EP1181306A1 - Compounds and methods for regulating apoptosis, and methods of making and screening for compounds that regulate apoptosis - Google Patents
Compounds and methods for regulating apoptosis, and methods of making and screening for compounds that regulate apoptosisInfo
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- EP1181306A1 EP1181306A1 EP00935853A EP00935853A EP1181306A1 EP 1181306 A1 EP1181306 A1 EP 1181306A1 EP 00935853 A EP00935853 A EP 00935853A EP 00935853 A EP00935853 A EP 00935853A EP 1181306 A1 EP1181306 A1 EP 1181306A1
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- acid sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4747—Apoptosis related proteins
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates generally to the field of cell physiology, and more particularly, to apoptosis or programmed cell death, a process whereby developmental or environmental stimuli activate a genetically programmed cascade of events that results in cell death. Specifically, the invention relates to the regulation of apoptosis, including the regulation of apoptosis resulting in cell survival, compounds therefor, and methods of making and screening for such compounds.
- the invention relates to mutants of BC1-X L /BC1-2 Associated Cell Death Regulator polypeptides ("BAD”), methods of making such mutants, and methods of screening for compounds that promote, induce, inhibit, or modulate apoptosis, or promote, induce, inhibit, or modulate cell survival.
- BAD BC1-X L /BC1-2 Associated Cell Death Regulator polypeptides
- apoptosis is one manner in which cells that are no longer needed or that no longer function normally can be eliminated.
- Apoptosis is believed to be a process actively regulated by the environment in which cells live. This process is critical to the normal development of all multicellular organisms and to the maintenance of homeostasis within such organisms (Raff, 1992). Moreover, apoptosis is vital in the defense against viral infection and in preventing the development of carcinogenesis. Amongst multicellular organisms, the apoptotic pathway leading to cell death is highly conserved (Hengartner, 1994).
- apoptotic pathway Many molecules that regulate the apoptotic pathway have been identified, including both positive regulators (agonists) and negative regulators (antagonists). Such regulators are often members of the same family of polypeptides, and can have roles important in the extracellular, cell surface, and/or intracellular steps of the apoptotic pathway (Oltvai and
- Bcl-2 family One such family of polypeptides, constituting an intracellular checkpoint in the apoptotic pathway, is the Bcl-2 family of polypeptides ("Bcl-2 family").
- This important family of apoptotic regulators can be divided into two classes: those that suppress cell death (apoptotic antagonists) (e.g., Bcl-2, BC1-X L , MCL-1, and Al) and those that appear to promote apoptosis (apoptotic agonists) (e.g., BAX, BAK, Bcl-Xs, and BAD).
- Bcl-2 a cell death inhibitor encoded by the bcl-2 proto-oncogene, initially isolated from cells of a follicular lymphoma (Bakhshi et al., 1985; Tsujimoto et al, 1985; Cleary and Sklar, 1985).
- Bcl-2 is a 26 kD integral membrane polypeptide localized to the mitochondria that extends or promotes the survival of many different cell types by inhibiting apoptosis induced by a variety of cell death-inducing stimuli
- the Bcl-2 family contains members that are structurally and functionally related to
- Bcl-2 is defined by polypeptides having amino acid sequence homology to one or more of four conserved motifs, termed Bcl-homology (BH1, BH2, BH3, and BH4) domains (for reviews see Reed, 1997 and Chittenden, 1998).
- Bcl-homology domains have been shown to be important in the formation of homodimers and heterodimers, both among and between Bcl-2 family members.
- BAD BC1-X /BC1-2 Associated Cell Death Regulator polypeptide
- Bcl-2 only within the BH3 domain.
- BAD is an unique pro-apoptotic member of the Bcl-2 family in that its function is regulated by phosphorylation (Yang et al, 1995), suggesting an important connection between extracellular apoptosis regulatory agents, intracellular signaling pathways and the function of this Bcl-2 family member.
- BAD is believed to play a role in the apoptotic signaling pathway through an association with Bcl-2 family members, chiefly the cell death inhibitors BC1-X and Bcl-2 (Yang et al, 1995).
- BC1-X and Bcl-2 Upon being dephosphorylated, BAD is active and forms heterodimers with BC1-X L and Bcl-2, thereby displacing BAX and promoting cell death.
- the death-promoting activity of BAD can be inhibited by the phosphorylation of either of two serine residues, corresponding to the serines at position 112 and position 136 in the amino acid sequence of murine BAD (SEQ ID NO:2).
- SEQ ID NO:2 The amino acid sequence of murine BAD
- BAD no longer binds BC1-X L or Bcl-2 and instead, is thought to be bound by the phosphoserine-binding protein 14-3-3, thereby allowing Bcl-X L and Bcl-2 to perform their anti-apoptotic functions (Zha et al, 1996).
- polypeptide was labeled with ⁇ 3 P-ATP in vitro and used as a probe to screen an oligo (dT)-
- the members of the 14-3-3 family are highly conserved and ubiquitously expressed. They bind to and regulate a variety of proteins, including a number of proteins involved in signal transduction. Family members recognize and bind sequences containing a conserved phosphoserine motif (Muslin et al,
- Muslin et al. identified a number of polypeptides, including BAD, that contain this motif and postulated that if these other polypeptides were appropriately phosphorylated, 14-3-
- BAD is the only known pro-apoptotic member of the Bcl-2 family whose function is regulated by phosphorylation.
- the serine/threonine kinase Akt a downstream effector of PI 3-kinase, phosphorylates murine BAD on the serine at position 136 (also called “Serl36” or "serine- 136"), thereby preventing murine BAD from associating with Bcl-2 or Bcl-X L , and freeing these proteins to promote cell survival.
- Serl36 serine/threonine kinase
- Serl 12 or "serine-112"
- Serl36 Although phosphorylation of Serl36 was sufficient to prevent BAD from binding to Bcl-X L , phosphorylation of Serl 12 appeared critical for cellular survival in some cell types but not in others (Zha et al, 1996; Datta et al, 1997).
- Several candidate enzymes have been proposed to be responsible for the phosphorylation of Serl 12, including PKA, c-Raf, and MEK (Harada et al, 1999; Wang and Reed, 1998; Scheid and
- Akt does not appear to phosphorylate BAD on Serl 12.
- Some disease conditions may be related to the development of a defective down- regulation (i.e., inhibition or modulation) of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals and apoptosis is thereby down- regulated.
- DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host's cellular machinery to drive their own replication and at the same time inhibit or modulate apoptosis, thereby repressing cell death and allowing the host cell to continue reproducing the virus.
- certain other disease conditions such as lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases, and cancers, including drug-resistant cancers, may result from a down-regulation (e.g., inhibition or modulation) of apoptosis. In such disease conditions it would be desirable to promote or induce apoptosis.
- BAD could be altered to promote or induce its binding to Bcl-X L and/or Bcl-2 and, thereby, diminish (or inhibit or modulate) the cell-survival promoting activity of these cell death inhibitors.
- BAD could be altered to block its ability to bind BC1-X L and/or Bcl-2 and, thereby, promote or induce the cell death repressor, or anti-apoptotic, activity of these cell death inhibitors.
- the present invention relates to the discovery of a novel phosphorylation site of BAD that is regulated differentially from other known phosphorylation events of BAD.
- the novel phosphorylation site is at the serine at position 155 (also called “Serl55” or “serine-155") of SEQ ID NO:2 of a murine BAD ("longer murine BAD") that corresponds to the serine at position 118 (also called “Serl 18" and “serine- 118") of SEQ ID NO:l of a human BAD, and the serine at position 113 (also called “Serl 13" or Serine- 113") of SEQ ID NO:3 of a murine BAD ("shorter murine BAD").
- a particularly significant aspect of the present invention relates to the discovery that the phosphorylation of BAD at the novel phosphorylation site renders BAD unable to bind to Bcl-X L , and promotes, induces, or modulates cell survival (and/or inhibits or modulates apoptosis). Moreover, the phosphorylation of BAD at the novel site is not dependent on the activation of PI 3-kinase and Akt. Rather, the phosphorylation of BAD at the novel phosphorylation site is regulated by the cAMP-dependent protein kinase ("PKA").
- PKA cAMP-dependent protein kinase
- the phosphorylation of endogenous BAD at Serl55 in Rat-1 fibroblasts and several tumor cell lines, such as A275 and A431, can be induced by growth factors at physiological levels in a PKA-dependent manner, but not in a PI 3-kinase dependent and Akt-dependent manner. Consistent with these findings, cells treated with L-epinephrine, a G-protein-coupled receptor ligand that induces elevation of intracellular cAMP levels, exhibit phosphorylation of BAD at Serl55.
- the present invention relates to the discovery that, both in vitro and in vivo, Serl 55 is the only major site of BAD phosphorylation by PKA, and that the phosphorylation of BAD at Serl 55 contributes significantly to the overall phosphorylation of BAD.
- the present invention further relates to the discovery that BAD, having a mutation wherein Serl 55 is changed to an alanine, promotes, agonizes, induces or modulates apoptotic activity in HeLa cells, as compared to the naturally-occurring or wild-type mammalian BAD.
- BAD having a mutation wherein Serl 55 is changed to an alanine
- promotes, agonizes induces or modulates apoptotic activity in HeLa cells, as compared to the naturally-occurring or wild-type mammalian BAD.
- the elevation of the level of intracellular cAMP promotes, induces, or modulates cell survival (and/or inhibits or modulates apoptosis) in HeLa cells expressing the naturally- occurring or wild-type mammalian BAD, but not in HeLa cells expressing the BAD having a mutation at Serl 55.
- the change of serine 155 to an aspartic acid which mimics the phospho-Serl55 BAD, showed
- the object of the present invention is to provide compositions and methods that regulate apoptosis and/or cell survival (apoptotic agonists and antagonists), for example, by promoting, inducing, inhibiting, or modulating apoptosis, or promoting, inducing, inhibiting, or modulating cell survival, and to provide methods of making and screening for such compositions.
- apoptosis and/or cell survival apoptotic agonists and antagonists
- an object of the present invention is to provide novel compounds and methods that can alter the phosphorylation of BAD and/or binding of BAD to members of the Bcl-2 family, such as BC1-X L and Bcl-2, and thereby regulate apoptosis and/or cell survival, for example, by promoting, inducing, inhibiting, or modulating apoptosis, or promoting, inducing, inhibiting, or modulating cell survival, and to provide methods of making and screening for such compounds.
- Another object of the present invention is to provide methods that utilize such novel compounds as a basis for treatment of disease conditions involving either inappropriate inhibition or inappropriate acceleration of cell death.
- novel compounds include, for example, polypeptides and polynucleotides, fragments of full-length polypeptides and polynucleotides, including mutants and homologs thereof, and chemical compounds, including peptide mimetics.
- An embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of said isolated or synthetic polypeptides comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments wherein the amino acid sequence of said mutant BAD, or said fragment, is identical to or substantially identical to either SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does not contain a serine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO :1, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of the mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- an embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity, and/or having the ability to bind BC1-X L and/or Bcl-2.
- the binding of said isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or said fragments may occur, for example, through a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD.
- the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments is derived from a naturally-occurring or wild-type mammalian BAD, contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does not contain a serine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- An embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments is derived from a naturally-occurring or wild-type mammalian BAD, contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and contains an alanine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of the mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- An embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments is derived from a naturally-occurring or wild-type mammalian BAD, contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does not contain a glycine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of the mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- An embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments is derived from a naturally-occurring or wild-type mammalian BAD, contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does not contain an alanine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively. More particularly, an embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments comprises the amino acid sequence corresponding to positions 103-123 of SEQ ID NO:l, positions 140-160 of SEQ ID NO:2, or positions 98-118 of SEQ ID NO:3, contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, and does not contain a serine at a position corresponding to position 118 of SEQ ID NO: 1, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of the mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- An embodiment of the present invention provides isolated or synthetic polypeptides comprising an amino acid sequence of a mutant BAD, or fragments of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- the amino acid sequence of the isolated or synthetic polypeptides of a mutant BAD, or said fragments contains a domain that is substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, wherein the amino acid sequence of the naturally-occurring or wild-type mammalian BAD is SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, and does not contain a serine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of the mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Another embodiment of the present invention provides methods for making polypeptides of a mutant BAD comprising an amino acid sequence of a naturally-occurring or wild-type mammalian BAD, or said fragments comprising a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD.
- the methods comprise first selecting an amino acid sequence of a naturally-occurring or wild-type mammalian BAD, or selecting a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD, comprising a BH3 domain substantially identical to the BH3 domain encoded by the amino acids at positions 114-122 of SEQ ID NO:l, positions 151-159 of SEQ ID NO:2, or positions 109-117 of SEQ ID NO:3.
- the BH3 domain of the naturally-occurring or wild-type mammalian BAD, or the BH3 domain of said fragment is identified by alignment of the amino acid sequence of the naturally-occurring or wild-type mammalian BAD, or the amino acid sequence of said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the amino acid of the amino acid sequence of the naturally- occurring or wild-type mammalian BAD, or said fragment, at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is changed to an amino acid other than serine.
- polypeptides of a mutant BAD, or said fragments comprising the amino acid sequence of a naturally-occurring or wild-type mammalian BAD having a mutation of the amino acid corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3, are made.
- an embodiment of the present invention provides methods for making polypeptides of a mutant BAD comprising an amino acid sequence of a naturally-occurring or _ wild-type mammalian BAD, or said fragments comprising a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD.
- the methods comprise first selecting an amino acid sequence of a naturally-occurring or wild-type mammalian BAD, or selecting a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD, comprising a BH3 domain substantially identical to the BH3 domain encoded by the amino acids at positions 114-122 of SEQ ID NO:l, positions 151-159 of SEQ ID NO:2, or positions 109-117 of SEQ ID NO:3.
- the BH3 domain of the naturally-occurring or wild-type mammalian BAD, or the BH3 domain of said fragment is identified by alignment of the amino acid sequence of the naturally-occurring or wild-type mammalian BAD, or the amino acid sequence of said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the amino acid of the amino acid sequence of the naturally- occurring or wild-type mammalian BAD, or said fragment, at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is changed to alanine.
- an embodiment of the present invention provides methods for making polypeptides of a mutant BAD comprising an amino acid sequence of the naturally- occurring or wild-type mammalian BAD encoded by SEQ ID NO:l , SEQ ID NO:2, or SEQ ID NO:3, or said fragments comprising a less than full-length amino acid sequence of the naturally-occurring or wild-type mammalian BAD encoded by SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- the methods comprise first selecting an amino acid sequence of a naturally-occurring or wild-type mammalian BAD, or selecting a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD, comprising a BH3 domain substantially identical to the BH3 domain encoded by the amino acids at positions 114-122 of SEQ ID NO:l, positions 151-159 of SEQ ID NO:2, or positions 109-117 of SEQ ID NO:3.
