Spatially selective MRLC regulation is absent in dedifferentiated vascular smooth muscle cells but is partially induced by fibronectin and Klf4.

The phosphorylation state of myosin regulatory light chain (MRLC) is central to the regulation of contractility that in turn impacts cellular homeostasis and fate decisions. Rho-kinase (ROCK) and myosin light chain kinase (MLCK) are major kinases for MRLC documented to selectively regulate MRLC in a subcellular position-specific manner; specifically, MLCK in some nonmuscle cell types works in the cell periphery to promote migration, while ROCK does at the central region to sustain contractility. However, it remains unclear whether or not the spatially selective regulation of the MRLC kinases is universally present in other cell types including dedifferentiated vascular smooth muscle cells (SMCs). Here, we demonstrate the absence of the spatial regulation in dedifferentiated SMCs using both cell lines and primary cells, thus distinct from previous reports on cells with migratory potential. We also observed that the spatial regulation is partly induced upon fibronectin stimulation and Klf4 overexpression. To find clues to the mechanism, we revealed how the phosphorylation state of MRLC is determined within dedifferentiated A7r5 SMCs under the enzymatic competition among three major regulators ROCK, MLCK, and MRLC phosphatase (MLCP). We show that ROCK but not MLCK predominantly regulates the MRLC phosphorylation in a manner distinct from previous in vitro-based and in silico-based reports. In this ROCK-dominating cellular system, the contractility at physiological conditions was regulated at the level of MRLC diphosphorylation because its monophosphorylation is already saturated. Thus, the present study provides insights into the molecular basis underlying the absence of spatial MRLC regulation in dedifferentiated SMCs.