Site-directed mutagenesis of the cAMP-binding sites of the recombinant type I regulatory subunit of cAMP-dependent protein kinase.
The type I regulatory subunit (R-I) of rat brain cAMP-dependent protein kinase was expressed in E. coli and site-directed mutagenesis was used to substitute amino acids in the putative cAMP-binding sites. The wild-type recombinant R-I bound 2 mol of cAMP/mol subunit, while two mutant R-Is with a single amino acid substitution in one of the two intrachain cAMP-binding sites (clone N153:a glutamate for Gly-200, and clone C254:an aspartate for Gly-324) bound 1 mol of cAMP/mol subunit. When these two substitutions were made in one mutant, cAMP did not bind to this mutant, indicating that binding of cAMP to N153 or C254 was to their nonmutated sites. Competition experiments with site-selective analogs and dissociation of bound cAMP from mutant R-Is provided evidence for strong intrachain interactions between the two classes of cAMP-binding sites in R-I.
Kuno
T
T
Department of Pharmacology, Kobe University School of Medicine, Japan.
Shuntoh
H
H
Sakaue
M
M
Saijoh
K
K
Takeda
T
T
Fukuda
K
K
Tanaka
C
C
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D000242:Cyclic AMP / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases