Characterization of major phosphoproteins in the cGMP-mediated protein phosphorylation system of vascular smooth muscle membranes.
G(0) (215-250 kD) and G(1) (120-140 kD), the unidentified major phosphoproteins in the cGMP-mediated protein phosphorylation system of vascular smooth muscle membranes, were compared for biochemical and immunological properties with the type 1 inositol 1,4, 5-trisphosphate receptor (InsP(3)R, 240 kD) and the myosin-binding subunit (MBS, 138 kD) of myosin phosphatase, both of them substrates for cGMP-dependent protein kinase. Two microsomal proteins that were immunoreactive with antibodies to InsP(3)R and MBS were detected, and comigrated with G(0) and G(1), respectively, on SDS-PAGE. When thiophosphorylated G(0) and G(1) were subjected to immunoprecipitation, MBS antibody induced the precipitation of a 138-kD phosphoprotein, but did not significantly affect the amount of G(1) remaining in the supernatant, while InsP(3)R antibody precipitated G(0) almost completely. Unexpectedly, InsP(3)R antibody coprecipitated a large portion of G(1), which did not cross-react with either antibody to MBS or InsP(3)R. Just like InsP(3)R, G(0) bound to the calmodulin column in a Ca(2+)-dependent manner, and, again, a large portion of G(1) was copurified with G(0). These results suggest that G(0) is identical to InsP(3)R, while G(1) consists of several phosphoproteins, including the 138-kD protein associated with InsP(3)R as a major component. MBS is not G(1) or may represent only a minor component of it.
Fujita
S
S
Department of Pharmacology, Niigata University School of Medicine, Niigata, Japan.
Yoshida
Y
Y
Islam
O
O
Ma
X
X
Fujioka
M
M
Ito
M
M
Nakano
T
T
Aizawa
Y
Y
Imai
S
S
Higuchi
H
H
D000818:Animals; D015220:Calcium Channels / Q000032:analysis*; D006152:Cyclic GMP / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis; D015544:Inositol 1,4,5-Trisphosphate; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism* / Q000648:ultrastructure; D043263:Myosin-Light-Chain Phosphatase; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis*; D013552:Swine; D014661:Vasoconstriction / Q000502:physiology
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008565:Membrane Proteins; D010750:Phosphoproteins; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D019204:GTP-Binding Proteins; D006152:Cyclic GMP