|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,800,000 (Direct Cost : ¥1,800,000)
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
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