- BH3 domain of said fragment is identified by alignment of the amino acid sequence of the naturally-occurring or wild-type mammalian BAD, or the amino acid sequence of said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the amino acid of the amino acid sequence of the naturally-occurring or wild-type mammalian BAD, or said fragment, at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is changed to an amino acid other than serine.
- Another embodiment of the present invention provides methods for making polypeptides of a mutant BAD comprising an amino acid sequence of a naturally-occurring or wild-type mammalian BAD, or fragments comprising a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD.
- the methods comprise first selecting an amino acid sequence of a naturally-occurring or wild-type mammalian BAD, or selecting a less than full-length amino acid sequence of a naturally-occurring or wild-type mammalian BAD, comprising a BH3 domain substantially identical to the BH3 domain encoded by the amino acids at positions 114-122 of SEQ ID NO:l, positions 151-159 of SEQ ID NO:2, or positions 109-117 of SEQ ID NO:3.
- the BH3 domain of the naturally-occurring or wild-type mammalian BAD, or the BH3 domain of said fragment is identified by alignment of the amino acid sequence of the naturally-occurring or wild-type mammalian BAD, or the amino acid sequence of said fragment, to SEQ ID NO: 1 , SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Second the amino acid of the amino acid sequence of the naturally- occurring or wild-type mammalian BAD, or said fragment, at a position corresponding to position 118 of SEQ ID NO: 1, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is changed to an amino acid other than serine.
- the polypeptide of the mutant BAD, or said fragment is expressed in a host cell, wherein the host cell is transformed with a polynucleotide comprising the amino acid sequence of the mutant BAD, or comprising the amino acid sequence of said fragment, respectively.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes apoptosis.
- the methods comprise first contacting a candidate drug with a sample comprising a mammalian BAD, or fragment of a mammalian BAD, and a kinase, to form a reacted fraction.
- the mammalian BAD, or said fragment comprises an amino acid sequence containing a serine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the kinase has phosphorylation activity capable of phosphorylating the mammalian BAD.
- the reacted fraction is compared to a control fraction to determine whether the candidate drug inhibits the phosphorylation activity of the kinase and, thereby, has activity that promotes apoptosis, by assaying for the amount of the mammalian BAD, or said fragment, that is unphosphorylated at the identified serine in the reacted fraction as compared to a control fraction.
- the reacted fraction is compared to a control fraction to determine whether the candidate drug inhibits the phosphorylation activity of the kinase and, thereby, has activity that promotes apoptosis, by assaying the isolated fraction as compared to a control fraction, for an amount of the mammalian BAD, or said fragment, that is bound to Bcl-X L and/or Bcl-2.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except the mammalian BAD, or said fragment, in the control fraction is not contacted with the candidate drug.
- Another embodiment of the present invention provides methods of inducing apoptosis in a cell expressing a mammalian BAD, or fragment of a mammalian BAD.
- the methods comprise first preparing a culture containing a cell line expressing the mammalian BAD, or said fragment.
- the mammalian BAD, or said fragment comprises an amino acid sequence containing a serine at a position corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the cultured cells are contacted with an extracellular agent, and/or an intracellular agent is induced in the cultured cells, to form a reacted fraction, wherein the extracellular and/or the intracellular agent is capable of inhibiting the phosphorylation activity of a kinase in the cell that is capable of phosphorylating the serine at position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- An example of such an extracellular or intracellular agent is inhibitor H89, wherein H89 inhibits the phosphorylation activity of an intracellular kinase.
- Kinase inhibition can also be achieved, for example, by the binding of a polypeptide or a polynucleotide to the kinase, thereby inhibiting the phosphorylation activity of the kinase, or by the binding of a polypeptide or polynucleotide to a polynucleotide that encodes the kinase, thereby preventing the expression of the kinase.
- a kinase is PKA, the cyclic AMP (cAMP)-dependent protein kinase.
- the cultured cells are contacted with an extracellular agent, and/or an intracellular agent is induced in the cultured cells, wherein the extracellular and/or intracellular agent is capable of activating the phosphatase activity of a phosphatase in the cell that is capable of dephosphorylating the mammalian BAD, or said fragment, that is phosphorylated at the serine corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- treated cells in the reacted fraction are compared with untreated control cells to determine whether apoptosis is induced in the treated cells by assaying for the amount of the mammalian
- BAD or said fragment, that is unphosphorylated and/or dephosphorylated in each.
- the treated cells in the reacted fraction are compared to untreated control cells to determine whether apoptosis is induced in the treated cells at a higher level by monitoring indicia of apoptosis in each.
- treatment of the control cells is essentially identical to that of the treated cells, except the control cells are not contacted with an extracellular agent and/or an intracellular agent is not induced in the control cells.
- Another embodiment of the present invention provides methods of assaying a candidate compound for phosphatase activity capable of dephosphorylating a mammalian BAD, or fragment of a mammalian BAD, at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the methods comprise first contacting the candidate compound with the mammalian BAD, or said fragment, to form a reacted fraction, wherein the mammalian BAD, or said fragment, is de-phosphorylated at the specified serine. Second, the reacted fraction is compared to a control fraction to determine whether the candidate compound has phosphatase activity by assaying for the amount of the mammalian BAD, or said fragment, that is bound to Bcl-X L and/or Bcl-2 in the reacted fraction as compared to a control fraction.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except the control fraction is not contacted with the candidate compound.
- Another embodiment of the present invention provides methods of assaying a candidate compound for phosphatase activity capable of dephosphorylating a mammalian BAD, or fragment of a mammalian BAD, at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the methods comprise first contacting the candidate compound with the mammalian BAD, or said fragment, to form a reacted fraction, wherein the mammalian BAD, or said fragment, is de-phosphorylated at the specified serine. Second, the reacted fraction is compared to a control fraction to determine whether the candidate compound has phosphatase activity by assaying for an amount of the mammalian BAD, or said fragment, that is dephosphorylated at the serine in the reacted fraction as compared to the control fraction.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except the control fraction is not contacted with the candidate compound.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes cell survival.
- the methods comprise first contacting the candidate drug with a mammalian BAD, or fragment of a mammalian BAD, and, optionally, a kinase, to form a reacted fraction.
- the mammalian BAD, or said fragment is capable of being phosphorylated at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, corresponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, respectively.
- the reacted fraction is compared to a control fraction to determine whether the candidate drug has activity that promotes cell survival by assaying for the amount of the mammalian BAD, or said fragment, that is phosphorylated at the serine in the reacted fraction as compared to the control fraction.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except the control fraction is not contacted with the candidate drug.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes cell survival.
- the methods comprise first contacting the candidate drug with a mammalian BAD, or fragment of a mammalian BAD, and, optionally, a kinase, to form a reacted fraction.
- the mammalian BAD, or said fragment is capable of being phosphorylated at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2 and SEQ ID NO:3, respectively.
- the reacted fraction is contacted with Bcl-X L and/or Bcl-2.
- the reacted fraction is compared to a control fraction to determine whether the candidate drug has activity that promotes cell survival by assaying for the amount of the mammalian BAD bound to Bcl-X L and/or Bcl-2, or the amount of said fragment bound to Bcl-X L and/or Bcl-2.
- the reacted fraction is compared to a control fraction to determine whether the candidate drug has activity that promotes cell survival by assaying for the amount of the mammalian BAD, or said fragment, that is phosphorylated at the specified serine in the reacted fraction as compared to the control fraction.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except that the control fraction is not contacted with the candidate drug.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes cell survival.
- the methods comprise first preparing a cell culture containing a cell line expressing a mammalian BAD, or fragment of a mammalian BAD, wherein the cell line has activity that promotes apoptosis, or is capable of having activity that promotes apoptosis.
- the mammalian BAD, or said fragment comprises an amino acid sequence containing a serine at a position co ⁇ esponding to position 118 of
- the cell culture is contacted with the candidate drug to form a reacted fraction.
- the cells in the reacted fraction are compared to cells of a control culture to determine whether the candidate drug has activity promoting cell survival by monitoring the viability of the cells in the reacted fraction as compared to the cells of the control culture.
- treatment of the control cell culture is essentially identical to that of the cell culture of the reacted fraction, except that the control cell culture is not contacted with the candidate drug.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes cell survival.
- the methods comprise first preparing a cell culture containing a cell line expressing a mammalian BAD, or fragment of a mammalian BAD, wherein the cell line has activity that promotes apoptosis, or is capable of having activity that promotes apoptosis.
- the mammalian BAD, or said fragment comprises an amino acid sequence containing a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or fragment of the mammalian BAD, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the cell culture is contacted with the candidate drug to form a reacted fraction.
- cultured cells in the reacted fraction are compared to cells of a control culture, to determine whether the candidate drug has activity promoting cell survival, by monitoring the viability of the cells in the reacted fraction as compared to the control cells.
- the cells in the reacted fraction can be compared to the control cells, in order to determine whether the candidate drug has activity promoting cell survival, by further contacting the cells of both cultures with at least one antibody specific for: 1) the mammalian BAD, or said fragment, that is phosphorylated at the serine; or 2) the mammalian BAD, or said fragment, that is unphosphorylated at the serine; and then assaying for the amount of the antibody binding to the mammalian BAD, or said fragment.
- treatment of the control cells is essentially identical to treatment of the cells in the reacted fraction, except that the control cells are not contacted with the candidate drug.
- Another embodiment of the present invention provides methods of inhibiting apoptosis in a cell expressing a mammalian BAD, or fragment of a mammalian BAD.
- the methods comprise first preparing a cell culture containing a cell line expressing a mammalian BAD, or said fragment.
- the mammalian BAD, or said fragment comprises an amino acid sequence containing a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or fragment of the mammalian BAD, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the cultured cells are contacted with an extracellular agent, and/or an intracellular agent is induced, to form a reacted fraction and, thereby, a kinase in the cells is activated, wherein the kinase is capable of phosphorylating the mammalian BAD, or said fragment, at the specified serine.
- the cells in the reacted fraction are compared to a control cell line to determine whether apoptosis is inhibited in the cells in the reacted fraction, by 1) assaying for the amount of the mammalian BAD, or said fragment, that is phosphorylated at the specified serine in the cells in the reacted fraction as compared to the control cells; and/or 2) monitoring indicia of apoptosis in the cells in the reacted fraction as compared to the control cells.
- treatment of the control cells is essentially identical to treatment of the cells in the reacted fraction, except that the control cells do not express a mammalian BAD, or said fragment, that is capable of being phosphorylated by the kinase.
- Examples of a serine kinase capable of phosphorylating the mammalian BAD, or said fragment, at the serine include PKA. Further examples of serine kinases capable of phosphorylating the mammalian BAD, or fragment of the mammalian BAD, include a heterologous kinase. Examples of a mammalian BAD include a heterologous mammalian BAD, and examples of a fragment of a mammalian BAD include a fragment of a heterologous mammalian BAD. Examples of an extracellular agent and/or said intracellular agent include a ligand of a G-protein-coupled receptor, such as L-epinephrine.
- Another embodiment of the present invention provides methods of assaying a candidate compound for a kinase activity capable of phosphorylating a mammalian BAD, or fragment of a mammalian BAD, at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l; position 155 of SEQ ID NO:2; or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or fragment of mammalian BAD, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the methods comprise first contacting the candidate compound with the mammalian BAD, or said fragment, to form a reacted fraction. Second, to determine whether the candidate compound has kinase activity capable of phosphorylating the mammalian BAD, or said fragment, the reacted fraction is assayed for the amount of the mammalian BAD, or said fragment, that is phosphorylated at the specified serine.
- the reacted fraction is assayed, for example, by detecting: 1) the amount of radioactive label on the serine, wherein the cell is contacted with radioactive label and the radioactive label is attached to the serine when the serine is phosphorylated; 2) a difference in the electrophoretic mobility of the mammalian BAD, or said fragment, that is phosphorylated at the serine as compared to the mammalian BAD, or said fragment, that is unphosphorylated at the serine; or 3) the amount of the mammalian BAD, or said fragment, bound to an antibody specific for the mammalian BAD, or said fragment, that is phosphorylated at the serine.
- an antibody specific for the mammalian BAD, or said fragment, that is phosphorylated at the serine include a monoclonal antibody.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes the phosphorylation of a mammalian BAD, or fragment of a mammalian BAD, at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the methods comprise first contacting the candidate drug with a sample comprising BC1-X L and the mammalian BAD, or said fragment, to form a reacted fraction, wherein the mammalian BAD, or said fragment is capable of being phosphorylated at the serine. Second, the reacted fraction is compared to a control fraction to determine whether the candidate drug has activity that promotes phosphorylation, by assaying for: 1) the amount of mammalian BAD, or said fragment, that is not bound to BC1-X in the reacted fraction as compared to the control fraction; and/or 2) the amount of the mammalian BAD, or said fragment, that is phosphorylated at the serine in the reacted fraction as compared to the control fraction.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except the mammalian BAD, or fragment of the mammalian BAD, in the control fraction is not contacted with the candidate drug, and/or the control fraction contains a mammalian BAD, or said fragment, that is not capable of being phosphorylated at the specified serine.
- Another embodiment of the present invention provides methods of screening a candidate drug for an activity that promotes the phosphorylation of a mammalian BAD, or fragment of a mammalian BAD, at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- the methods comprise first contacting a candidate drug with a sample comprising the mammalian BAD, or said fragment, and a kinase, to form a reacted fraction, wherein the mammalian BAD, or said fragment, is capable of being phosphorylated by the kinase. Second, the reacted fraction is compared to a control fraction to determine whether the candidate drug has activity that promotes phosphorylation by the kinase by assaying for the amount of the mammalian BAD, or said fragment, that is phosphorylated at the specified serine in the reacted fraction as compared to the control fraction.
- treatment of the control fraction is essentially identical to that of the reacted fraction, except the control fraction is not contacted with the candidate drug and/or the control fraction contains a mammalian BAD, or said fragment, that is not capable of being phosphorylated at the specified serine.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that promotes phosphorylation in a cell of a mammalian BAD, or fragment of a mammalian BAD, at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position
- SEQ ID NO:2 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:
- the methods comprise first preparing a cell culture containing a cell line expressing the mammalian BAD, or said fragment, wherein the cell line has activity that promotes apoptosis, or is capable of having activity that promotes apoptosis. Second, the cells are contacted with a candidate drug to form a reacted fraction.
- the cells in the reacted fraction are compared to a control cell line to determine whether the candidate drug has activity that promotes phosphorylation by assaying for the amount of the mammalian BAD, or said fragment, that is phosphorylated at the specified serine, in the cells in the reacted fraction as compared to the control cells, and/or monitoring indicia of apoptosis in the cells in the reacted fraction as compared to the control cells.
- the cells in the reacted fraction are compared to control cells to determine whether the candidate drug has activity that promotes phosphorylation by further contacting cells of both fractions with at least one antibody, wherein the antibody is selected from a group consisting of at least one antibody specific for the mammalian BAD, or said fragment, that is phosphorylated at the serine, or at least one antibody specific for the mammalian BAD, or said fragment, that is unphosphorylated at the serine.
