Stat5 dephosphorylation by Shp-2

描述(申请人提供)：信号转导和转录因子5激活因子(STAT5)在广谱细胞因子的功能中起着非常重要的作用。当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(在Stat5a的第694位)，形成二聚体，转位到细胞核，并启动各种细胞因子诱导的基因。虽然人们对Stat5的激活过程知之甚少，但对酪氨酸磷酸化的Stat5失活的机制却知之甚少。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。利用与Stat5a酪氨酸磷酸化位点相对应的多肽，我们鉴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与Stat5特异地相互作用。SHP-2的过度表达减弱了细胞因子诱导的Stat5的酪氨酸磷酸化，而SHP-2的缺失显著延迟了Stat5的去磷酸化。SHP-2通常以非活性形式存在于细胞中，其N端SH2结构域与其催化结构域的结合抑制了SHP-2的活性。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多肽，我们与SHP-2共纯化了受体蛋白CrkL。在细胞内，CrkL能与Stat5和SHP-2结合。此外，初步数据表明，CrkL在细胞中的过表达加速了Star5的去磷酸化。基于这些发现，我们假设SHP-2是一种特异性的Stat5磷酸酶，它被募集到酪氨酸磷酸化的Stat5中，并通过适配蛋白CrkL从非活性状态释放到活性状态。在细胞因子刺激下，激活的JAKS磷酸化STAT5和适配蛋白CrkL。酪氨酸磷酸化的Stat5和CrkL形成复合体，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招募到复合体中，导致Shp2的激活。因此，激活的SHP-2访问并去磷酸化酪氨酸磷酸化的Stat5。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。这项研究将有助于理解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，并有助于确定特定治疗的靶点。

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.