KR20090055157A - Method for diagnosis of cancer by measurement of tyrosine 9 phosphorylation of pdk1 - Google Patents

Method for diagnosis of cancer by measurement of tyrosine 9 phosphorylation of pdk1 Download PDF

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KR20090055157A
KR20090055157A KR1020070121931A KR20070121931A KR20090055157A KR 20090055157 A KR20090055157 A KR 20090055157A KR 1020070121931 A KR1020070121931 A KR 1020070121931A KR 20070121931 A KR20070121931 A KR 20070121931A KR 20090055157 A KR20090055157 A KR 20090055157A
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pdk1
cancer
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박종선
허강민
양금진
석정호
홍장희
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Abstract

A method for diagnosing cancer by measuring tyrosine 9 phosphorylation of PDK1 is provided to examine the activation of PDK1 by measuring the expression degree of PDK1 and Hsp90 protein. A method for diagnosing cancer by measuring tyrosine 9 phosphorylation of PDK1 (PDK1 Try9) comprises comparing phosphorylation amount of PDK1 Try9 between normal cell and sample cell. The phosphorylation amount of PDK1 Try9 is measured through the fluorescence staining by using anti-Y9K antibody. The cancer is gastric cancer, colon cancer, lung cancer or breast cancer. The method for diagnosing cancer further contains comparing the expression level of PDK1 and/or Hsp90 between normal cell and sample cell.

Description

ＰＤＫ１의 티로신９ 인산화 측정에 의한 암 진단 방법{Method for diagnosis of cancer by measurement of tyrosine ９ phosphorylation of ＰＤＫ１}Method for diagnosis of cancer by measurement of tyrosine ９ phosphorylation of PDX1

본 발명은 PDK1의 Tyr9 인산화도 측정에 의한 암 진단 방법에 관한 것으로, 보다 상세하게는 암의 발생 및 전이와 관련된 PDK1의 활성화가 PDK1의 Tyr9의 인산화로부터 기인함을 밝히고 이를 이용하여 암을 진단하는 방법에 관한 것이다.The present invention relates to a method for diagnosing cancer by measuring the degree of Tyr9 phosphorylation of PDK1, and more particularly, the activation of PDK1 associated with the occurrence and metastasis of cancer originates from the phosphorylation of Tyr9 of PDK1 and diagnoses cancer using the same. It is about a method.

3-phosphoinoitide-dependent protein kinase-1(PDK1)은 protein kinase B (PKB/Akt)의 상위 카이네이즈로 알려진 이후, 많은 세포 신호전달 과정에 관여하는 대표적인 카이네이즈로 알려져 왔다. 3-phosphoinoitide-dependent protein kinase-1 (PDK1) has been known as a top kinase of protein kinase B (PKB / Akt) and has been known as a representative kinase involved in many cellular signaling processes.

PDK1은 N-말단의 카이네이즈 도메인과 C-말단의 pleckstrin homology (PH) 도메인으로 구성되어있다. PDK1에 의한 PKB의 인산화는 phosphoinositide 3-kinase (PI3K)가 먼저 활성화 됨으로써 두 번째 신호전달자인 PtdIns[3, 4, 5]P3과 PtdIns[3, 4]P2 가 생성되어서 PDK1과 PKB가 PH 도메인에 결합하여 일어난다. 지질은 PKB나 PDK1을 활성화시키는 대신에 효소들을 세포막으로 불러들이고 같은 곳에서 만나도록 하여서 PDK1이 PKB를 활성화시키도록 한다. PDK1 consists of a N-terminal kinase domain and a C-terminal pleckstrin homology (PH) domain. Phosphorylation of PKB by PDK1 resulted in the activation of phosphoinositide 3-kinase (PI3K) first, resulting in the formation of a second signal carrier, PtdIns [3, 4, 5] P 3 and PtdIns [3, 4] P 2 , Occurs in conjunction with the domain. Instead of activating PKB or PDK1, lipids bring enzymes into the cell membrane and meet in the same place, allowing PDK1 to activate PKB.

PKB와는 달리 p70 ribosomal protein S6 kinase (p70S6K), serum and glucocorticoid-inducible kinase (SGK), p90 ribosomal protein S6 kinase (RSK)과 같은 다른 PDK1 기질들은 인지질과 결합하지는 않는다. 대신에 hydrophobic motif (HM)로 알려진 카이네이즈 도메인에서 이 효소들의 C-말단에 위치한 영역을 인산화 시키는 것에 의해 이 효소들을 인산화한다.Unlike PKB, other PDK1 substrates such as p70 ribosomal protein S6 kinase (p70S6K), serum and glucocorticoid-inducible kinase (SGK), and p90 ribosomal protein S6 kinase (RSK) do not bind phospholipids. Instead, they phosphorylate these enzymes by phosphorylating the C-terminal region of the enzymes in a kinase domain known as a hydrophobic motif (HM).

PDK1이 인슐린 신호전달 과정에 중요한 역할을 한다는 것은 잘 알려져 있지만 PDK1 활성이 조절되는 기전은 아직 확실하지 않다. 세포막에서 PDK1은 최적의 활성을 위해서 적절한 3차 구조를 이루고 있다. 기존의 보고에 따르면 활성화 고리에 존재하는 Ser241의 인산화가 PDK1 활성화에 필요하다고 한다(Biochem. J. 342(Pt 2), 287-292 (1999)). 또한 PDK1이 여러 가지 성장 조절인자에 의해서 tyrosine 잔기에 인산화된다는 확실한 증거가 있다. HEK293 세포에서 tyrosine phosphatase 저해제인 pervanadate를 이용한 실험에서 PDK1이 100% 활성화되기 위해서는 Tyr373/376에 인산화가 필요하다는 결과가 있었다(J. Biological Chem. 276(40), 37459-37471 (2001)). 또한 HEK293 세포에서 인슐린에 의한 PDK1 활성 조절이 Src 카이네이즈군에 의해서 이루어진다는 것도 보고되었다. 그러나 어떻게 Src 카이네이즈가 tyrosine 인산화에 의한 PDK1의 활성화에 기여하는지에 대한 분 자적 기전은 밝혀지지 않았다. It is well known that PDK1 plays an important role in insulin signaling, but the mechanism by which PDK1 activity is regulated is still unclear. PDK1 has a proper tertiary structure in the cell membrane for optimal activity. Previous reports indicate that phosphorylation of Ser241 in the activation ring is required for PDK1 activation (Biochem. J. 342 (Pt 2), 287-292 (1999)). There is also clear evidence that PDK1 is phosphorylated at tyrosine residues by several growth regulators. Experiments using pervanadate, a tyrosine phosphatase inhibitor in HEK293 cells, indicated that Tyr373 / 376 required phosphorylation for 100% activation of PDK1 (J. Biological Chem. 276 (40), 37459-37471 (2001)). It has also been reported that the regulation of PDK1 activity by insulin in HEK293 cells is achieved by the Src kinase group. However, the molecular mechanism of how Src kinase contributes to the activation of PDK1 by tyrosine phosphorylation is unknown.

한편, PDK1이 여러 가지 암의 진행과 전이에 관련되어 있다는 보고가 증가하고 있어, 암에 대한 신약의 개발 시 PI3K/PDK1/PKB 신호전달에 관련된 체계를 목표로 하고 있다. 예를 들어, Ras 활성화와 PTEN 비활성화는 PI3K/PDK1/PKB 신호를 자극함으로써 갑상선 암의 진행에 중요한 작용을 한다 (Motti et al, 2005, Am J Pathol, 166, 737-749)고 하며, PKB는 인간의 갑상선 암과 갑상선 암 세포에서 세포 이동과 전이에 중요한 조절자임이 조사되었다 (Vasko et al, 2004, J Med Genet, 41, 161-170; Vasko et al, 2007, Endocr Dev, 10, 140-172). 또한 PDK1 저해제인 UCN-01 (Facchinetti et al, 2004, Cancer Res, 15, 3629-3637) 등은 대장암, 위암, 간암, 유방암 등에 효과가 있다고 보고되고 있다. On the other hand, there are increasing reports that PDK1 is involved in the progression and metastasis of various cancers, aiming for a system related to PI3K / PDK1 / PKB signaling in the development of new drugs for cancer. For example, Ras activation and PTEN inactivation play an important role in the progression of thyroid cancer by stimulating PI3K / PDK1 / PKB signaling (Motti et al, 2005, Am J Pathol, 166, 737-749). It has been investigated as an important regulator of cell migration and metastasis in human thyroid and thyroid cancer cells (Vasko et al, 2004, J Med Genet, 41, 161-170; Vasko et al, 2007, Endocr Dev, 10, 140- 172). In addition, PDK1 inhibitor UCN-01 (Facchinetti et al, 2004, Cancer Res, 15, 3629-3637) is reported to be effective in colorectal cancer, gastric cancer, liver cancer, breast cancer and the like.

그러나 PDK1의 활성화에 대한 분자적 기전이 잘 알려져 있지 않기 때문에 암의 진행과 PDK1의 어떻게 관련되어 있는지는 아직 명확히 이해되고 있지 않다. 따라서, PDK1의 분자적 기전에 대한 연구는 암의 진단, 더 나아가 암의 예방 및 치료에도 적용이 가능할 것으로 기대된다. However, since the molecular mechanisms for PDK1 activation are not well known, it is not yet clearly understood how cancer progression relates to PDK1. Therefore, the study of the molecular mechanism of PDK1 is expected to be applicable to the diagnosis of cancer, further to the prevention and treatment of cancer.

본 발명은 상기와 같은 종래기술의 문제점을 해결하기 위한 것으로, PDK1의 활성화에 대한 분자적 기전에 대해 이해함으로써, 암의 진단 및 예방, 치료에 응용할 수 있는 방법을 제공하는 것을 목적으로 한다.The present invention is to solve the above problems of the prior art, by understanding the molecular mechanisms for the activation of PDK1, it is an object of the present invention to provide a method that can be applied to the diagnosis, prevention, treatment of cancer.

전술한 목적을 달성하기 위한 본 발명은 정상세포와 샘플세포의 PDK1 Tyr9의 인산화도를 측정하여 비교하는 것을 특징으로 하는 암 진단 방법에 관한 것이다. 상기 PDK1 Try9의 인산화도의 측정은 anti-Y9K 항체를 이용한 형광 염색법을 이용할 수 있다.The present invention for achieving the above object relates to a method for diagnosing cancer, characterized in that by comparing the degree of phosphorylation of PDK1 Tyr9 of normal cells and sample cells. The phosphorylation of PDK1 Try9 can be measured by fluorescence staining using anti-Y9K antibody.

본 발명의 암 진단의 정확성을 높이기 위해서, 상기 PDK1 Try9의 인산화도 측정과 더불어 세포내 PDK1 및/또는 Hsp90의 발현 레벨을 추가로 비교할 수 있다. 암 세포에서 PDK1 및 Hsp90의 발현은 정상 세포에 비해 크게 증가하므로 PDK1 Try9의 인산화도와 함께 암의 표지로서 사용이 가능하다. In order to increase the accuracy of cancer diagnosis of the present invention, the expression level of intracellular PDK1 and / or Hsp90 can be further compared with the phosphorylation of PDK1 Try9. The expression of PDK1 and Hsp90 in cancer cells is significantly increased compared to normal cells, and thus can be used as a cancer marker with the phosphorylation degree of PDK1 Try9.

이하에서, 본 발명에 대하여 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.

인슐린이나 pervanadate를 세포에 처리하면 PDK1이 인산화가 된다는 것은 보고된 바 있다. 또한 HEK293 세포나 L6 근육종세포에 v-Src 또는 IR을 발현시키면 PDK1의 tyrosine 잔기에 인산화가 유도된다는 것도 보고되었다. 이러한 결과들을 토대로 인산화된 tyrosine 잔기와 결합하는데 관여하는 SH2 도메인을 가지고 있는 여러 가지 단백질들과 tyrosine 잔기가 인산화된 PDK1과 사이에 상호작용이 있는지를 조사하여, Src, Crk, GAP의 SH2 도메인이 in vitro에서 tyrosine 잔기가 인산화된 PDK1과 결합하는 것을 관찰하였다. PI3K 신호전달 과정에서 PDK1의 조절 메 커니즘을 연구하는 데에 주로 사용되는 HEK293 세포에서 Src의 부분삭제 돌연변이들을 사용해서 Src의 SH2 도메인이 PDK1과 Src이 결합하는데 관여한다는 것을 재확인하였다. It has been reported that treatment of insulin or pervanadate with cells results in phosphorylation of PDK1. It has also been reported that expression of v-Src or IR in HEK293 cells or L6 myoma cells induces phosphorylation at tyrosine residues in PDK1. To investigate whether the interaction between and the various proteins and tyrosine residues phosphorylated PDK1 with SH2 domains are involved in binding with phosphorylated tyrosine residues on the basis of these results, Src, Crk, the SH2 domain of GAP in In vitro , tyrosine residues were observed to bind to phosphorylated PDK1. Src partial deletion mutations were used in HEK293 cells mainly used to study the regulatory mechanism of PDK1 during PI3K signaling, reconfirming that the SH2 domain of Src is involved in binding PDK1 and Src.

