroacetic acid precipitation is washed 2 times with 4 ml of ethanohethyl ether (1:4; v/v) and then 2 additional times with 4 ml acidified ethanohethyl ether (1:4; 0.1 N HCl). The washed phoshohistone pellet is allowed to air dry and resuspended in 5 mM Tris HCl (pH 7.4). Phosphorylase α is prepared essentially according to the methods described in Honkanen et al, Mol. Pharmacol. 40, 577-83 (1991). Briefly, ³² P-phosphorylase α is prepared by the phosphorylation of phosphorylase β with phosphorylase kinase using 30 mg of phosphorylase b, 1.4 mCi of γ³² P-ATP (to give 1 x 10⁴ cpm pmole^"1) and 100 U of phosphorylase kinase. The phosphorylation reaction is carried out for 1.5 hour at pH 8.2 and 30 °C. After termination of the reaction, phosphorylase α is crystallized by adjustment of the pH to 6.8 and placing the mixture on ice. The crystals are collected by centrifugation and washed extensively with 20 mM Tris-HCL, 50 mM 2-mercaptoethanol, pH 6.8.

Determination of protein phosphatase activity. Protein phosphatase activity against phosphohistone is determined by the quantification of liberated P from phosphohistone according to previously established methods (see Honkanen et al., J.

Biol Chem. 265, 19401-04, 1990; Honkanen et al, Mol Pharmacol 40, 577-83,

1991; Critz & Honkanen, Neuroprotocols 6, 78-83, 1995). Assays (80 μl final volume) are conducted in 50 mM Tris-buffer (pH 7.4) containing 0.5 mM DTT, 4 mM EDTA, and phosphoprotein (2 μM PO₄). The assay is initiated by the addition of substrate (30 μl) to a 1.5 ml microfuge tube containing 50 μl of dilute homogenate.

Assays are conducted at 30°C for 10 minutes and are stopped by the addition of

100 μl of IN H₂SO₄ containing 1 mM K₂HPO₄.

³² P-Phosphate liberated by the enzyme is then extracted as a phosphomolybdate complex and measured according to the methods of Killilea et al, Arch. Biochem. Biophys. 191, 638-46, 1978). Briefly, free phosphate is extracted by adding 20 μl of ammonium molybdate (7.5% w/v in 1.4 N H SO₄) and 250 μl of isobutanokbenzene (1:1, v/v) to each tube. The tubes are mixed vigorously for approximately 10 seconds followed by centrifugation at 14,000 x g for 2 minutes. Aliquots of the upper phase (100 μl) are removed for counting, and radioactivity is quantified with a scintillation counter.

For inhibition studies, fostriecin or okadaic acid or a test compound is added to the enzyme mixture 10 minutes before the reaction is initiated with the addition of substrate. Controls receive solvent alone, and in all experiments the amount of enzyme is diluted to ensure that the samples are below the titration endpoint. The titration endpoint is defined as the concentration of enzyme after which further dilution no longer affects the IC₅o of the toxin, and represents a point where the concentration of enzyme used in the assay no longer approaches that of the toxin. This ensures that IC₅o represents the potency of the inhibitor alone and is not representative of a combination of potency of the toxin and titration artifacts of the assay system. Preliminary assays are performed to ensure the dephosphorylation reaction is linear with respect to enzyme concentration and time.

EXAMPLE 5

Identification of a test compound which decreases protein phosphatase IIC ABI2 activity

Cellular extracts from cells comprising human protein phosphatase IIC ABI2 are contacted with test compounds from a small molecule library and assayed for protein phosphatase IIC ABI2 activity. Control extracts, in the absence of a test compound, also are assayed. Human protein phosphatase IIC ABI2 activity can be measured, for example, as described in Example 3, above. A test compound which decreases protein phosphatase IIC ABI2 activity of the extract relative to the control extract by at least 20% is identified as a protein phosphatase IIC ABI2 inhibitor.

EXAMPLE 6

Identification of a test compound which decreases protein phosphatase IIC ABI2 gene expression

A test compound is administered to a culture of the breast tumor cell line MDA-468 and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells incubated for the same time without the test compound provides a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P -labeled protein phosphatase IIC ABI2-specific probe at 65°C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of nucleotides 215-1270 of SEQ ED NO.17. A test compound which decreases the protein phosphatase IIC ABI2 -specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of protein phosphatase IIC ABI2 gene expression.

EXAMPLE 7

Inhibition of apoptosis by inhibiting human protein phosphatase IIC AB12 with antisense oligonucleotides

The cell line used for testing is the human colon cancer cell line HCT116. Cells are cultured in RPMI-1640 with 10-15% fetal calf serum at a concentration of 10,000 cells per milliliter in a volume of 0.5 ml and kept at 37°C in a 95% air/5% CO₂ atmosphere. Phosphorothioate oligoribonucleotides are synthesized on an Applied Biosystems Model 380B DNA synthesizer using phosphoroamidite chemistry. The test oligonucleotide is a sequence of 24 bases: 5'-CAG-AGT-TGT-CGA-CGG-AAT- TAA-TGA-3 ' (complementary to the nucleotides at positions 1 -24 of SEQ ED NO.1).

As a control, another (random) sequence 5'-TCA-ACT-GAC-TAG-ATG-TAC-ATG- GAC-3 ' is used. Following assembly and deprotection, oligonucleotides are ethanol- precipitated twice, dried, and suspended in phosphate buffered saline at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electro- phoresis and ion exchange HPLC. The purified oligonucleotides are added to the culture medium at a concentration of 10 μM.

The addition of the test oligonucleotide for seven days results in significantly reduced expression of the phosphatase IIC AB12 as determined by Western blotting. This effect is not observed with the control oligonucleotide. After 3 to 7 days, the number of cells is counted using an automatic cell counter. The microscopic appearance of cells in cultures treated with the control oligonucleotide is compared with the microscopic appearance of cells in cultures treated with the test oligonucleotide. Nuclei of cells in the test dishes are largely intact, whereas in the control dishes, changes in morphology characteristic of apoptosis has occurred, indicating that inhibition of human protein phosphatase IIC AB12 prevents apoptosis.

EXAMPLE 8

Treatment of breast cancer with a reagent which specifically binds to a protein phosphatase IICABI2 gene product

Synthesis of antisense protein phosphatase IIC ABI2 oligonucleotides comprising at least 11 contiguous nucleotides selected from the complement of nucleotides 215-

1270 of SEQ ED NO.17 is performed on a Pharmacia Gene Assembler series synthesizer using the phosphoramidite procedure (Uhlmann et al, Chem. Rev. 90, 534-83, 1990). Following assembly and deprotection, oligonucleotides are ethanol- precipitated twice, dried, and suspended in phosphate-buffered saline (PBS) at the desired concentration. Purity of these oligonucleotides is tested by capillary gel electrophoreses and ion exchange HPLC. Endotoxin levels in the oligonucleotide preparation are determined using the Limulus Amebocyte Assay (Bang, Biol. Bull. (Woods Hole, Mass.) 105, 361-362, 1953).

An aqueous composition containing the antisense oligonucleotides at a concentration of 0.1-100 μM is administered directly to a patient's breast tumor by injection. The size of the tumor is thereby decreased.

EXAMPLE 9

Tissue-specific expression of protein phosphatase IIC ABI2 - I.

To establish a role for protein phosphatase IIC ABI2 in the pathogenesis of COPD, expression profiling of the gene was done using real-time quantitative PCR with RNA samples from human respiratory tissues and inflammatory cells relevant to COPD. The panel consisted of total RNA samples lung (adult and fetal), trachea, freshly isolated alveolar type II cells, cultured human bronchial epithelial cells, cultured small airway epithelial cells, cultured bronchial sooth muscle cells, cultured H441 cells (Clara-like), freshly isolated neutrophils and monocytes, and cultured monocytes (macrophage-like). Expression of protein phosphatase IIC ABI2 also was evaluated in a range of human tissues using total RNA panels obtained from

Clontech Laboratories, UK, Ltd.. The tissues were adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, mammary gland, pancreas, prostate, salivary gland, skeletal muscle, small infesting, spleen, stomach, testis, thymus, trachea, thyroid, and uterus. Real-time quantitative PCR. Expression profiling of the target gene was performed using real-time quantitative PCR, a development of the kinetic analysis of PCR first described in Higuchi et al., BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.

PCR amplification is performed in the presence of an oligonucleotide probe (TaqMan probe) that is complementary to the target sequence and labeled with a fluorescent reporter dye and a quencher dye. During the extension phase of PCR, the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase, releasing the fluorophore from the effect of the quenching dye (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission increases in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 6, 995-1001, 1996).

Real-time quantitative PCR was done using an ABI Prism 7700 Sequence Detector. The C_T value generated for each reaciton was used to determine the initial template concentration (copy number) by interpolation from a universal standard curve. The level of expression of the target gene in each sample was calculated relative to the sample with the lowest expression of the gene.

RNA extraction and cDNA preparation. Total RNA from each of the respiratory tissues and inflammatory cell types listed above were isolated using Qiagen' s RNeasy system according to the manufacturer's protocol (Crawley, West Sussex, UK). The concentration of purified RNA was determined using a RiboGreen RNA quantitation kit (Molecular Probes Europe, The Netherlands). For the preparation of cDNA, 1 μg of total RNA was reverse transcribed in a final volume of 20 μl, using

200 U of SUPERSCRIPT™ RNase H^" Reverse Transcriptase (Life Technologies, Paisley, UK), 10 mM dithiothreitol, 0.5 mM of each dNTP and 5 μM random hexamers (Applied Biosystems, Warrington, Cheshire, UK) according to the manufacturer's protocol.

TaqMan quantitative analysis. Specific primers and probe were designed according to the recommendations of PE Applied Biosystems; a FAM (6-carboxy-fluorescein)- labeled probe was used.

Quantification PCR was performed with 5 ng of reverse transcribed RNA from each sample. Each determination is done in duplicate.

The assay reaction mix was as follows: IX final TaqMan Universal PCR Master Mix (from 2X stock) (PE Applied Biosystems, CA); 900 nM forward primer; 900 nM reverse primer; 200 nM probe; 5 ng cDNA; and water to 25 μl.

Each of the following steps were carried out once: pre PCR, 2 minutes at 50°C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation, 15 seconds at 95°C, annealing/extension, 1 minute at 60°C.

All experiments were performed using an ABI Prism 7700 Sequence Detector (PE

Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR were processed as described in the ABI Prism 7700 user's manual to achieve better background subtraction as well as signal linearity with the starting target quantity.

Tables 1 and 2 show the results of expression profiling for protein phosphatase EC ABI2 using the indicated cell and tissue samples. For Table 1, the cells are defined as follows: HBEC, cultured human bronchial epithelial cells; H441, a Clara-like cell line; SAE, cultured small airway epithelial cells; SMC, cultured airway smooth muscle cells; All, freshly isolated human alveolar type II cells; Neut, freshly isolated circulating neutrophils; Mono, freshly isolated monocytes; and CM, cultured monocytes. Other letters identify the donor. The results are shown graphically in FIGS. 22 and 23.

Table 1

Tissue Relative expression

Adrenal gland 31.53247064

Bone Marrow 159.4391609

Brain 806.1781069

Colon 70.67991198

Heart 65.48985065

HL60 1

Kidney 182.204039

Liver 15.08627431

Lung 335.376015

Mammary gland 44.44326944

Pancreas 160.4556448

Prostate 723.6173978

Salivary gland 335.376015

Skeletal Muscle 155.4368548

Sm Intest 49.20032822

Spleen 361.9536829

Stomach 157.425108

Testis 1195.531435

Thymus 547.0940606

Thyroid 150.5750444

Uterus 12.78842321 Table 2

Tissue Relative expression

Lung 3801.084988

Trachea 1114.806725

HBEC 1 104.8155168

HBEC 2 14.2475628

H441 1455.88

SMC 50.78899042

SAE 205.5898076

AE 405.822928

Foetal lung 609.516659

COPD Neut 1 2.964724757

COPD Neut 2 1

COPD Neut 4 1.25708713

GAP Neut 40.14621259

AEM Neut 2.529186063

AT Neut 8.845646508

KNNeut 1.888053864

SM Mono 12.00097635

DLF Mono 22.80297862

DS Mono 79.75163218

RLH CM 26.3921384

CTP CM 26.3921384 EXAMPLE 10

Tissue-specific expression of protein phosphatase IICABI2 — II.

Expression profiling is based on a quantitative polymerase chain reaction (PCR) analysis, also called kinetic analysis, first described in Higuchi et al., 1992 and Higuchi et al., 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies. Using this technique, the expression levels of particular genes, which are transcribed from the chromosomes as messenger RNA (mRNA), are measured by first making a DNA copy (cDNA) of the mRNA, and then performing quantitative PCR on the cDNA, a method called quantitative reverse transcription-polymerase chain reaction (quantitative RT-PCR).

Quantitative RT-PCR analysis of RNA from different human tissues was performed to investigate the tissue distribution of protein phosphatase IIC ABI2. 25 μg of total RNA from various tissues (Human Total RNA Panel I-V, Clontech Laboratories, Palo Alto, CA, USA) was used as a template to synthsize first-strand cDNA using the SUPERSCRIPT™ First-Strand Synthesis System for RT-PCR (Life Technologies,

Rockville , MD, USA). First-strand cDNA synthesis was carried out according to the manufacturer's protocol using oligo (dT) to hybridize to the 3' poly A tails of mRNA and prime the synthesis reaction. 10 ng of the first-strand cDNA was then used as template in a polymerase chain reaction. The polymerase chain reaction was performed in a LightCycler (Roche Molecular Biochemicals, Indianapolis, IN, USA), in the presence of the DNA-binding fluorescent dye SYBR Green I which binds to the minor groove of the DNA double helix, produced only when double-stranded DNA is successfully synthesized in the reaction (Morrison et al., 1998). Upon binding to double-stranded DNA, SYBR Green I emits light that can be quantitatively measured by the LightCycler machine. The polymerase chain reaction was carried out using oligonucleotide primers ABI2-L1 (SEQ ID NO.20) and ABI2- R2 (SEQ ID NO.21) and measurements of the intensity of emitted light were taken following each cycle of the reaction when the reaction had reached a temperature of 84°C. Intensities of emitted light were converted into copy numbers of the gene transcript per nanogram of template cDNA by comparison with simultaneously reacted standards of known concentration.

To correct for differences in mRNA transcription levels per cell in the various tissue types, a normalization procedure was performed using similarly calculated expression levels in the various tissues of five different housekeeping genes: glyceraldehyde-3-phosphatase (G3PDH), hypoxanthine guanine phophoribosyl transferase (HPRT), beta-actin, porphobilinogen deaminase (PBGD), and beta-2- microglobulin. The level of housekeeping gene expression is considered to be relatively constant for all tissues (Adams et al., 1993, Adams et al., 1995, Liew et al., 1994) and therefore can be used as a gauge to approximate relative numbers of cells per .mu.g of total RNA used in the cDNA synthesis step. Except for the use of a slightly different set of housekeeping genes and the use of the LightCycler system to measure expression levels, the normalization procedure was essentially the same as that described in the RNA Master Blot User Manual, Apendix C (1997, Clontech Laboratories, Palo Alto, CA, USA). In brief, expression levels of the five housekeeping genes in all tissue samples were measured in three independent reactions per gene using the LightCycler and a constant amount (25 .mu.g) of starting RNA. The calculated copy numbers for each gene, derived from comparison with simultaneously reacted standards of known concentrations, were recorded and converted into a percentage of the sum of the copy numbers of the gene in all tissue samples. Then for each tissue sample, the sum of the percentage values for each gene was calculated, and a normalization factor was calculated by dividing the sum percentage value for each tissue by the sum percentage value of one of the tissues arbitrarily selected as a standard. To normalize an experimentally obtained value for the expression of a particular gene in a tissue sample, the obtained value was multiplied by the normalization factor for the tissue tested. Results are given in FIG. 26, showing the experimentally obtained copy numbers of mRNA per 10 ng of first-strand cDNA on the left and the normalized values on the right. RNAs used for the cDNA synthesis, along with their supplier and catalog numbers are shown in Table 3.

