Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine

Wang HH, Nakamura A, Matsumoto A, Yoshiyama S, Qin X, Ye LH, Xie C, Zhang Y, Gao Y, Ishikawa R, Kohama K. Nonkinase activity of MLCK in elongated filopodia formation and chemotaxis of vascular smooth muscle cells toward sphingosylphosphorylcholine. Am J Physiol Heart Circ Physiol 296: H1683-H1693, 2009. First published February 20, 2009; doi:10.1152/ajpheart.00965.2008.-The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin-and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration.