Correlation of photolabeling with occupancy of cAMP binding sites in the regulatory subunit of cAMP-dependent protein kinase I

Abstract

Each regulatory subunit of the cAMP-dependent protein kinase contains two in-tandem cAMP binding sites. Photolabelling of holoenzyme I with 8-azidoadenosine 3'',5''-monophosphate (8-N3-cAMP) leads to the covalent modification of two residues, Trp-260 and Tyr-371. In order to correlate photolabeling of these two residues with occupancy of each specific cAMP binding site, photolabeling was carried out in the presence of various analogues of cAMP that bind preferentially to one site. Photolabeling of holoenzyme I after dissociation of 60% of 8-N3-[3H]cAMP with an excess of N6-monobutyryl-cAMP nearly abolished the incorporation of 8-N3-cAMP into Trp-260, whereas the modification of Tyr-371 was reduced by 49%. When 8-N3-[32P]cAMP was bound under equilibrium conditions in the presence of various cAMP analogues, N 6-monbutyryl-cAMP also selectively abolished incorporation of radioactivity into Trp-260, whereas 8-(methylamino)-cAMP preferentially reduced the covalent modification of Tyr-371. Photolabeling with trace amounts of 8-N3-[32P]cAMP in the presence of saturating amounts of N6-monobutyryl-cAMP led to the covalent modification of only Tyr-371. In addition, photolabeling of Tyr-37 was enhanced synergistically in the presence of N6-monobutyryl-cAMP. MgATP reduced the covalent modification of both Trp-260 and Tyr-371 but showed no selectivity for either site. These studies support a model that correlates photolabeling of Trp-260 with occupancy of cAMP binding site A and photolabeling of Tyr-371 with occupancy of cAMP binding site B. Thus, Trp-260, although it lies at the boundary between domain A and domain B, must be in close contact with the cyclic nucleotide that is bound to domain A. The results also establish unambiguously that N6-substituted analogues of cAMP which are selective for the fast dissociation site preferentially bind to the first cAMP binding site in the linear sequence (site A), whereas C-8-substituted analogues which are selective for the slow dissociation site preferentially bind to the second site (site B). These two sites are correlated with other features that are known to distinguish the two cAMP binding sites.