In situ reassociation of the regulatory and catalytic subunits of 3',5'-cyclic adenosine monophosphate-dependent protein kinase isoenzymes in AtT20 cells.
Dissociation and reassociation of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinases I and II were studied in intact AtT20 cells. Cells were stimulated with 50 microM forskolin to raise intracellular cAMP levels and induce complete dissociation of R and C subunits. After the removal of forskolin from the incubation medium cAMP levels rapidly declined to basal levels. Reassociation of R and C subunits was monitored by immunoprecipitation of cAMP-dependent protein kinase activity using anti-R immunoglobulins. The time course for reassociation of R and C subunits paralleled the loss of cellular cAMP. Total cAMP-dependent protein kinase activity and the ratio of protein kinase I to protein kinase II seen 30 min after the removal of forskolin was the same as in control cells. Similar results were seen using crude AtT20 cell extracts treated with exogenous cAMP and Mg2+. Our data showed that after removal of a stimulus from AtT20 cells inactivation of both cAMP-dependent protein kinase isoenzymes occurred by the rapid reassociation of R and C subunits to form holoenzyme. Our studies also showed that half of the type I regulatory subunit (RI) present in control cells contained bound cAMP. This represented approximately 30% of the cellular cAMP in nonstimulated cells. The cAMP bound to RI was resistant to hydrolysis by cyclic nucleotide phosphodiesterase but was dissociated from RI in the presence of excess purified bovine heart C. The RI subunits devoid of C may function to sequester cAMP and, thereby, prevent the activation of cAMP-dependent protein kinase activity in nonstimulated AtT20 cells.
Weiss
A
A
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Erlichman
J
J
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D011494:Protein Kinases / Q000276:immunology / Q000378:metabolism*
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C015482:protein kinase modulator; D005576:Colforsin; D003346:Corticotropin-Releasing Hormone; D000242:Cyclic AMP; D011494:Protein Kinases