PDK1의 특정서열 돌연변이들을 사용하여서는 Tyr9을 Phe로 치환하면 PDK1/Src 복합체 형성이 완전히 저해되고 Tyr373/376을 Phe로 치환하면 PDK1과 Src의 결합이 다소 감소하는 것으로 나왔다. Phosphopeptide binding assay 결과들을 바탕으로 PDK1의 Tyr9 인산화는 Src과 결합하는 데에 필요한 교두보 역할을 한다는 것을 의미하고 이로 인하여 PDK1 활성화가 유도된다는 것을 말한다. Using specific sequence mutations in PDK1, substitution of Tyr9 with Phe completely inhibited PDK1 / Src complex formation, and substitution of Tyr373 / 376 with Phe reduced PDK1 and Src binding slightly. Based on the results of the phosphopeptide binding assay, Tyr9 phosphorylation of PDK1 means that it acts as a bridgehead for Src binding, which induces PDK1 activation.

기존의 보고와 마찬가지로 Hsp90 저해제인 geldanamycin은 PDK1 단백질량을 감소시켰다. 이러한 PDK1 단백질의 불안정화 효과는 PDK1-Y9F가 발현하는 HEK293 세포에서는 일부 감소하였다. Hsp90를 동시에 발현시키면 PDK1/Src 복합체 형성이 다시 증가되었고 PDK1 활성도도 증가하였으며 이로 인하여 AGC 카이네이즈들의 인산화가 향상되었다. 더욱이 siRNA를 통해서 Hsp90를 제거하면 PDK1 활성이 감소하였다. 이러한 결과들로부터 PDK1 활성은 Src의 SH2 도메인에 의해서 Src/PDK1/Hsp90 복합체 형성을 유도한다고 할 수 있다.As previously reported, geldanamycin, an Hsp90 inhibitor, reduced the amount of PDK1 protein. The destabilizing effect of PDK1 protein was partially reduced in HEK293 cells expressing PDK1-Y9F. Simultaneous expression of Hsp90 increased PDK1 / Src complex formation and increased PDK1 activity, thereby improving phosphorylation of AGC kinase. Furthermore, removing Hsp90 through siRNA decreased PDK1 activity. These results suggest that PDK1 activity induces Src / PDK1 / Hsp90 complex formation by the SH2 domain of Src.

PDK1이 암의 발생 및 전이와 관련이 있을 것이라는 기존의 보고와 상기와 같은 PDK1의 작용 기작으로부터 PDK1 Tyr9의 인산화 정도가 암의 발생 및 전이의 표지로서 작용할 수 있을 것으로 예상하고, 위암, 폐암, 결장암 및 유방암 세포에서 대조군인 정상 세포에 비해 PDK1 Try9의 인산화가 크게 증가한 것을 확인할 수 있었다. 또한, 각 결장암 세포에서 PDK1과 Hsp90 단백질 레벨 역시 크게 증가하여 암 진단에 응용할 수 있음을 알 수 있었다. From previous reports that PDK1 may be involved in the development and metastasis of cancer and the mechanism of action of PDK1, it is expected that the degree of phosphorylation of PDK1 Tyr9 could serve as a marker of the development and metastasis of gastric cancer, lung cancer and colon cancer. And it was confirmed that the phosphorylation of PDK1 Try9 was significantly increased compared to the normal control cells in breast cancer cells. In addition, PDK1 and Hsp90 protein levels in each colon cancer cells also increased significantly, it can be seen that it can be applied to cancer diagnosis.

현재 Hsp90과 PDK1의 저해제로 임상적으로 쓰이고 있는 약물들이 존재한다. 본 실험에서도 사용한 Hsp90 저해제인 17-AAG (Banerji et al, 2005; Neckers et al, 2005), PDK1 저해제인 UCN-01 (Facchinetti et al, 2004) 또는 PI3K 저해제인 LY294002 (Newton, 2004; Wei et al, 2005) 등은 대장암, 위암, 간암, 유방암 등에 효과가 있다고 보고되고 있다. 이와 같이 PI3K/PDK1/PKB 신호 전달 메커니즘은 암의 진행뿐만 아니라 암 치료에 있어서 암에 대한 환자의 반응에도 영향을 준다고 보고되고 있다 (Vara et al, 2004). 따라서 PDK1과 Hsp90 단백질의 발현 정도를 측정하거나 유도된 PDK1의 활성도 정도를 알 수 있는 Tyr9 인산화 정도를 통해서 암을 진단함으로써 암의 원인이 되는 과정을 알 수 있다.Currently, there are drugs that are used clinically as inhibitors of Hsp90 and PDK1. Hsp90 inhibitor 17-AAG (Banerji et al , 2005; Neckers et al , 2005), PDK1 inhibitor UCN-01 (Facchinetti et al, 2004), or PI3K inhibitor LY294002 (Newton, 2004; Wei et al) , 2005) have been reported to be effective in colorectal cancer, stomach cancer, liver cancer and breast cancer. As such, it has been reported that PI3K / PDK1 / PKB signaling mechanisms affect not only cancer progression but also patient response to cancer in cancer treatment (Vara et al, 2004). Therefore, the cancer-causing process can be known by measuring the expression level of PDK1 and Hsp90 proteins or diagnosing the cancer through Tyr9 phosphorylation, which indicates the degree of PDK1 activity.

이상과 같이 본 발명에 의하면 암의 원인이 되는 PDK1과 Hsp90 단백질의 발현 정도를 측정하거나 유도된 PDK1의 활성도 정도를 알 수 있는 Tyr9 인산화 정도를 통해서 암을 진단함으로써 보다 정확하게 암을 판별할 수 있으며 많은 환자, 소량의 시료로 동시에 또한 용이하게 조기 진단 할 수 있다. As described above, according to the present invention, cancer can be more accurately discriminated by measuring the expression level of PDK1 and Hsp90 proteins that cause cancer, or by diagnosing cancer through Tyr9 phosphorylation level, which indicates the degree of activity of induced PDK1. The patient can be diagnosed early and easily at the same time with a small sample.

이하 실시예를 통하여 본 발명을 상세하게 설명한다. 그러나, 이들 실시예 는 예시적인 목적일 뿐 본 발명이 이에 한정되는 것은 아니다.The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

실시예Example

실험방법Experiment method

하기 실시예에 사용된 시약 및 구체적인 실험방법은 다음과 같다.The reagents used in the following examples and specific experimental methods are as follows.

1) 시약1) Reagent

Anti-Myc 9E10(이하 anti-Myc로 약칭)과 anti-HA 12CA5(이하 anti-HA로 약칭) 단일 클론 항체는 하이브리도마(ATCC)로부터 생산하였다. Anti-PDK1과 anti-PKB와 anti-pSGK (T256) 항체는 Upstate-Biotechnology로부터, Anti-GST와 anti-Hsp90 항체는 Stressgene으로부터, Anti-Flag과 anti-actin 항체는 Sigma로부터, Anti-pPKB (S473) 항체는 Cell Signaling으로부터 각각 구입하였다. Anti-pPDK1 (Y9 or Y373/376) 항체는 The Journal of Biololgical Chemistry 276(40), 37459 (2001)에 기재된 방법에 의해 얻었다. HRP 결합 anti-mouse IgG 및 anti-rabbit IgG는 Calbiochem에서, HRP 결합 anti-sheep IgG는 Sigma에서 구입하였다. Sodium orthovanadate(Na3VO4)와 암모늄 sulfate ((NH4)2SO4)는 Sigma로부터 구매하여 사용하였다. Anti-Myc 9E10 (hereinafter referred to as anti-Myc) and anti-HA 12CA5 (hereinafter referred to as anti-HA) monoclonal antibodies were produced from hybridomas (ATCC). Anti-PDK1 and anti-PKB and anti-pSGK (T256) antibodies from Upstate-Biotechnology, Anti-GST and anti-Hsp90 antibodies from Stressgene, Anti-Flag and anti-actin antibodies from Sigma, and Anti-pPKB (S473) ) Antibodies were purchased from Cell Signaling, respectively. Anti-pPDK1 (Y9 or Y373 / 376) antibodies were obtained by the methods described in The Journal of Biololgical Chemistry 276 (40), 37459 (2001). HRP binding anti-mouse IgG and anti-rabbit IgG were purchased from Calbiochem and HRP binding anti-sheep IgG was purchased from Sigma. Sodium orthovanadate (Na 3 VO 4 ) and ammonium sulfate ((NH 4 ) 2 SO 4 ) were purchased from Sigma.

2) 발현벡터의 구축2) Construction of Expression Vectors

Myc-PDK1과 그 인산화 위치의 돌연변이체 Myc-PDK1 Y9F 및 Myc-PDK1 Y373/376F는 The Journal of Biololgical Chemistry 276(40), 37459 (2001)에 보고된 방법에 의해 제작하였다. Myc-PDK1 and the mutants Myc-PDK1 Y9F and Myc-PDK1 Y373 / 376F at their phosphorylation sites were prepared by the methods reported in The Journal of Biololgical Chemistry 276 (40), 37459 (2001).

HA-Src는 HEK293 셀 (ATCC) 의 cDNA를 프라이머 5'-CG GGA TCC ATG GGG AGC AGC AAG(서열번호 1)와 3'-G GAA TTC CTA TAG GTT CTC TCC(서열번호 2)로 증폭하여 제조하였다. 증폭된 PCR 산물을 포유동물의 발현벡터 pcDNA3-HA의 BamH I과 EcoR I 사이에 서브클론하였다. HA-Src was prepared by amplifying the cDNA of HEK293 cells (ATCC) with primers 5'-CG GGA TCC ATG GGG AGC AGC AAG (SEQ ID NO: 1) and 3'-G GAA TTC CTA TAG GTT CTC TCC (SEQ ID NO: 2) It was. The amplified PCR product was subcloned between BamH I and EcoR I of mammalian expression vector pcDNA3-HA.

Src의 결실변이주(HA-Src-ΔSH2 및 HA-Src-ΔSH3)는 pcDNA3-HA 발현 벡터로부터 표준 PCR-cloning 방법에 의해 구성하였다. HA-Src-ΔSH2 및 HA-Src-ΔSH3의 개열지도는 도 1에 각각 도시하였다.Deletion mutants of Src (HA-Src-ΔSH2 and HA-Src-ΔSH3) were constructed by standard PCR-cloning methods from pcDNA3-HA expression vectors. Cleavage maps of HA-Src-ΔSH2 and HA-Src-ΔSH3 are shown in FIG. 1, respectively.

Lys295 (kinase-deficient HA-Src KD K295M)와 Tyr527 (constitutive-active HA-Src-CA Y527F)의 변이체는 QuikChangeTM Site-Directed Mutagenesis Kit (Stratagene)를 사용하여 pcDNA3 HA-Src-WT를 주형으로 매뉴얼에 따라 제조하였다. Flag-Hsp90은 Carcinogenesis (2006, 28; 71-80)에 기재된 방법에 의해 제조하였다. pTaq2에서 Flag-Hsp40과 Flag-Hsp27은 Dr. YM Kim(NIH, USA)로부터 제공받았다. 전기의 모든 발현벡터들은 DNA 오토시퀀싱에 의해 확인하고 각 서열을 서열번호 3~14에 기재하였다.Lys295 (kinase-deficient HA-Src KD K295M) and Tyr527 (constitutive-active HA-Src -CA Y527F) mutant of the QuikChange TM Site-Directed Mutagenesis Kit using (Stratagene) pcDNA3 HA-Src- WT the man as a template It was prepared according to. Flag-Hsp90 is a carcinogenesis (2006, 28; 71-80). Flag-Hsp40 and Flag-Hsp27 in Dr. YM Kim (NIH, USA). All the expression vectors mentioned above were confirmed by DNA autosequencing and each sequence was set forth in SEQ ID NOs: 3 to 14.

3) 세포 배양 및 자극 3) Cell Culture and Stimulation

Myc tagged PDK1 WT, PDK1 Y9F 또는 PDK1 Y373/3736F를 발현하는 안정한 세 포주는 HEK 293 세포로부터 The Journal of Biololgical Chemistry 276(40), 37459 (2001)에서 보고한 것과 같이 유도하였다. 상기 세포들은 10% FBS, 2 mM 글루타민, 100 unit/ml 페니실린, 100 μg/ml 스트렙토마이신 (Life Technologies), 1 μg/ml puromycine이 보충된 DMEM(Dulbecco's modified Eagle's Medium)에 보관하고, 제조자 매뉴얼에 따라 jetPEI(Q-biogene) 또는 Lipofectamine(Invitrogen)을 사용하여 감염(transfect)시겼다. 감염(transfection) 혼합물은 24 h 인큐베이션 후 제거하였으며, 세포는 0.2 mM H2O2로 제조된 0.1 M pervanadate로 15분, 또는 5 μg/ml Geldanamycin (GA; Calbiochem)이나 1μM 17-allylamino-17-demethoxygeldanamycin(17-AAG; Calbiochem)으로 각 표시된 시간 동안 stimulation 전에 16시간 동안 serum-starved 하였다. 세포 자극 전에 1μM latacystin (Alexis), 1 μM PSI (Alexis) 또는 5 nM MG132 (Calbiochem)로 1시간 동안 전처리하였다. RNA 간섭(interference)을 위해, 인간 Hsp90α/β siRNA와 대조군으로서 scrambled RNA duplex를 Santa Cruz Biotechnology로부터 입수하였다. HEK293 세포는 Lipofectamine (Invitrogen)을 사용하여 Hsp90α/β siRNA 또는 대조군 siRNA로 감염시켰다.Stable cells expressing Myc tagged PDK1 WT, PDK1 Y9F or PDK1 Y373 / 3736F were derived from HEK 293 cells as reported in The Journal of Biololgical Chemistry 276 (40), 37459 (2001). The cells were stored in Dulbecco's modified Eagle's Medium (DMEM) supplemented with 10% FBS, 2 mM glutamine, 100 unit / ml penicillin, 100 μg / ml streptomycin (Life Technologies), 1 μg / ml puromycine, and in the manufacturer's manual. Therefore, the infection was performed using jetPEI (Q-biogene) or Lipofectamine (Invitrogen). The transfection mixture was removed after 24 h incubation and the cells were removed for 15 min at 0.1 M pervanadate prepared with 0.2 mM H 2 O 2 , or 5 μg / ml Geldanamycin (GA; Calbiochem) or 1 μM 17-allylamino-17- Demethoxygeldanamycin (17-AAG; Calbiochem) was serum-starved for 16 hours prior to stimulation for each indicated time. Prior to cell stimulation it was pretreated for 1 hour with 1 μM latacystin (Alexis), 1 μM PSI (Alexis) or 5 nM MG132 (Calbiochem). For RNA interference, human Hsp90α / β siRNA and scrambled RNA duplex as a control were obtained from Santa Cruz Biotechnology. HEK293 cells were infected with Hsp90α / β siRNA or control siRNA using Lipofectamine (Invitrogen).

