Biphasic Regulation of Myosin Light Chain Phosphorylation by p21-activated Kinase Modulates Intestinal Smooth Muscle Contractility

• Published in: The Journal of biological chemistry 2013-01, Vol.288 (2), p.1200-1213
• Main Authors: Chu, Ji, Pham, Ngoc T., Olate, Nicole, Kislitsyna, Karina, Day, Mary-Clare, LeTourneau, Phillip A., Kots, Alexander, Stewart, Randolph H., Laine, Glen A., Cox, Charles S., Uray, Karen
• Format: Article
• Language: English
• Subjects: Animals; Biphasic Regulation; Cell Motility; Cells, Cultured; Gastrointestinal Motility; Humans; Intestines - physiology; Male; Mechanotransduction; MLC Phosphatase Targeting Subunit (MYPT1); Molecular Bases of Disease; Muscle Contraction; Muscle, Smooth - physiology; Myosin; Myosin Light Chain (MLC); Myosin Light Chains - metabolism; p21-activated Kinase; p21-Activated Kinases - metabolism; Phosphoprotein Phosphatase; Phosphorylation; Rats; Rats, Sprague-Dawley; Smooth Muscle
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M112.370718

Supraphysiological mechanical stretching in smooth muscle results in decreased contractile activity. However, the mechanism is unclear. Previous studies indicated that intestinal motility dysfunction after edema development is associated with increased smooth muscle stress and decreased myosin light chain (MLC) phosphorylation in vivo, providing an ideal model for studying mechanical stress-mediated decrease in smooth muscle contraction. Primary human intestinal smooth muscle cells (hISMCs) were subjected to either control cyclical stretch (CCS) or edema (increasing) cyclical stretch (ECS), mimicking the biophysical forces in non-edematous and edematous intestinal smooth muscle in vivo. ECS induced significant decreases in phosphorylation of MLC and MLC phosphatase targeting subunit (MYPT1) and a significant increase in p21-activated kinase (PAK) activity compared with CCS. PAK regulated MLC phosphorylation in an activity-dependent biphasic manner. PAK activation increased MLC and MYPT1 phosphorylation in CCS but decreased MLC and MYPT1 phosphorylation in hISMCs subjected to ECS. PAK inhibition had the opposite results. siRNA studies showed that PAK1 plays a critical role in regulating MLC phosphorylation in hISMCs. PAK1 enhanced MLC phosphorylation via phosphorylating MYPT1 on Thr-696, whereas PAK1 inhibited MLC phosphorylation via decreasing MYPT1 on both Thr-696 and Thr-853. Importantly, in vivo data indicated that PAK activity increased in edematous tissue, and inhibition of PAK in edematous intestine improved intestinal motility. We conclude that PAK1 positively regulates MLC phosphorylation in intestinal smooth muscle through increasing inhibitory phosphorylation of MYPT1 under physiologic conditions, whereas PAK1 negatively regulates MLC phosphorylation via inhibiting MYPT1 phosphorylation when PAK activity is increased under pathologic conditions. Background: Myosin light chain (MLC) phosphorylation is the driving force of cell contractility. Results: p21-activated kinase (PAK) can either activate or inhibit myosin light chain phosphorylation, depending on the level of PAK activation. Conclusion: Inhibition of intestinal motility by supraphysiological mechanical stretch is attributable to increased PAK activity. Significance: The biphasic regulation of PAK may provide an explanation for the conflicting observations on PAK-mediated MLC phosphorylation.