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 (MLC20) 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+/-3.52 and 42.19+/-3.85% nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12+/-46.60% and 290.59+/-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 MLC20.
Patil
Suresh B
SB
Division of Pediatric Gastroenterology, University of Michigan Medical School, 1150 W. Medical Center Dr., MSRB 1, Rm. A520, Ann Arbor, MI 48109-0656, USA.
Bitar
Khalil N
KN
D000109:Acetylcholine / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D006360:Heat-Shock Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D009130:Muscle, Smooth / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D015398:Signal Transduction; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D000577:Amides; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D009281:Naphthalenes; D011725:Pyridines; C108830:Y 27632; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D051571:Protein Kinase C-alpha; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; C058819:calphostin C; D000109:Acetylcholine