The Involvement of Fyn Tyrosine Kinase in the Signal Transduction of Ca2+ sensitization of Vascular Smooth Muscle Contraction Mediated by Rho-kinase

Abstract

Rho-kinase (ROK)-mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction plays a critical role in abnormal VSM contraction such as vasospasm. Previously we found that sphingosylphosphorylcholine (SPC) induces the ROK-mediated Ca2+ sensitization through the activation of Src family tyrosine kinase (Src-TK) independently of a protein kinase C pathway. Since western blot analysis showed the presence of Fyn and c-Src among Src-TKs in VSM, we aimed to clarify which Src-TK is truly involved in the Ca2+ sensitization mediated by the SPC/ROK pathway. Immunofluorescent study showed that SPC induced the translocation of Fyn, but not c-Src, to plasma membrane in cultured VSM cells, which was blocked by eicosapentaenoic acid, a specific inhibitor of the SPC-induced Ca2+ sensitization. The siRNA-mediated knockdown of Fyn diminished the SPC-induced contraction remarkably in cultured VSM cells. In β-escin-permeabilized VSM strips, constitutively- active Fyn, which was produced by a baculovirus expression system, induced the Ca2+ sensitization, which was blocked by Y27632 (a ROK inhibitor). Dominant-negative Fyn blocked the Ca2+ sensitization induced by SPC, GTPγS (a direct activator of G-protein), and a GPCR agonist + GTP. Overexpression of constitutively-active and dominant-negative Fyn induced contraction and relaxation of cultured VSM cells, respectively. Furthermore, in order to identify the target(s) of Fyn, tyrosine-phosphorylated proteins were immunoprecipitated from the VSM cells which were treated with or without SPC. Subsequently, nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) was used to identify the SPC-induced tyrosine-phosphorylated and tyrosine-dephosphorylated proteins and their phosphorylation sites. These findings clearly indicate that Fyn tyrosine kinase plays an essential role in the ROK-mediated Ca2+ sensitization of VSM contraction. Functional proteomic approach has been useful to reveal possible targets of Fyn, and their functional roles are currently under investigation.