Single amino acids determine specificity of binding of protein kinase A regulatory subunits by protein kinase A anchoring proteins

Cyclic AMP-dependent protein kinase is tethered to protein kinase A anchori ng proteins (AKAPs) through regulatory subunits (R) by RI alpha-specific, R II alpha-specific, or RI alpha/RII alpha dual-specific binding. Ala- and Va t-scanning mutagenesis determined that hydrophobic amino acids at three hom ologous positions are required for binding of RI alpha to FSC1/AKAP82 domai n B and RII alpha to AKAP Ht31. A mutation at the middle position reversed the binding specificity of both AKAPs, and mutations at this same position of the dual-specific domain A of FSC1/AKAP82 converted it into either an RI alpha or RII alpha binding domain. This suggests that hydrophobic amino ac ids at three conserved positions within the primary sequence and an amphipa thic helix of AKAPs are required for cyclic AMP-dependent protein kinase bi nding, with the size of the aliphatic side chain at the middle position det ermining RI alpha or RII alpha binding specificity.