ESSENTIAL ROLES OF LYN IN FIBRONECTIN-MEDIATED FILAMENTOUS ACTIN ASSEMBLY AND CELL MOTILITY IN MAST-CELLS

Although the requirement for c-Src in extracellular matrix (ECM)-media ted fibroblast motility has been well established, the roles of hemopo ietic Src family protein tyrosine kinases in leukocyte migration have not been fully elucidated. To address the issue, we analyzed fibronect in (Fn)-mediated adhesion signaling in rat basophilic leukemia (RBL) 2 H3 cells overexpressing 1) Csk, 2) a membrane-anchored, gain-of-functi on Csk (mCsk), and 3) a kinase-defective mCsk (mCsk(-)), Parent RBL2H3 cells, expressing autoactivated c-kit, readily adhered to Fn-coated s urface, developed typical leukocyte adhesion machinery (podosome), and migrated toward Fn without cytokine priming, thus provided a simple e xperimental 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 alpha(5) integrin recruitment to th e attachment sites, Csk suppressed and mCsk almost abolished Fn-mediat ed tyrosine phosphorylation of paxillin, filamentous actin assembly to podosomes, and cell migration, but mCsk(-) did not. Coexpression of L ynA devoid of C-terminal negative regulatory tyrosine in mCsk cells su ccessfully restored Fn-mediated podosome formation and cell migration. Coexpression of c-Src lacking the C-terminal tyrosine reconstructed p odosomes, but could not restore the cell migration regardless of its e xpression level, Collectively, these observations provide evidence tha t Src family protein tyrosine kinases are required, and that Lyn could transmit sufficient signal for Fn-mediated cytoskeletal changes leadi ng to cell locomotion in RBL2H3 cells, and they suggest that Lyn and c -Src are differentially involved in cell motility.