Distinct roles of Chk and Csk in regulation of Src family kinases in hematopoietic cells

Src家族蛋白酪氨酸激酶在调节包括造血细胞在内的多种细胞类型的增殖和分化中起着至关重要的作用。Src家族激酶的活性受到酪氨酸磷酸化和去磷酸化事件的严格调控。c端Src激酶（Csk）普遍表达，已被证明可以磷酸化Src家族激酶的c端负调节酪氨酸残基并抑制其激酶活性。造血细胞中表达的Csk家族的第二个成员最近被确定为Csk同源激酶（Chk）。为了了解Chk和Csk在造血细胞中共表达的意义，我们检测了这两种蛋白的亚细胞定位。Chk而不是Csk定位在CD36（膜糖蛋白IV）锚定的Lyn附近，并且Lyn的激酶活性被选择性抑制。在凝血酶刺激下，Chk快速而完全地从cd36锚定的Lyn转移。在蛋白酪氨酸磷酸酶的作用下，Lyn的c端负调节酪氨酸被去磷酸化。我们提出Chk而不是Csk在凝血素诱导的血小板活化中作为cd36 -锚定Lyn的易位控制负调节因子。为了进一步研究Chk在造血细胞中的作用，我们在巨核细胞系Dami中过表达Chk。在整个纤维连接蛋白刺激过程中，Chk的过表达抑制了VLA5整合素介导的细胞扩散，而不是细胞附着。这种抑制依赖于负责膜锚定的SH3结构域和激酶活性。由膜锚定Chk调控的Lyn持续激活是vla5介导的细胞在纤维连接蛋白底物上扩散所必需的。因此，这些结果表明，与Csk不同，Chk在体内对Src家族激酶具有选择性的负调控作用，并在造血细胞中发挥良好的调节作用。少

Src family protein-tyrosine kinases play crucial roles in regulating proliferation and differentiation of multiple cell types including hematopoietic cells. The activity of Src family kinases is tightly regulated by tyrosine phosphorylation and dephosphorylation events. The C-terminal Src kinase (Csk), which is expressed ubiquitously, has been shown to phosphorylate the C-terminal negative regulatory tyrosine residue of Src family kinases and suppress their kinase activity. A second member of the Csk family expressed in hematopoietic cells was recently identified as the Csk homologous kinase (Chk).To understand the significance of co-expression of Chk and Csk in hematopoietic cells, we examined the subcellular localization of each protein. Chk but not Csk was localized close to CD36 (membrane glycoprotein IV)-anchored Lyn, and the kinase activity of Lyn was selectively suppressed. Upon stimulation with thrombin, the rapid and complete translocation of Chk away from CD36-anchored Lyn ca … More used concomitant activation of Lyn. The activation was accompanied by dephosphorylation of Lyn at its C-terminal negative regulatory tyrosine in cooperation with a protein tyrosine phosphatase. We propose that Chk but not Csk functions as a translocation-controlled negative regulator of CD36-anchored Lyn in thrombin-induced platelet activation.To further examine die role of Chk in hematopoietic cells, we overexpressed Chk in the megakaryocytic cell line Dami. Overexpression of Chk suppressed VLA5 integrin-mediated cell spreading, but not cell attachment, throughout fibronectin stimulation. This suppression was dependent upon both the SH3 domain, which is responsible for membrane anchoring, and kinase activity. Sustained activation of Lyn, which is regulated by membrane-anchored Chk, was required for VLA5-mediated cell spreading on a fibronectin substrate.Thus, these results suggest that Chk, unlike Csk, negatively regulates Src family kinases in vivo with selectivity toward Lyn and acts as a fine regulator in hematopoietic cells. Less