Phosphorylation of RII subunit and attenuation of cAMP-dependent protein kinase activity by proline-directed protein kinase.
Previous studies identified proline-directed protein kinase (PDPK) as a growth factor-sensitive serine/threonine protein kinase that is active in the cytosol of proliferative cells and tissues during interphase. In this communication, we report that the regulatory subunit (RII) of bovine cardiac muscle cAMP-dependent protein kinase (PKA) is a putative substrate for the multifunctional PDPK. Purified RII is readily phosphorylated by PDPK in vitro in a time-dependent, enzyme-dependent manner to a stoichiometry approaching 0.7 mol phosphate/mol RII subunit protein. The major RII phosphorylation site is identified as a threonine residue located within a large hydrophobic tryptic peptide that is predicted to contain the cAMP binding domains. In contrast to the reported effects of RII autophosphorylation, kinetic analysis of RII function following phosphorylation by PDPK indicates that the inhibitory potency of RII toward the catalytic subunit of PKA in a reassociation assay is increased in proportion to the degree of phosphorylation. Further studies indicate that the cAMP-dependent activation of the RII2C2 holoenzyme is inhibited by PDPK phosphorylation. Taken together, the results of these studies indicate that phosphorylation of RII by PDPK attenuates the activity of PKA. This antagonistic interaction suggests a biochemical mechanism by which a growth factor-activated signaling system may function to modulate cAMP-dependent cellular responses.
Braun
R K
RK
Department of Veterinary Pharmacology and Toxicology, School of Veterinary Medicine, University of California, Davis 95616.
Vulliet
P R
PR
Carbonaro-Hall
D A
DA
Hall
F L
FL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D038461:Proline-Directed Protein Kinases; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin; D014407:Tumor Cells, Cultured
D010446:Peptide Fragments; D010769:Phosphothreonine; D000242:Cyclic AMP; D011494:Protein Kinases; D038461:Proline-Directed Protein Kinases; D014357:Trypsin