Studies on the function of a protein-tyrosine kinase CAKβ/PYK2 and a protein Hic-5 binding to CAKβ

蛋白酪氨酸激酶 CAKβ/PYK2 和蛋白 Hic-5 与 CAKβ 结合的功能研究

• 批准号: 13670127
• 负责人: SASAKI Terukatsu
• 金额: $ 2.3万
• 依托单位: Sapporo Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670127/
• 关键词: protein-tyrosine kinase; CAKβ; PYK2; nuclear translocation; SUMO ligase; SH3 domain; tyrosine-phosphorylation; Peptide Mass Fingerprinting法; Graf; アンドロゲン受容体

We have been studying a non-receptor protein-tyrosine kinase of the focal adhesion kinase (FAK) family CAKβ/PYK2. Although CAKβ has been shown to participate in the signal transduction regulating cell movement and cell adhesion, CAKβ is known to localize in the cytoplasm in addition to the focal adhesion. When we expressed a P859A mutant CAKβ in cultured cells, the mutant CAKβ exclusively localized in the cell nucleus, which markedly contrasts to the localization of the wild-type CAKβ. This point mutation disrupts one of the two PXXP motifs in CAKβ important as ligands for the SH3 domain. We obtained evidence indicating that the wild-type CAKβ also shuttles between the cytoplasm and the nucleus. It remains unknown the function of CAKβ in the nucleus. In order to know more about the signaling pathways where CAKβ participates, we tried to find new CAKβ-binding proteins by a screening in the yeast two-hybrid system. One CAKβ-binding protein thus identified was a protein with SUMO ligase E3 activity against transcription factors and others. When this protein was exogenously expressed in H1299 cells, the amount of intracellular p53 significantly increased and the transcription of a p53 reporter was also enhanced. Co-expression of CAKβ with this newly identified protein in H1299 cells resulted in a decrease in the amount of p53 in the cells and also resulted in a suppression of the transcription activity by p53. These results indicate that CAKβ has some function in the nucleus and suggest a functional difference of CAKβ and FAK. It has already been shown that Hic-5, a CAKβ binding protein, functions as a coactivator enhancing the transcriptional activity of the androgen receptor.

我们一直在研究粘着斑激酶（FAK）家族CAKβ/PYK 2的一种非受体蛋白酪氨酸激酶。尽管CAKβ已被证明参与调节细胞运动和细胞粘附的信号转导，但已知CAKβ除了局灶性粘附之外还定位于细胞质中。当我们在培养细胞中表达P859 A突变型CAKβ时，突变型CAKβ仅定位于细胞核，这与野生型CAKβ的定位形成鲜明对比。该点突变破坏了CAKβ中作为SH 3结构域配体的两个重要PXXP基序之一。我们获得的证据表明，野生型CAKβ也穿梭于细胞质和细胞核之间。CAKβ在细胞核中的功能尚不清楚。为了进一步了解CAKβ参与的信号通路，我们尝试通过酵母双杂交系统筛选新的CAKβ结合蛋白。由此鉴定的一种CAKβ结合蛋白是具有针对转录因子和其它因子的SUMO连接酶E3活性的蛋白。当该蛋白在H1299细胞中外源表达时，细胞内p53的量显著增加，并且p53报告基因的转录也增强。在H1299细胞中共表达CAKβ与这种新鉴定的蛋白质导致细胞中p53的量减少，并且还导致p53的转录活性受到抑制。这些结果提示CAKβ在细胞核内具有一定的功能，并提示CAKβ与FAK在功能上存在差异。已经表明，Hic-5，一种CAKβ结合蛋白，作为一种共激活剂，增强雄激素受体的转录活性。

项目成果

Ishino, M., Ohba, T., Aoto, H., Sasaki, H. and Sasaki, T.: "Quantitative analysis of protein-protein interactions by competitive dot-far western blotting"Recent Res.Devel.Anal.Biochem.. 2. 239-248 (2002)

Ishino, M.、Ohba, T.、Aoto, H.、Sasaki, H. 和 Sasaki, T.：“通过竞争性远点免疫印迹对蛋白质-蛋白质相互作用进行定量分析”最近的 Res.Devel.Anal.Biochem。

Hua Tang, et al.: "PYK2/CAKβ Tyrosine Kinase Activity-mediated Angiogenesis of Pulmonary Vascular Endothelial Cells"The Journal of Biological Chemistry. 277・7. 5441-5447 (2002)

唐华等：“PYK2/CAKβ酪氨酸激酶活性介导的肺血管内皮细胞的血管生成”《生物化学杂志》277·7（2002）。

Tang, H., Hao, Q., Fitzgerald, T., Sasaki, T., Landon, E.J. and Inagami, T.: "PYK2/CAK-βtyrosine kinase activity-mediated angiogenesis of pulmonary vascular endothelial cells"J.Biol.Chem.. 277(7). 5441-5447 (2002)

Tang, H.、Hao, Q.、Fitzgerald, T.、Sasaki, T.、Landon, E.J. 和 Inagami, T.：“PYK2/CAK-β 酪氨酸激酶活性介导的肺血管内皮细胞血管生成”J.Biol。化学.277(7).5441-5447(2002)

Takagi, C., Ueki, K., Ikeuchi, H., Kuroiwa, T., Kaneko, Y., Tsukada, Y., Maezawa, A., Mitaka, T., Sasaki, T. and Nojima, Y.: "Increased expression of cell adhesion kinase β in human and rat crescentric glomerulonephritis"American Journal of Kidney Disease

Takagi, C.、Ueki, K.、Ikeuchi, H.、Kuroiwa, T.、Kaneko, Y.、Tsukada, Y.、Maezawa, A.、Mitaka, T.、Sasaki, T. 和 Nojima, Y.： “人类和大鼠新月体肾小球肾炎中细胞粘附激酶 β 的表达增加”美国肾脏病杂志

Masaho Ishino, et al.: "Identification of proteins that associate with CAKβ/PYK2"Tumor Research. 37. 23-31 (2002)

Masaho Ishino 等人：“与 CAKβ/PYK2 相关的蛋白质的鉴定”肿瘤研究 37. 23-31 (2002)。