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 against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.
Sahara
S
S
Graduate School of Science and Technology, Biosignal Research Center, Kobe University, Japan.
Sato
K
K
Kaise
H
H
Mori
K
K
Sato
A
A
Aoto
M
M
Tokmakov
A A
AA
Fukami
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002384:Catalysis; D002417:Cattle; D003067:Coenzymes / Q000276:immunology / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism*; D011487:Protein Conformation; D017510:Protein Folding; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D003067:Coenzymes; D010446:Peptide Fragments; D017868:Cyclic AMP-Dependent Protein Kinases