BIOCHEMICAL-EVIDENCE FOR THE INTERACTION OF REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE WITH IDA (INTER-DFG-APE) REGION OF CATALYTIC SUBUNIT

To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised again st a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG -APE) region, which lies between the kinase subdomains VII and VIII. A lthough the C-subunit immunoprecipitated with anti-IDA antibodies coul d not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhib itor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- a nd C-subunits, which is distinct from that mediated through the substr ate site and substrate binding site, In accordance with this idea, ass ociation of synthetic IDA peptides with the R-subunit was directly dem onstrated by resonance mirror analysis, The calculated association con stants of IDA peptides were high enough to suggest a possible involvem ent of the IDA region in the initial step of holoenzyme formation.