Mutational analysis of the A-kinase anchoring protein (AKAP)-binding site on RII - Classification of side chain determinants for anchoring and isoform selective association with AKAPs

Compartmentalization of the type II cAMP-dependent protein kinase is conferred by interaction of the regulatory subunit (RII) with (A) under bar-(K) under bar inase (A) under bar nchoring (P) under bar roteins (AKAPs). The AKAP-binding site involves amino-terminal residues on each RII protomer and is formed through dimerization. A site-directed mutagenesis strategy was utilized to assess the contribution of individual residues in either RII isoform, RII alpha or RII beta, for interaction with various anchoring proteins. Substitution of long-chain or bulky hydrophobic groups (leucines or phenylalanines) for isoleucines at positions 3 and 5 in RII alpha decreased AKAP-binding up to 24 +/- 3 (n = 8)-fold, whereas introduction of valines had minimal effects. Replacement with hydrophilic residues (serine or asparigine) at both positions abolished AKAP binding. Mutation of proline 6 in RII alpha reduced binding for four AKAPs (Ht31, MAP2, AKAP79, and AKAP95) from 2.3 to 20-fold (n = 4) whereas introduction of an additional proline at position 6 in RII beta increased or conferred binding toward these anchoring proteins. Therefore, we conclude thats-branched side chains at positions 3 and 5 are favored determinants for AKAP-binding and pro lines at positions 6 and 7 increase or stabilize RII alpha interaction with selected anchoring proteins.