Stat5 dephosphorylation by Shp-2

描述（申请人提供）：信号转导和转录激活因子5（Stat 5）在广谱细胞因子的功能中起着非常重要的作用。在细胞因子与它们的受体结合后，Stat 5被激活的Janus蛋白酪氨酸激酶（Jaks）酪氨酸磷酸化（在Stat 5A的Tyr 694处），形成二聚体，易位到细胞核，并开启多种精氨酸诱导的基因。虽然对Stat 5的活化过程了解很多，但对酪氨酸磷酸化Stat 5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Stat 5的失活是通过去磷酸化。使用对应于Stat 5A酪氨酸磷酸化位点的肽，我们鉴定了蛋白酪氨酸磷酸酶Shp-2作为Stat 5磷酸酶。Shp-2在生理条件下以酪氨酸磷酸化依赖的方式与Stat 5特异性相互作用。Shp-2的过度表达减弱了精氨酸诱导的Stat 5的酪氨酸磷酸化，而Shp-2的缺乏则显著延迟了Stat 5的去磷酸化。Shp-2通常以非活性形式存在于细胞中，通过其自身的N-末端SH 2结构域与其催化结构域的结合来抑制。我们的初步数据表明，SH 2结构域的Shp-2不直接与酪氨酸磷酸化的Stat 5。使用酪氨酸磷酸化的Stat 5A肽，我们共纯化的衔接蛋白，CrkL，与Shp-2。CrkL能够与细胞中的Stat 5和Shp-2结合。此外，初步数据显示，CrkL在细胞中的过表达加速了Star 5的去磷酸化。基于这些发现，我们假设Shp-2是一种特异性Stat 5磷酸酶，它被酪氨酸磷酸化Stat 5募集，并通过接头蛋白CrkL从非活性形式释放到活性形式。在细胞因子刺激后，活化的Jaks使Stat 5和衔接蛋白CrkL磷酸化。酪氨酸磷酸化的Stat 5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SH 2结构域的相互作用将Shp-2募集到复合物中，导致Shp 2的活化。因此，活化的Shp-2接近并去磷酸化酪氨酸磷酸化的Stat 5。为了检验我们的假设，我们将1）鉴定Stat 5和Shp-2的结构域，其是Stat 5与Shp-2相互作用所需的，2）确定CrkL在Stat 5与Shp-2相互作用和由Shp-2去磷酸化中的作用，和3）研究Stat 5去磷酸化的生理作用。拟议的研究将有助于理解Stat 5失活的机制，为包括血液恶性肿瘤在内的许多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。

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.