4) 면역침강 및 PDK1의 in vitro kinase assay 4) immunoprecipitation and in vitro kinase assay of PDK1

HEK 293 세포를 얼음에 넣고 pH 7.5의 50mM Tris-HCl, 1% v/v NP-40, 120mM NaCl, 25mM sodium Fluoride, 40mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine 및 2μM microcystin-LR을 함유하는 lysis 완충액으로 추출하였다. Lysate를 12,000g에서 15분간 원심분리하고 Myc-PDK1 단백질을 셀을 제거한 추출물 500μg으로부터 protein G-Sepharose(Amersham Pharmacia)에 고정된 anti-Myc 9E10 단일클론항체로 면역침강시켰다. 면역복합체는 0.5M NaCl을 함유하는 lysis 완충액으로 한번 세척한 후 kinase assay 완충액(pH 7.5, 50mM Tris-HCl, 0.1% v/v 2-mercaptehanol)로 최종 세척하였다. In vitro kinase assay는 kinase 완충액에 30㎕ 면역침강물, 기질로서 100μM Suntide(RRKDGATMKTFCGTPE), 10mM MgCl2, 1μM protein kinase A inhibitor peptide(Alexis) 및 100μM [γ-32P]ATP(Amersham; 1000-2000 cpm/pmol)로 이루어진 반응물 50㎕에 대해 30℃에서 60분간 수행하였다. 반응물에 EDTA를 50mM 농도가 되도록 추가하여 반응을 중지시키고 The Journal of Biololgical Chemistry 276(40), 37459 (2001)에 기재한 방법에 의해 처리하였다. 단백질의 농도는 BSA를 표준물질로 사용하여 Bradford(BioRad)의 방법에 의해 측정하였다.Put HEK 293 cells on ice, 50 mM Tris-HCl, 1% v / v NP-40, 120 mM NaCl, 25 mM sodium fluoride, 40 mM β-glycerol phosphate, 0.1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine at pH 7.5 And lysis buffer containing 2 μM microcystin-LR. Lysate was centrifuged at 12,000 g for 15 min and Myc-PDK1 protein was immunoprecipitated with anti-Myc 9E10 monoclonal antibody immobilized on protein G-Sepharose (Amersham Pharmacia) from 500 μg of cell-free extract. The immunocomplex was washed once with lysis buffer containing 0.5M NaCl and finally washed with kinase assay buffer (pH 7.5, 50mM Tris-HCl, 0.1% v / v 2-mercaptehanol). In vitro kinase assay was performed with 30 μl immunoprecipitate in kinase buffer, 100 μM Suntide (RRKDGATMKTFCGTPE), 10 mM MgCl 2 , 1 μM protein kinase A inhibitor peptide (Alexis) and 100 μM [γ- 32 P] ATP (Amersham; 1000-2000 cpm) as substrate. / pmol) was carried out for 60 minutes at 30 ° C for 50 μl of the reaction. The reaction was stopped by adding EDTA to a 50 mM concentration in the reaction and treated by the method described in The Journal of Biololgical Chemistry 276 (40), 37459 (2001). Protein concentrations were measured by the method of Bradford (BioRad) using BSA as a standard.

5) 통계처리5) Statistical Processing

Tina 2.1 Program (Raytest Isotopenmegerate)을 사용하여 웨스턴 블럿 분석의 정량화하였다. 즉, 밴드의 상대적인 강도(area x density)는 densitometer에 의해 정량화하였다. 블랭크 밴드 (blank band)로 부터의 기본 값은 공재하였다. 결과는 해당하는 대조 밴드와 비교하여 비율 변화로서 계산하고, 데이타는 세번의 독립된 실험의 평균±표준 편차(SD)로 나타내었으며 Student's t-test에 의해 분석하였다(SPSS 12.0 Software, SPSS Inc). 대조값과 비교하여 *p < 0.05는 유의한 것으로, **p < 0.01은 매우 유의한 것으로 간주하였다.Western blot analysis was quantified using the Tina 2.1 Program (Raytest Isotopenmegerate). That is, the area intensity (area x density) of the band was quantified by a densitometer. Default values from blank bands were shared. Results were calculated as percentage changes compared to the corresponding control bands, and data were expressed as mean ± standard deviation (SD) of three independent experiments and analyzed by Student's t-test (SPSS 12.0 Software, SPSS Inc). Compared to the control, * p <0.05 was considered significant and ** p <0.01 was considered very significant.

실시예 1 : Tyrosine 잔기가 인산화된 PDK1과 SH2 도메인을 가지고 있는 단백질 간의 in vitro 결합Example 1 In Vitro Binding Between PDK1 and SH2 Domain Phosphorylated with Tyrosine Residue

The EMBO Journal 15(18), 4940-4948 (1996)에 기재된 방법에 의해 다양한 단백질들(Src, Lck, Fyn2, PLC gamma, p85, Crk, Syp, GAP, Nck, Grb2, Vav)의 SH-2 도메인을 GST-융합 단백질로 발현시킨 후 pervanadate를 처리한 세포로부터 면역침강법으로 얻은 Myc-PDK1과 반응시켜 결합능을 조사하였다. SH-2 of various proteins (Src, Lck, Fyn2, PLC gamma, p85, Crk, Syp, GAP, Nck, Grb2, Vav) by the method described in The EMBO Journal 15 (18), 4940-4948 (1996). The domains were expressed with GST-fusion protein and then reacted with Myc-PDK1 obtained by immunoprecipitation from cells treated with pervanadate to investigate their binding capacity.

보다 구체적으로, GST-SH2 융합 단백질을 GST-SH2 발현벡터로 형질전환된 BL21DE3 세포로부터 분리하였다. 각 단백질들의 발현벡터는 Dr. T. Pawson(University of Toronto, Canada)로부터 입수하였다. 박테리아는 먼저 37℃에서 2 시간 동안 배양하였고(OD600 = 0.5 - 0.7), 이후 0.5 mM isopropyl-thio-beta-D-galactopyranoside(IPTG; Promega)를 추가하여 induction하였다. 세포는 lysis 완충액 (0.5% Nonidet P-40, 50 mM Tris-HCl (pH 8), 120 mM NaCl, 7 mM DTT, 0.1 mM PMSF)에서 sonicator (Beckman)를 사용하여 lysis하였다. GST-SH2 융합 단백질은 glutathione agarose 비드(Amersham Bioscience)에서 EMBO J, 18, 3024 (1999)에 기재된 방법에 의해 정제하였다. More specifically, GST-SH2 fusion protein was isolated from BL21DE3 cells transformed with GST-SH2 expression vector. The expression vector of each protein is Dr. T. Pawson (University of Toronto, Canada). The bacteria were first incubated at 37 ° C. for 2 hours (OD600 = 0.5−0.7), followed by induction by adding 0.5 mM isopropyl-thio-beta-D-galactopyranoside (IPTG; Promega). Cells were lysed using sonicator (Beckman) in lysis buffer (0.5% Nonidet P-40, 50 mM Tris-HCl, pH 8), 120 mM NaCl, 7 mM DTT, 0.1 mM PMSF). GST-SH2 fusion proteins were purified by glutathione agarose beads (Amersham Bioscience) by the method described in EMBO J, 18, 3024 (1999).

Myc-PDK1을 과발현하는 HEK 293 세포는 상기 실험방법에 기재된 방법에 의해 15분간 100μM pervanadate와 함께 처리하였다. HEK 293 cells overexpressing Myc-PDK1 were treated with 100 μM pervanadate for 15 minutes by the method described in the above experimental method.

GST-pull-down assay를 위하여 Myc-PDK1 WT를 과발현한 lysate 500μg을 상기의 정제된 2μg GST 융합 단백질과 함께 4℃에서 2시간 동안 배양한 후, 비드를 세척 완충액(100 mM NaCl, 1 mM EDTA, 10 mM Tris-HCl (pH8.0), 0.5% NP-40 및 0.05% SDS)에서 세 번 세척하였다. 최종 세척의 상등액을 제거한 후, 샘플을 5X SDS 샘플 완충액에 재 현탁하고 5분간 끓인 후 비드에 남아있는 단백질을 SDS-PAGE에 용해시켰다. 500 μg of lysate overexpressing Myc-PDK1 WT was incubated for 2 hours at 4 ° C. with the purified 2 μg GST fusion protein for GST-pull-down assay, and the beads were washed with wash buffer (100 mM NaCl, 1 mM EDTA). , 10 mM Tris-HCl (pH 8.0), 0.5% NP-40 and 0.05% SDS). After removing the supernatant of the final wash, the sample was resuspended in 5X SDS sample buffer and boiled for 5 minutes before the proteins remaining in the beads were dissolved in SDS-PAGE.

상기 반응액을 웨스턴블럿 후 anti-PDK1 항체 및 anti-GST 항체로 분석한 결과를 도 2에 도시하였다. 도 2에서 확인할 수 있듯이 PDK1은 Src, Crk와 GAP의 SH2 도메인과 in vitro에서 결합하였으며(도 2, lanes 3, 11, 15), 다른 단백질들의 SH2 도메인이나 GST 자체와는 결합하지 않았다.The reaction solution was analyzed by Western blot, anti-PDK1 antibody and anti-GST antibody, and the results are shown in FIG. 2. As can be seen in Figure 2 PDK1 was coupled in vitro with the SH2 domain of Src, Crk and GAP (FIG. 2, lanes 3, 11, 15), and did not bind to the SH2 domain of other proteins or GST itself.

실시예 2 : PDK1 Tyr9의 인산화가 Src-PDK1의 결합에 미치는 영향Example 2 Effect of Phosphorylation of PDK1 Tyr9 on Binding of Src-PDK1

Src-SH2 도메인과 tyrosine 잔기가 인산화된 PDK1의 결합을 더 명확히 확인하기 위해서, HA-Src WT과 Myc-PDK1 WT이 in vitro와 in vivo에서 반응하는지를 조사하였다. In order to more clearly identify the binding of PDK1 to which the Src-SH2 domain and tyrosine residues were phosphorylated, we investigated whether HA-Src WT and Myc-PDK1 WT react in vitro and in vivo.

단계적 결합을 위해서, HEK-293 셀을 HA-Src WT로 감염시킨 후 100μM pervanadate로 15분간 처리하였다. 먼저 HA-Src WT는 anti-HA 12CA5 단일클론 항체를 사용하여 셀 추출물 500㎍으로부터 면역침강하였다. 면역침강된 HA-Src는 Myc-PDK1 WT 세포를 pervanadate 처리한 lysate와 함께 배양하고 다시 anti-HA 항체를 사용하여 면역침강하였다(Seq-bind).For step binding, HEK-293 cells were infected with HA-Src WT and then treated with 100 μM pervanadate for 15 minutes. First HA-Src WT was immunoprecipitated from 500 μg of cell extract using anti-HA 12CA5 monoclonal antibody. Immunoprecipitated HA-Src was incubated with pervanadate-treated lysate of Myc-PDK1 WT cells and again immunoprecipitated using anti-HA antibody (Seq-bind).

한편 동시 결합을 위해서는 HA-Src 및 Myc-PDK1이 각각 과발현된 lysate를 혼합하고 인큐베이션하여 결합 PDK1을 HA-Src 면역침강으로 회수하였다(Co-bind). Meanwhile, for simultaneous binding, lysate overexpressed HA-Src and Myc-PDK1 were mixed and incubated to recover binding PDK1 by HA-Src immunoprecipitation (Co-bind).

동시 발현을 위해서는, HA-Src WT를 Myc-PDK WT, Y9F 또는 Y373/376F를 안정적으로 과발현하는 HEK 293 셀에 감염시켰다. 세포를 100μM pervanadate로 15분간 처리하였다. HA-Src 면역침강에 결합된 Myc-PDK는 anti-Myc 9E10 단일클론 항체를 사용하여 검출하였다.For co-expression, HA-Src WT was infected with HEK 293 cells stably overexpressing Myc-PDK WT, Y9F or Y373 / 376F. Cells were treated with 100 μM pervanadate for 15 minutes. Myc-PDK bound to HA-Src immunoprecipitation was detected using anti-Myc 9E10 monoclonal antibody.

