Effect of phosphorylation on the regulatory subunit of the type I cAMP-dependent protein kinase.

Abstract

The regulatory subunit of the type I cAMP-dependent protein kinase (RI), isolated from bovine or rat skeletal muscle, can be phosphorylated both in vitro (Geahlen, R. L., and Krebs, E. G. (1980) J. Biol. Chem. 255, 1164-1169) and in vivo (Geahlen, R. L., and Krebs, E. G. (1980) J. Biol. Chem. 255, 9375-9379). The effects of each modification on the ability of RI to associate with the catalytic subunit (C) and with cAMP are compared. The phosphorylation of bovine RI in vitro by cGMP-dependent protein kinase results in a loss of inhibitory activity toward C and in the loss of one of two cAMP binding sites per RI monomer. Inhibitory activity can be regained upon removal of the phosphate with potato acid phosphatase. Similar effects are not observed for the subunits phosphorylated in vivo. A comparison of unmodified bovine RI with RI modified in vivo reveals no differences in their interactions with either C or cAMP. Dephosphorylation of purified rat RI also does not affect its association with C or subsequent activation by cAMP. Dephosphorylated rat RI is not a substrate for C but can be slowly phosphorylated by cGMP-dependent protein kinase. The phosphorylation of RI in isolated rat soleus muscles is not inhibited by cycloheximide, ruling out the possibility that only nascent polypeptide chains are substrates for phosphorylation in vivo.