Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms I alpha and II beta to the catalytic subunit

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzym e results in a proteolytic-resistant Delta (1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits e xtends beyond the autoinhibitory site in the R subunit at the NR, terminus. Sequence alignment of the two R subunit isoforms, Rf and RII, reveals a si gnificantly sequence diversity at this specific region. To determine whethe r this sequence diversity is functionally important for interaction with th e catalytic subunit, specific mutations, R133A and D328A, are introduced in to sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg(133) with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining sim ilar affinities for Rn as compared with the wild-type catalytic subunit. Th ese results suggest that sequence immediately NH2-terminal to the consensus inhibition site in RI and RII interacts with different sites at the proxim al region of the active site cleft in the catalytic subunit. These isoform- specific differences would dictate a significantly different domain organiz ation in the type I and type II holoenzymes.