Correlation of the cAMP binding domain with a site of autophosphorylation on the regulatory subunit of cAMP-dependent protein kinase II from porcine skeletal muscle.

Abstract

The regulatory subunit of CAMP-dependent protein kinase II from porcine skeletal muscle (subunit M, = 55,000) is labile to specific and limited proteolysis. Both the phosphorylated and dephosphorylated form of the subunit are cleaved by trypsin and chymotrypsin to yield a 37,000 molecular weight CAMP-binding frag- ment and a 16,000 to 17,000 molecular weight fragment. Thermolysin yielded the same proteolytic fragments from the dephosphorylated regulatory subunit; how- ever, the phosphorylated regulatory subunit was rela- tively resistant to proteolysis with thermolysin. Ex- tended proteolysis with trypsin indicated that even smaller CAMP-binding fragments could be generated. If the regulatory subunit was autophosphorylated with [Y-~‘P]ATP or covalently labeled with the photoaf- finity reagent 8-azido-[32P]cAMP, the radioactivity in both cases was retained exclusively with the 37,000 molecular weight fragment following proteolysis with chymotrypsin. This fragment, although retaining the CAMP binding site, was monomeric in comparison to the highly asymmetric dimeric structure of the native regulatory subunit. In contrast to the native regulatory subunit which has a blocked NH2-terminal residue, dansylation of the 37,000 molecular weight fragment gave an NH2-termi- nal aspartic acid, indicating that this fragment corre- sponds to the COOH-terminal end of the native poly- peptide chain. Further sequencing of this fragment yielded the following sequence: Asx-Arg-Arg-Val- P I Ser-Val with 3zP from the autophosphorylated subunit released at Step 5. This sequence indicated that at least one site of autophosphorylation is located near the proteolytically labile site and may explain why the phosphorylated regulatory subunit was resistant to thermolytic cleavage. A model for defining the func- tional domains of the regulatory subunit is presented. Two major forms of CAMP-dependent protein kinases have been identified and are distinguished most frequently by their elution from DEAE-cellulose (l-4). In addition to electrostatic differences, the two types of CAMP-dependent protein kinase, by convention referred to as protein kinase I and II, differ immunologically (5) and genetically (6). Differences are also apparent in the dissociation and reassociation of catalytic and regulatory subunits for the two forms of protein kinase (7-9).