Modulation of protein phosphorylation by a factor purified from adipocytes.
1. A factor which modulates the activity of cyclic AMP-dependent protein kinase copurifies from rat adipocytes with an inhibitor of adenylate cyclase. Purification and stability studies suggest that both effects reside in a single factor previously referred to as a feedback regulator. 2. The magnitude and direction of the feedback regulator effect on cyclic AMP-dependent protein kinase activity was dependent on the concentration of feedback regulator and the concentration and type of protein substrate. Using histone type IIA as substrate, feedback regulator was inhibitory at low histone concentrations and stimulatory at high concentrations. Preincubation of protein kinase with feedback regulator resulted in inhibition at all histone concentrations. With some protein substrates, e.g. histone f2b and casein, inhibition was observed at all histone concentrations. 3. The stimulation of histone type IIA phosphorylation resulted from an increased V with no effect on either the apparent Ka for cyclic AMP or the Km for ATP. Time course studies suggest that feedback regulator increased the rate of phosphorylation without increasing the total number of phosphorylation sites. Increased histone phosphorylation was observed regardless of whether the cyclic AMP-dependent protein kinase was peak I or peak II (off Deae-cellulose), isolated from bovine or rabbit skeletal muscle or rat heart. A small stimulation was observed using cyclic GMP-dependent protein kinase. 4. These results indicate that feedback regulator can inhibit or stimulate protein kinase, an effect which is probably substrate directed, and depends on the reaction conditions. Whether feedback regulator modulated protein phosphorylation in vivo in addition to its inhibition of adenylate cyclase is unknown. However, stimulation of protein kinase activity in the presence of cyclic AMP is a valuable and rapid assay for monitoring feedback regulator fractions during purification procedures.
D000273:Adipose Tissue / Q000201:enzymology*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D004837:Epinephrine / Q000494:pharmacology; D005246:Feedback; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D006656:Protamine Kinase / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000302:isolation & purification / Q000502:physiology*; D051381:Rats; D013696:Temperature
D011506:Proteins; D002110:Caffeine; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase; D004837:Epinephrine