- treatment of the control cell line is essentially identical to that of the cells in the reacted fraction, except the control cells are not contacted with the candidate drug.
- Another embodiment of the present invention provides methods of screening a candidate drug for activity that modulates apoptosis promoting activity in a cell.
- the methods comprise first preparing a cell culture containing a cell line expressing a mammalian BAD, or fragment of a mammalian BAD.
- the mammalian BAD, or said fragment comprises an amino acid sequence containing a serine at a position co ⁇ esponding to position 118 of SEQ
- the cells are contacted with an apoptosis promoting substance, wherein the cell line has activity that promotes apoptosis, or is capable of having activity that promotes apoptosis.
- the cells are contacted with the candidate drug to form a reacted fraction.
- the cells in the reacted fraction are compared to a control cell line to determine whether the candidate drug has activity that modulates apoptosis promoting activity by: 1) assaying for the amount of the mammalian BAD, or said fragment, that is phosphorylated or unphosphorylated at the specified serine in the cells in the reacted fraction as compared to the control cells; or 2) monitoring indicia of apoptosis in the cells in the reacted fraction as compared to the control cells.
- treatment of the control cell line is essentially identical to that of the cells in the reacted fraction, except that the control cells are not contacted with the candidate drug.
- Another embodiment of the present invention provides antibodies that specifically bind to a mammalian BAD, or fragment of a mammalian BAD, phosphorylated at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- an embodiment of the present invention provides monoclonal antibodies that specifically bind to a mammalian BAD, or fragment of a mammalian BAD, phosphorylated at a serine at a position in the amino acid sequence of the mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ
- an embodiment of the present invention provides antibodies that specifically bind to a mammalian BAD, or fragment of a mammalian BAD, unphosphorylated at a serine at a position in the amino acid sequence of a mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- an embodiment of the present invention provides monoclonal antibodies that specifically bind to a mammalian BAD, or fragment of a mammalian BAD, unphosphorylated at a serine at a position in the amino acid sequence of a mammalian BAD, or said fragment, co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the serine is identified by alignment of the amino acid sequence of the mammalian BAD, or said fragment, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Another embodiment of the present invention provides polynucleotides encoding at least one isolated or synthetic polypeptide comprising an amino acid sequence of a mutant BAD, or at least one fragment of an isolated or synthetic polypeptide comprising a less than full-length amino acid sequence of a mutant BAD, having cell death promoting activity.
- the amino acid sequence of the encoded isolated or synthetic polypeptides of a mutant BAD, or fragment of a mutant BAD is: 1) derived from a naturally-occurring or wild-type mammalian BAD; 2) contains a domain that is substantially identical to a BH3 domain of a naturally-occu ⁇ ing or wild-type mammalian BAD; and 3) does not contain a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- position co ⁇ esponding to position 118 of SEQ ID NO: 1, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 is determined by alignment of the amino acid sequence of the isolated or synthetic polypeptides of the mutant BAD, or said fragments, to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Fig. 1 is a western blot.
- Whole cell lysates were prepared from COS-7 cells transiently expressing the double mutant HA-BAD SI 12A/S136A. Lysates were treated ("+") with lambda phosphatase or a buffer control ("-") (lanes 1 and 2). Separate aliquots of the lysate were incubated with GST-BCI-X L ("+") or a buffer control ("-”) and precipitated with glutathione beads (lanes 3 and 4). Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- Fig. 2(A) is a western blot.
- Whole cell lysates were prepared from COS-7 cells transiently expressing empty pcDNA3 vector ("mock") (lane 1), the double mutant HA-BAD SI 12A/S136A (lane 2), the triple mutant HA-BAD SI 12A/S134A/S136A (lanes 3 and 4), or the triple mutant HA-BAD SI 12A/S136A/S155A (lanes 5 and 6). Lysates were treated ("+") with lambda phosphatase or a buffer control ("-”) and precipitated with an anti-HA antibody. Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- 2(B) is a western blot.
- Whole cell lysates were prepared from HeLa cells transiently expressing empty pcDNA3 vector ("mock") (lane 1), shorter murine HA-BAD (SEQ ID NO:3) ("wildtype") (lane 2), HA-BAD SI 12A (lane 3), HA-BAD S136A (lane 4), or HA-BAD SI 55 A (lane 5). Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- Fig. 2(C) is a western blot.
- Whole cell lysates were prepared from serum-starved HeLa cells expressing HA-BAD ("wildtype") or HA-BAD SI 55 A, pretreated with epidermal growth factor ("EGF"), fetal calf serum (“FCS”) or buffer control. Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- EGF epidermal growth factor
- FCS fetal calf serum
- Fig. 3(A) is an amino acid sequence alignment of BH3 domains of the Bel-family members, against the BH3 domain of BAD (SEQ ID NO: 1).
- the open rectangle, encompassing the closed rectangle, represents the amino acid sequence of the longer murine BAD (SEQ ID NO:l) protein.
- the closed rectangle represents the BH3 domain of the longer murine BAD (SEQ ID NO:l) protein.
- SI 12 S136
- S155 indicate the location of serine residues at positions 112, 136 and 155 of the longer murine BAD (SEQ ID NO:l) protein, respectively.
- the amino acid sequences are those of BAD (SEQ ID NO:4), BAK (SEQ ID NO:5), BAX (SEQ ID NO:6), BIK (SEQ ID NO:7), BID (SEQ ID NO:8), HRK (SEQ ID NO:9), BOK (SEQ ID NO: 10), and BIM (SEQ ID NO: 11).
- Residues su ⁇ ounded by a black box are identical.
- Residues su ⁇ ounded by a gray box are homologous among BH3 domains.
- A alanine
- C cysteine
- D aspartic acid
- E glutamic acid
- F phenylalanine
- G glycine
- H histidine
- I Isoleucine
- K lysine
- L leucine
- M methionine
- N asparagine
- P proline
- R arginine
- Q glutamine
- S serine
- T threonine
- V valine
- W tryptophan
- Y tyrosine.
- Fig. 3(B) is a graphical representation of the results of an in vitro competition binding assay.
- Recombinant GST-BCI-X was incubated with a BAD BH3 peptide (residues 143- 168) phosphorylated on Serl55 ("BAD BH3-P"), a BAD BH3 peptide (residues 143-168) unphosphorylated on Serl 55 (“BAD BH3”), or a BAK BH3 peptide (residues 71-89) as a positive control, at the indicated concentrations.
- the reaction mixtures were then added to microtiter plates pre-coated with BAK BH3 peptide.
- the amount of bound GST-BCI-X L was determined by ELISA using an anti-GST primary antibody and a horse-radish peroxidase- conjugated anti-mouse IgG secondary antibody with ABTS as substrate.
- Fig. 3(C) is two autoradiographs.
- Full-length BAD wildtype
- mutant BAD SI 55 A mutant BAD SI 55 A
- the labeled proteins were incubated with PKA ("+”) or a buffer control (“-"), and then incubated with either GST or GST-BCI-X L , followed by capture on glutathione-agarose beads. Proteins bound to the beads ("Bound”) (lower panel) and samples of the reactions collected prior to incubation with the beads (“Total”) (upper panel) were analyzed by SDS- PAGE followed by autoradiography.
- Fig. 4 is a western blot and an autoradiograph.
- Whole cell lysates were prepared from COS-7 cells expressing GST-tagged shorter murine BAD (SEQ ID NO:3) ("wildtype") (lane 1), GST-BAD SI 12A ("S112A") (lane 2), GST-BAD S136A ("S136A”) (lane 3), GST-BAD SI 55 A (“SI 55 A”) (lane 4), GST-BAD S112A/S136A ("S112A/S136A”) (lane 5), and GST- BAD S112A/S136A/S155A (“S112A/S136A/S155A”) (lane 6). Lysates were precipitated with glutathione beads, and the purified GST-tagged polypeptides were incubated with PKA
- Fig. 5(A) is a western blot.
- Whole cell lysates were prepared from HeLa cells transiently expressing empty expression vector ("mock") (lane 1), HA-BAD S112A/S136A (lanes 2, 3 and 4), and HA-BAD SI 12A/S136A/S155A (lanes 5 and 6), pretreated with Forskolin ("+") or a buffer control ("-") and then lambda phosphatase ("+”) or a buffer control ("-”) prior to lysis. Proteins were separated by SDS-PAGE.
- Fig. 5(B) is a western blot.
- Whole cell lysates were prepared from HeLa cells transiently expressing empty expression vector ("mock") (lane 1), or HA-BAD SI 12A/S136A (lanes 2-9), treated with Forskolin ("Fk”) for the indicated time periods prior to lysis. Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- Fig. 5(C) is a series of western blots.
- Whole cell lysates were prepared from HeLa cells transiently expressing HA-BAD ("wildtype") (lanes 1 and 2), HA-BAD SI 55 A ("SI 55 A”) (lanes 3 and 4), HA-BAD S112A/S136A ("S112A/S136A”) (lanes 5 and 6), or HA-BAD SI 12A/S136A/S155A (“SI 12A/S136A/S155A ”) (lanes 7 and 8), pretreated with Forskolin ("+”) or a buffer control ("-”) prior to lysis. Proteins were separated by SDS- PAGE. Blots were probed with anti-phospho-Serl55 specific BAD antibody ("anti-pS155 Ab probe”) (upper panels), followed by stripping and reprobing with an anti-BAD antibody (“anti-BAD Ab probe”) (lower panels).
- Fig. 6(A) is a western blot.
- Whole cell lysates were prepared from HeLa cells transiently expressing empty expression vector ("-") (lane 1), or HA-BAD SI 12A/S136A
- TSH thyroid stimulating hormone
- L-epi L-epinephrine
- ACTH adrenocorticotropic hormone
- TSH L-epi ACTH a combination of thyroid stimulating hormone, L-epinephrine and adrenocorticotropic hormone
- Fig. 6(B) is a western blot.
- Whole cell lysates were prepared from HeLa cells transiently expressing HA-BAD ("wildtype") (lanes 1 and 2), HA-BAD SI 12A ("SI 12 A”) (lanes 3 and 4), HA-BAD S136A (“S136A”) (lanes 5 and 6) and HA-BAD S155A (“S155A”) (lane 7 and 8), pretreated with L-epinephrine ("+”) or a buffer control ("-”) prior to lysis. Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- Fig. 7(A) is a western blot.
- Fig. 8(A) is two western blots.
- HeLa cells transiently expressing HA-BAD SI 12A/S136A and either an empty expression vector ("vector") (lanes 1 and 2) or HA-PKI ("PKI") (lanes 3 and 4) were treated with L-epinephrine ("+") or a buffer control ("-").
- Whole cell lysates were prepared and proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- the upper panel displays proteins co ⁇ esponding, in size, to HA-BAD SI 12A/S136A.
- the lower panel displays proteins co ⁇ esponding, in size, to HA- PKI.
- Fig. 8(B) is a western blot.
- Fig. 9(B) is two western blots.
- Rat-1 cells transiently expressing HA-BAD SI 12A/S136A were treated with a buffer control, H89 (PKA inhibitor) or Wortmannin (PI 3- kinase inhibitor), prior to stimulation with Forskolin or platelet-derived growth factor ("PDGF").
- H89 PKA inhibitor
- Wortmannin PI 3- kinase inhibitor
- Whole cell lysates were then prepared and proteins were separated by SDS- PAGE. Blots were separately probed with an anti-phospho-S473 Akt antibody (the kinase that phosphorylates Serl 36) (upper panel) and an anti-BAD antibody (lower panel).
- Fig. 9(C) is two western blots.
- FIG. 10(A) is two western blots.
- Whole cell lysates were prepared from serum- starved HeLa cells co-transfected with HA-BAD SI 12A/S136 and either the PKI expression vector ("PKI") (lanes 3 and 4) or empty expression vector (“vector”) (lanes 1 and 2), and then treated with EGF or a buffer control, prior to lysis. Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody (upper panel) or an anti-PKI antibody (lower panel).
- PKI PKI expression vector
- vector empty expression vector
- Fig. 10(B) is a series of western blots. Serum-starved HeLa cells expressing HA- BAD SI 55 A, were pretreated with a buffer control (lanes 1 and 2), AG1478 ("AG”) (lane 3), Wortmannin ("Wm”) (lane 4), or H89 (lane 5). The cell cultures were then stimulated with a buffer control (lane 1) or EGF (lanes 2-5). Whole cell lysates were made from the cultures and the proteins were separated by SDS-PAGE.
- Fig. 10(C) is a western blot. Serum-starved Rat-1 cells were pretreated with a buffer control (lanes 1 and 2), H89 (lane 3) or Wortmannin ("Wm") (lane 4), followed by stimulation with PDGF. Whole cell lysates were prepared, endogenous BAD was immunoprecipitated and the resulting proteins were separated by SDS-PAGE. The blot was probed with an anti-phospho-Serl55 BAD antibody.
- Fig. 11(A) is a graphic representation of the results of an enzyme-linked
- ELISA immunosorbent assay
- Fig. 11(B) is a western blot.
- Whole cell lysates were prepared as described above in Fig. 11(a).
- Empty vector (“mock") (lane 1), wild-type BAD ("wild-type") (lane 2), BAD S112A ("S112A") (lane 3), BAD S136A ("SI 36 A”) (lane 4), BAD S155A ("S155A”) (lane 5), BAD S112A/S136A ("S112A/S136A”) (lane 6), or BAD S112A/S136A/S155A ("SI 12A/S136A/S155A”) (lane 7). Proteins were separated by SDS-PAGE. Blots were probed with an anti-HA antibody.
- Fig. 12(A) is a graphic representation of the results of an ELISA. HeLa cells were co-
- Wild-type BAD (“WT") (lanes 3 and 4), BAD SI 55 A (“SI 55 A”) (lanes 5 and 6), BAD S112A/S136A (“AA”) (lanes 7 and 8), or BAD S112A/S136A/S155A (“AAA”) (lanes 9 and 10).
- WT wild-type BAD
- SI 55 A BAD SI 55 A
- AA BAD S112A/S136A
- AAA BAD S112A/S136A/S155A
- Fig. 12(B) is a graphic representation of the results of a ⁇ -galactosidase assay.
- SI 55 A BAD SI 55 A
- S112A/S136A HA-BAD S112A/S136A
- AAA HA-BAD SI 12A/S136A/S155A
- Fig. 12(C) is a graphic representation of the results of a ⁇ -galactosidase assay.
- WT wild-type BAD
- S155A BAD S155A mutant
- S155D BAD S155D mutant
- Fig. 13 is a western blot.
- Whole cell lysates were prepared from COS-7 cells expressing empty vector ("mock") (lane 1, upper panel), the 204 amino acid form of murine BAD (longer murine BAD) ("204 aa”) (lanes 2 and 3, upper panel) or the 162 amino acid form of murine BAD (shorter murine BAD) ("162 aa”) (lanes 4 and 5, upper panel). Proteins were separated by SDS-PAGE. Blots were probed with an anti-BAD antibody.
- the amino acid sequence of the human BAD of SEQ ID NO:l and of the murine BAD of SEQ ID NO:3 are aligned against the amino acid sequence of the murine BAD of SEQ ID NO:2 in Table 1, below, illustrating the co ⁇ esponding positions of the amino acids.
- the serine phosphorylation sites at positions 112, 136, and 155 of SEQ ID NO:2 are denoted with an asterisk, and the co ⁇ esponding positions in the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:l are given below the serine at positions 112, 136, and 155 of SEQ ID NO:2.
- the positions having an amino acid residue that is common between all three BAD amino acid sequences are denoted in bold.
- SEQ ID NO: 2 50 EPSEQEDASATDRGLGPSLTEDQPGP YLAPGLLGSNIHQQGRAA
- SEQ ID NO: 3 8 EPSEQEDASATDRGLGPSLTEDQPGP YLAPGLLGSNIHQQGRAA
- BAD refers to a Bcl-X L /Bcl-2 Associated Cell Death Regulator polypeptide, and includes any polypeptide of any origin that is a cell death promoter, that is substantially identical to and/or biologically equivalent to BAD, and that binds to BC1-X and/or Bcl-2 in competition with the cell death promoters BAX and/or BAK to inhibit the cell death repressor activity of BC1-X L and/or Bcl-2.
- mutant BAD refers to a BAD having at least an amino acid sequence in which the serine at a position co ⁇ esponding to position 118 of SEQ ID NO: 1 , position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3, has been replaced with an amino acid other than serine.
- a fragment of the present invention is characterized as having biological activity such as, e.g., cell death promoting activity and/or the ability to bind to BC1-X L and/or Bcl-2.
- a fragment of a BAD comprises an amino acid sequence of a less than full-length BAD.
- a fragment of a BAD contains a domain substantially identical to a BH3 domain of a naturally-occu ⁇ ing or wild-type mammalian BAD, wherein the amino acid sequence of the fragment has a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- a fragment of a mutant BAD comprises an amino acid sequence of a less than full-length mutant BAD.
- a fragment of a mutant BAD contains a domain substantially identical to a BH3 domain of a naturally-occurring or wild-type mammalian BAD, wherein the amino acid sequence of the fragment of a mutant BAD has an amino acid other than serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the position of the amino acid co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3, is identified by alignment of the amino acid sequence of the fragment to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Naturally-occurring or wild-type refer to a polynucleotide or polypeptide that can be found in nature and is present in an organism (including viruses) although not necessarily in a discrete or isolated form, which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory.
- BH3 domain refers to the amino acids comprising from approximately residue 143 to residue 168 of the murine BAD of SEQ ID NO:2, or any portion thereof, or any sequence of amino acids that co ⁇ esponds to this sequence when aligned with the murine BAD of SEQ ID NO:2, or any portion thereof.
- the BAD, mutant BAD, and fragments of a BAD or mutant BAD of the present invention in one embodiment are provided in an isolated form.
- isolated refers to the fact that the object species such as a BAD, mutant BAD, or fragment of a BAD or mutant BAD, is substantially free of other substances which are not the object species, i.e. not in a natural environment.
- a substantially pure composition will comprise more than about 80 to 90 percent of all macromolecular species present in the composition.
- the object species is purified to essential homogeneity such that contaminant species cannot be detected in the composition by standard methods of detection and wherein the composition consists essentially of a single macromolecular species.
- Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
- the BAD, mutant BAD, and fragments of BAD or mutant BAD of the present invention may be derived from any naturally-occurring or wild-type BAD native to any tissue or species.
- the biological activity e.g., cell death promoting activity and/or binding to Bcl-X L and/or Bcl-2, of such BAD, mutant BAD, and fragments of BAD or mutant BAD, can be characterized using any number of biological assay systems known to those skilled in the art.
- activity that promotes apoptosis and “cell death promoting activity” as used herein mean functional activity of a protein, such as BAD, mutant BAD, and fragments of BAD or mutant BAD of the present invention, that induces, instigates, causes or triggers apoptosis or cell death, or the signal transduction pathway that leads to apoptosis or cell death.
- monitoring indicia of apoptosis means examining culture cells, whether by assay or visual inspection, for one or more of loss of cellular junctions and microvilli, cytoplasmic condensation and nuclear chromatin margination, nuclear fragmentation, cytoplasmic contraction, mitochondrial and ribosomal compaction, endoplasmic reticulum dilation and fusion with the plasma membrane, and cellular break up into membrane-bound apoptotic bodies (Wyllie, 1980).
- biologically equivalent means that the BAD, mutant BAD, or fragments of a BAD or mutant BAD, of the present invention are capable of demonstrating some or all of the same biological activity of a naturally-occurring or wild-type BAD, e.g., cell death promoting activity, and/or binding to BC1-X L and/or Bcl-2, although not necessarily to the same degree as the naturally-occurring or wild-type BAD.
- the biological activity of a BAD, mutant BAD, or fragment of a BAD or mutant BAD, of the present invention can be determined using assays known to those skilled in the art.
- the percent viability of cells expressing a BAD, mutant BAD, or fragment of a BAD or mutant BAD, of the present invention can be monitored as an indication of the cell death promoting activity of the expressed polypeptide.
- a mutant BAD, or fragment of the mutant BAD can show increased or decreased cell death promoting activity compared to, for example, the naturally-occurring or wild-type BAD from which the mutant BAD, or fragment of the mutant BAD, was derived, depending upon the particular mutant.
- percent sequence identity As used herein are intended to mean the percentage of the same residues or nucleotides between two or more amino acid sequences or nucleic acid sequences, respectively (Higgins et al, 1992). The percentage of the same residues or nucleotides between multiple amino acid or nucleic acid sequences can be determined by aligning the amino acid or nucleic acid sequences, respectively, using sequence analysis software such as, for example, the Lasergene biocomputing software (DNASTAR, Inc., Madison, Wis.). The amino acid residue weight table used for the Lasergene alignment program is PAM250 (Dayhoff et al, 1978).
- Sequence alignment allows identification of regions of sequence homology, such as, for example, the BH3 domain and, in particular, it allows identification of the serine, or other amino acid, at a position co ⁇ esponding to the serine at position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, and position 113 of SEQ ID NO:3.
- the term "substantially identical" as used herein is intended to mean that there is at least 75%, preferably 85%, and more preferably 90 to 95% identity between two or more amino acid sequences or between two or more nucleotide sequences, and preferably the amino acid sequence includes a BH3 domain, or the nucleotide sequence encodes a BH3 domain.
- Reference to a mutant BAD herein preferably includes a mutant BAD having an amino acid sequence that is substantially identical to at least one of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- mutant BAD herein includes a mutant BAD having an amino acid sequence with at least 85 percent sequence identity with at least one of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- mutant BAD herein includes a mutant BAD having an amino acid sequence with at least 90-95 percent sequence identity with at least one of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- the BAD, mutant BAD, and fragments of BAD and mutant BAD, of the present invention can also include synthetic, derivative, heterologous, hybrid, fusion, and modified forms of such polypeptides.
- the different forms of such polypeptides include forms in which certain amino acids have been deleted, added, replaced, or substituted; one or more amino acids has/have been changed to an amino acid analogue; and/or there are glycosylations of such polypeptides.
- Such polypeptides are characterized as having biological activity, e.g., cell death promoting activity and/or the ability to bind to BC1-X L and/or Bcl-2.
- BAD mutant BAD, or fragment of a BAD or mutant BAD, having an amino acid substitution at a position co ⁇ esponding to the serine at position 118 of SEQ ID NO:l, 155 of SEQ ID NO:2, or 113 of SEQ ID NO:3, such biological activity may be blocked by the phosphorylation of the amino acid at such a co ⁇ esponding position.
- the polynucleotides of the present invention encoding BAD, mutant BAD, or a fragment of BAD or mutant BAD include, for example, isolated or synthetic DNA, genomic DNA, mRNA, and cDNA.
- the isolated polynucleotides may be isolated, for example, through hybridization with the complementary sequence of genomic, subgenomic DNA, cDNA, or mRNA encoding BAD, mutant BAD, or fragments of BAD or mutant BAD.
- the polynucleotides may encode BAD, mutant BAD, or fragments of BAD or mutant BAD, having substituted serine residues and/or phosphorylated serine residues. It will also be appreciated by one skilled in the art that degenerate DNA sequences can encode BAD, mutant BAD, or fragments of BAD or mutant BAD having serine substitutions or having serines which can be phosphorylated.
- Also intended to be included within the present invention are those polynucleotides encoding allelic variants of BAD and serine substituted and/or serine phosphorylated derivatives of such BAD.
- Polynucleotides of the present invention encoding a BAD, mutant BAD, or fragment of a BAD or mutant BAD may include sequences that facilitate RNA transcription (expression sequences) and protein translation of the coding sequences, such that the encoded polypeptide product is produced. Methods for construction of such polynucleotides are known to those skilled in the art and are described, for example, in Sambrook et al, Molecular Cloning, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989.
- polynucleotides may include a promoter, a transcription termination site, polyadenylation site, ribosome binding site, an enhancer for the use in eukaryotic expression hosts, and/or, optionally, sequences necessary for replication of a vector.
- a typical eukaryotic expression vector may include a polynucleotide sequence encoding a BAD, mutant BAD, or fragment of a BAD or mutant BAD, inserted in- frame and downstream of a promoter such as, e.g., the herpes simplex virus thymidine kinase ("HSV-tk”) promoter or the phosphoglycerate kinase (“pgk”) promoter, optionally linked in-frame to an enhancer and a downstream polyadenylation site (e.g., the SV40 large T polyadenylation site).
- a promoter such as, e.g., the herpes simplex virus thymidine kinase (“HSV-tk”) promoter or the phosphoglycerate kinase (“pgk”) promoter, optionally linked in-frame to an enhancer and a downstream polyadenylation site (e.g., the SV40 large T polyadenylation site).
- the degeneracy of the genetic code gives a finite set of polynucleotide sequences encoding these amino acid sequences of the BAD, mutant BAD, and fragments of the BAD or mutant BAD of the present invention.
- This set of degenerate sequences may be readily generated by one skilled in the art, by hand or by computer using commercially available software.
- Isolated polynucleotides encoding a BAD, mutant BAD, or fragment of a BAD or mutant BAD are typically less than approximately 10,000 nucleotides, more preferably less than approximately 3,000 nucleotides, still more preferably less than approximately 1,500 nucleotides, and most preferably approximately 600 nucleotides.
- Prefe ⁇ ed polynucleotides are those polynucleotides encoding a mutant BAD, or fragment of a mutant BAD, having an amino acid sequence that is substantially identical to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- Prefe ⁇ ed polynucleotides also include those polynucleotides capable of hybridizing, under stringent conditions, with a polynucleotide encoding a naturally-occurring or wild-type mammalian BAD.
- stringent conditions are known to, and/or can be determined by standard methods, by those skilled in the art. Examples of such stringent conditions are provided in Sambrook et al, 1989.
- Such polynucleotides may be further screened, under the stringent conditions described above, to select for polynucleotides that do not hybridize to polynucleotides encoding any other members of the Bcl-2 family. Such selective hybridization can be performed using standard cross-hybridization tests known to those skilled in the art.
- Polynucleotides encoding a fragment of a BAD or mutant BAD of the present invention may be short oligonucleotides, for example oligonucleotides that are 20-100 nucleotides in length, wherein such oligonucleotides have biological activity, for example, have cell death promoting activity and/or the ability to bind to BC1-X L and/or Bcl-2.
- BAD of the present invention may also comprise part of a larger polynucleotide, such as, for example, a cloning vector.
- polynucleotides encoding a BAD, mutant BAD, or fragment of a BAD or mutant BAD, of the present invention may be fused in- frame, by polynucleotide linkage, to another polynucleotide sequence encoding a different polypeptide such as, for example, a polynucleotide encoding a heterologous polypeptide such as the Tat polypeptide (YGRKKRRQRRRG) (SEQ ID NO:20) which facilitates intracellular delivery of the BAD, mutant BAD, or fragment of the BAD or mutant BAD.
- YGRKKRRQRRRG Tat polypeptide
- the encoded polypeptide can be a fusion polypeptide, such as, for example, a BAD, mutant BAD, or fragment of a BAD or mutant BAD, fused to a heterologous polypeptide such as, for example, the Tat polypeptide (YGRKKRRQRRRG) (SEQ ID NO:20).
- a fusion polypeptide such as, for example, a BAD, mutant BAD, or fragment of a BAD or mutant BAD, fused to a heterologous polypeptide such as, for example, the Tat polypeptide (YGRKKRRQRRRG) (SEQ ID NO:20).
- the polynucleotides encoding a mutant BAD, or fragment of a mutant BAD comprise at least 25 consecutive nucleotides which are substantially identical to the polynucleotide sequence encoding a naturally-occurring or wild-type mammalian BAD and encoding a codon for an amino acid substitution of the serine at a position co ⁇ esponding to position 118 of SEQ ID NO: 1, 155 of SEQ ID NO:2, or 113 of SEQ ID NO:3.
- the polynucleotides encoding a mutant BAD, or fragment of a mutant BAD comprise at least 50 to 100 consecutive nucleotides, and still more preferably at least 500 to 550 consecutive nucleotides, which are substantially identical to the polynucleotide sequence encoding a naturally-occu ⁇ ing or wild-type mammalian BAD and encoding an amino acid substitution of the serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, 155 of SEQ ID NO:2, or 113 of SEQ ID NO:3.
- a polynucleotide encoding a mutant BAD, or fragment of a mutant BAD can be used to construct transgenes for expressing such polypeptides at high levels, and/or under the transcriptional control of transcription control sequences which do not naturally occur adjacent to a naturally-occurring or wild-type Bad gene.
- a constitutive promoter e.g., a HSV-tk or pgk promoter
- a cell-lineage specific transcriptional regulatory sequence e.g., a CD4 or CD8 gene promoter/enhancer
- tissue- specific transcriptional regulatory sequence may be operably linked to a polynucleotide encoding a mutant BAD, or fragment of a mutant BAD, to form a transgene (typically in combination with a selectable marker such as, e.g., a neo gene expression cassette).
- a selectable marker such as, e.g., a neo gene expression cassette
- Transgenic cells and/or transgenic nonhuman animals may be used to generate models of diseases involving overexpression or inappropriate expression of BAD and to screen for agents to treat such diseases as, for example, immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like.
- Polynucleotides encoding a BAD, or fragment of a BAD can also be used to construct, express, and use a transgene in the same manner as described above for a mutant BAD, or fragment of a mutant BAD.
- the present invention provides a method for producing a mutant BAD, or fragment of a mutant BAD, with increased cell death promoting activity, relative to a co ⁇ esponding wild-type BAD or mutant BAD (i.e. one having the same sequence except for the particular amino acid changes) or with the ability to modulate cell death promoting activity.
