Stat5 dephosphorylation by Shp-2

Shp-2 使 Stat5 去磷酸化

• 批准号: 6879605
• 负责人: DEMIN WANG
• 金额: $ 27.37万
• 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/6879605
• 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）的信号换能器和激活器在多种细胞因子的功能中起着非常重要的作用。当细胞因子与受体结合时，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，易位到细胞核，并开启多种细胞因子诱导基因。虽然我们对Stat5的激活过程了解很多，但对酪氨酸磷酸化的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的Stat5的失活是通过去磷酸化实现的。利用Stat5A酪氨酸磷酸化位点对应的肽，我们确定了蛋白酪氨酸磷酸酶Shp-2为Stat5磷酸酶。Shp-2在生理条件下以酪氨酸磷酸化依赖的方式特异性地与Stat5相互作用。过表达Shp-2会减弱细胞因子诱导的Stat5酪氨酸磷酸化，而缺乏Shp-2则会显著延迟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。利用酪氨酸磷酸化的Stat5A肽，我们与Shp-2共同纯化了适配蛋白CrkL。在细胞中，CrkL能够与Stat5和Shp-2结合。此外，初步数据显示，细胞中CrkL的过表达加速了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，它被酪氨酸磷酸化的Stat5招募，并被适配蛋白CrkL从非活性形式释放到活性形式。在细胞因子刺激下，活化的Jaks使Stat5和适配蛋白CrkL磷酸化。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的唯一磷酸化酪氨酸与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，活化的Shp-2进入并使酪氨酸磷酸化的Stat5去磷酸化。为了验证我们的假设，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相互作用和被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。本研究将有助于理解Stat5失活的机制，为包括血液系统恶性肿瘤在内的多种疾病的分子发病机制提供更多线索，并有助于确定特异性治疗靶点。