Ca2+‐independent thr18 regulatory myosin light chain phosphorylation and contraction in vascular smooth muscle

Abstract

Evidence for a Ca2+-independent contraction in vascular smooth muscle (VSM) involving phosphorylation of regulatory myosin light chain (rMLC) by kinases other than myosin light chain kinase (MLCK) has been suggested. In this study we used both immunoblot and tandem mass spectrometry (FT-MS) techniques to investigate non-MLCK-dependent contraction in rat mesenteric VSM. Comparison of signals obtained using non-specific rMLC antibody, and specific phos-ser19 and phos-thr18 rMLC antibodies, indicated that Ca2+-dependent VSM contraction was accompanied primarily by increases in ser19 rMLC phosphorylation, which was sensitive to the MLCK inhibitor wortmannin (WOR). In contrast, Ca2+-independent contraction in the presence of the MLC phosphatase inhibitor calyculin A was accompanied primarily by increases in thr18 rMLC phosphorylation which was insensitive to WOR, but sensitive to the non-specific kinase inhibitor staurosporine (STR). Calyculin A, in either Ca2+or Ca2+ free conditions, also resulted in prominent diphos-thr18/ser19 rMLC bands, which were sensitive only to STR. Analysis of the FT-MS data obtained from in situ trypsin digested rMLC antibody-identified bands confirmed the identity of the rMLC proteins, as well as phos-ser19 in the presence of Ca2+ and phos-thr18 in the absence of Ca2+. These data suggest that a Ca2+- and MLCK-independent contraction pathway involving thr18 phosphorylation of rMLC is unmasked in the presence of calyculin. This work was supported by NIH R01GM055849 (to WAB).