상기 모든 조건에서 결합된 PDK1의 tyrosine 인산화의 변화를 anti-pY9 또는 anti-pY373/376 항체를 사용하여 분석하고 그 결과를 도 2에 도시하였다. 각 PDK1 돌연변이 단백질의 input은 anti-Myc 항체를 사용하여 검출하고 도 3에 함께 도시하였다. Changes in tyrosine phosphorylation of PDK1 bound under all these conditions were analyzed using anti-pY9 or anti-pY373 / 376 antibodies and the results are shown in FIG. 2. The input of each PDK1 mutant protein was detected using anti-Myc antibody and shown together in FIG. 3.

도 3에서 확인할 수 있듯이 HA-Src WT와 Myc-PDK1 WT이 각각 과발현시킨 세포의 추출물을 섞었을 때에는 Src과 PDK1 간의 결합이 이루어지지 않았다 (레인2, 3). 반면에 HA-Src WT와 Myc-PDK1 WT을 한 세포에서 과발현시켰을 때에는 tyrosine 잔기가 인산화된 PDK1이 Src과 결합하였다 (Co-expression; 레인 4). 이것은 Src과 PDK1의 결합이 간접적이라는 것이고 in vivo에서 두 단백질 간의 결합을 촉진시키기 위한 또 다른 단백질들이 필요하다는 것을 의미한다. Y9F-PDK1은 Src과 한 세포에서 과발현시키면 Src/PDK1 복합체의 형성이 이루어지지 않았으나 Y373/376F-PDK1은 복합체 형성이 부분적으로 저해되었다 (레인 5, 6). 더욱이 Y373/376F- PDK1와의 결합에서는 Tyr9 인산화는 여전히 보여지지만 Y9F-PDK1과의 결합에서는 Tyr3737/376 인산화가 완전히 저해되었다. 이것은 Tyr9 인산화가 Src/PDK1 복합체 형성에 중요하다는 것을 의미한다. As can be seen in Figure 3 HA-Src WT and Myc-PDK1 WT when the extract of the cells overexpressed, respectively, when the mixture between the Src and PDK1 was not made (lane 2, 3). On the other hand, when HA-Src WT and Myc-PDK1 WT were overexpressed in one cell, PDK1 phosphorylated with tyrosine residues bound to Src (Co-expression; lane 4). This means that the binding of Src to PDK1 is indirect and that other proteins are needed to promote the binding between the two proteins in vivo. Overexpression of Y9F-PDK1 in Src and one cell did not result in the formation of the Src / PDK1 complex, whereas Y373 / 376F-PDK1 partially inhibited the complex formation (lanes 5 and 6). Moreover, Tyr9 phosphorylation was still seen in binding to Y373 / 376F-PDK1, but Tyr3737 / 376 phosphorylation was completely inhibited in binding to Y9F-PDK1. This means that Tyr9 phosphorylation is important for Src / PDK1 complex formation.

이러한 특정 서열에 관한 결합력에 대해서 더 증명하기 위해서 synthetic-phosphopeptide beads를 사용하여 확인하였다. 즉, 각각 pY9 또는 pY373/376 잔기를 포함한 인산화된 펩타이드 RTTSQLpYDAVPIQS (서열번호 15) 및 EDDEDCpYGNpYDNLLSQF(서열번호 16)를 합성하여 준비한 다음에 Affi-Gel 15에 부착시킨 후, 일부 GST-SH2 단백질들 (Src, Fyn2, Crk)과 반응시켰다. 각 GST-SH2 단백질과 함께 결합된 단백질을 anti-GST 항체를 이용하여 웨스턴 블럿에서 분석하여 도 4에 도시하였다. 도 2의 결과와 마찬가지로 도 4에서도 phosphopeptide beads에 결합한 GST-SH2 도메인을 통해서 Src과 Crk가 pY9 펩타이드와 강한 결합력이 있음을 확인할 수 있었다. 그러나 pY373/376 펩타이드와의 결합은 관찰되지 않았다. In order to further demonstrate the binding capacity for this particular sequence was confirmed using synthetic-phosphopeptide beads. That is, after synthesis and preparation of the phosphorylated peptides RTTSQLpYDAVPIQS (SEQ ID NO: 15) and EDDEDCpYGNpYDNLLSQF (SEQ ID NO: 16) containing pY9 or pY373 / 376 residues, respectively, and then attached to Affi-Gel 15, some GST-SH2 proteins (Src , Fyn2, Crk). Proteins bound with each GST-SH2 protein were shown in Figure 4 by analysis in Western blot using anti-GST antibodies. As in the result of FIG. 2, Src and Crk were strongly bound to the pY9 peptide through the GST-SH2 domain bound to the phosphopeptide beads in FIG. 4. However, no binding to the pY373 / 376 peptide was observed.

실시예 3 : Src와 Myc-PDK1의 결합에서 SH2 도메인의 역할Example 3 Role of SH2 Domain in Src and Myc-PDK1 Binding

Src/PDK1 복합체 형성에 Src의 각 도메인에 역할을 규명하기 위해서 SH2 또는 SH3 도메인을 제거한 돌연변이들 (HA-Src-△SH2 또는 HA-Src-△SH3), 카이네이즈 기능을 없앤 돌연변이 (HA-Src-KD), 항상 활성화 형태로 존재하는 돌연변이 (HA-Src-CA)를 제작하였다 (도 5의 A). 일시적으로 유전자를 삽입하면 발현량이 동일하지 않기 때문에 지속적으로 Myc-PDK1이 발현하는 HEK293 세포를 사용하였다. Mutations that remove the SH2 or SH3 domain (HA-Src-ΔSH2 or HA-Src-ΔSH3), mutations that lack kinase function (HA-Src-) to characterize each domain of Src in Src / PDK1 complex formation KD), a mutation (HA-Src-CA) was always present in the activated form (FIG. 5A). Since the expression level is not the same when the gene is temporarily inserted, HEK293 cells continuously expressing Myc-PDK1 were used.

Myc-PDK1을 안정적으로 과발현하는 HEK293 세포에 이 돌연변이체들을 감염시키고 100μM pervanadate로 15분간 처리하였다. Myc-PDK1을 사용하여 면역침강시킨 후 결합된 Src 돌연변이를 anti-HA 항체를 사용하여 immunoblotting하여 그 결과를 도 5의 B에 도시하였다. 도 5의 B에서 확인할 수 있듯이, Src의 SH2 도메인은 Src과 PDK1이 결합하는 데에 필요하다는 것을 알 수 있었다(도 5의 B, 레인 5). 또한, Src-ΔSH3와 Myc-PDK1과의 결합은 Src-WT과 비교하여 감소하였다 (도 5의 B, 레인 2, 6). 특히, Src-CA는 Src-WT과 비교하여 Myc-PDK1과 강한 결합을 보였다(도 5의 B, 레인3, 4). 반대로, Src-KD와 Myc-PDK1 사이의 결합은 거의 보여지지 않았다. HEK293 cells stably overexpressing Myc-PDK1 were infected with these mutants and treated with 100 μM pervanadate for 15 minutes. After immunoprecipitation using Myc-PDK1, bound Src mutants were immunoblotting using anti-HA antibody and the results are shown in B of FIG. 5. As can be seen in FIG. 5B, it was found that the SH2 domain of Src is required for Src and PDK1 to bind (B, lane 5 in FIG. 5). In addition, the binding of Src-ΔSH3 with Myc-PDK1 was reduced compared to Src-WT (B, lanes 2 and 6 in FIG. 5). In particular, Src-CA showed a strong binding to Myc-PDK1 compared to Src-WT (B, lanes 3 and 4 of Figure 5). In contrast, little binding between Src-KD and Myc-PDK1 was seen.

이는 Src과 PDK1과의 결합에는 Src의 SH2 도메인뿐만 아니라 Src의 활성도도 필요하다는 것을 말한다.This suggests that the binding of Src and PDK1 requires the activity of Src as well as the SH2 domain of Src.

실시예 4 : Hsp90 억제제에 의한 Hsp90/PDK1 복합체 형성 저해Example 4 Inhibition of Hsp90 / PDK1 Complex Formation by Hsp90 Inhibitors

PDK1/Src 복합체를 이루는 데에 Hsp90가 관여하는지 확인하기 위하여 Hsp90의 저해제로 알려진 geldanamycin이 PDK1 안정성에 영향을 주는지를 조사하였다. To determine whether Hsp90 is involved in the PDK1 / Src complex, we investigated whether geldanamycin, known as an inhibitor of Hsp90, affects PDK1 stability.

PDK1-WT, PDK1-Y9F 또는 PDK1-Y373/376F을 발현하는 HEK293 세포에 각각 10μM geldanamycin을 0, 12 또는 24시간 동안 처리하고 관찰하였다. 도 6의 A는 전체의 lysate를 anti-PDK1 항체로 immunoblotting에 의해 분석한 결과를, B는 이를 densitometry로 측정한 결과를 보여준다. A의 하단은 대조군으로서 동일한 양의 단백질을 anti-actin 항체로 immunoblotting하여 확인한 결과이다. PDK1의 안정성 은 PDK1-WT과 PDK1-Y373/376F에서는 geldanamycin 처리 시 24시간째에 약 30% 가량 감소하였으나, PDK1-Y9F 세포에서는 크게 영향을 미치지 않았다. HEK293 cells expressing PDK1-WT, PDK1-Y9F or PDK1-Y373 / 376F were treated with 10 μM geldanamycin for 0, 12 or 24 hours, respectively, and observed. Figure 6 A shows the result of the analysis of the whole lysate by anti-PDK1 antibody by immunoblotting, B shows the result measured by densitometry. The bottom of A is the result of immunoblotting of the same amount of protein with anti-actin antibody as a control. The stability of PDK1 decreased about 30% at 24 hours after geldanamycin treatment in PDK1-WT and PDK1-Y373 / 376F, but not in PDK1-Y9F cells.

동일 조건에서 셀 추출물로부터의 PDK1 면역침강물에 대하여 anti-Hsp90 항체로 웨스턴 블럿 분석을 실시하여 결합된 Hsp90의 분석으로부터 PDK1/Hsp90 복합체가 형성되는 정도를 측정하였다(도 6의 C, D). PDK1의 안정성과 마찬가지로 PDK1-WT 또는 PDK1-Y373/376F와 Hsp90와의 결합능은 geldanamycin의 처리에 의해서 감소하였는데 반해, PDK1-Y9F의 경우에서는 거의 변화가 없었다. Western blot analysis was performed with anti-Hsp90 antibody on PDK1 immunoprecipitates from cell extracts under the same conditions to determine the extent of formation of PDK1 / Hsp90 complex from analysis of bound Hsp90 (C, D of FIG. 6). As with PDK1, PDK1-WT or PDK1-Y373 / 376F binding to Hsp90 was decreased by geldanamycin treatment, whereas PDK1-Y9F showed little change.

실시예 5 : PDK1/Src 복합체와 Src에 의한 PDK1 활성화에 대한 Hsp90의 역할Example 5 Role of Hsp90 on PDK1 Activation by PDK1 / Src Complex and Src

PDK1/Src 결합에서 Hsp90가 직접적으로 관여하는지를 in vitro에서 검토하기 위해서, Myc-PDK1과 항상 활성화 상태로 존재하는 돌연변이 HA-Src-CA를 각각 준비하였다.To examine in vitro whether Hsp90 is directly involved in PDK1 / Src binding, Myc-PDK1 and mutant HA-Src-CA, which are always active, were prepared.

Myc-PDK1 WT를 안정적으로 발현하는 HEK 293 셀에 HA-Src-CA를 감염시켰다(co-exp). 한편, HA-Src-CA와 대조군 vector, Flag-Hsp90, Flag-Hsp40 또는 Flag-Hsp27을 serum 존재하에 감염시켰다(co-binding). Myc-PDK1 WT으로부터 pervanadate 처리된 lysate를 혼합하고 해당 lysate와 인큐베이션하고 anti-HA 항체로 면역침강한 후 결합된 Myc-PDK1을 anti-Myc 항체를 사용하여 검출하였다(도 7의 위). 각각을 anti-Myc, anti-HA 또는 anti-Flag 항체를 사용하여 체크하여 도 7에 함께 도시하였다. 도 7에서 확인할 수 있듯이, Hsp40이나 Hsp27이 아닌 Hsp90를 같이 발현시켜 주었을 때에는 in vitro에서도 PDK1/Src 복합체 형성이 증가하였 다(레인 3 위). HEK 293 cells stably expressing Myc-PDK1 WT were infected with HA-Src-CA (co-exp). Meanwhile, HA-Src-CA and the control vector, Flag-Hsp90, Flag-Hsp40 or Flag-Hsp27 were infected in the presence of serum (co-binding). Pervanadate treated lysate from Myc-PDK1 WT was mixed, incubated with the lysate, immunoprecipitated with anti-HA antibody and bound Myc-PDK1 was detected using anti-Myc antibody (above FIG. 7). Each was checked with anti-Myc, anti-HA or anti-Flag antibodies and shown together in FIG. 7. As can be seen in Figure 7, when the expression of Hsp90, but not Hsp40 or Hsp27 together, PDK1 / Src complex formation increased in vitro (lane 3).

