RhoA- and PKC-[alpha]-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain ([MLC.sub.20]) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-[alpha]. Presently, we examined the cross talk between RhoA and PKC-[alpha] in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-[alpha], CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 [+ or -] 3.52 and 42.19 [+ or -] 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 [+ or -] 46.60% and 290.59 [+ or -] 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-[alpha] by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-[alpha] pathways, suggesting cross talk between RhoA and PKC-[alpha] resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of [MLC.sub.20]. myosin light chain; contraction; CPI-17; acetylcholine; Rho kinase; colon; phosphatase; protein kinase C; heat shock protein