Rho-associated kinase of chicken gizzard smooth muscle

Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was p urified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrop horesis) and identified as the ROK alpha isoform, Several substrates were p hosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myos in, and the 20-kDa myosin light chain were higher than other substrates, Th iophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphory lation of myosin at serine 19 increased actin-activated Mg+-ATPase activity , i.e, similar to myosin light chain kinase. Myosin phosphorylation was inc reased at higher ionic strengths, possibly by formation of 6 S myosin, Phos phorylation of the isolated light chain and myosin phosphatase was decrease d by increasing ionic strength. Rhokinase was stimulated 1.5-2-fold by guan osine 5'-O-3-(thio)triphosphate.RhoA whereas limited tryptic hydrolysis cau sed a 5-6-fold activation, independent of RhoA. Several kinase inhibitors w ere screened and most effective were Y-27632, staurosporine, and H-89, Seve ral 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 contractil e process via phosphorylation of MYPT1 and myosin, Activation by arachidoni c acid presents a possible regulatory mechanism for Rho-kinase.