- the method comprises preparation of a mutant BAD, or fragment of a mutant BAD, having an amino acid substitution at a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- the mutant BAD, or fragment of a mutant BAD can be made based upon the complete or partial sequence of a BAD from any source, for example a mammalian BAD such as the BAD of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- a mammalian BAD such as the BAD of SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3.
- the position of the serine, or other amino acid, at a position co ⁇ esponding to position 118 of SEQ ID NO: 1, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO: 3 is identified by alignment of the sequence of the mutant BAD, or fragment of a mutant BAD, with SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Such a mutant BAD, or fragment of a mutant BAD can be prepared using standard methods known to those skilled in the art.
- a mutant BAD, or fragment of a mutant BAD may be made by expression of the DNA sequence encoding the mutant BAD, or fragment of a mutant BAD, in a suitable transformed host cell.
- the DNA encoding the mutant BAD, or fragment of a mutant BAD can be prepared and inserted into an expression vector, transformed into a host cell, and suitable conditions established for expression of the mutant BAD, or fragment of the mutant BAD, in the transformed cell.
- a BAD, or fragment of a BAD can also be produced and expressed in the same manner described above for the mutant BAD, and fragment of a mutant BAD.
- Any suitable expression vector may be employed to produce a recombinant BAD, mutant BAD, or fragment of a BAD or mutant BAD, such as, for example, the mammalian expression vector pCB6 (Brewer, 1994) or the E. coli pET expression vectors, for example, pET-30a (Studier et al, 1990).
- suitable expression vectors for expression in mammalian and bacterial cells that are known in the art are, for example, expression vectors for use in yeast or insect cells.
- Baculovirus vectors and expression systems can be employed.
- a BAD, mutant BAD, or fragments of a BAD or mutant BAD can also be prepared by chemical synthesis, by expression in in vitro translation systems using a polynucleotide template, or by isolation from biological samples.
- Chemical synthesis of a polypeptide can be performed, for example, by the classical Me ⁇ ifeld method of solid phase peptide synthesis (Me ⁇ ifeld, 1963), or by the FMOC strategy on a Rapid Automated Multiple Peptide Synthesis System (DuPont Company, Wilmington, Del.) (Caprino and Han, 1972). Fragments, analogs, and modified forms of a BAD or mutant BAD can also be constructed or synthesized using methods well known in the art.
- analog refers to polypeptides which are comprised of a segment of at least 10 amino acids that have substantial identity to a portion of the sequence of a naturally-occurring or wild-type polypeptide, for example, a naturally-occurring or wild-type BAD.
- analog polypeptides comprise a conservative amino acid substitution (or addition or deletion) with respect to a naturally- occurring or wild-type polypeptide sequence, or a mutant polypeptide, for example, the serine-substituted mutant BAD or fragment of a mutant BAD of the present invention.
- Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer up to the length of a full-length naturally-occurring or wild-type polypeptide.
- the discovery of the inhibitory effect of the phosphorylation of BAD on the binding of BAD to BC1-X L and/or Bcl-2 provides a new site for intervention in the modulation of apoptosis or programmed cell death.
- Such intervention can involve the administration of, for example, a mutant BAD, fragment of a mutant BAD, analog of a BAD, or fusion thereof, having an amino acid other than serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3, and preferably, having an amino acid sequence that is substantially identical to SEQ ID NO:l, SEQ ID NO:2, or SEQ ID NO:3, respectively.
- Modulation of the phosphorylation of BAD within a cell can be accomplished by altering the intracellular phosphorylation state of BAD.
- the phosphorylation state of many polypeptides is dynamically controlled by both protein kinases and protein phosphatases (Cohen, 1989).
- the present work shows that both protein kinases and protein phosphatases can alter the phosphorylation state of BAD.
- Phosphatases that dephosphorylate serine residues in polypeptides have been extensively studied and both inhibitors and activators have been reported (for reviews, see Wera and Hemmings, 1995; Shenolikar, 1995).
- inhibitors or activators can be used to modulate, increase, or decrease, including diminish, the ability of intracellular phosphatase to remove the phosphate from the serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- phosphatase inhibitors and activators are known and these can be readily screened by one skilled in the art for activity, for example, by determining the effect of a test agent on the in vitro or in vivo cleavage, by phosphatase activity, of a radiolabeled phosphate group from the serine at position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3 (see, e.g., Matthews, 1995; Shenolikar, 1995). It is prefe ⁇ ed that the inhibitors and activators have selective actions on the phosphatase(s) acting upon the BAD, or fragments of BAD, of the present invention.
- kinase inhibitors or activators of protein kinases are known and can be used as therapeutic agents (see, e.g., Levitski, 1994).
- either kinase inhibitors or activators can act to modulate, increase, or decrease, including diminish, the action of intracellular kinases which phosphorylate a BAD, or fragment of a BAD, at a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3. It is prefe ⁇ ed that the inhibitors and activators have selective actions on the kinase(s) acting upon the BAD, or fragment of BAD, of the present invention.
- kinases act selectively and that neither phosphokinase C (PKC) nor RAFl could phosphorylate BAD, in vitro, at the serine at position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, and position 113 of SEQ ID NO:3.
- PLC phosphokinase C
- RAFl RAFl
- BAD heart muscle kinase
- HMK heart muscle kinase
- inhibitors and activators effective in modulating, increasing, or decreasing, including diminishing, the phosphorylation state of BAD could be tested for their effect on BAD phosphorylation in vitro using HMK, for example, as a selective serine kinase that phosphorylates a serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, or position 113 of SEQ ID NO:3.
- in vivo testing could be done using a number of experimental approaches, and one example, as reported herein, utilizes the endogenous phosphorylation of BAD upon re-addition of IL-3 after withdrawal for two hours.
- Such standard testing systems could be used to test candidate compounds as inhibitors or activators of BAD phosphorylation.
- PMA promotes BAD phosphorylation
- staurosporin inhibits BAD phosphorylation.
- Such treatments can involve administration of BAD serine phosphatase inhibitors or selective serine kinase activators that phosphorylate the serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, 155 of SEQ ID NO:2, and/or 113 of SEQ ID NO:3 to decrease the ability of BAD to bind to Bcl-X L and/or Bcl-2.
- Such treatment would be useful in diseases such as, for example, immunodeficiency diseases, senescence, neurodegenerative disease, ischemic cell death, reperfusion cell death, infertility and wound-healing.
- BAD serine phosphatase activators or inhibitors of kinase that phosphorylate the serine at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, and/or position 113 of SEQ ID NO:3 to increase the ability of BAD to bind to Bcl-X L and/or Bcl-2.
- diseases such as, for example, cancer, viral infections, lymphoproliferative conditions, arthritis, infertility, inflammation and autoimmune diseases.
- the BAD, mutant BAD, and fragments of BAD or mutant BAD, of the present invention can be prepared by chemical synthesis; in recombinant cells transformed with a polynucleotide encoding at least one of such polypeptides; by expression in in vitro translation systems using a polynucleotide template encoding at least one of such polypeptides; or by isolation of such polypeptides from biological samples.
- Phosphorylation of one or more amino acid residues such as to produce the phosphorylated serine-containing polypeptides of the present invention, can be accomplished by well known methods in the art.
- the amino acid residue(s) can be phosphorylated prior to polypeptide synthesis. In other methods the amino acid(s) is phosphorylated after synthesis of the polypeptide, such as in the case when a kinase such as HMK is used.
- Derivatives of the BAD, mutant BAD, fragments of BAD or mutant BAD, and/or phosphorylated forms of such polypeptides, of the present invention can include non-peptide substances possessing the biological properties of the BAD, mutant BAD, fragments of BAD or mutant BAD, and/or phosphorylated forms of such polypeptides, in inducing and/or promoting an apoptotic state and/or binding to BC1-X or Bcl-2.
- the techniques for making peptide mimetics are well known to those skilled in the art (see, e.g., Navia and Peattie, 1993; Olson et al, 1993).
- making peptide mimetics involves identification and characterization of the polypeptide target site as well as the polypeptide ligand using X-ray crystallography and nuclear magnetic resonance.
- amino acid sequence of two murine BAD (SEQ ID NO:2 and SEQ ID NO:3) and a human BAD (SEQ ID NO:l), containing a BH3 domain and containing a phosphorylation site at the serine at position 155 of SEQ ID NO: 1 , position 118 of SEQ NO:2, and position 113 of SEQ ID NO:3, respectively has been identified.
- a pharmacophore hypothesis can be developed and compounds can be made and tested in an assay system as described herein or known in the art.
- the BAD, mutant BAD, and fragment of BAD or mutant BAD, of the present invention can also be used to detect new polypeptides as well as non-peptide compositions capable of associating with or binding to BC1-X L and/or Bcl-2 and thereby acting as inhibitors to the binding of BAD to Bcl-X L and/or Bcl-2, for example, by using a standard radioligand assay system (see, e.g., Bylund and Toews, 1993). Such inhibitors could serve to remove any apoptotic inducing or modulating effect that the binding of BAD to BC1-X and/or Bcl-2 might have.
- the inhibitors can be polypeptides that also contain the conserved serine residues as described above, including BAD, or a fragment of BAD. It is also possible to utilize as inhibitory polypeptides having a modified or substituted amino acid or non-amino acid residue at a position co ⁇ esponding to position 118 of SEQ ID NO:l, position 155 of SEQ ID NO:2, and/or position 113 of SEQ ID NO:3, for example, in place of a serine residue, such that the polypeptide containing the modified amino acid or non-amino acid residue functions in the same way as dephosphorylated BAD, in that it binds to Bcl-X L and/or Bcl-2, and thereby displaces BAD.
- the radioligand assays useful in screening for inhibitors of the binding of BAD to Bcl-X L and/or Bcl-2 can involve preparation of a radiolabeled form of BAD, mutant BAD, or fragment of BAD or mutant BAD, capable of binding to Bcl-X L and/or Bcl-2 using, for example, either a 3 H or 125 I according to standard methods.
- the Bolton Hunter Reagent can be used (ICN Chemicals, Radioisotope Division, Irvine, Calif).
- the radiolabeled BAD ligand binds to the Bcl-X L and/or Bcl-2 immobilized to a substrate such as in a standard ELISA-style plate assay.
- the amount of bound and/or free radiolabeled ligand is then measured (see, e.g., Slack et al, 1989; Dower et al, 1989).
- the Bcl-X L and/or Bcl-2 can be radiolabeled and the BAD, mutant BAD, or fragment of BAD or mutant BAD, immobilized to a substrate.
- the binding assay is performed with soluble, non- immobilized BAD, mutant BAD, or fragments of BAD or mutant BAD, and BC1-X L and/or Bcl-2.
- Competitive inhibition of the binding of the radiolabeled BAD ligand to BC1-X L and/or Bcl-2 on addition of a test compound can be evaluated by standard methods of analysis (see, e.g., Rovati, 1993).
- the present invention also includes therapeutic or pharmaceutical compositions comprising an active agent which is: a phosphatase inhibitor or activator; a kinase inhibitor or activator; or a BAD, mutant BAD, fragment of a BAD or mutant BAD; or a phosphorylated BAD, mutant BAD, or fragment of a BAD; for treating diseases or disease conditions in which the propensity for cell death can be advantageously modulated, and methods of making and using such compositions.
- an active agent which is: a phosphatase inhibitor or activator; a kinase inhibitor or activator; or a BAD, mutant BAD, fragment of a BAD or mutant BAD; or a phosphorylated BAD, mutant BAD, or fragment of a BAD
- compositions and methods are useful for treating a number of diseases such as, for example, neoplasia, certain viral infections (e.g., Epstein-Ban virus), lymphoproliferative conditions, arthritis, inflammation, autoimmune diseases and the like resulting from an inappropriate decrease in cell death as well as diseases such as, for example, immunodeficiency diseases, senescence, neurodegenerative disease, ischemic cell death, reperfusion cell death, infertility, wound- healing and the like resulting from an inappropriate increase in cell death.
- diseases such as, for example, immunodeficiency diseases, senescence, neurodegenerative disease, ischemic cell death, reperfusion cell death, infertility, wound- healing and the like resulting from an inappropriate increase in cell death.
- Treatment can also involve administration to affected cells ex vivo.
- compositions of the present invention can be administered by any suitable route known in the art including for example intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of a slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that the active agent be administered to cells in the central nervous system, administration can be with one or more agents capable of promoting penetration of the active agent across the blood-brain barrier (see, e.g., Friden et al, 1993).
- CSF cerebrospinal fluid
- BAD mutant BAD, fragments of BAD or mutant BAD, and serine- phosphorylated forms of such polypeptides
- a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties (see, e.g., Davis et al, 1978; Burnham, 1994).
- the active agent can be in a composition which aids in delivery into the cytosol of a cell.
- the peptide may be conjugated with a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
- a carrier moiety such as a liposome that is capable of delivering the peptide into the cytosol of a cell.
- the active agent can be modified to include specific transit peptides or fused to such transit peptides which are capable of delivering the BAD, mutant BAD, or fragment of BAD or mutant BAD, of the present invention into a cell.
- such polypeptides can be delivered directly into a cell by microinjection.
- phosphatase inhibitors and activators can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties as described above, such as the coupling of the active substance to a compound which promotes penetration or transport across the blood-brain ba ⁇ ier or stably linking the active substance to a polymer to obtain desirable properties of solubility, stability, half-life and the like.
- compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art.
- One prefe ⁇ ed preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable earners such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition.
- Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
- the primary solvent can be aqueous or alternatively non- aqueous.
- the active agent can also be incorporated into a solid or semi-solid biologically compatible matrix which can be implanted into tissues requiring treatment.
- the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
- the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
- excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.
- Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used. It is also contemplated that certain formulations containing the active agent may be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
- suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpy ⁇ olidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
- the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
- compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
- the formulations can also contain substances that diminish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents.
- the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
- the dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in assay preparations of target cells. Exact dosages are determined in conjunction with standard dose- response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration.
- the formulations and methods herein can be suitably prepared and used for veterinary applications in addition to human applications and the term "patient” as used herein is intended to include human and veterinary patients.
- the term "patient” as used herein is intended to include human and veterinary patients.
- a high level of non-phosphorylated BAD, or a high ratio of non- phosphorylated BAD to phosphorylated BAD might indicate an increase in apoptotic activity in the cell, or progression of the state of apoptosis in the cell, and could indicate the need for treatment to decrease the non-phosphorylated BAD, or the ratio of non-phosphorylated BAD to phosphorylated BAD.
- low levels of non-phosphorylated BAD or a low ratio of non-phosphorylated BAD to phosphorylated BAD could indicate the need to increase either the non-phosphorylated BAD or the ratio of non-phosphorylated BAD to phosphorylated BAD.
