Overexpression of C-terminal Src Kinase Homologous Kinase Suppresses Activation of Lyn Tyrosine Kinase Required for VLA5-mediated Dami Cell Spreading*
- From the Department of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Kumamoto 860-0811, Japan
Abstract
The Csk homologouskinase (Chk), which is co-expressed with C-terminal Src kinase (Csk) in hematopoietic cells, negatively regulates Src family kinases in vitro with selectivity toward Lyn but not c-Src in platelets. To explore the role of Src family kinases in hematopoietic cell adhesion, we overexpressed Chk in the megakaryocytic cell line Dami and established clones exhibiting a 10-fold increase in the amount of Chk. Overexpression of Chk was found to suppress VLA5 integrin-mediated cell spreading, but not cell attachment, throughout fibronectin (FN) stimulation. Deletion and point mutagenesis analyses of Chk showed that this suppression was dependent upon both the SH3 domain, which is responsible for membrane anchoring, and kinase activity. FN-induced cell spreading accompanied a sustained increase in Lyn activity with coincidental kinetics and the activation of Lyn was also suppressed by overexpression of Chk but not a Chk mutant lacking the SH3 domain. Expression of a truncated Lyn mutant lacking the kinase domain inhibited both cell spreading and Lyn activation upon stimulation with FN. These results suggest that sustained activation of Lyn, which is regulated by membrane-anchored Chk, plays a crucial role in VLA5-mediated cell spreading but not cell attachment to a FN substrate.
Footnotes
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↵* This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ Supported by a Research Fellowship of the Japan Society for the Promotion of Sciences for Young Scientists.
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↵§ To whom correspondence should be addressed: Dept. of Cell Differentiation, Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, Honjo 2-2-1, Kumamoto 860-0811, Japan. Tel.: 81-96-373-5330; Fax: 81-96-373-5332; E-mail:bunseini{at}kaiju.medic.kumamoto-u.ac.jp.
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↵1 The abbreviations used are: Csk, C-terminal Src kinase; Chk, Csk homologous kinase; FN, fibronectin; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting.
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↵2 A. Hirao, X.-L. Huang, T. Suda, and N. Yamaguchi, unpublished observations.
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- Received December 2, 1997.
- Revision received February 10, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
