Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Identification of a specific interchain disulfide bond.
The catalytic (C) subunit and the type II regulatory (RII) subunit of cAMP-dependent protein kinase can be cross-linked by interchain disulfide bonding. This disulfide bond can be catalyzed by cupric phenanthroline and also can be generated by a disulfide interchange using either RII-subunit or C-subunit that has been modified with either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-4(azidophenylthio)phthalimide (APTP). When the 2 cysteine residues of the C-subunit are reacted with DTNB prior to incubation with the RII-subunit, interchain disulfide bonding occurs. Similar observations are seen with C-subunit that had been modified with APTP. Interchain disulfide bonds also form when the RII-subunit is modified with DTNB prior to incubation with the C-subunit. The presence of cAMP facilitates this cross-linking while autophosphorylation of the RII-subunit retards the rate at which the interchain disulfide bond forms. Interchain disulfide bonds also form spontaneously when the RII-subunit and the C-subunit are dialyzed at pH 8.0 in the absence of reducing agents. The specific amino acid residues that participate in intersubunit disulfide bonding have been identified as Cys-97 in the RII-subunit and Cys-199 in the C-subunit. Based on the sequence homologies of the RII-subunit with other kinase substrates and on the proximity of Cys-97 to the catalytic site, a model is proposed in which the autophosphorylation site of the RII-subunit occupies the substrate-binding site in the holoenzyme. The model also proposes that this same site may be occupied by the region flanking Cys-199 in the C-subunit when the C-subunit is dissociated.
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
Bubis
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Taylor
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D000595:Amino Acid Sequence; D001386:Azides / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D010797:Phthalimides / Q000494:pharmacology; D011494:Protein Kinases / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D001386:Azides; D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010797:Phthalimides; C033168:N-((4-azidophenyl)thio)phthalimide; D004228:Dithionitrobenzoic Acid; D011494:Protein Kinases; D014357:Trypsin; D004229:Dithiothreitol