Submitted on September 4, 2008
Revised on February 12, 2009
Accepted on February 18, 2009
Non-kinase Activity of MLCK in Elongated Filopodia Formation and Chemotaxis of Vascular Smooth Muscle Cells toward Sphingosylphosphorylcholine
Hong Hui Wang1, Akio Nakamura, Atsushi Matsumoto, Shinji Yoshiyama, Xiaoran Qin2, Li-Hong Ye2, Ce Xe, Yue Zhang, Ying Gao3, Ryoki Ishikawa, and Kazuhiro Kohama1*
1 Gunma University Graduate School of Medicine
2 College of Life Sciences, Nankai University
3 Dalian Medical University
* To whom correspondence should be addressed. E-mail: kohamak{at}med.gunma-u.ac.jp.
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, non-kinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (Biochem. Biophys. Res. Commun. 369: 135-143, 2008). We previously showed that non-kinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (Li et al., Am. J. Physiol. Heart Circ. Physiol. 291: H1262-1272, 2006). To explore the regulatory role of non-kinase 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 down-regulated 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 down-regulated 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 non-kinase activity plays a major role for SPC-induced migration.