Hsp90이 PDK1/Src 복합체 형성에서 하는 역할을 보다 구체적으로 확인하기 위하여 Myc-PDK1 WT 셀을 HA-Src WT (및 Flag-Hsp90)과 함께 감염시킨 후 100μM pervanadate로 15분간 처리하고, Myc-PDK1 면역침감체에 결합된 HA-Src와 단백질의 발현을 anti-HA, anti-Myc, anti-HA 및 anti-Hsp90 항체를 사용하여 각각 분석하여 도 8에 도시하였다. 도 8에서 Van은 pervanadate의 처리를 나타낸다. pervanadate를 처리한 경우, Myc-PDK1 면역침강체에 Src 결합이 다시 이루어진 것으로부터 대조군에 비해 Hsp90을 과발현하는 HEK293 세포에서 PDK1/Src 복합체 형성이 현저하게 증가함을 알 수 있다. To more specifically identify the role of Hsp90 in PDK1 / Src complex formation, Myc-PDK1 WT cells were infected with HA-Src WT (and Flag-Hsp90), treated with 100 μM pervanadate for 15 minutes, and subjected to Myc-PDK1 immunity. The expression of HA-Src and protein bound to the degenerate body was analyzed using anti-HA, anti-Myc, anti-HA, and anti-Hsp90 antibodies, respectively, and shown in FIG. 8. In Fig. 8, Van represents the processing of pervanadate. In the case of pervanadate treatment, the Sc binding to Myc-PDK1 immunoprecipitates showed that PDK1 / Src complex formation was markedly increased in HEK293 cells overexpressing Hsp90 compared to the control group.

PDK1/Src 복합체 형성 증가가 직접적으로 PDK1 활성화에까지 영향을 주는지를 검증하기 위해서, 면역침강된 PDK1의 tyrosine 인산화의 변화를 anti-pY9 또는 anti-pY373/376 항체를 사용하여 웨스턴 블럿에 의해 측정하고 actin으로 normalize하여 도 9의 A에 도시하였다. 또한, 기질로서 Suntide를 이용하여 면역침강시킨 Myc-PDK1의 카이네이즈 활성도를 측정하여(The Journal of Biololgical Chemistry 276(40), 37459 (2001)) 도 9의 B에 나타내었다. 도 9의 A와 B에서 볼 수 있듯이, Hsp90 발현은 PDK1 활성도를 1.5-2배 증가시켰을 뿐만 아니라 PDK1의 Tyr9과 Tyr373/376 잔기들의 tyrosine 인산화도 증가시켰다. To verify that increased PDK1 / Src complex formation directly affects PDK1 activation, changes in tyrosine phosphorylation of immunoprecipitated PDK1 were measured by Western blot using anti-pY9 or anti-pY373 / 376 antibodies and actin Normalized to FIG. 9A. In addition, the kinase activity of Myc-PDK1 immunoprecipitated using Suntide as a substrate (The Journal of Biololgical Chemistry 276 (40), 37459 (2001)) is shown in FIG. 9B. As can be seen from A and B of Figure 9, Hsp90 expression not only increased the PDK1 activity 1.5-2 fold, but also increased the tyrosine phosphorylation of Tyr9 and Tyr373 / 376 residues of PDK1.

더 나아가 Hsp90에 의한 PDK1 활성을 알아보기 위해서, HEK293 세포에 Hsp90을 표적으로 하는 siRNA(Hsp90 siRNA) 또는 대조군 siRNA(co siRNA)를 주입한 후 아래의 방법에 의해 원형질막 분획을 얻어 PDK1의 활성을 조사하였다.Furthermore, in order to examine PDK1 activity by Hsp90, after injecting sisp (Hsp90 siRNA) or control siRNA (co siRNA) targeting Hsp90 to HEK293 cells, plasma membrane fractions were obtained by the following method to investigate PDK1 activity. It was.

HEK 293 B를 얼음에 넣고 냉각된 PBS로 세척한 후 20 mM HEPES-NaOH (pH 7.4), 250 mM 자당, 25 mM 불화나트륨, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 2 μM microcystin LR, 1 mM PMSF와 1 mM benzamidine이 포함된 냉각된 fractionation 완충액 500㎕에서 파쇠한 후, 1 ml Dounce homogenizer로 15회 통과하여 균질화하였다. Organelles(pellet)에서 cytosolic 부분(상등액)을 분리하기 위해 균질물(Homogenates)을 3000g에서 10 분간 원심분리하였다. 얻어진 상등액을 다시 Beckman TLA 100.3 rotor를 사용하여 106,000g에서 60분간 원심분리하여 원형질막 P100 부분 (pellet)과 cytosolic S100 부분 (상등액)을 분리하였다. P100 pellet은 1 % (v/v) NP40과 단백질 농축물을 포함하는 fractionation 완충액에서 재현탁하였다. 각 fraction으로부터 측정된 단백질 농축물을 kinase assay와 anti-Hsp90 항체에 대해 immunoblotting하여 그 결과를 도 10에 도시하였다. Hsp90-siRNA를 주입한 세포에서 얻은 원형질막 분획에 존재하는 PDK1 활성도는 두드러지게 감소하였는데 반해서 대조군-siRNA를 주입한 세포에서는 거의 차이가 없었다. HEK 293 B was put on ice and washed with cold PBS, 20 mM HEPES-NaOH (pH 7.4), 250 mM sucrose, 25 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.1 mM sodium orthovanadate, 2 μM microcystin LR, 1 mM After crushing in 500 μl of the cooled fractionation buffer containing PMSF and 1 mM benzamidine, the mixture was homogenized by 15 passes with a 1 ml Dounce homogenizer. Homogenates were centrifuged at 3000 g for 10 minutes to separate cytosolic parts (supernatant) from organelles (pellet). The obtained supernatant was again centrifuged at 106,000 g for 60 minutes using a Beckman TLA 100.3 rotor to separate the plasma membrane P100 (pellet) and cytosolic S100 (supernatant). P100 pellet was resuspended in fractionation buffer containing 1% (v / v) NP40 and protein concentrate. Protein concentrates measured from each fraction were immunoblotting against kinase assay and anti-Hsp90 antibody and the results are shown in FIG. 10. PDK1 activity in plasma membrane fractions obtained from cells injected with Hsp90-siRNA was markedly decreased, whereas there was little difference in cells injected with control-siRNA.

PDK1은 PKB의 Thr308 잔기와 SGK의 Ser256 잔기를 인산화한다고 보고된 것처럼 PDK1의 활성화 변화가 PDK1의 하위 기질에까지 영향을 주는지 확인하였다. PKB와 SGK의 인산화는 anti-pPKB (Thr308) 또는 anti-pSGK(Thr256) 항체를 사용하여 immunoblotting에 의해 분석하였고 각 단백질의 총양 또한 anti-PKB 또는 anti-actin 항체로 분석하였다(도 11의 A). 인산화-PKB와 인산화-SGK의 통계적 차이를 각 lane에서 PKB와 actin의 값을 비교하여 결정하고 도 10의 B에 도시하였다. 도 11의 A와 B에서 확인할 수 있듯이 Hsp90 과발현은 PKB와 SGK 모두의 인산화를 상승시켰다. 즉 이러한 결과는 Hsp90가 PDK1의 안정화와 PDK1/Src 복합체 형성을 증진시킴으로써 in vitro와 in vivo에서 모두 PDK1의 하위 기질의 활성화를 유도한다는 것을 강력하게 시사한다.PDK1 confirmed that the activation change of PDK1 affects the underlying substrate of PDK1 as reported to phosphorylate the Thr308 residue of PKB and the Ser256 residue of SGK. Phosphorylation of PKB and SGK was analyzed by immunoblotting using anti-pPKB (Thr308) or anti-pSGK (Thr256) antibodies and the total amount of each protein was also analyzed by anti-PKB or anti-actin antibody (FIG. 11A). . Statistical difference between phosphorylated-PKB and phosphorylated-SGK was determined by comparing the values of PKB and actin in each lane and are shown in B of FIG. 10. As can be seen from A and B of Figure 11 Hsp90 overexpression increased the phosphorylation of both PKB and SGK. These results strongly suggest that Hsp90 induces PDK1 sub-substrate activation both in vitro and in vivo by enhancing PDK1 stabilization and PDK1 / Src complex formation.

실시예 6 : 여러가지 암에서 PDK1의 Tyr9 인산화 증가Example 6 Increased Tyr9 Phosphorylation of PDK1 in Various Cancers

PDK1과 여러 가지 암의 진행 및 전이와의 관련이 PDK1이 Try9의 인산화와 연관되어 있는지 확인하였다. The association of PDK1 with the progression and metastasis of various cancers was confirmed to be related to the phosphorylation of Try9.

BioMax로부터 파라핀으로 구획된 사람의 암 조직 array 슬라이드를 구매하고, Histostain-Plus Kits (Zymed)는 조직 array (Am J Clin Pathol 92(1) 62 (1989))의 면역조직화학에 대한 사용자 매뉴얼에 따라 사용하였다. 즉, 파라핀 구획을 xylene으로 녹여내고 ethanol에서 rehydrate하였다. 슬라이드를 peroxidase quenching 용액에 10분간 담근 후 PBS로 5분간 두 번 세척하고 blocking을 위하여 Reagent A를 2방울 가하여 30분 동안 인큐베이션하였다. 다시 PBS로 2회 세척한 후, 일차 항체 Anti-pY9 항체를 4℃에서 밤새 적용하였다. 그 후 PBS로 다시 세척한 후 biotinylation 된 2차 항체 Reagent B를 가하여 상온에서 1 시간 동안 인큐베이션하였다. PBS로 세척 후 Reagent C가 결합된 효소를 떨어뜨리고 다시 PBS, DAB chromogen, Reagent D1, D2, D3의 혼합물을 가하여 형광 현미경으로 관련신호를 관찰하였다. 증류수로 고정하고 X20의 배율로 형광 현미경으로 사진을 찍어 도 12의 A에 도시하였다. anti-Tyr9 항체로 면역조직 염색법을 하여서 비교해보면, Tyr9 인산화 정도가 폐, 간, 결장, 유방조직에서는 정상 조직에 비하여 질병이 있는 조직에서 현저하게 증가되었다.Purchasing human cancer tissue array slides with paraffin sections from BioMax, and Histostain-Plus Kits (Zymed) were prepared according to the user manual for immunohistochemistry of tissue arrays (Am J Clin Pathol 92 (1) 62 (1989)). Used. In other words, the paraffin compartment was dissolved in xylene and rehydrated in ethanol. The slide was soaked in peroxidase quenching solution for 10 minutes, washed twice with PBS for 5 minutes, and incubated for 30 minutes by adding 2 drops of Reagent A for blocking. After washing twice with PBS again, the primary antibody Anti-pY9 antibody was applied overnight at 4 ° C. After washing with PBS again, the biotinylated secondary antibody Reagent B was added and incubated for 1 hour at room temperature. After washing with PBS, the enzyme bound to Reagent C was dropped, and a mixture of PBS, DAB chromogen, Reagent D1, D2, and D3 was added thereto, and the related signal was observed under a fluorescence microscope. Fixed with distilled water and photographed with a fluorescence microscope at a magnification of X20 is shown in FIG. In comparison with immunohistostaining with anti-Tyr9 antibodies, Tyr9 phosphorylation was significantly increased in diseased tissues compared to normal tissues in lung, liver, colon and breast tissues.

결과를 보다 명확히 검증하기 위해서 4명의 악성 결장암 환자의 수술 중에 얻어지는 정상 조직과 암 조직으로부터 웨스턴 블럿에 의해 PDK1과 Hsp90의 단백질 레벨을 분석하여 도 12의 B에 도시하였다. Anti-actin 항체를 사용하여 로딩 양을 normalize 하였다. 도 12의 B에서 확인할 수 있듯이 모든 시료에서 결장 암 조직들은 정상 조직보다 두드러지게 높은 PDK1 단백질 발현을 보였다. 또한 이러한 현상은 Hsp90 단백질 발현에서도 동일하게 관찰되었다. To more clearly verify the results, protein levels of PDK1 and Hsp90 were analyzed by Western blot from normal and cancerous tissues obtained during surgery of four malignant colon cancer patients, and are shown in FIG. Anti-actin antibody was used to normalize loading amount. As shown in FIG. 12B, colon cancer tissues showed significantly higher PDK1 protein expression than normal tissues in all samples. This phenomenon was also observed in the expression of Hsp90 protein.

도 1은 HA-Src-△SH2 및 HA-Src-△SH3의 개열지도.1 is a cleavage map of HA-Src-ΔSH2 and HA-Src-ΔSH3.

도 2는 Tyrosine 잔기가 인산화된 PDK1과 SH2 도메인을 가지고 있는 단백질 간의 in vitro 결합을 보여주는 웨스턴 블럿의 사진.FIG. 2 is a photograph of Western blot showing in vitro binding between PDK1 and SH2 domains with phosphorylated Tyrosine residues. FIG.

도 3은 HA-Src WT과 Myc-PDK1 WT이 in vitro에서 결합하는 것을 보여주는 웨스턴 블럿의 사진.Figure 3 is a photograph of Western blot showing HA-Src WT and Myc-PDK1 WT binding in vitro.

도 4는 pY9 또는 pY373/376 잔기를 포함하는 인산화 펩타이드 비드와 Src, Fyn2 및 C가 단백질과의 반응을 보여주는 웨스턴 블럿의 사진.4 is a photograph of Western blot showing the reaction of phosphorylated peptide beads containing pY9 or pY373 / 376 residues with Src, Fyn2 and C proteins.

