Rho-associated kinase of chicken gizzard smooth muscle

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROK alpha isoform, Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates, Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e, similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin, Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rhokinase was stimulated 1.5-2-fold by guanosine 5'-O-3-(thio)triphosphate.RhoA whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89, Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 mu M) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin, Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.