Stat5 dephosphorylation by Shp-2

Shp-2 使 Stat5 去磷酸化

• 批准号: 6717687
• 负责人: DEMIN WANG
• 金额: $ 27.08万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6717687
• 关键词: JAK kinase; T lymphocyte; binding proteins; cytokine; enzyme activity; enzyme mechanism; gene deletion mutation; immunoprecipitation; laboratory mouse; phosphorylation; protein protein interaction; protein structure function; protein tyrosine phosphatase; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant): The signal transducer and activator of transcription factor 5 (Stat5) plays a very important role in functions of a broad spectrum of cytokines. Upon binding of cytokines to their receptors, Stat5 is tyrosine-phosphorylated (at Tyr694 of Stat5A) by activated Janus protein tyrosine kinases (Jaks), forms a dimer, translocates to the nucleus, and turns on a variety of cytokine-inducible genes. Although much is known about the process of Stat5 activation, little is known about the mechanism by which the tyrosine-phosphorylated Stat5 is inactivated. Our recent studies demonstrate that inactivation of the tyrosine-phosphorylated Stat5 is via dephosphorylation. Using peptides corresponding to the Stat5A tyrosine phosphorylation site, we identified the protein-tyrosine phosphatase, Shp-2, as a Stat5 phosphatase. Shp-2 specifically interacts with Stat5 under physiological conditions in a tyrosine-phosphorylation-dependent manner. Over-expression of Shp-2 attenuates cytokine-induced tyrosine phosphorylation of Stat5, whereas Shp-2 deficiency dramatically delays the dephosphorylation of Stat5. Shp-2 normally exists as an inactive form in cells, inhibited by binding of its own N-terminal SH2 domain to its catalytic domain. Our preliminary data show that the SH2 domains of Shp-2 do not directly interact with tyrosine-phosphorylated Stat5. Using the tyrosine-phosphorylated Stat5A peptides, we have co-purified the adapter protein, CrkL, with Shp-2. CrkL is able to associate with both Stat5 and Shp-2 in cells. Moreover, preliminary data show that over-expression of CrkL in cells accelerates the dephosphorylation of Star5. Based on these findings, we hypothesize that Shp-2 is a specific Stat5 phosphatase that is recruited to tyrosine-phosphorylated Stat5 and released from an inactive form to an active one by the adapter protein CrkL. Upon cytokine stimulation, activated Jaks phosphorylate Stat5 and the adapter protein CrkL. Tyrosine-phosphorylated Stat5 and CrkL form a complex, and subsequently, tyrosine-phosphorylated CrkL recruits Shp-2 to the complex through interaction of the sole phosphotyrosine of CrkL with SH2 domains of Shp-2, leading to activation of Shp2. Consequently, the activated Shp-2 accesses and dephosphorylates tyrosine-phosphorylated Stat5. To test our hypothesis, we will 1) identify the domains of Stat5 and Shp-2 that are required for Stat5 interaction with Shp-2, 2) determine the role of CrkL in Stat5 interaction with, and dephosphorylation by, Shp-2, and 3) study the physiological role of Stat5 dephosphorylation. The proposed research will contribute to understanding of the mechanism of Stat5 inactivation, provide more clues to the molecular pathogenesis of numerous diseases including hematological malignancies, and help identify targets for specific therapies.

描述（由申请人提供）：转录因子5（Stat5）的信号转导器和激活剂在多种细胞因子的功能中发挥着非常重要的作用。细胞因子与其受体结合后，Stat5 被激活的 Janus 蛋白酪氨酸激酶 (Jaks) 酪氨酸磷酸化（Stat5A 的 Tyr694），形成二聚体，易位至细胞核，并开启多种细胞因子诱导基因。尽管人们对 Stat5 激活的过程了解甚多，但对酪氨酸磷酸化 Stat5 失活的机制知之甚少。我们最近的研究表明，酪氨酸磷酸化 Stat5 的失活是通过去磷酸化实现的。使用与 Stat5A 酪氨酸磷酸化位点相对应的肽，我们将蛋白质酪氨酸磷酸酶 Shp-2 鉴定为 Stat5 磷酸酶。在生理条件下，Shp-2 以酪氨酸磷酸化依赖性方式与 Stat5 特异性相互作用。 Shp-2 的过度表达会减弱细胞因子诱导的 Stat5 酪氨酸磷酸化，而 Shp-2 缺乏会显着延迟 Stat5 的去磷酸化。 Shp-2 通常以非活性形式存在于细胞中，通过其自身 N 末端 SH2 结构域与其催化结构域的结合而受到抑制。我们的初步数据表明，Shp-2 的 SH2 结构域不直接与酪氨酸磷酸化的 Stat5 相互作用。使用酪氨酸磷酸化的 Stat5A 肽，我们与 Shp-2 共同纯化了接头蛋白 CrkL。 CrkL 能够与细胞中的 Stat5 和 Shp-2 结合。此外，初步数据表明，细胞中CrkL的过度表达会加速Star5的去磷酸化。基于这些发现，我们假设Shp-2是一种特定的Stat5磷酸酶，它被招募到酪氨酸磷酸化的Stat5中，并通过衔接蛋白CrkL从非活性形式释放为活性形式。在细胞因子刺激后，激活的 Jaks 磷酸化 Stat5 和接头蛋白 CrkL。酪氨酸磷酸化的 Stat5 和 CrkL 形成复合物，随后，酪氨酸磷酸化的 CrkL 通过 CrkL 的唯一磷酸酪氨酸与 Shp-2 的 SH2 结构域相互作用将 Shp-2 招募到复合物中，从而激活 Shp2。因此，激活的 Shp-2 会接触酪氨酸磷酸化的 Stat5 并使其去磷酸化。为了检验我们的假设，我们将 1) 确定 Stat5 与 Shp-2 相互作用所需的 Stat5 和 Shp-2 结构域，2) 确定 CrkL 在 Stat5 与 Shp-2 相互作用以及由 Shp-2 去磷酸化中的作用，以及 3) 研究 Stat5 去磷酸化的生理作用。拟议的研究将有助于了解 Stat5 失活的机制，为包括血液恶性肿瘤在内的多种疾病的分子发病机制提供更多线索，并帮助确定特定治疗的靶点。