도 5는 Src의 결실변이와 PDK1과 SH2 도메인에 의해 결합함을 보여주는 immunoblotting 사진.FIG. 5 is an immunoblotting photograph showing binding of Src deletion by PDK1 and SH2 domains.

도 6은 Hsp90 억제제에 의한 Hsp90/PDK1 복합체 형성 저해되는 것을 보여주는 immunoblotting 사진.6 is an immunoblotting photograph showing that Hsp90 / PDK1 complex formation is inhibited by Hsp90 inhibitors.

도 7은 PDK1/Src 복합체 형성에 Hsp90이 관여함을 보여주는 immunoblotting 사진.7 is an immunoblotting photo showing that Hsp90 is involved in PDK1 / Src complex formation.

도 8은 Myc-PDK1 면역침강물에 결합된 HA-Src를 anti-HA 항체로 분석한 결과를 보여주는 사진.Figure 8 is a photograph showing the results of analysis of the HA-Src bound to Myc-PDK1 immunoprecipitate with an anti-HA antibody.

도 9는 면역침강된 PDDK1에서 tyrosine 인산화의 변화를 검출한 웨스턴 블럿 사진.9 is a Western blot photograph detecting changes in tyrosine phosphorylation in immunoprecipitated PDDK1.

도 10은 Hsp90 SiTNA 또는 대조군 siRNA로 감염된 HEK293 cell에서 PDK1 활성을 측정한 결과를 보여주는 그래프.10 is a graph showing the results of measuring PDK1 activity in HEK293 cells infected with Hsp90 SiTNA or control siRNA.

도 11은 PDK1에 대한 downstream target의 인산화를 anti-pPKB (Thr308) 또는 anti-pSGK(Thr256) 항체를 사용하여 immunoblotting에 의해 분석한 사진 및 그래프.FIG. 11 is a photograph and graph of phosphorylation of downstream targets for PDK1 by immunoblotting using anti-pPKB (Thr308) or anti-pSGK (Thr256) antibodies. FIG.

도 12는 여러 가지 암에서 PDK1의 Try 인산화 및 Hsp90 발현을 측정이 크게 증가함을 보여주는 웨스턴 블런 사진.FIG. 12 is a Western bloon photograph showing a significant increase in measuring Try phosphorylation and Hsp90 expression of PDK1 in various cancers.

<110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Method for diagnosis of cancer by measurement of tyrosine 9 phosphorylation of PDK1 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cgggatccat ggggagcagc aag 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggaattccta taggttctct cc 22 <210> 3 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1 WT <400> 3 atggccagga ccaccagcca gctgtatgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctatg gcaattatga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat actaaagatg 1380 ggcccagtgg ataagcggaa gggtttattt gcaagacgac gacagctgtt gctcacagaa 1440 ggaccacatt tatattatgt ggatcctgtc aacaaagttc tgaaaggtga aattccttgg 1500 tcacaagaac ttcgaccaga ggccaagaat tttaaaactt tctttgtcca cacgcctaac 1560 aggacgtatt atctgatgga ccccagcggg aacgcacaca agtggtgcag gaagatccag 1620 gaggtttgga ggcagcgata ccagagccac ccggacgccg ctgtgcagtg a 1671 <210> 4 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> HA-Src WT <400> 4 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat caaaaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 gactacttca cgtccaccga gccccagtac cagcccgggg agaacctcta g 1611 <210> 5 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1 Y9F <400> 5 atggccagga ccaccagcca gctgtttgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctatg gcaattatga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat actaaagatg 1380 ggcccagtgg ataagcggaa gggtttattt gcaagacgac gacagctgtt gctcacagaa 1440 ggaccacatt tatattatgt ggatcctgtc aacaaagttc tgaaaggtga aattccttgg 1500 tcacaagaac ttcgaccaga ggccaagaat tttaaaactt tctttgtcca cacgcctaac 1560 aggacgtatt atctgatgga ccccagcggg aacgcacaca agtggtgcag gaagatccag 1620 gaggtttgga ggcagcgata ccagagccac ccggacgccg ctgtgcagtg a 1671 <210> 6 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1 Y373/376F <400> 6 atggccagga ccaccagcca gctgtatgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctttg gcaattttga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat actaaagatg 1380 ggcccagtgg ataagcggaa gggtttattt gcaagacgac gacagctgtt gctcacagaa 1440 ggaccacatt tatattatgt ggatcctgtc aacaaagttc tgaaaggtga aattccttgg 1500 tcacaagaac ttcgaccaga ggccaagaat tttaaaactt tctttgtcca cacgcctaac 1560 aggacgtatt atctgatgga ccccagcggg aacgcacaca agtggtgcag gaagatccag 1620 gaggtttgga ggcagcgata ccagagccac ccggacgccg ctgtgcagtg a 1671 <210> 7 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1-PH <400> 7 atggccagga ccaccagcca gctgtatgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctatg gcaattatga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat acta 1374 <210> 8 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-CA <400> 8 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat caaaaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 gactacttca cgtccaccga gccccagttt cagcccgggg agaacctcta g 1611 <210> 9 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-KD <400> 9 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat catgaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 gactacttca cgtccaccga gccccagtac cagcccgggg agaacctcta g 1611 <210> 10 <211> 1377 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-SH2 <400> 10 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatccg cctgcagcag ctggtggcct actactccaa acacgccgat 480 ggcctgtgcc accgcctcac caccgtgtgc cccacgtcca agccgcagac tcagggcctg 540 gccaaggatg cctgggagat ccctcgggag tcgctgcggc tggaggtcaa gctgggccag 600 ggctgctttg gcgaggtgtg gatggggacc tggaacggta ccaccagggt ggccatcaaa 660 accctgaagc ctggcacgat gtctccagag gccttcctgc aggaggccca ggtcatgaag 720 aagctgaggc atgagaagct ggtgcagttg tatgctgtgg tttcagagga gcccatttac 780 atcgtcacgg agtacatgag caaggggagt ttgctggact ttctcaaggg ggagacaggc 840 aagtacctgc ggctgcctca gctggtggac atggctgctc agatcgcctc aggcatggcg 900 tacgtggagc ggatgaacta cgtccaccgg gaccttcgtg cagccaacat cctggtggga 960 gagaacctgg tgtgcaaagt ggccgacttt gggctggctc ggctcattga agacaatgag 1020 tacacggcgc ggcaaggtgc caaattcccc atcaagtgga cggctccaga agctgccctc 1080 tatggccgct tcaccatcaa gtcggacgtg tggtccttcg ggatcctgct gactgagctc 1140 accacaaagg gacgggtgcc ctaccctggg atggtgaacc gcgaggtgct ggaccaggtg 1200 gagcggggct accggatgcc ctgcccgccg gagtgtcccg agtccctgca cgacctcatg 1260 tgccagtgct ggcggaagga gcctgaggag cggcccacct tcgagtacct gcaggccttc 1320 ctggaggact acttcacgtc caccgagccc cagtaccagc ccggggagaa cctctag 1377 <210> 11 <211> 1425 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-SH3 <400> 11 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgccc 240 tccgactcca tccaggctga ggagtggtat tttggcaaga tcaccagacg ggagtcagag 300 cggttactgc tcaatgcaga gaacccgaga gggaccttcc tcgtgcgaga aagtgagacc 360 acgaaaggtg cctactgcct ctcagtgtct gacttcgaca acgccaaggg cctcaacgtg 420 aagcactaca agatccgcaa gctggacagc ggcggcttct acatcacctc ccgcacccag 480 ttcaacagcc tgcagcagct ggtggcctac tactccaaac acgccgatgg cctgtgccac 540 cgcctcacca ccgtgtgccc cacgtccaag ccgcagactc agggcctggc caaggatgcc 600 tgggagatcc ctcgggagtc gctgcggctg gaggtcaagc tgggccaggg ctgctttggc 660 gaggtgtgga tggggacctg gaacggtacc accagggtgg ccatcaaaac cctgaagcct 720 ggcacgatgt ctccagaggc cttcctgcag gaggcccagg tcatgaagaa gctgaggcat 780 gagaagctgg tgcagttgta tgctgtggtt tcagaggagc ccatttacat cgtcacggag 840 tacatgagca aggggagttt gctggacttt ctcaaggggg agacaggcaa gtacctgcgg 900 ctgcctcagc tggtggacat ggctgctcag atcgcctcag gcatggcgta cgtggagcgg 960 atgaactacg tccaccggga ccttcgtgca gccaacatcc tggtgggaga gaacctggtg 1020 tgcaaagtgg ccgactttgg gctggctcgg ctcattgaag acaatgagta cacggcgcgg 1080 caaggtgcca aattccccat caagtggacg gctccagaag ctgccctcta tggccgcttc 1140 accatcaagt cggacgtgtg gtccttcggg atcctgctga ctgagctcac cacaaaggga 1200 cgggtgccct accctgggat ggtgaaccgc gaggtgctgg accaggtgga gcggggctac 1260 cggatgccct gcccgccgga gtgtcccgag tccctgcacg acctcatgtg ccagtgctgg 1320 cggaaggagc ctgaggagcg gcccaccttc gagtacctgc aggccttcct ggaggactac 1380 ttcacgtcca ccgagcccca gtaccagccc ggggagaacc tctag 1425 <210> 12 <211> 2175 <212> DNA <213> Artificial Sequence <220> <223> Flag-Hsp90 <400> 12 atgcctgagg aagtgcacca tggagaggag gaggtggaga cttttgcctt tcaggcagaa 60 attgcccaac tcatgtccct catcatcaat accttctatt ccaacaagga gattttcctt 120 cgggagttga tctctaatgc ttctgatgcc ttggacaaga ttcgctatga gagcctgaca 180 gacccttcga agttggacag tggtaaagag ctgaaaattg acatcatccc caaccctcag 240 gaacgtaccc tgactttggt agacacaggc attggcatga ccaaagctga tctcataaat 300 aatttgggaa ccattgccaa gtctggtact aaagcattca tggaggctct tcaggctggt 360 gcagacatct ccatgattgg gcagtttggt gttggctttt attctgccta cttggtggca 420 gagaaagtgg ttgtgatcac aaagcacaac gatgatgaac agtatgcttg ggagtcttct 480 gctggaggtt ccttcactgt gcgtgctgac catggtgagc ccattggcag gggtaccaaa 540 gtgatcctcc atcttaaaga agatcagaca gagtacctag aagagaggcg ggtcaaagaa 600 gtagtgaaga agcattctca gttcataggc tatcccatca ccctttattt ggagaaggaa 660 cgagagaagg aaattagtga tgatgaggca gaggaagaga aaggtgagaa agaagaggaa 720 gataaagatg atgaagaaaa acccaagatc gaagatgtgg gttcagatga ggaggatgac 780 agcggtaagg ataagaagaa gaaaactaag aagatcaaag agaaatacat tgatcaggaa 840 gaactaaaca agaccaagcc tatttggacc agaaaccctg atgacatcac ccaagaggag 900 tatggagaat tctacaagag cctcactaat gactgggaag accacttggc agtcaagcac 960 ttttctgtag aaggtcagtt ggaattcagg gcattgctat ttattcctcg tcgggctccc 1020 tttgaccttt ttgagaacaa gaagaaaaag aacaacatca aactctatgt ccgccgtgtg 1080 ttcatcatgg acagctgtga tgagttgata ccagagtatc tcaattttat ccgtggtgtg 1140 gttgactctg aggatctgcc cctgaacatc tcccgagaaa tgctccagca gagcaaaatc 1200 ttgaaagtca ttcgcaaaaa cattgttaag aagtgccttg agctcttctc tgagctggca 1260 gaagacaagg agaattacaa gaaattctat gaggcattct ctaaaaatct caagcttgga 1320 atccacgaag actccactaa ccgccgccgc ctgtctgagc tgctgcgcta tcatacctcc 1380 cagtctggag atgagatgac atctctgtca gagtatgttt ctcgcatgaa ggagacacag 1440 aagtccatct attacatcac tggtgagagc aaagagcagg tggccaactc agcttttgtg 1500 gagcgagtgc ggaaacgggg cttcgaggtg gtatatatga ccgagcccat tgacgagtac 1560 tgtgtgcagc agctcaagga atttgatggg aagagcctgg tctcagttac caaggagggt 1620 ctggagctgc ctgaggatga ggaggagaag aagaagatgg aagagagcaa ggcaaagttt 1680 gagaacctct gcaagctcat gaaagaaatc ttagataaga aggttgagaa ggtgacaatc 1740 tccaatagac ttgtgtcttc accttgctgc attgtgacca gcacctacgg ctggacagcc 1800 aatatggagc ggatcatgaa agcccaggca cttcgggaca actccaccat gggctatatg 1860 atggccaaaa agcacctgga gatcaaccct gaccacccca ttgtggagac gctgcggcag 1920 aaggctgagg ccgacaagaa tgataaggca gttaaggacc tggtggtgct gctgtttgaa 1980 accgccctgc tatcttctgg cttttccctt gaggatcccc agacccactc caaccgcatc 2040 tatcgcatga tcaagctagg tctaggtatt gatgaagatg aagtggcagc agaggaaccc 2100 aatgctgcag ttcctgatga gatcccccct ctcgagggcg atgaggatgc gtctcgcatg 2160 gaagaagtcg attag 2175 <210> 13 <211> 1443 <212> DNA <213> Artificial Sequence <220> <223> Flag-Hsp40 <400> 13 atggctgcgc ggtgctccac acgctggttg ctggtggttg tggggacccc gcggctgccg 60 gctatatcgg gtagaggggc ccggccgccc agggagggcg tggtgggggc atggctgagc 120 cgcaagctga gcgtccccgc ctttgcgtct tccctgacct cttgcggccc ccgagcgctg 180 ctgacattga gacctggtgt cagcctcaca ggaacaaaac attacccttt catttgtact 240 gcctccttcc acacgagtgc ccctttggcc aaagaagatt attatcagat attaggagtg 300 cctcgaaatg ccagccagaa agagatcaag aaagcctatt atcagcttgc caagaagtat 360 caccctgaca caaataagga tgatcccaaa gccaaggaga agttctccca gctggcagaa 420 gcctatgagg ttttgagtga tgaggtgaag aggaagcagt acgatgccta cggctctgca 480 ggcttcgatc ctggggccag cggctcccag catagctact ggaagggagg ccccactgtg 540 gaccccgagg agctgttcag gaagatcttt ggcgagttct catcctcttc atttggagat 600 ttccagaccg tgtttgatca gcctcaggaa tacttcatgg agttgacatt caatcaagct 660 gcaaaggggg tcaacaagga gttcaccgtg aacatcatgg acacgtgtga gcgctgcaac 720 ggcaagggga acgagcccgg caccaaggtg cagcattgcc actactgtgg cggctccggc 780 atggaaacca tcaacacagg cccttttgtg atgcgttcca cgtgtaggag atgtggtggc 840 cgcggctcca tcatcatatc gccctgtgtg gtctgcaggg gagcaggaca agccaagcag 900 aaaaagcgag tgatgatccc tgtgcctgca ggagtcgagg atggccagac cgtgaggatg 960 cctgtgggaa aaagggaaat tttcattacg ttcagggtgc agaaaagccc tgtgttccgg 1020 agggacggcg cagacatcca ctccgacctc tttatttcta tagctcaggc tcttcttggg 1080 ggaacagcca gagcccaggg cctgtacgag acgatcaacg tgacgatccc ccctgggact 1140 cagacagacc agaagattcg gatgggtggg aaaggcatcc cccggattaa cagctacggc 1200 tacggagacc actacatcca catcaagata cgagttccaa agaggctaac gagccggcag 1260 cagagcctga tcctgagcta cgccgaggac gagacagatg tggaggggac ggtgaacggc 1320 gtcaccctca ccagctctgg tggcagcacc atggatagct ccgcaggaag caaggctagg 1380 cgtgaggctg gggaggacga ggagggattc ctttccaaac ttaagaaaat gtttacctca 1440 tga 1443 <210> 14 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> Flag-Hsp27 <400> 14 atgaccgagc gccgcgtccc cttctcgctc ctgcggggcc ccagctggga ccccttccgc 60 gactggtacc cgcatagccg cctcttcgac caggccttcg ggctgccccg gctgccggag 120 gagtggtcgc agtggttagg cggcagcagc tggccaggct acgtgcgccc cctgcccccc 180 gccgccatcg agagccccgc agtggccgcg cccgcctaca gccgcgcgct cagccggcaa 240 ctcagcagcg gggtctcgga gatccggcac actgcggacc gctggcgcgt gtccctggat 300 gtcaaccact tcgccccgga cgagctgacg gtcaagacca aggatggcgt ggtggagatc 360 accggcaagc acgaggagcg gcaggacgag catggctaca tctcccggtg cttcacgcgg 420 aaatacacgc tgccccccgg tgtggacccc acccaagttt cctcctccct gtcccctgag 480 ggcacactga ccgtggaggc ccccatgccc aagctagcca cgcagtccaa cgagatcacc 540 atcccagtca ccttcgagtc gcgggcccag cttgggggcc cagaagctgc aaaatccgat 600 gagactgccg ccaagtaa 618 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> oligopetide <220> <221> MOD_RES <222> (7) <223> PHOSPHORYLATION, <400> 15 Arg Thr Thr Ser Gln Leu Tyr Asp Ala Val Pro Ile Gln Ser 1 5 10 <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> oligopeptide <220> <221> MOD_RES <222> (7) <223> PHOSPHORYLATION, <400> 16 Glu Asp Asp Glu Asp Cys Tyr Gly Asn Tyr Asp Asn Leu Leu Ser Gln 1 5 10 15 Phe <110> The Industry & Academic Cooperation in Chungnam National University (IAC) <120> Method for diagnosis of cancer by measurement of tyrosine 9          phosphorylation of PDK1 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cgggatccat ggggagcagc aag 23 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggaattccta taggttctct cc 22 <210> 3 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1 WT <400> 3 atggccagga ccaccagcca gctgtatgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctatg gcaattatga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat actaaagatg 1380 ggcccagtgg ataagcggaa gggtttattt gcaagacgac gacagctgtt gctcacagaa 1440 ggaccacatt tatattatgt ggatcctgtc aacaaagttc tgaaaggtga aattccttgg 1500 tcacaagaac ttcgaccaga ggccaagaat tttaaaactt tctttgtcca cacgcctaac 1560 aggacgtatt atctgatgga ccccagcggg aacgcacaca agtggtgcag gaagatccag 1620 gaggtttgga ggcagcgata ccagagccac ccggacgccg ctgtgcagtg a 1671 <210> 4 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> HA-Src WT <400> 4 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat caaaaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 gactacttca cgtccaccga gccccagtac cagcccgggg agaacctcta g 1611 <210> 5 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1 Y9F <400> 5 atggccagga ccaccagcca gctgtttgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctatg gcaattatga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat actaaagatg 1380 ggcccagtgg ataagcggaa gggtttattt gcaagacgac gacagctgtt gctcacagaa 1440 ggaccacatt tatattatgt ggatcctgtc aacaaagttc tgaaaggtga aattccttgg 1500 tcacaagaac ttcgaccaga ggccaagaat tttaaaactt tctttgtcca cacgcctaac 1560 aggacgtatt atctgatgga ccccagcggg aacgcacaca agtggtgcag gaagatccag 1620 gaggtttgga ggcagcgata ccagagccac ccggacgccg ctgtgcagtg a 1671 <210> 6 <211> 1671 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1 Y373 / 376F <400> 6 atggccagga ccaccagcca gctgtatgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctttg gcaattttga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat actaaagatg 1380 ggcccagtgg ataagcggaa gggtttattt gcaagacgac gacagctgtt gctcacagaa 1440 ggaccacatt tatattatgt ggatcctgtc aacaaagttc tgaaaggtga aattccttgg 1500 tcacaagaac ttcgaccaga ggccaagaat tttaaaactt tctttgtcca cacgcctaac 1560 aggacgtatt atctgatgga ccccagcggg aacgcacaca agtggtgcag gaagatccag 1620 gaggtttgga ggcagcgata ccagagccac ccggacgccg ctgtgcagtg a 1671 <210> 7 <211> 1374 <212> DNA <213> Artificial Sequence <220> <223> Myc-PDK1-PH <400> 7 atggccagga ccaccagcca gctgtatgac gccgtgccca tccagtccag cgtggtgtta 60 tgttcctgcc catccccatc aatggtgagg acccagactg agtccagcac gccccctggc 120 attcctggtg gcagcaggca gggccccgcc atggacggca ctgcagccga gcctcggccc 180 ggcgccggct ccctgcagca tgcccagcct ccgccgcagc ctcggaagaa gcggcctgag 240 gacttcaagt ttgggaaaat ccttggggaa ggctcttttt ccacggttgt cctggctcga 300 gaactggcaa cctccagaga atatgcgatt aaaattctgg agaagcgaca tatcataaaa 360 gagaacaagg tcccctatgt aaccagagag cgggatgtca tgtcgcgcct ggatcacccc 420 ttctttgtta agctttactt cacatttcag gacgacgaga agctgtattt cggccttagt 480 tatgccaaaa atggagaact acttaaatat attcgcaaaa tcggttcatt cgatgagacc 540 tgtacccgat tttacacggc tgagatcgtg tctgctttag agtacttgca cggcaagggc 600 atcattcaca gggaccttaa accggaaaac attttgttaa atgaagatat gcacatccag 660 atcacagatt ttggaacagc aaaagtctta tccccagaga gcaaacaagc cagggccaac 720 tcattcgtgg gaacagcgca gtacgtttct ccagagctgc tcacggagaa gtccgcctgt 780 aagagttcag acctttgggc tcttggatgc ataatatacc agcttgtggc aggactccca 840 ccattccgag ctggaaacga gtatcttata tttcagaaga tcattaagtt ggaatatgac 900 tttccagaaa aattcttccc taaggcaaga gacctcgtgg agaaactttt ggttttagat 960 gccacaaagc ggttaggctg tgaggaaatg gaaggatacg gacctcttaa agcacacccg 1020 ttcttcgagt ccgtcacgtg ggagaacctg caccagcaga cgcctccgaa gctcaccgct 1080 tacctgccgg ctatgtcgga agacgacgag gactgctatg gcaattatga caatctcctg 1140 agccagtttg gctgcatgca ggtgtcttcg tcctcctcct cacactccct gtcagcctcc 1200 gacacgggcc tgccccagag gtcaggcagc aacatagagc agtacattca cgatctggac 1260 tcgaactcct ttgaactgga cttacagttt tccgaagatg agaagaggtt gttgttggag 1320 aagcaggctg gcggaaaccc ttggcaccag tttgtagaaa ataatttaat acta 1374 <210> 8 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-CA <400> 8 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat caaaaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 gactacttca cgtccaccga gccccagttt cagcccgggg agaacctcta g 1611 <210> 9 <211> 1611 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-KD <400> 9 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatcca ggctgaggag tggtattttg gcaagatcac cagacgggag 480 tcagagcggt tactgctcaa tgcagagaac ccgagaggga ccttcctcgt gcgagaaagt 540 gagaccacga aaggtgccta ctgcctctca gtgtctgact tcgacaacgc caagggcctc 600 aacgtgaagc actacaagat ccgcaagctg gacagcggcg gcttctacat cacctcccgc 660 acccagttca acagcctgca gcagctggtg gcctactact ccaaacacgc cgatggcctg 720 tgccaccgcc tcaccaccgt gtgccccacg tccaagccgc agactcaggg cctggccaag 780 gatgcctggg agatccctcg ggagtcgctg cggctggagg tcaagctggg ccagggctgc 840 tttggcgagg tgtggatggg gacctggaac ggtaccacca gggtggccat catgaccctg 900 aagcctggca cgatgtctcc agaggccttc ctgcaggagg cccaggtcat gaagaagctg 960 aggcatgaga agctggtgca gttgtatgct gtggtttcag aggagcccat ttacatcgtc 1020 acggagtaca tgagcaaggg gagtttgctg gactttctca agggggagac aggcaagtac 1080 ctgcggctgc ctcagctggt ggacatggct gctcagatcg cctcaggcat ggcgtacgtg 1140 gagcggatga actacgtcca ccgggacctt cgtgcagcca acatcctggt gggagagaac 1200 ctggtgtgca aagtggccga ctttgggctg gctcggctca ttgaagacaa tgagtacacg 1260 gcgcggcaag gtgccaaatt ccccatcaag tggacggctc cagaagctgc cctctatggc 1320 cgcttcacca tcaagtcgga cgtgtggtcc ttcgggatcc tgctgactga gctcaccaca 1380 aagggacggg tgccctaccc tgggatggtg aaccgcgagg tgctggacca ggtggagcgg 1440 ggctaccgga tgccctgccc gccggagtgt cccgagtccc tgcacgacct catgtgccag 1500 tgctggcgga aggagcctga ggagcggccc accttcgagt acctgcaggc cttcctggag 1560 gactacttca cgtccaccga gccccagtac cagcccgggg agaacctcta g 1611 <210> 10 <211> 1377 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-SH2 <400> 10 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgggc 240 ccgctggccg gtggagtgac cacctttgtg gccctctatg actatgagtc taggacggag 300 acagacctgt ccttcaagaa aggcgagcgg ctccagattg tcaacaacac agagggagac 360 tggtggctgg cccactcgct cagcacagga cagacaggct acatccccag caactacgtg 420 gcgccctccg actccatccg cctgcagcag ctggtggcct actactccaa acacgccgat 480 ggcctgtgcc accgcctcac caccgtgtgc cccacgtcca agccgcagac tcagggcctg 540 gccaaggatg cctgggagat ccctcgggag tcgctgcggc tggaggtcaa gctgggccag 600 ggctgctttg gcgaggtgtg gatggggacc tggaacggta ccaccagggt ggccatcaaa 660 accctgaagc ctggcacgat gtctccagag gccttcctgc aggaggccca ggtcatgaag 720 aagctgaggc atgagaagct ggtgcagttg tatgctgtgg tttcagagga gcccatttac 780 atcgtcacgg agtacatgag caaggggagt ttgctggact ttctcaaggg ggagacaggc 840 aagtacctgc ggctgcctca gctggtggac atggctgctc agatcgcctc aggcatggcg 900 tacgtggagc ggatgaacta cgtccaccgg gaccttcgtg cagccaacat cctggtggga 960 gagaacctgg tgtgcaaagt ggccgacttt gggctggctc ggctcattga agacaatgag 1020 tacacggcgc ggcaaggtgc caaattcccc atcaagtgga cggctccaga agctgccctc 1080 tatggccgct tcaccatcaa gtcggacgtg tggtccttcg ggatcctgct gactgagctc 1140 accacaaagg gacgggtgcc ctaccctggg atggtgaacc gcgaggtgct ggaccaggtg 1200 gagcggggct accggatgcc ctgcccgccg gagtgtcccg agtccctgca cgacctcatg 1260 tgccagtgct ggcggaagga gcctgaggag cggcccacct tcgagtacct gcaggccttc 1320 ctggaggact acttcacgtc caccgagccc cagtaccagc ccggggagaa cctctag 1377 <210> 11 <211> 1425 <212> DNA <213> Artificial Sequence <220> <223> HA-Src-SH3 <400> 11 atgggtagca acaagagcaa gcccaaggat gccagccagc ggcgccgcag cctggagccc 60 gccgagaacg tgcacggcgc tggcgggggc gctttccccg cctcgcagac ccccagcaag 120 ccagcctcgg ccgacggcca ccgcggcccc agcgcggcct tcgcccccgc ggccgccgag 180 cccaagctgt tcggaggctt caactcctcg gacaccgtca cctccccgca gagggcgccc 240 tccgactcca tccaggctga ggagtggtat tttggcaaga tcaccagacg ggagtcagag 300 cggttactgc tcaatgcaga gaacccgaga gggaccttcc tcgtgcgaga aagtgagacc 360 acgaaaggtg cctactgcct ctcagtgtct gacttcgaca acgccaaggg cctcaacgtg 420 aagcactaca agatccgcaa gctggacagc ggcggcttct acatcacctc ccgcacccag 480 ttcaacagcc tgcagcagct ggtggcctac tactccaaac acgccgatgg cctgtgccac 540 cgcctcacca ccgtgtgccc cacgtccaag ccgcagactc agggcctggc caaggatgcc 600 tgggagatcc ctcgggagtc gctgcggctg gaggtcaagc tgggccaggg ctgctttggc 660 gaggtgtgga tggggacctg gaacggtacc accagggtgg ccatcaaaac cctgaagcct 720 ggcacgatgt ctccagaggc cttcctgcag gaggcccagg tcatgaagaa gctgaggcat 780 gagaagctgg tgcagttgta tgctgtggtt tcagaggagc ccatttacat cgtcacggag 840 tacatgagca aggggagttt gctggacttt ctcaaggggg agacaggcaa gtacctgcgg 900 ctgcctcagc tggtggacat ggctgctcag atcgcctcag gcatggcgta cgtggagcgg 960 atgaactacg tccaccggga ccttcgtgca gccaacatcc tggtgggaga gaacctggtg 1020 tgcaaagtgg ccgactttgg gctggctcgg ctcattgaag acaatgagta cacggcgcgg 1080 caaggtgcca aattccccat caagtggacg gctccagaag ctgccctcta tggccgcttc 1140 accatcaagt cggacgtgtg gtccttcggg atcctgctga ctgagctcac cacaaaggga 1200 cgggtgccct accctgggat ggtgaaccgc gaggtgctgg accaggtgga gcggggctac 1260 cggatgccct gcccgccgga gtgtcccgag tccctgcacg acctcatgtg ccagtgctgg 1320 cggaaggagc ctgaggagcg gcccaccttc gagtacctgc aggccttcct ggaggactac 1380 ttcacgtcca ccgagcccca gtaccagccc ggggagaacc tctag 1425 <210> 12 <211> 2175 <212> DNA <213> Artificial Sequence <220> <223> Flag-Hsp90 <400> 12 atgcctgagg aagtgcacca tggagaggag gaggtggaga cttttgcctt tcaggcagaa 60 attgcccaac tcatgtccct catcatcaat accttctatt ccaacaagga gattttcctt 120 cgggagttga tctctaatgc ttctgatgcc ttggacaaga ttcgctatga gagcctgaca 180 gacccttcga agttggacag tggtaaagag ctgaaaattg acatcatccc caaccctcag 240 gaacgtaccc tgactttggt agacacaggc attggcatga ccaaagctga tctcataaat 300 aatttgggaa ccattgccaa gtctggtact aaagcattca tggaggctct tcaggctggt 360 gcagacatct ccatgattgg gcagtttggt gttggctttt attctgccta cttggtggca 420 gagaaagtgg ttgtgatcac aaagcacaac gatgatgaac agtatgcttg ggagtcttct 480 gctggaggtt ccttcactgt gcgtgctgac catggtgagc ccattggcag gggtaccaaa 540 gtgatcctcc atcttaaaga agatcagaca gagtacctag aagagaggcg ggtcaaagaa 600 gtagtgaaga agcattctca gttcataggc tatcccatca ccctttattt ggagaaggaa 660 cgagagaagg aaattagtga tgatgaggca gaggaagaga aaggtgagaa agaagaggaa 720 gataaagatg atgaagaaaa acccaagatc gaagatgtgg gttcagatga ggaggatgac 780 agcggtaagg ataagaagaa gaaaactaag aagatcaaag agaaatacat tgatcaggaa 840 gaactaaaca agaccaagcc tatttggacc agaaaccctg atgacatcac ccaagaggag 900 tatggagaat tctacaagag cctcactaat gactgggaag accacttggc agtcaagcac 960 ttttctgtag aaggtcagtt ggaattcagg gcattgctat ttattcctcg tcgggctccc 1020 tttgaccttt ttgagaacaa gaagaaaaag aacaacatca aactctatgt ccgccgtgtg 1080 ttcatcatgg acagctgtga tgagttgata ccagagtatc tcaattttat ccgtggtgtg 1140 gttgactctg aggatctgcc cctgaacatc tcccgagaaa tgctccagca gagcaaaatc 1200 ttgaaagtca ttcgcaaaaa cattgttaag aagtgccttg agctcttctc tgagctggca 1260 gaagacaagg agaattacaa gaaattctat gaggcattct ctaaaaatct caagcttgga 1320 atccacgaag actccactaa ccgccgccgc ctgtctgagc tgctgcgcta tcatacctcc 1380 cagtctggag atgagatgac atctctgtca gagtatgttt ctcgcatgaa ggagacacag 1440 aagtccatct attacatcac tggtgagagc aaagagcagg tggccaactc agcttttgtg 1500 gagcgagtgc ggaaacgggg cttcgaggtg gtatatatga ccgagcccat tgacgagtac 1560 tgtgtgcagc agctcaagga atttgatggg aagagcctgg tctcagttac caaggagggt 1620 ctggagctgc ctgaggatga ggaggagaag aagaagatgg aagagagcaa ggcaaagttt 1680 gagaacctct gcaagctcat gaaagaaatc ttagataaga aggttgagaa ggtgacaatc 1740 tccaatagac ttgtgtcttc accttgctgc attgtgacca gcacctacgg ctggacagcc 1800 aatatggagc ggatcatgaa agcccaggca cttcgggaca actccaccat gggctatatg 1860 atggccaaaa agcacctgga gatcaaccct gaccacccca ttgtggagac gctgcggcag 1920 aaggctgagg ccgacaagaa tgataaggca gttaaggacc tggtggtgct gctgtttgaa 1980 accgccctgc tatcttctgg cttttccctt gaggatcccc agacccactc caaccgcatc 2040 tatcgcatga tcaagctagg tctaggtatt gatgaagatg aagtggcagc agaggaaccc 2100 aatgctgcag ttcctgatga gatcccccct ctcgagggcg atgaggatgc gtctcgcatg 2160 gaagaagtcg attag 2175 <210> 13 <211> 1443 <212> DNA <213> Artificial Sequence <220> <223> Flag-Hsp40 <400> 13 atggctgcgc ggtgctccac acgctggttg ctggtggttg tggggacccc gcggctgccg 60 gctatatcgg gtagaggggc ccggccgccc agggagggcg tggtgggggc atggctgagc 120 cgcaagctga gcgtccccgc ctttgcgtct tccctgacct cttgcggccc ccgagcgctg 180 ctgacattga gacctggtgt cagcctcaca ggaacaaaac attacccttt catttgtact 240 gcctccttcc acacgagtgc ccctttggcc aaagaagatt attatcagat attaggagtg 300 cctcgaaatg ccagccagaa agagatcaag aaagcctatt atcagcttgc caagaagtat 360 caccctgaca caaataagga tgatcccaaa gccaaggaga agttctccca gctggcagaa 420 gcctatgagg ttttgagtga tgaggtgaag aggaagcagt acgatgccta cggctctgca 480 ggcttcgatc ctggggccag cggctcccag catagctact ggaagggagg ccccactgtg 540 gaccccgagg agctgttcag gaagatcttt ggcgagttct catcctcttc atttggagat 600 ttccagaccg tgtttgatca gcctcaggaa tacttcatgg agttgacatt caatcaagct 660 gcaaaggggg tcaacaagga gttcaccgtg aacatcatgg acacgtgtga gcgctgcaac 720 ggcaagggga acgagcccgg caccaaggtg cagcattgcc actactgtgg cggctccggc 780 atggaaacca tcaacacagg cccttttgtg atgcgttcca cgtgtaggag atgtggtggc 840 cgcggctcca tcatcatatc gccctgtgtg gtctgcaggg gagcaggaca agccaagcag 900 aaaaagcgag tgatgatccc tgtgcctgca ggagtcgagg atggccagac cgtgaggatg 960 cctgtgggaa aaagggaaat tttcattacg ttcagggtgc agaaaagccc tgtgttccgg 1020 agggacggcg cagacatcca ctccgacctc tttatttcta tagctcaggc tcttcttggg 1080 ggaacagcca gagcccaggg cctgtacgag acgatcaacg tgacgatccc ccctgggact 1140 cagacagacc agaagattcg gatgggtggg aaaggcatcc cccggattaa cagctacggc 1200 tacggagacc actacatcca catcaagata cgagttccaa agaggctaac gagccggcag 1260 cagagcctga tcctgagcta cgccgaggac gagacagatg tggaggggac ggtgaacggc 1320 gtcaccctca ccagctctgg tggcagcacc atggatagct ccgcaggaag caaggctagg 1380 cgtgaggctg gggaggacga ggagggattc ctttccaaac ttaagaaaat gtttacctca 1440 tga 1443 <210> 14 <211> 618 <212> DNA <213> Artificial Sequence <220> <223> Flag-Hsp27 <400> 14 atgaccgagc gccgcgtccc cttctcgctc ctgcggggcc ccagctggga ccccttccgc 60 gactggtacc cgcatagccg cctcttcgac caggccttcg ggctgccccg gctgccggag 120 gagtggtcgc agtggttagg cggcagcagc tggccaggct acgtgcgccc cctgcccccc 180 gccgccatcg agagccccgc agtggccgcg cccgcctaca gccgcgcgct cagccggcaa 240 ctcagcagcg gggtctcgga gatccggcac actgcggacc gctggcgcgt gtccctggat 300 gtcaaccact tcgccccgga cgagctgacg gtcaagacca aggatggcgt ggtggagatc 360 accggcaagc acgaggagcg gcaggacgag catggctaca tctcccggtg cttcacgcgg 420 aaatacacgc tgccccccgg tgtggacccc acccaagttt cctcctccct gtcccctgag 480 ggcacactga ccgtggaggc ccccatgccc aagctagcca cgcagtccaa cgagatcacc 540 atcccagtca ccttcgagtc gcgggcccag cttgggggcc cagaagctgc aaaatccgat 600 gagactgccg ccaagtaa 618 <210> 15 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> oligopetide <220> <221> MOD_RES <222> (7) <223> PHOSPHORYLATION, <400> 15 Arg Thr Thr Ser Gln Leu Tyr Asp Ala Val Pro Ile Gln Ser   1 5 10 <210> 16 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> oligopeptide <220> <221> MOD_RES <222> (7) <223> PHOSPHORYLATION, <400> 16 Glu Asp Asp Glu Asp Cys Tyr Gly Asn Tyr Asp Asn Leu Leu Ser Gln   1 5 10 15 Phe      

