THE STRUCTURAL DOMAINS OF CAMP-DEPENDENT PROTEIN KINASE-I - CHARACTERIZATION OF 2 SITES OF PROTEOLYTIC CLEAVAGE AND HOMOLOGIES TO CAMP-DEPENDENT PROTEIN KINASE-II

  • 1 January 1980
    • journal article
    • research article
    • Vol. 255  (20) , 9706-9712
Abstract
The regulatory subunit of [porcine skeletal muscle] cAMP-dependent protein kinase I was cleaved proteolytically into 2 structurally independent domains. The larger domain (35K with trypsin or thermolysin and 31K with chymotrypsin) corresponded to the COOH-terminal end of the polypeptide chain and retained the cAMP binding site(s). The smaller domain (11 to 12K with trypsin), corresponding to the NH2-terminal region of the regulatory subunit, contained the region of dimer interaction. In the absence of reducing reagent the 2 protomers of the native regulatory subunit and of the smaller domain could be covalently cross-linked by a disulfide bond. In addition to the 2 major domains, a 15-residue peptide that links the 2 domains was isolated and partially characterized. Two major sites on the type I regulatory subunit were susceptible to proteolytic degradation. Site 1 was susceptible to cleavage by both trypsin and thermolysin. Cleavage at this site generated a 35K cAMP-binding fragment. Site 2 contained a chymotryptic cleavage site as well as a secondary tryptic site; cleavage generated a 31K cAMP-binding fragment. Both sites contained 2 consecutive basic amino acid residues similar to the corresponding sequence in the type II regulatory subunit; in the case of the type I regulatory subunit, the serine at Site 1 does not serve as a site of autophosphorylation. The holoenzyme is partially protected from proteolytic degradation.

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