- compositions containing BAD can be administered exogenously and it would likely be desirable to achieve certain target levels of BAD, as well as a ratio of non-phosphorylated to phosphorylated BAD in sera, in any desired tissue compartment or in the affected cells or tissue. It would, therefore, be advantageous to be able to monitor the levels of non-phosphorylated and phosphorylated BAD in a patient or in a biological sample, including a tissue biopsy sample obtained from a patient and, in some cases, it might also be desirable to monitor the level of other members of the Bcl-2 family, including BC1-X L and/or Bcl-2.
- the present invention also provides methods for detecting the presence of BAD, and the ratio of non-phosphorylated to phosphorylated BAD, in a cell or a population of cells or in a sample from a patient.
- detection as used herein, in the context of detecting the presence of non- phosphorylated and phosphorylated BAD in a patient, is intended to include the ability to determine the amount of, or distinguish, non-phosphorylated and phosphorylated BAD; to determine the amount of, or distinguish from other polypeptides, expressed and/or post- translationally modified BAD; distinguish non-phosphorylated and phosphorylated forms of BAD from each other, and from other members of the Bcl-2 family; estimate the probable outcome of a disease involving non-phosphorylated and phosphorylated BAD; estimate the prospect for recovery; determine the level of non-phosphorylated and phosphorylated BAD over a period of time as a measure of the status of a disease or condition; and/or monitor the phosphorylated
- a sample is obtained from the population of cells or from the patient.
- the sample can be a population of cells, a tissue biopsy sample, a sample of blood, or a cell fraction from blood, plasma or the like.
- any of a variety of tissues known to express BAD can serve as the source of cells for testing as can a sample or biopsy from a diseased tissue such as a neoplasia.
- the sample can be a sample of cells obtained from blood or a cell- free sample such as plasma or serum.
- the present invention further provides for methods to detect the presence of the non- phosphorylated and phosphorylated forms of BAD in a sample obtained from a patient.
- Any method known in the art for detecting proteins can be used. Such methods include, but are not limited to immunodiffusion, immunoelectrophoresis, immunochemical methods, binder- ligand assays, immunohistochemical techniques, agglutination and complement assays (for example see Sites and Ten, eds., 1991).
- Prefe ⁇ ed are binder-ligand immunoassay methods, including reacting antibodies with an epitope or epitopes of BAD and competitively displacing a labeled BAD or derivative thereof.
- the measurement of levels of phosphorylated BAD can be by any of a variety of methods.
- a particularly useful method based on the work described herein would involve the determination of the amount of phosphorylated BAD bound to BC1-X L and/or Bcl-2. This can be done by immunoprecipitation and western blot analysis using, for example, anti-BAD, anti-serine- phosphorylated-BAD, anti-Bcl-X L , and/or anti -Bcl-2 antibodies, all of which can be prepared by known methods.
- recombinant BAD tagged with the hemagglutinin ("HA") epitope can be immunoprecipitated or probed on a western blot using anti-HA antibody.
- recombinant BAD tagged with a glutathione S-transferase (“GST”) moiety can be precipitated using glutathione beads.
- GST glutathione S-transferase
- serine-phosphorylated and - unphosphorylated BAD can be determined by using anti-BAD and anti-serine- phosphorylated-BAD antibodies in an immunoassay method as described below. Such methods of detection described above can also be used to detect the phosphorylation state and/or the presence of a mutant BAD, or fragment of a BAD or mutant BAD, including a recombinant form thereof.
- Polyclonal antibodies can be prepared by immunizing rabbits or other animals by injecting antigen followed by subsequent boosts at appropriate intervals. The animals are bled and sera assayed against the purified BAD, mutant BAD, or fragments of the BAD or mutant BAD of the present invention, usually by enzyme-linked immunosorbent assay (ELISA) or by bioassay based upon the ability to accelerate apoptosis in cells.
- Monoclonal antibodies can be prepared by methods known in the art, such as by the method of Milstein and Kohler by fusing splenocytes from immunized mice with continuously replicating tumor cells such as myeloma or lymphoma cells (Milstein and Kohler, 1975; Gulfre and Milstein, 1981). The hybridoma cells formed in this manner are then cloned by limiting dilution methods and the supernatants assayed for antibody production ELISA, radioimmunoassay (RIA), or bioassay.
- ELISA enzyme
- Antibodies employed in such assays may be unlabeled, for example as used in agglutination tests, or labeled for use in a wide variety of assay methods.
- Labels that can be used include, for example, radionucleides, enzymes, fluorescers, chemiluminescers, enzyme substrates or co-factors, enzyme inhibitors, particles, dyes and the like for use in RIA, enzyme immunoassays, e.g., ELISA, fluorescent immunoassays and the like.
- Polyclonal or monoclonal antibodies directed against a BAD, mutant BAD, a fragment of a BAD or mutant BAD, or an epitope thereof, can be made for use in immunoassays by any of a number of methods known in the art.
- Immunoprecipitating BAD heterodimers from cells expressing BAD and other polypeptides that bind to BAD, for example, members of the Bcl-2 family, including BC1-X L , and Bcl-2, can be accomplished by methods well known in the art.
- a polynucleotide encoding a BAD can be inserted into a plasmid expression vector encoding the GST moiety and the amino acid sequence of the heart muscle kinase (HMK) phosphorylation target sequence, so that the encoded BAD is operably linked, in-frame, to the GST moiety and HMK target sequence.
- Cells for example E.
- coli can then be transformed with such a plasmid vector and produce a BAD which is fused to a GST moiety and the HMK target sequence.
- the resulting polypeptide can be purified after overexpression in E. coli by standard methods, for example, using GST agarose beads.
- the purified GST-BAD can then be labeled in vitro, for example, with ⁇ - P-ATP and heart muscle kinase to produce radiolabeled, phosphorylated GST-BAD.
- Such methods for immunoprecipitation as described above can also be used to immunoprecipitate heterodimers in cells expressing a mutant BAD, or fragments of a BAD or mutant BAD, and polypeptides that bind to such a mutant BAD, or fragments of a BAD or mutant BAD, and forming heterodimers.
- An expression library such as a cDNA expression library, can then be screened with the radiolabeled, phosphorylated GST-BAD for clones that produce a polypeptide which binds to the GST-BAD (see Neilsen, 1991).
- the identified clone can then be isolated and the DNA sequence of the clone determined by standard methods known in the art. One skilled in the art would know how to predict the amino acid sequence of a potential BAD from the DNA sequence of the clone (Muslin et al, 1996).
- Such methods for screening, cloning, and sequencing as described above can also be applied to phosphorylate and screen with a radiolabeled mutant BAD, or fragment of a BAD or mutant BAD.
- cells can be labeled, for example, either in phosphate-free media (e.g., RPMI 1640 media) with 32 P-orthophosphate (e.g., 1 mCi/10 6 cells) or in methionine-free media (e.g., RPMI 1640 media) with S-methionine (e.g., 200 mCi/10 cells).
- phosphate-free media e.g., RPMI 1640 media
- methionine-free media e.g., RPMI 1640 media
- S-methionine e.g. 200 mCi/10 cells
- the cells can then be lysed (e.g., in either in 137 mM NaCl, 20 mM Tris (pH 8.0), 1.5 mM MgCl 2 , 1 mM EDTA, 50 mM NaF, 0.2%-0.5% NP40 containing aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM phenylmethysulfonyl fluoride) for co-immunoprecipitation, or for direct immunoprecipitation (in RIP A buffer).
- the lysates are first cleared, for example, with protein A beads (e.g., 30 min), followed by incubation with an antibody (e.g., 1.5 hour, on ice).
- the antibody complexes are then captured with protein A beads (e.g., 1 hour).
- the immunoprecipitate is then washed (e.g., with 0.2% NP-40 lysis buffer), resuspended in loading buffer, and separated by SDS-PAGE.
- the gel is then treated, for example, with fluorography and visualized by autoradiography, or transfe ⁇ ed to a nitrocellulose membrane for further immunoblot analysis.
- lysates are separated by SDS-PAGE, and transfe ⁇ ed, typically, to a nitrocellulose membrane.
- the membrane is first blocked with a milk solution (e.g., 3% milk solution, 1 hour), followed by incubation with primary and secondary antibodies (e.g., for one hour each), and finally developed by enhanced chemiluminescence using, for example, a commercially available kit (e.g., a kit supplied by Amersham Pharmacia Biotech, Piscataway, NJ).
- a milk solution e.g., 3% milk solution, 1 hour
- primary and secondary antibodies e.g., for one hour each
- enhanced chemiluminescence e.g., a commercially available kit (e.g., a kit supplied by Amersham Pharmacia Biotech, Piscataway, NJ).
- Plasmids capable of expressing a fusion polypeptide comprising a naturally-occurring or wild-type BAD, mutant BAD, or fragment of a BAD or mutant BAD; or a fusion polypeptide capable of binding to a BAD, mutant BAD, or fragment of a BAD or mutant BAD, for example, BC1-X L or Bcl-2, can be constructed by inserting a polynucleotide encoding the amino acid sequence of at least one of such polypeptides into an expression vector.
- a fusion polypeptide can be constructed, for example, by inserting a polynucleotide encoding the amino acid sequence of a first polypeptide into an expression vector containing a polynucleotide encoding the amino acid sequence of a second polypeptide, such that the amino acid sequence of the first polypeptide is in- frame with, and operably linked to the amino acid sequence of the second polypeptide.
- the amino acid sequence of a BAD, mutant BAD, or fragment of a BAD or mutant BAD can be operably linked to the amino acid sequence of the HA epitope.
- a plasmid is constructed that is capable of expressing a fusion polypeptide that comprises a BAD, mutant BAD, or fragment of a BAD or mutant BAD, that is tagged with the HA epitope.
- plasmids capable of expressing a fusion polypeptide that comprises a polypeptide that can bind to a BAD, mutant BAD, or fragment of a BAD or mutant BAD, and is operably linked with the HA epitope or other polypeptide can be constructed.
- polypeptides that bind to BAD are BC1-X L and Bcl-2.
- plasmids capable of expressing a naturally- occu ⁇ ing or wild-type BAD, or polypeptide that binds to a naturally-occurring or wild-type BAD can be constructed by inserting a polynucleotide encoding the amino acid sequence of at least one of such polypeptides into an expression vector.
- the methods and vectors for constructing such plasmids are well known to those skilled in the art.
- cells e.g., FL 5 . 12 cells
- cells can be transformed (e.g. , using electroporation) with the expression plasmid containing the fusion polypeptide. More particularly, cells can be co-transformed with a plasmid capable of expressing a BAD, mutant BAD, or fragment of a BAD or mutant BAD, and another plasmid capable of expressing a polypeptide capable of binding to a BAD, mutant BAD, or fragment of a BAD or mutant BAD. Thereafter, (e.g., forty-eight hours after transformation) a limited dilution of the cells can be performed in selection medium (e.g., in medium containing G418).
- selection medium e.g., in medium containing G418).
- Single- cell clones expressing the polypeptide encoded by the polynucleotide contained in the plasmid used to transform the cells and detected by western blot analysis, can be selected several days later (e.g., 7-10 days later). Lysates from cells expressing the fusion polypeptide can be analyzed by immunoprecipitation and/or western blot analysis using, for example, anti-BAD antibody, anti-HA antibody, anti-Bcl-X L , and/or anti-Bcl-2 antibody.
- the phosphorylation of a BAD, mutant BAD, or fragment of a BAD or mutant BAD can be detected in vivo, for example, by radiolabeling the BAD, mutant BAD, or fragment of a BAD or mutant BAD expressed in cells.
- the cells are first transformed with a plasmid capable of expressing BAD, mutant BAD, or fragment of a BAD or mutant BAD, and then, using standard methods, the polypeptides expressed in the transformed cells are labeled, for example, with 32 P-orthophosphate.
- the cells are lysed and the BAD, mutant BAD, or fragment of a BAD or mutant BAD, expressed in the transformed cells can be immunoprecipitated with, for example, anti-BAD antibody, resolved on an SDS-PAGE gel, and analyzed, for example, by fluorography.
- a sample of the labeled cell lysate can be analyzed by western blot with an anti-BAD antibody.
- the serine phosphorylation of a BAD, mutant BAD, or fragment of a BAD or mutant BAD can be detected, in vivo, using the methods described above.
- 32 P-radiolabeled BAD, mutant BAD, or fragment of a BAD or mutant BAD, bound to another polypeptide, for example, BC1-X L or Bcl-2, can be immunoprecipitated from cells expressing BAD, mutant BAD, or a fragment of a BAD or mutant BAD, and a polypeptide that binds to BAD, mutant BAD, or fragment of a BAD or mutant BAD as described above.
- the immunoprecipitated BAD, mutant BAD, or fragment of a BAD or mutant BAD can then be treated with phosphatase.
- protein A-BAD complexes can be suspended in an appropriate buffer (e.g., 500 ml of 40 mM PIPES piperazine-N,N'-bis(2-thansulfonic acid) buffer (pH 6.0) containing 1 mM DTT, aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM phenylmethysulfonyl fluoride) and treated with a commercially available phosphatase (e.g., potato acid phosphatase, Sigma).
- an appropriate buffer e.g., 500 ml of 40 mM PIPES piperazine-N,N'-bis(2-thansulfonic acid) buffer (pH 6.0) containing 1 mM DTT, aprotinin (0.15 U/ml), 20 mM leupeptin, and 1 mM phenylmethysulfonyl fluoride
- Samples containing phosphatase inhibitors can be supplemented with, for example, 50 mM sodium fluoride, 5 mM sodium phosphate, 10 mM sodium pyrophosphate, 10 mM ammonium molybdate, 5 mM EDTA and 5 mM EGTA.
- the protein A beads can then be pelleted by centrifugation, washed (e.g., with NP-40 lysis buffer), resuspended in a gel loading buffer, and the bound proteins can be examined by western blot analysis.
- BAD can resolve on a gel as a doublet; phosphorylated BAD resolves as a higher molecular weight band relative to unphosphorylated or dephosphorylated BAD, which migrates as a lower molecular weight relative to phosphorylated BAD.
- Treatment of immunoprecipitated BAD with potato acid phosphatase (PAP) can eliminate the higher molecular weight band by converting the higher molecular weight phosphorylated BAD to the lower molecular weight dephosphorylated BAD.
- PAP potato acid phosphatase
- the immunoprecipitated 32 P-radiolabeled BAD, mutant BAD, or fragment of a BAD or mutant BAD can be separated by SDS-PAGE and transfe ⁇ ed to a nitrocellulose membrane.
- Membrane slices containing either the phosphorylated, dephosphorylated or unphosphorylated BAD, mutant BAD, or fragment of a BAD or mutant BAD can be isolated from the membrane, and digested with trypsin under appropriate conditions. Standard methods known to those skilled in the art can be used to isolate the nitrocellulose membrane- bound BAD, mutant BAD, or fragment of a BAD or mutant BAD, and digest it with commercially available trypsin (e.g., Worthington Biochemicals). The peptides resulting from the trypsin digest can then be dried, washed, hydrolyzed, and resolved by TLC, using standard methods known to those skilled in the art (see Boyle et al, 1991). The location of phosphoamino acids can then be determined, for example, by ninhydrin staining and autoradiography. Phosphoamino acid analysis of the BAD doublet revealed that BAD was exclusively phosphorylated at serine residues.