Table 3 Whole-body-screen tissues

>ABI2-Ll:acccatgaagctgaccattgacca >ABI2-R2: ttccatctgtggtgaggaccagga References

Higuchi, R., Dollinger, G., Walsh, P.S. and Griffith, R. (1992) Simultaneous amplification and detection of specific DNA sequences. BioTechnology 10:413-417.

Higuchi, R., Fockler, C, Dollinger, G. and Watson, R. (1993) Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. BioTechnology 11:1026-1030.

T.B. Morrison, J.J. Weis & CT. Wittwer .(1998) Quantification of low-copy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954-962.

Adams, M. D., Kerlavage, A. R., Fields, C. & Venter, C. (1993) 3,400 new expressed sequence tags identify diversity of transcripts in human brain. Nature Genet. 4:256-

265.

Adams, M. D., et al. (1995) Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence. Nature 377 suρp:3-174.

Liew, C. C, Hwang, D. M., Fung, Y. W., Laurenson, C, Cukerman, E., Tsui, S. & Lee, C. Y. (1994) A catalog of genes in the cardiovascular system as identified by expressed sequence tags. Proc. Natl. Acad. Sci. USA 91:10145-10649.

Claims

1. An isolated polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide and being selected from the group consisting of:

a) a polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide comprising an amino acid sequence selected form the group consisting of:

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 2; the amino acid sequence shown in SEQ ID NO.

2; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ED NO. 18; the amino acid sequence shown in SEQ ID NO. 18; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 19; the amino acid sequence shown in SEQ ED NO. 19. a polynucleotide comprising the sequence of SEQ ED NO. 1;

d) a polynucleotide the sequence of which deviates from the poly- nucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code; and

e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a to (d).

An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified protein phosphatase IIC ABI2 polypeptide encoded by a polynucleotide of claim 1.

5. A method for producing a protein phosphatase IIC ABI2 polypeptide, wherein the method comprises the following steps:

a) culturing the host cell of claim 3 under conditions suitable for the expression of the protein phosphatase IIC ABI2 polypeptide; and

b) recovering the protein phosphatase EC ABI2 polypeptide from the host cell culture.

6. A method for detection of a polynucleotide encoding a protein phosphatase IIC ABI2 polypeptide in a biological sample comprising the following steps:

a) hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and .

b) detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a protein phosphatase IIC ABI2 polypeptide of claim 4 comprising the steps of: contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the protein phosphatase EC ABI2 polypeptide.

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

5

10. A method of screening for agents which decrease the activity of a protein phosphatase IIC ABI2, comprising the steps of:

contacting a test compound with any protein phosphatase EC ABI2 10. polypeptide encoded by any polynucleotide of claiml ;

detecting binding of the test compound to the protein phosphatase EC ABI2 polypeptide, wherein a test compound wliich binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a 15 protein phosphatase IIC ABI2.

11. A method of screening for agents which regulate the activity of a protein phosphatase EC ABI2, comprising the steps of:

20 contacting a test compound with a protein phosphatase IIC ABI2 polypeptide encoded by any polynucleotide of claim 1; and

detecting a protein phosphatase IIC ABI2 activity of the polypeptide, wherein a test compound which increases the protein phosphatase IIC ABI2 activity is

25 identified as a potential therapeutic agent for increasing the activity of the protein phosphatase IIC ABI2, and wherein a test compound which decreases the protein phosphatase IIC ABI2 activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the protein phosphatase IIC ABI2.

30

12. A method of screening for agents which decrease the activity of a protein phosphatase EC ABI2, comprising the steps of:

contacting a test compound with any polynucleotide of claim 1 and detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of protein phosphatase EC ABI2.

13. A method of reducing the activity of protein phosphatase EC ABI2, comprising the steps of:

contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any protein phosphatase IIC ABI2 polypeptide of claim 4, whereby the activity of protein phosphatase IIC ABI2 is reduced.

14. A reagent that modulates the activity of a protein phosphatase EC ABI2 polypeptide or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A pharmaceutical composition, comprising:

the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the pharmaceutical composition of claim 15 for modulating the activity of a protein phosphatase EC ABI2 in a disease.

