Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores.
The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo.
D000345:Affinity Labels / Q000494:pharmacology; D001386:Azides; D001754:Blastocladiella / Q000201:enzymology*; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D005658:Fungi / Q000201:enzymology*; D008970:Molecular Weight; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D013172:Spores, Fungal / Q000201:enzymology
D000345:Affinity Labels; D001386:Azides; D010761:Phosphorus Radioisotopes; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases