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*
Department of Internal Medicine and Physical Therapy, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan;
Division of Protein Metabolism, Institute for Protein Research, Osaka University, Yamadaoka, Suita, Osaka, Japan; and
Department of Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan
Although the requirement for c-Src in extracellular matrix
(ECM)-mediated fibroblast motility has been well established, the roles
of hemopoietic Src family protein tyrosine kinases in leukocyte
migration have not been fully elucidated. To address the issue, we
analyzed fibronectin (Fn)-mediated adhesion signaling in rat basophilic
leukemia (RBL) 2H3 cells overexpressing 1) Csk, 2) a membrane-anchored,
gain-of-function Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(-)).
Parent RBL2H3 cells, expressing autoactivated c-kit,
readily adhered to Fn-coated surface, developed typical leukocyte
adhesion machinery (podosome), and migrated toward Fn without cytokine
priming, thus provided a simple experimental system to analyze
Fn-mediated outside-in signaling. While overexpression of Csk or the
Csk mutants did not significantly affect cell adhesion to the Fn
surface or 
5 integrin recruitment to the attachment
sites, Csk suppressed and mCsk almost abolished Fn-mediated tyrosine
phosphorylation of paxillin, filamentous actin assembly to podosomes,
and cell migration, but mCsk(-) did not. Coexpression of LynA devoid
of C-terminal negative regulatory tyrosine in mCsk cells successfully
restored Fn-mediated podosome formation and cell migration.
Coexpression of c-Src lacking the C-terminal tyrosine reconstructed
podosomes, but could not restore the cell migration regardless of its
expression level. Collectively, these observations provide evidence
that Src family protein tyrosine kinases are required, and that Lyn
could transmit sufficient signal for Fn-mediated cytoskeletal changes
leading to cell locomotion in RBL2H3 cells, and they suggest that Lyn
and c-Src are differentially involved in cell
motility.
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