Activation of type I cyclic AMP-dependent protein kinases is impaired by a point mutation in cyclic AMP binding sites.
The type I regulatory (R-I) subunit of cyclic AMP-dependent protein kinase (A-kinase) was expressed in E. coli, and a single amino acid substitution in cyclic AMP binding sites A or B was introduced by site-directed mutagenesis. The cyclic AMP binding activity and cyclic AMP-stimulated phosphotransferase activity of the holoenzymes formed by wild-type or mutant R-Is and the purified bovine catalytic subunit of A-kinase were then examined. The wild-type holoenzyme was activated by low concentrations of cyclic AMP, a finding in accord with its high-affinity binding to cyclic AMP. In contrast, although the two mutant holoenzymes showed high-affinity cyclic AMP binding at their non-mutated sites, both holoenzymes were resistant to activation by cyclic AMP. Thus, binding of cyclic AMP to the non-mutated cyclic AMP binding site is not sufficient to dissociate the catalytic subunit from the mutant R-Is upon cyclic AMP binding. These results suggest that both A and B cyclic AMP binding sites are required for efficient coupling between cyclic AMP binding and activation of the enzyme.
Kuno
T
T
Department of Pharmacology, Kobe University School of Medicine, Japan.
Shuntoh
H
H
Takeda
T
T
Ito
A
A
Sakaue
M
M
Hirai
M
M
Ando
H
H
Tanaka
C
C
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D007289:Cyclic IMP / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005786:Gene Expression Regulation; D066298:In Vitro Techniques; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis; D014982:Xenopus laevis
D011953:Receptors, Cyclic AMP; D011994:Recombinant Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D007289:Cyclic IMP; D010770:Phosphotransferases; D011494:Protein Kinases