Abstract
It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.
Highlights
It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA
The mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation
The expression of a constitutively active form of Rho-kinase induced stress fiber and focal adhesion formation in fibroblasts and an increase in the level of MLC20 phosphorylation [17]. These results in smooth muscle and in non-muscle cells are attributed to an increase in MLC20 phosphorylation and this is thought to reflect the inhibition of MP
Summary
It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr695 and this recognized only phosphorylated Thr695 in both native and recombinant MYPT1 Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr695 phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. The expression of a constitutively active form of Rho-kinase induced stress fiber and focal adhesion formation in fibroblasts and an increase in the level of MLC20 phosphorylation [17] These results in smooth muscle and in non-muscle cells are attributed to an increase in MLC20 phosphorylation and this is thought to reflect the inhibition of MP. It is was suggested that the phosphorylation site(s) for Rho-kinase was in the sequence 753 to 1004 of gizzard MYPT1 [18]
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