Abstract
Using RNA interference method, we previously showed that Fyn, a member of Src family tyrosine kinase, was involved in the sphingosylphosphorylcholine (SPC)-induced formation of stress fibers. The siRNA-mediated down-regulation of Fyn tyrosine kinase partially inhibited the SPC-induced stress fiber formation. In further study, here we investigated the effects of over-expression of Fyn constructs on the assembly of actin stress fibers. The cDNAs encoding constitutively active form (ca-Fyn; FynY530F), dominant negative form (dn-Fyn; FynK298M), and wild type of Fyn (FynWT) were cloned into pcDNA6/myc-His A vector. The constructs were verified by DNA sequencing. Using Nucleofector, NIH3T3 fibroblasts were transfected with myc-tagged Fyn constructs, and their over-expressions were confirmed in western blotting using the antibody against c-myc (9E10). SPC actions were mimicked by ca-Fyn (FynY530F) and were blocked by dn-Fyn (FynK298M). Immunofluoresence analysis showed that stress fiber formation was induced by ca-Fyn (FynY530F), but was blocked by dn-Fyn (FynK298M). Stimulation of NIH3T3 fibroblasts with SPC induced stress fiber formation in the cells transfected with empty vector or FynWT, but not in the FynK298M-overexpressed cells. These findings suggest that Fyn tyrosine kinase plays an important role in actin stress fiber formation induced by SPC. [J Physiol Sci. 2007;57 Suppl:S124]