17. Use of claim 16 wherein the disease is Asthma, COPD, peripheral or central nervous system disease including neurodenerative disease, a disorder associated with an increase in apoptosis, including AIDS and other infectious or genetic immunodeficiency, myelodysplasia, ischemic injury, toxin-induced disease, wasting disease, viral infection and osteoporosis.

18. Use of claim 16 wherein the disease is disorder associated with a decrease in apoptosis, including cancer and inflammatory disorder.

19. A cDNA encoding a polypeptide comprising the amino acid sequence shown in SEQ ED NO.2, 18, 19.

20. The cDNA of claim 19 which comprises SEQ ED NO.1.

21. The cDNA of claim 19 which consists of SEQ ID NO. 1.

22. An expression vector comprising a polynucleotide which encodes a polypeptide comprising the amino acid sequence shown in SEQ ED NO. 2, 18,

19.

23. The expression vector of claim 22 wherein the polynucleotide consists of SEQ ID NO. 1.

24. A host cell comprising an expression vector which encodes a polypeptide comprising the amino acid sequence shown in SEQ ED NO. 2, 18, 19.

25. The host cell of claim 24 wherein the polynucleotide consists of SEQ ED NO. 1.

26. A purified polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2, 18, 19.

27. The purified polypeptide of claim 26 which consists of the amino acid sequence shown in SEQ ID NO. 2, 18, 19.

28. A fusion protein comprising a polypeptide having the amino acid sequence shown in SEQ ED NO. 2, 18, 19.

29. A method of producing a polypeptide comprising the amino acid sequence shown in SEQ ED NO. 2, 18, 19 comprising the steps of:

culturing a host cell comprising an expression vector which encodes the polypeptide under conditions whereby the polypeptide is expressed; and

isolating the polypeptide.

30. The method of claim 29 wherein the expression vector comprises SEQ ED NO. l.

31. A method of detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2, 18, 19 comprising the steps of:

hybridizing a polynucleotide comprising 11 contiguous nucleotides of SEQ ID NO.l, 3, 5, 7, 9, 11, 13, or 17 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and

detecting the hybridization complex.

32. The method of claim 31 further comprising the step of amplifying the nucleic acid material before the step of hybridizing.

33. A kit for detecting a coding sequence for a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2, 18, 19 comprising: a polynucleotide comprising 11 contiguous nucleotides of SEQ ED NO. 1, 3, 5, 7, 9, 11, 13, or 17; and

instructions for the method of claim 31.

34. A method of detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2, 18, 19, comprising the steps of:

contacting a biological sample with a reagent that specifically binds to the polypeptide to form a reagent-polypeptide complex; and

detecting the reagent-polypeptide complex.

35. The method of claim 34 wherein the reagent is an antibody.

36. A kit for detecting a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2, 18, 19, comprising:

an antibody which specifically binds to the polypeptide; and

instructions for the method of claim 34.

37. A method of screening for agents which can modulate the activity of a human protein phosphatase IIC ABI2, comprising the steps of:

contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 15, 16, 18, and 19 and (2) the amino acid sequence shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 15, 16, 18 and 19; and detecting binding of the test compound to the polypeptide, wherein a test compound which binds to the polypeptide is identified as a potential agent for regulating activity of the human protein phosphatase EC ABI2.

38. The method of claim 37 wherein the step of contacting is in a cell.

39. The method of claim 37 wherein the cell is in vitro.

40. The method of claim 37 wherein the step of contacting is in a cell-free system.

41. The method of claim 37 wherein the polypeptide comprises a detectable label.

42. The method of claim 37 wherein the test compound comprises a detectable label.

43. The method of claim 37 wherein the test compound displaces a labeled ligand which is bound to the polypeptide.

44. The method of claim 37 wherein the polypeptide is bound to a solid support.

45. The method of claim 37 wherein the test compound is bound to a solid support.

46. A method of screening for agents which modulate an activity of a human protein phosphatase IIC ABI2, comprising the steps of:

contacting a test compound with a polypeptide comprising an amino acid sequence selected from the group consisting of: (1) amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 15, 16, 18 and 19 and (2) the amino acid sequence shown in SEQ ED NO. 2, 4, 6, 8, 10, 12, 14, 15, 16, 18 and 19, and

detecting an activity of the polypeptide, wherein a test compound which increases the activity of the polypeptide is identified as a potential agent for increasing the activity of the human protein phosphatase EC ABI2, and wherein a test compound which decreases the activity of the polypeptide is identified as a potential agent for decreasing the activity of the human protein phosphatase IIC ABI2.