- two-dimensional tryptic peptide mapping it is possible to identify the specific amino acids and their positions in the amino acid sequence of a BAD, mutant BAD, or fragment of a BAD or mutant BAD, that were phosphorylated in vivo. For example, by generating two dimensional tryptic peptidic maps of a BAD the precise sites of serine phosphorylation can be identified.
- the tryptic peptides can be separated, horizontally, in the first dimension (e.g., in pH 8.9 buffer; see Boyle et al, 1991) by TLC (e.g., utilizing a HTLE-7000 apparatus and electrophoresing for
- Separation in the second dimension can be performed by ascending chromatography (e.g., in 37.5% n-butanol, 25% pyridine and 7.5% acetic acid for 10 hours).
- the 32 P-phosphopeptides can then be visualized by autoradiography and the non- radiolabeled phosphopeptides can be visualized, for example, with ninhydrin staining.
- the labeled peptides can then be eluted (e.g., in pH 1.9 buffer) from the TLC plates, conjugated to membrane (e.g., Sequelon-AA membrane (Perspective Biosystem, Framingham, Mass.),
- eluted peptides can then be either directly applied onto a TLC plate or treated with performic acid to uniformly oxidize the peptides before application to the TLC plate.
- the peptides are then separated according to charge in the first dimension, followed by hydrophobicity in the second dimension, as above.
- the 2D maps of the upper and lower molecular bands, representing phosphorylated and unphosphorylated or dephosphorylated BAD species can then be resolved.
- the resolved 32 P-labeled tryptic peptides can then be subjected to manual Edman degradation performed as previously described (Boyle et al, 1991; Luo, et al, 1991) and the identity and position of the specific amino acids in the BAD amino acid sequence determined.
- mammalian expression vectors encoding the shorter murine BAD of SEQ ID NO:3 or a mutant BAD derived from the shorter murine BAD of SEQ ID NO:3 were constructed, wherein the amino acid sequence of the mutant BAD contained an amino acid substitution at the serine at a position co ⁇ esponding to the serine at position 112 ("Serl 12") and/or position 136 ("Serl36”) of SEQ ID NO:2.
- the mutant BAD SI 12A has an amino acid sequence wherein the serine at a position co ⁇ esponding to Serl 12 is substituted with alanine; the mutant BAD S136A has an amino acid sequence wherein the serine at a position co ⁇ esponding to Serl36 is substituted with alanine; and the mutant BAD SI 12A/S136A has an amino acid sequence wherein the serines at positions co ⁇ esponding to both Serl 12 and Serl 36 are substituted with alanine.
- Mouse FL 5 . ⁇ 2 cells were then transfected with the expression vector encoding the shorter murine BAD of SEQ ID NO:3 or one of the three BAD mutants.
- Total mRNA was isolated from the transfected FL 5.12 cells, and reverse transcribed into cDNA.
- the cDNA was then subcloned into the expression vector pcDNA3 so that the amino acid sequence of the BAD or mutant BAD, encoded by the subcloned cDNA, was in-frame and operably linked to an N-terminal hemagglutinin ("HA") epitope encoded by the expression vector.
- the expression vectors encoding the HA-tagged shorter murine BAD and mutant BAD were expressed in COS-7 cells.
- WCL Whole cell lysates
- the phosphorylation of the shorter murine BAD of SEQ ID NO: 3 and mutant BAD were detected by a western blot of the WCL, probed with an anti- HA antibody ("anti-HA antibody").
- anti-HA antibody an anti- HA antibody
- each of the BAD mutants resolved as a doublet on a 16% SDS-PAGE, even though in the amino acid sequence of each of the BAD mutants one or both of the serines co ⁇ esponding to Serl 12 and Serl36 were mutated, i.e., substituted with alanine.
- the higher molecular weight band of the doublet comi grated on the SDS-PAGE with the band representing phosphorylated shorter murine BAD, and the lower molecular weight band of the doublet comigrated on the SDS-PAGE with the band representing unphosphorylated shorter murine BAD (results not shown).
- COS-7 cells transfected with the HA-BAD S 112A/S 136A double mutant were lysed and treated with lambda phosphatase or a buffer control.
- the proteins were separated via SDS-PAGE, transfer to nitrocellulose and probed with an anti-HA antibody.
- HA-BAD triple mutants were constructed.
- the triple mutant HA-BAD SI 12A/S134A/S136A has an amino acid sequence, wherein the serines at positions co ⁇ esponding to Serl 12 and Serl36, and the serine at position 134 ("Serl34"), of SEQ ID NO:2, are substituted with alanine.
- the triple mutant HA-BAD SI 12A/S136A/S155A has an amino acid sequence, wherein the serines at positions co ⁇ esponding to Serl 12 and Serl36, as well as the serine at position 155 ("Serl 55") of SEQ ID NO:2, are substituted with alanine.
- the co ⁇ ect DNA sequence encoding BAD or a mutant BAD was confirmed by DNA sequencing.
- the DNA encoding BAD or a mutant BAD was then inserted into the vector pcDNA3, between the BamBI and Ec ⁇ RI restriction enzyme sites, so that the encoded amino acid sequence of the BAD or mutant BAD was in- frame with and operably linked to an HA epitope at the amino terminus of the encoded BAD or mutant BAD.
- COS-7 cells were transfected with the expression vector encoding the BAD or a mutant BAD.
- a western blot was performed on WCL prepared from the transfected COS-7 cells expressing HA-BAD S112A/S136A, HA-BAD S112A/S134A/S136A, or HA-BAD SI 12A/S136A/S155A and treated with phosphatase or a buffer control. The western blot was probed with anti-HA antibody.
- a western blot was performed on WCL prepared from cells expressing the shorter murine BAD (SEQ ID NO:3), HA-BAD SI 12A, HA-BAD S136A, or HA-BAD S155A, and probed with anti-HA antibody.
- the results of the western blot analysis demonstrated that a single mutation of the amino acid sequence of the shorter murine BAD (SEQ ID NO:3), at the serine at a position co ⁇ esponding to Serl 55, dramatically reduced the phosphorylation of the murine BAD in HeLa cells (Fig. 2(b), lane 5), as compared to a single mutation of the serine at a position co ⁇ esponding to Serl 12 or Serl 36 (Fig. 2(b), lanes 3 and 4, respectively).
- These results indicate that phosphorylation of the serine at a position co ⁇ esponding to Serl 55 contributed significantly to the overall phosphorylation of BAD.
- HeLa cells transiently expressing HA-BAD or mutant HA-BAD S 155 A were cultured in the presence of epidermal growth factor (EGF), fetal calf serum (FCS), or a buffer control.
- EGF epidermal growth factor
- FCS fetal calf serum
- a western blot was performed on whole cell lysates prepared from the cultures, and probed with anti-HA antibody.
- BAD BH3 peptide encompassing BAD residues 143 to 168, was incubated with recombinant GST-BCI-X L at the indicated concentrations (Fig. 3(b)).
- the affinity of the BAD BH3 peptide for GST-BCI-X L was measured as the ability of the peptide to block subsequent binding of GST-Bcl-X L to a BAK BH3 peptide when the reacted mixture was added to a microtiter plate pre-immobilized with a BAK BH3 peptide, as detected by ELISA (triplicate samples, +/- standard deviation). Both phosphorylated (BAD BH3-P) and unphosphorylated (BAD BH3) BAD were assayed, as was a BAK BH3 peptide (residues 71-89) as a positive control.
- Serl55 of BAD is phosphorylated by PKA in vitro
- the amino acid sequence su ⁇ ounding Serl 55 of the longer murine BAD was examined for the presence of serine-threonine kinase recognition motifs.
- the amino acid sequence LRRMSD (SEQ ID NO: 19) matched well with the consensus recognition sequence of mammalian PKA, which is XRRXSX (Kemp and Pearson, 1990). Based on this finding, PKA was tested to determine whether it could phosphorylate BAD, in vitro, at the serine at a position co ⁇ esponding to Serl 55.
- Expression vectors encoding fusion polypeptides of the shorter murine BAD (SEQ ID NO:3) and BAD mutants derived from the shorter murine BAD, were constructed such that the encoded amino acid sequence of the BAD or mutant BAD was fused in- frame and operably linked to the GST moiety.
- the amino acid sequence of GST-BAD encodes a GST- tagged shorter murine BAD.
- the amino acid sequence of GST-BAD SI 12A encodes a GST- tagged mutant BAD, wherein the serine at a position co ⁇ esponding to Serl 12 is substituted with alanine.
- the amino acid sequence of GST-BAD S136A encodes a GST-tagged mutant BAD, wherein the serine at a position co ⁇ esponding to Serl 36 is substituted with alanine.
- the amino acid sequence of GST-BAD SI 55 A encodes a GST-tagged mutant BAD, wherein the serine at a position co ⁇ esponding to Serl 55 is substituted with alanine.
- the amino acid sequence of GST-BAD S112A/S136A encodes a GST-tagged mutant BAD, wherein the serines at positions co ⁇ esponding to Serl 12 and Serl36 are substituted with alanine.
- the amino acid sequence of GST-BAD SI 12A/S136A/S155A encodes a GST-tagged mutant BAD, wherein the serines at positions co ⁇ esponding to position 112, position 136, and position 155 of SEQ ID NO:2 are substituted with alanine.
- the GST-tagged BAD and BAD mutants were then expressed individually in cells and the purified GST-tagged polypeptides incubated with purified PKA, in the presence of P radiolabel.
- the GST-tagged BAD and BAD mutants phosphorylated by PKA could be detected directly by autoradiography due to the incorporation of 32 P radiolabel when the polypeptides were phosphorylated (Fig. 4, upper panel), whereas the total amount of BAD and mutant BAD present in the in vitro reaction was detected by the anti-BAD antibody probe (Fig. 4, lower panel).
- Forskolin could induce the phosphorylation BAD or mutant BAD.
- the results of the western blot demonstrated that treatment with Forskolin induced a significant band- shift of HA-BAD SI 12A/S136A (Fig. 5(a), lanes 2 and 3) that was rapid and sustained (Fig. 5(b)).
- Forskolin is a known activator of the pathway leading to PKA activation, these results indicate that cellular PKA is responsible for phosphorylation of Serl55 of BAD.
- treatment of the cells with the cell permeant cAMP analog, 6-Bnz- cAMP, in place of Forskolin also led to Serl55 phosphorylation of BAD (see Fig. 9(a)).
- An anti-phospho-Serl55 specific BAD antibody (see Materials and Methods) was used to provide direct evidence for BAD phosphorylation on Serl55.
- HeLa cells transiently expressing HA-BAD, HA-BAD SI 55 A, HA-BAD SI 12A S136A, or HA-BAD SI 12A/S136A/S155A were treated with Forskolin or a buffer control.
- a western blot analysis was performed on lysates prepared from these cells and probed with either anti- phospho-Serl55 specific BAD antibody (Fig. 5(c), upper panels) or an anti-BAD antibody (Fig. 5(c), lower panels).
- the HA-BAD SI 12A/S136A double mutant was transiently expressed in HeLa cells, and transfected cell cultures were treated with each of the GPCR ligands, individually and as a group.
- a western blot analysis, probed with anti-HA antibody, was performed on lysates produced from the treated and untreated (negative control) cells.
- the results of the western blot demonstrated that treatment with L-epinephrine, either alone, or in combination with TK and ACTH, induced a significant band-shift of HA-BAD SI 12A/S136A (Fig. 6(a) lanes 4 and 6).
- neither TK (Fig. 6(a), lane 3) nor ACTH Fig.
- HA-BAD constructs HA-BAD, HA-BAD SI 12A, HA-BAD S136A, and HA-BAD S155A
- samples of the transfected cells were treated with L-epinephrine.
- Untreated controls received no L-epinephrine.
- L-epinephrine receptor i.e. the ⁇ -adrenergic receptor (Linder
- BAD Serl 55 phosphorylation was an artifact of BAD overexpression, the effects of these agents on endogenous BAD were examined.
- Non-trans feet ed HeLa cells were treated with Forskolin or L-epinephrine, followed by western blot analysis of cell lysates using anti-BAD antibodies.
- a portion of BAD exhibited retarded migration, suggesting that there was some basal BAD phosphorylation in quiescent cells (Fig. 7(a), lane 1).
- treatment with Forskolin or L-epinephrine resulted in a new band-shift, seen as a third band, as shown in the time course in Fig. 7(a).
- Fig. 7(b) demonstrate that the band-shift was sensitive to the PKA inhibitor, H89 (Fig. 7(b), lane 4), but not to Wortmannin, a PI 3-kinase inhibitor (Fig. 7(b) lane 5). Furthermore, both upper bands were sensitive to phosphatase, indicating that they represent phosphorylated BAD (Fig. 7(b), lane 3).
- Rat-1 cells transiently expressing HA- BAD S112A/S136A were treated with platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), N 6 -Benzoyl-Adenosine 3', 5'- cyclic monophosphate (6-Bnz-cAMP) and Forskolin (Fig. 9(a)).
- PDGF platelet-derived growth factor
- EGF epidermal growth factor
- IGF-1 insulin-like growth factor 1
- 6-Bnz-cAMP N 6 -Benzoyl-Adenosine 3', 5'- cyclic monophosphate
- IGF-1, EGF and PDGF induced a band-shift in the BAD double mutant in Rat-1 fibroblasts.
- the magnitude of phosphorylation was weaker and may be more transient, than the effect of Forskolin (Fig. 9(a)).
- Serl55 phosphorylation following IGF-1, EGF, or PDGF stimulation was not detected in HeLa cells (results not shown).
- Akt kinase activity the serine kinase shown to phosphorylate BAD Serl 36, is induced by growth factors and is dependent on PI 3-kinase activation (Datta et al, 1997; del Peso et al, 1997).
- Rat-1 cells were pretreated with either the PKA inhibitor H89, or the PI 3-kinase inhibitor Wortmannin (available, e.g., from Sigma, St. Louis, MO), followed by stimulation with Forskolin (also available from Sigma) or PDGF.
- Resulting western blot analyses of cell lysates demonstrated that a PDGF-induced BAD band-shift was significantly reduced by H89 pretreatment. This indicated the shift was PKA dependent (Fig.
- HeLa cells were transfected with the mutant HA-BAD SI 55 A and pretreated with Wortmannin, an inhibitor of PI 3-kinase. These cells were then stimulated with epidermal growth factor (EGF), lysed and analyzed by western blot analysis. Wortmannin treatment prevented the activation of endogenous Akt by PI-3 kinase following EGF stimulation (Fig. 10(b), compare lanes 2 and 4, panel B), but did not impair EGF- induced phosphorylation of BAD on Serl 55 (Fig. 10(b), compare lanes 2 and 4, panel D).
