S. Sahara et al., BIOCHEMICAL-EVIDENCE FOR THE INTERACTION OF REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE WITH IDA (INTER-DFG-APE) REGION OF CATALYTIC SUBUNIT, FEBS letters, 384(2), 1996, pp. 138-142
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.