Evidence of cyclic GMP may regulate the association of myosin II heavy chain with the cytoskeleton by inhibiting its phosphorylation.
Previous studies have implicated cyclic GMP in the regulation of myosin II heavy chain (MHC) association with the cytoskeleton in Dictyostelium discoideum. Here we provide evidence that cyclic GMP may regulate MHC association with the cytoskeleton through MHC phosphorylation. Comparative data are presented of MHC phosphorylation in the wild-type strain NC4, the parental strain XP55 and streamer mutants NP368 and NP377. Using an anti-MHC monoclonal antibody to immunoprecipitate MHC from [32P]phosphate-labelled developing cells, we found that cyclic AMP stimulation of the wild-type strain NC4 and parental strain XP55 induced MHC phosphorylation in vivo. A peak of phosphorylation was observed at 30-40 s, followed by a gradual decrease to basal level at 160 s. In contrast, in both of the streamer mutants NP368 and NP377 (which have prolonged cyclic GMP accumulation and prolonged MHC association with the cytoskeleton), the phosphorylation of MHC was delayed and did not form a peak until 60-80 s after cyclic AMP stimulation. We also found that cytoskeletal MHC showed only minor phosphorylation, the majority of the phosphorylated MHC being found in the cytosol. We present a model to account for these results in which cyclic GMP regulates MHC association with the cytoskeleton by regulating the phosphorylation/dephosphorylation cycle of MHC in these cells.
Liu
G
G
Department of Biochemistry, University of Oxford, UK.
Newell
P C
PC
D000818:Animals; D000911:Antibodies, Monoclonal; D000242:Cyclic AMP; D006152:Cyclic GMP / Q000378:metabolism*; D003599:Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000737:chemistry; D004023:Dictyostelium / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D006825:Hybridomas; D015151:Immunoblotting; D008954:Models, Biological; D009218:Myosins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation
D000911:Antibodies, Monoclonal; D000242:Cyclic AMP; D009218:Myosins; D006152:Cyclic GMP