Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain
Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain
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DOI：
10.1074/jbc.m204255200
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发表时间：
2002-11-22
影响因子：
4.8
通讯作者：
Smithgall, TE
中科院分区：
文献类型：
--
作者：
Schreiner, SJ;Schiavone, AP;Smithgall, TE
STAT3是具有Src同源性2 （SH2）结构域的转录因子家族中的一员，在多种细胞因子和生长因子的作用下被酪氨酸磷酸化激活。在这项研究中，我们研究了非受体酪氨酸激酶Src家族激活STAT3的机制，该家族在正常和转化细胞类型中都与STAT激活有关。利用Sf-9昆虫细胞，我们证实了Src激酶家族的5个成员（Src、Hck、Lyn、Fyn和Fgr）对STAT3酪氨酸的直接磷酸化和DNA结合活性的刺激。我们还在该系统中观察到稳定的STAT3-Src家族激酶复合物的形成。重组Src家族激酶SH3结构域足以与STAT3相互作用，这表明Src激酶-STAT3相互作用的机制基础。为了在体内测试Src家族激酶SH3结构域对STAT3募集和激活的贡献，我们使用了表达髓系Src家族成员Hck激活突变体的大鼠-2成纤维细胞。缺乏负调控尾酪氨酸残基（Hck- yf）的激活Hck突变体转化成纤维细胞诱导内源性STAT3的强DNA结合活性。通过Ala取代保守的Trp残基（W93A突变体）使Hck SH3功能失活，完全消除了Hck- yf对STAT3的激活，并且在不影响Hck激酶活性的情况下降低了50%的转化活性。最后，在没有细胞外信号的情况下，STAT3在Rat-2细胞中的过表达会短暂地刺激Hck和c-Src激酶活性，这种作用依赖于STAT3中假定的SH3结合基序。这些结果支持Src家族激酶通过sh3依赖机制招募STAT3的模型，导致短暂的激酶激活和STAT3磷酸化。
STAT3 is a member of a family of transcription factors with Src homology 2 (SH2) domains that are activated by tyrosine phosphorylation in response to a wide variety of cytokines and growth factors. In this study, we investigated the mechanism of STAT3 activation by the Src family of nonreceptor tyrosine kinases, which have been linked to STAT activation in both normal and transformed cell types. Using Sf-9 insect cells, we demonstrate direct STAT3 tyrosine phosphorylation and stimulation of DNA binding activity by five members of the Src kinase family (Src, Hck, Lyn, Fyn, and Fgr). We also observed stable STAT3-Src family kinase complex formation in this system. Recombinant Src family kinase SH3 domains were sufficient for interaction with STAT3, suggesting a mechanistic basis for the Src kinase-STAT3 interaction. To test the contribution of Src family kinase SH3 domains to the recruitment and activation of STAT3 in vivo, we used Rat-2 fibroblasts expressing activated mutants of the myeloid Src family member Hck. Transformation of fibroblasts by an activated Hck mutant lacking the negative regulatory tail tyrosine residue (Hck-YF) induced strong DNA binding activity of endogenous STAT3. Inactivation of Hck SH3 function by Ala replacement of a conserved Trp residue (W93A mutant) completely abolished STAT3 activation by Hck-YF and reduced transforming activity by 50% without affecting Hck kinase activity. Finally, overexpression of STAT3 in Rat-2 cells transiently stimulated Hck and c-Src kinase activity in the absence of extracellular signals, an effect that was dependent upon a putative SH3 binding motif in STAT3. These results support a model in which Src family kinases recruit STAT3 through an SH3-dependent mechanism, resulting in transient kinase activation and STAT3 phosphorylation.