The involvement of Fyn tyrosine kinase and membrane rafts in the signal transduction of abnormal vascular smooth muscle contraction

Abstract

Rho-kinase (ROK)-mediated Ca²⁺ sensitization plays a pivotal role in abnormal vascular smooth muscle (VSM) contraction such as vasospasm. Previously we identified sphingosylphosphorylcholine (SPC) and Src family tyrosine kinase (Src-TK) as an upstream signaling molecule of ROK-mediated Ca²⁺ sensitization. Since VSM contains both Fyn and c-Src among Src-TK, we analyzed which Src-TK was truly important for the Ca²⁺ sensitization mediated by SPC/ROK pathway. Immunofluorescent study showed that SPC induced the translocation of Fyn, but not c-Src, to plasma membrane in cultured VSM cells and eicosapentaenoic acid, a specific inhibitor of SPC-induced VSM contraction, blocked the translocation of Fyn. The siRNA which specifically knockdown Fyn diminished SPC-induced contraction remarkably in cultured VSM cells. In β-escin permeabilized VSM strips, constitutively-active Fyn, which was expressed by baculovirus system, induced Ca²⁺ sensitization and dominant-negative Fyn blocked Ca²⁺ sensitization. In confocal study, SPC induced the translocation of Fyn to plasma membrane where it colocalized with caveolin-1, a membrane-raft-associated protein. A functional proteomics approach identified p160 and its phosphorylation site as a possible target of Fyn. Those findings suggested that membrane rafts and its associated Fyn played an essential role in ROK-mediated Ca²⁺-sensitization of VSM contraction. [J Physiol Sci. 2006;56 Suppl:S41]