- EGF epidermal growth factor
- EGFR EGFR kinase inhibitor AG1478 (AG) blocked the phosphorylation of both Akt (Fig. 10(b), compare lanes 2 and 3, panel B) and BAD Serl 55 (Fig. 10(b), compare lanes 2 and 3, panel D).
- Akt Akt
- BAD Serl 55 Akt
- PDGF-induced Serl 55 phosphorylation of endogenous BAD was also significantly reduced by pretreatment of cells with the PKA inhibitor H89, but not the PI 3-kinase inhibitor Wortmannin (Fig. 10(c)).
- BAD Serl 55 phosphorylation like Serl 36 phosphorylation, prevents BAD from binding to Bcl-X L (Fig. 1), it was possible that by analogy, Serl 55 dephosphorylation promotes cell death.
- HeLa cells were co-transfected with BAD or various BAD serine mutants and a ⁇ -gal reporter plasmid. Twenty-four hours post-transfection, cell
- Beta-gal ELISAs were performed 24 hours post transfection. As shown in Fig. 12(a), Forskolin was able to reduce apoptosis in wild-type BAD and the BAD SI 12A/S136A double mutant, but not in the BAD S155A mutant or the BAD S112A/S136A/S155A triple mutant transfected cells. Thus, phosphorylation of Serl55 rescues cells from BAD-induced apoptosis, while substitution of Serl 55 with a nonphosphorylatable residue results in increased cell death and the failure of cells to be protected from induced PKA activity.
- Serl55-phosphorylated BAD is deficient in binding to BCI-X L -
- the BAD SI 55 A mutant showed enhanced apoptotic activity compared with the wild-type mammalian BAD, suggesting that phosphorylation on Serl 55 is anti-apoptotic (Figs. 11(a) and 12(a)).
- the BAD SI 12A/S136A/S155A triple mutant was more toxic to transfected HeLa cells than the SI 12A/S136A double mutant, suggesting the apoptotic effect of Serl 55 dephosphorylation is additive to those of Serl 12 and Serl 36 (Fig. 11(a)).
- HeLa cells were co-transfected with the ⁇ -galactosidase gene and either wild-
- BAD S155A BAD S112A/S136A or BAD S112A/S136A/S155A.
- SFM serum-free medium
- EGF EGF
- Serl 55 was replaced with aspartic acid (S155D) to mimic the negatively
- HeLa cells were co-transfected with ⁇ -gal and either an
- BAD should be minimal in untransfected or BAD mutant-transfected cells
- reporter ⁇ -gal were introduced into cells at a ratio of 5:1, therefore the exogenous BAD or
- BAD mutants were expected to be dominant over endogenous BAD.
- the transient expression of BAD-induced apoptosis appeared to depend on activation of caspases, treatment of the transfected cells with a caspase inhibitor z-VAD, efficiently protected the cells (data not shown).
- PDGF appears to activate both PKA- and Akt-mediated phosphorylation of BAD.
- the PKA inhibitor blocked a part of the growth factor-induced phosphorylation without interrupting the activation of PI 3-kinase and Akt; the PKA activator Forskolin induced BAD phosphorylation without activating Akt; and the PI 3-kinase inhibitor only partially blocked PDGF-induced BAD phosphorylation (Fig. 9(b)).
- the BAD kinases Akt and PKA are both activated by PDGF, but are likely to function independently.
- phosphorylation on Serl 12 or Serl 36 generates a consensus binding site for the cytosolic protein, 14-3-3, which may bind and alter the sub-cellular distribution of BAD to prevent interaction with BC1-X L at mitochondrial membranes.
- the discovery of the novel phosphorylation site, Serl55, of BAD, and the possible cellular regulatory mechanisms that lead to its phosphorylation, indicate that cells have the ability to protect themselves via multiple survival pathways. Which pathway is used could be dependent on the intrinsic properties of the cells, such as the distribution of cell membrane receptors, as well as the extracellular environment.
- the cDNA encoding the murine BAD of SEQ ID NO:2 (“longer murine BAD”) was obtained by the Reverse Transcription Polymerase Chain Reaction ("RT-PCR") of mRNA isolated from FL 5 12 cells using the primers:
- the open-reading frame of the RT-PCR product was predicted to encode a protein of 204 amino acids (aa) (Yang et al, 1995).
- the cDNA encoding the longer murine BAD was cloned into the pcDNA3 vector (Invitrogen), expressed in FL 5 12 cells, and the expressed longer murine BAD detected with an anti-BAD antibody (C-20, Santa Cruz).
- the expressed longer murine BAD was significantly larger in molecular weight than the endogenous BAD (approximately 30 kD and 23 kD, respectively) (Fig. 13, compare lane 1 against lanes 2 and 3, left panel).
- a second murine BAD cDNA construct was produced, the murine BAD of SEQ ID NO:3 ("shorter murine BAD").
- SEQ ID NO: 14 5'- TGGAGACCAGGATCCCAGAGTAGCT - 3' (SEQ ID NO: 14) and the same downstream primer as above (i.e., SEQ ID NO: 13) were used to generate the cDNA of SEQ ID NO:3, which was then cloned into the pcDNA3 vector.
- This construct when expressed in FL 5 12 cells, co-migrated with the endogenous BAD (Fig. 13, compare lanes 4 and 5, left panel, with lane 1, right panel), suggesting that the shorter murine BAD, which is 162 aa in length, is likely to be the major translation product of BAD in FL 5 12 cells.
- BAD mutants have been produced where one or more serine residues were changed to alanine residues using PCR-mediated mutagenesis (Ge and Rudolph, 1997).
- BAD mutants were also produced, where one or more of the serine residues at positions 112, 134, 136, and 155, co ⁇ esponding to the amino acid positions of the longer murine BAD of SEQ ID NO:2, were changed to alanine or aspartic acid. These positions co ⁇ espond to serine residues in positions 75, 99, 101 and 118 in SEQ ID NO:l and positions 70, 94, 96 and 113 in SEQ ID NO: 3.
- One of ordinary skill in the art would understand how to use PCR-mediated mutagenesis to make such changes.
- the cDNA encoding human protein kinase inhibitor (PKI) was isolated from total mRNA of HeLa cells by RT-PCR.
- the primers used for the reaction were:
- the product of the RT-PCR was then digested with the restriction enzymes BamBl and EcoRI, and the sequence encoding the PKI was inserted into the polylinker region of the pcDNA3 vector, so that the nucleotide sequence encoding PKI was in-frame with and operably linked to an HA epitope at the amino ("N") terminus of the encoded PKI.
- the co ⁇ ect DNA sequence encoding the HA-tagged PKI was confirmed by DNA sequencing. Expression and purification of glutathione s-transferase (GST) fusions of wild-type and mutant BAD
- HBS HEPES- buffered saline
- DMEM fetal calf serum
- GEBCO penicillin/streptomycin
- FL 5 12 cells were cultured in glutamine-free IMEM supplemented with 10% FCS, 10% WEHI-conditioned media (collected from WEHI cell cultures), 1% glutamine and 1% penicillin/streptomycin.
- Transfections were carried out by using the Superfection Kit from Qiagen or the GenePROTOR transfection reagent from Gene Therapy Systems.
- MCF-7 cells stably expressing BAD or mutant BAD were isolated by G418 selection at 1 mg/ml; G418 -resistant clones were then pooled and used for further assays.
- Anti-BAD antibody (C-20) was from Santa Cruz Biotechnology, Inc. and anti-HA antibody (3F10) was from Boehringer Mannheim.
- NH 2 - (GC)QRYGRELRRMpSDESVDSF - COOH (SEQ ID NO: 18) was synthesized at >70% purity and conjugated to keyhole limpet hemocyanin. This antigen was injected into two rabbits, from which serum was collected. The serum included pre- bleed, 6 th , 8 th and 10 th week bleeds. The GC in parenthesis represent the linker amino acids. When used at 1 :500 dilution, the 10 th week bleed from one of the animals was reactive with Serl55-phosphorylated BAD in western blot analysis. The pre-bleed was not reactive.
- Fig. 14 shows the results of a western blot probe with the polyclonal antibody.
- the anti-serum was found to recognize both forms of phosphorylated murine BAD and human BAD, as well as fragments of all three proteins and mutant BAD where the serine residue co ⁇ esponding to Serl 55 was not mutated to alanine.
- HeLa cells transiently expressing HA-BAD SI 12A/S136A were kept in culture for two days in the absence or presence of Forskolin. Lysates were then prepared. Following SDS-PAGE separation, lysate samples were probed with either the anti-HA antibody or the rabbit anti-serum. Lysates from cells prepared in the presence and absence of Forskolin show a band reactive with anti-HA antibody (M r ⁇ 30 kD).
- cells were washed with ice-cold PBS and lysed in RIPA buffer (50 mM HEPES, pH 7.5, 1% deoxycholate, 1% NP-40, 0.1% SDS, 150 mM
- Lysates were centrifuged at 16,000 x g for 15 min at 4°C and the cleared supernatant was transfe ⁇ ed to new tubes.
- Beads were washed with RIPA buffer, boiled in loading buffer (2% SDS, 10% glycerol, 5%
- Phosphoserine BADs can be used in assays to screen for inhibitors or activators of serine-phosphatase agents in which a test agent converts the serine-phosphorylated BAD to the non-phosphorylated BAD death promoter.
- the polypeptides can also be used to screen for and identify serine phosphatase agents that are capable of participating in the control of apoptosis.
- GST-BCI-X L (1 mg/ml final concentration) was added to cell lysates derived from BAD expressing cells, and incubated with rotation for two hours at 4°C. GST-BCI-X L and any associated BAD were then isolated using glutathione beads
- phosphatase treatments For phosphatase treatments, cells were lysed in RIPA buffer without phosphatase inhibitors. Lambda protein phosphatase (New England Biolabs) was added to the lysate at 4 U/ml, supplemented with MnCl 2 and phosphatase reaction buffer (50 mM Tris-HCl, pH 7.8, containing 5 mM dithiothreitol). The reaction proceeded for 30 min. at 30°C.
- Beta-gal ELISA assays were performed using the ⁇ -gal ELISA system from Boehringer Mannheim, according to manufacture's instructions. Cells were transfected with
- BAD BAD mutant
- ⁇ -gal ⁇ -gal in a ratio of 5:1.
- the transfected cells were trypsinized and distributed into 96-well tissue culture plates. Twenty-four hours after transfection, cell
- MTP anti- ⁇ -gal-coated microtiter plates
- gal activity was measured by the absorbance of the sample at 410 nM. Data were based on triplicate wells with standard deviations calculated.
- HeLa cells were plated in 12-well plates at 5 x IO 4
- transfection reagent Qiagen. Twenty-four hours after transfection, cells were cultured in serum-free medium (SFM) or in SFM plus EGF, for additional 12 hours. Beta-gal activity
- biotinylated at the amino terminus was added to the wells in 50 ⁇ l of 10 mM HEPES
- GST-BCI-X L fusion protein was produced in E. coli by a similar procedure described for the production of GST-BAD (see above).
Abstract
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US20040082509A1 (en) | 1999-10-12 | 2004-04-29 | Christophe Bonny | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
US8183339B1 (en) | 1999-10-12 | 2012-05-22 | Xigen S.A. | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
JP4142356B2 (en) | 2001-07-05 | 2008-09-03 | オイクロ ヨーロピアン コントラクト リサーチ ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディト ゲゼルシャフト | PP2C substrate |
EP1273654A1 (en) * | 2001-07-05 | 2003-01-08 | Schering Aktiengesellschaft | BAD as substrate for PP2C |
US20040235163A1 (en) * | 2001-08-22 | 2004-11-25 | Mitsuhiro Yokota | Myocardial cell apoptosis inhibitors |
WO2003042239A1 (en) * | 2001-11-12 | 2003-05-22 | Stichting Sanquin Bloedvoorziening | Peptides inhibiting signaling by ras-like gtpases |
DE60210919T2 (en) * | 2002-03-07 | 2007-05-10 | Institut Pasteur | A method for screening compounds that modulate apoptosis, compounds so identified, and use of the compounds as pharmaceutical agents |
WO2003082322A1 (en) * | 2002-03-28 | 2003-10-09 | Medvet Science Pty. Ltd. | A method of modulating cellular activity |
JP2004018524A (en) * | 2002-06-13 | 2004-01-22 | Eucro European Contract Research Gmbh & Co Kg | Method for treating arteriosclerosis |
EP1661912A1 (en) * | 2004-11-29 | 2006-05-31 | Xigen S.A. | Fusion protein comprising a BH3-domain of a BH3-only protein |
WO2007031098A1 (en) | 2005-09-12 | 2007-03-22 | Xigen S.A. | Cell-permeable peptide inhibitors of the jnk signal transduction pathway |
US8080517B2 (en) | 2005-09-12 | 2011-12-20 | Xigen Sa | Cell-permeable peptide inhibitors of the JNK signal transduction pathway |
WO2007035494A2 (en) * | 2005-09-16 | 2007-03-29 | The Regents Of The University Of California | Inducing expression of puma to reduce joint inflammation in the treatment of arthritis |
JP2010521183A (en) * | 2007-03-20 | 2010-06-24 | ザ ウォルター アンド エリザ ホール インスティテュート オブ メディカル リサーチ | Screening method |
WO2009143864A1 (en) | 2008-05-30 | 2009-12-03 | Xigen S.A. | Use of cell-permeable peptide inhibitors of the jnk signal transduction pathway for the treatment of chronic or non-chronic inflammatory digestive diseases |
WO2009143865A1 (en) | 2008-05-30 | 2009-12-03 | Xigen S.A. | Use of cell-permeable peptide inhibitors of the jnk signal transduction pathway for the treatment of various diseases |
WO2010072228A1 (en) | 2008-12-22 | 2010-07-01 | Xigen S.A. | Novel transporter constructs and transporter cargo conjugate molecules |
WO2011160653A1 (en) | 2010-06-21 | 2011-12-29 | Xigen S.A. | Novel jnk inhibitor molecules |
US9150618B2 (en) | 2010-10-14 | 2015-10-06 | Xigen Inflammation Ltd. | Use of cell-permeable peptide inhibitors of the JNK signal transduction pathway for the treatment of chronic or non-chronic inflammatory eye diseases |
WO2013091670A1 (en) | 2011-12-21 | 2013-06-27 | Xigen S.A. | Novel jnk inhibitor molecules for treatment of various diseases |
CA2903275A1 (en) | 2013-06-26 | 2014-12-31 | Xigen Inflammation Ltd. | New use of cell-permeable peptide inhibitors of the jnk signal transduction pathway for the treatment of various diseases |
WO2014206427A1 (en) | 2013-06-26 | 2014-12-31 | Xigen Inflammation Ltd. | New use of cell-permeable peptide inhibitors of the jnk signal transduction pathway for the treatment of various diseases |
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