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

Abstract

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.

Myosin Light Chain Kinase / Actin Interaction in Phorbol Dibutyrate–Stimulated Smooth Muscle Cells

“…FRET is a powerful tool to quantitatively assess the interactions between two different proteins in cells (33). Therefore, the FRET interaction between MLCK and two different actin isoforms, α-and β-actin, was examined with unstimulated and PDBu-stimulated A7r5 smooth muscle cells.…”

“…Figure 6 shows the . The filled circles were for a MLCK-deficient Gba-SM-4 cell line of which MLCK expression was silenced by RNAi as described in Materials and Methods (33). Both cell lines were stained by MLCK and α-actin antibodies.…”

“…Whole cell FRET analysis, as presently employed, has been used successfully to evaluate protein-protein interactions in smooth muscle cells (32,33,36). Because the donor/acceptor must be within 10-nm distance from each other for efficient energy transfer (37,38), this technique provides a measure of protein-protein distances compatible with molecular interaction.…”

“…Lentiviral siRNA silencing of endogenous MLCK named RNAi was generated according to the protocol published recently (33). Gba-SM-4 cells were incubated with virus-containing condition media (titer = 2.8 × 10 5 transducing unit/mL) supplemented with polybrene (6 μg/mL) for 14 days.…”

“…After the blasticidin-resistant colonies were isolated and expanded, the expressions of MLCK isoforms were evaluated by Western blot analysis. The Gba-SM-4 stable cell lines constitutively expressing RNAi for guinea-pig MLCK, and thus lacking the expression of both isoforms MLCK, were used as the MLCK-deficient cells (for the silencing mechanism, see For over-expression of kinase-deficient MLCK cells, we constructed a wild-type (WT) plasmid using the full length bovine stomach cDNA, pUC 118/ BS 155K, as previously described (33). The kinase inactive-MLCK named dATP was generated by the mutagenesis of the ATP-binding site.…”