Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKCalpha.
Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 +/- 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 +/-.9% decreases in cell length (n = 4)]. Furthermore, in 3D cells, translocation of both PKCalpha and of RhoA was greatly enhanced and resulted in a greater association of PKCalpha-RhoA in the membrane fraction. In 3G transfected cells, PKCalpha and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKCalpha-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKCalpha and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKCalpha-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.
Patil
Suresh B
SB
Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA.
Pawar
Mercy D
MD
Bitar
Khalil N
KN
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000502:physiology*; D015151:Immunoblotting; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D020742:rhoA GTP-Binding Protein; D000109:Acetylcholine