Phosphorylation of three proteins in the plasma membrane of Y-1 adrenal cells by a membrane-bound adenosine 3',5'-monophosphate-dependent protein kinase.
Highly purified plasma membranes from Y-1 adrenal tumor cells were incubated with [gamma-32P]ATP with and without cAMP to determine whether endogenous protein substrates are phosphorylated by a cAMP-dependent protein kinase. Three membrane proteins (mol wt 270,000, 35,000, and 17,000) were phosphorylated without cAMP and, to a greater extent, with the nucleotide (0.05-20 microM). Phosphorylation was rapid (less than 60 sec), specific for cAMP, and occurred exclusively at serine residues. Two of the three proteins (35,000 and 17,000) were phosphorylated in whole cells under the influence of cAMP when the cells were incubated with [32P]orthophosphate. The cAMP-dependent protein kinase of these plasma membranes was not extracted by Triton X-100 (0.5% wt/vol) nor by KCl (0.4 M) but was almost completely extracted by the two agents together. By means of photoactivation of 8-azido-[32P]cAMP, it was found that both regulatory subunits RI and RII are present in the membranes. To the extent that the second messenger acts only by way of protein kinase enzymes, these changes in the three proteins are likely to be important in the responses of the plasma membranes of adrenal cells to ACTH.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000311:Adrenal Glands / Q000187:drug effects / Q000201:enzymology*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010777:Photochemistry; D011494:Protein Kinases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D008565:Membrane Proteins; D010710:Phosphates; D010750:Phosphoproteins; C017042:8-azidoadenosine-3',5'-monophosphate; D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; D000242:Cyclic AMP; D011494:Protein Kinases