Subunit interaction sites between the regulatory and catalytic subunits of cAMP-dependent protein kinase. Heterobifunctional cross-linking reagents lead to photodependent and photoindependent cross-linking.
Heterobifunctional cross-linking reagents have been introduced into the catalytic subunit of cAMP-dependent protein kinase as potential probes for identifying specific points of contact between the catalytic (C)-subunit and the type II regulatory (RII) subunit in the holoenzyme complex. Since at least one of the 2 cysteine residues in the C-subunit is known to be in close proximity to the interaction site between the C-subunit and the RII-subunit, these cysteines were chosen initially as targets for covalent modification by two heterobifunctional cross-linking reagents, p-azidophenacyl bromide and N-4-(azidophenylthio)phthalimide. Treatment of the C-subunit with each reagent led to the stoichiometric modification of Cys-199 and Cys-343. In each case, the modified C-subunit was still capable of forming a stable complex with the RII-subunit. Both modified C-subunits also could be covalently cross-linked to the RII-subunit; however, the mechanisms for cross-linking differed. Catalytic subunit modified by p-azidophenacyl bromide was cross-linked to the RII-subunit in a photodependent manner by a mechanism that was maximal when holoenzyme was formed and cAMP was absent. In contrast, the C-subunit modified by N-4-(azidophenylthio)phthalimide was cross-linked to the RII-subunit by a mechanism that was independent of photolysis. In this case, cross-linking was enhanced by the presence of cAMP. This cross-linking was the result of a disulfide interchange between a modified cysteine in the C-subunit and an unmodified cysteine in the RII-subunit.
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
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D001386:Azides / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D046911:Macromolecular Substances; D008956:Models, Chemical; D010446:Peptide Fragments / Q000032:analysis; D010777:Photochemistry; D010782:Photolysis; D010797:Phthalimides / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*
D001386:Azides; D003432:Cross-Linking Reagents; D007461:Iodoacetates; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010797:Phthalimides; C033168:N-((4-azidophenyl)thio)phthalimide; D000242:Cyclic AMP; D011494:Protein Kinases; C011171:4-azidophenacyl bromide; D019807:Iodoacetic Acid