47. The method of claim 46 wherein the step of contacting is in a cell.

48. The method of claim 46 wherein the cell is in vitro.

49. The method of claim 46 wherein the step of contacting is in a cell-free system.

50. A method of screening for agents which modulate an activity of a human protein phosphatase IIC ABI2, comprising the steps of:

contacting a test compound with a product encoded by a polynucleotide which comprises the nucleotide sequence shown in SEQ ED NO. 1, 3, 5, 7, 9, 11, 13 or 17; and

detecting binding of the test compound to the product, wherein a test compound which binds to the product is identified as a potential agent for regulating the activity of the human protein phosphatase IIC ABI2.

51. The method of claim 50 wherein the product is a polypeptide.

52. The method of claim 50 wherein the product is RNA.

53. A method of reducing activity of a human protein phosphatase IIC ABI2, comprising the step of:

contacting a cell with a reagent which specifically binds to a product encoded by a polynucleotide comprising the nucleotide sequence shown in SEQ ED NO. 1, 3, 5, 7, 9, 11, 13 or 17, whereby the activity of a human protein phosphatase IIC ABI2 is reduced.

54. The method of claim 53 wherein the product is a polypeptide.

55. The method of claim 54 wherein the reagent is an antibody.

56. The method of claim 53 wherein the product is RNA.

57. The method of claim 56 wherein the reagent is an antisense oligonucleotide.

58. The method of claim 57 wherein the reagent is a ribozyme.

59. The method of claim 53 wherein the cell is in vitro.

60. The method of claim 53 wherein the cell is in vivo.

61. A pharmaceutical composition, comprising:

a reagent which specifically binds to a polypeptide comprising the amino acid sequence shown in SEQ ED NO. 2, 4, 6, 8, 10, 12, 14, 15, 16, 18 and 19, and

a pharmaceutically acceptable carrier.

62. The pharmaceutical composition of claim 61 wherein the reagent is an antibody.

63. A pharmaceutical composition, comprising:

a reagent which specifically binds to a product of a polynucleotide comprising the nucleotide sequence shown in SEQ ED NO. 1, 3, 5, 7, 9, 11, 13 or 17; and

a pharmaceutically acceptable carrier.

64. The pharmaceutical composition of claim 63 wherein the reagent is a ribozyme.

65. The pharmaceutical composition of claim 63 wherein the reagent is an antisense oligonucleotide.

66. The pharmaceutical composition of claim 63 wherein the reagent is an antibody.

67. A pharmaceutical composition, comprising:

an expression vector encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 15, 16, 18 and 19, and

a pharmaceutically acceptable carrier.

68. The pharmaceutical composition of claim 67 wherein the expression vector comprises SEQ ID NO. 1, 3, 5, 7, 9, 11, 13 or 17.

69. A method of treating a protein phosphatase EC ABI2 dysfunction related disease, comprising the step of:

administering to a patient in need thereof a therapeutically effective dose of a reagent that modulates a function of a human protein phosphatase EC ABI2, whereby symptoms of the protein phosphatase IIC ABI2 dysfunction related disease are ameliorated.

70. The method of claim 69 wherein the reagent is identified by the method of claim 37.

71. The method of claim 69 wherein the reagent is identified by the method of claim 46.

72. The method of claim 69 wherein the reagent is identified by the method of claim 50.

73. The method of claim 69 wherein the disease is Asthma, COPD, peripheral or central nervous system disease including neurodenerative disease, a disorder associated with an increase in apoptosis, including AEDS and other infectious or genetic immunodeficiency, myelodysplasia, ischemic injury, toxin-induced disease, wasting disease, viral infection and osteoporosis.

74. The method of claim 69 wherein the disease is disorder associated with a decrease in apoptosis, including cancer and inflammatory disorder.