Claims (4)

정상세포와 샘플세포의 PDK1 Tyr9의 인산화도를 측정하여 비교하는 것을 특징으로 하는 암 진단 방법.A method for diagnosing cancer, comprising measuring and comparing the phosphorylation levels of PDK1 Tyr9 between normal cells and sample cells. 제 1 항에 있어서,The method of claim 1, 상기 PDK1 Try9의 인산화도의 측정은 anti-Y9K 항체를 이용한 형광 염색법인 것을 특징으로 하는 암 진단 방법.Measuring the phosphorylation level of PDK1 Try9 is a cancer diagnostic method, characterized in that the fluorescent staining method using an anti-Y9K antibody. 제 1 항 또는 제 2 항에 있어서,The method according to claim 1 or 2, 상기 암은 위암, 결장암, 폐암 또는 유방암인 것을 특징으로 하는 암 진단 방법.The cancer is a cancer diagnosis method, characterized in that the stomach cancer, colon cancer, lung cancer or breast cancer. 제 1 항에 있어서,The method of claim 1, 정상세포와 샘플 세포내 PDK1 및/또는 Hsp90의 발현 레벨을 추가로 비교하는 것을 특징으로 하는 암 진단 방법.And comparing the expression levels of PDK1 and / or Hsp90 in normal cells and sample cells.
KR1020070121931A 2007-11-28 2007-11-28 Method for diagnosis of cancer by measurement of tyrosine 9 phosphorylation of pdk1 KR20090055157A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160025019A (en) * 2013-06-27 2016-03-07 칭화 유니버시티 Tumor biomarker

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160025019A (en) * 2013-06-27 2016-03-07 칭화 유니버시티 Tumor biomarker

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