| Popis: |
Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Iβ cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQSAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (∼0.8 phosphate/cGK monomer) ∼3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (∼67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Iβ cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75. |
