Deletion mutants as probes for localizing regions of subunit interaction in cAMP-dependent protein kinase.
The regulatory subunit of cAMP-dependent protein kinase has a well-defined domain structure, and recombinant DNA techniques have been used to define further the functional properties that are associated with each domain. Our initial question was to define the minimal structural unit that is required for forming a stable complex with the catalytic subunit that will still bind and hence be dissociated by cAMP. To answer these questions, the entire second cAMP-binding domain was deleted using oligonucleotide-directed mutagenesis to introduce a premature stop codon at Trp260. This mutation results in the expression of a stable protein with an Mr of 38,000 based on polyacrylamide gel electrophoresis. The resulting mutant protein is a dimer; and like the native R-subunit, the two protomers of the dimer are cross-linked by disulfide bonds at the amino terminus. The mutant R-subunit binds 1 mol of cAMP/monomer based on equilibrium dialysis. The Kd(cAMP) was 25 nM, which is slightly higher than the Kd(cAMP) for the native R-subunit. The removal of the second cAMP domain does not prevent aggregation with the catalytic subunit, and the inactive holoenzyme complex that is formed in the absence of cAMP can still be dissociated and consequently activated by cAMP. In conjunction with previous results based on limited proteolysis, it is concluded that the region extending from Arg94 to Lys259 constitutes a structural unit that will be sufficient to interact with the catalytic subunit in a cAMP-dependent manner.
Saraswat
L D
LD
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Ringheim
G E
GE
Bubis
J
J
Taylor
S S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003062:Codon; D000242:Cyclic AMP / Q000378:metabolism; D005796:Genes; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D013552:Swine
D003062:Codon; D046911:Macromolecular Substances; D000242:Cyclic AMP; D011494:Protein Kinases