Characterization of the isolated cAMP-binding B domain of cAMP-dependent protein kinase.
A 14.4-kDa cAMP-binding fragment was generated during bacterial expression and purification of recombinant bovine cAMP-dependent protein kinase type I alpha regulatory subunit (RI alpha). The full-length RI alpha from which the fragment was derived contained a point mutation allowing its B domain to bind both cAMP and cGMP with high affinity while leaving its A domain highly cAMP selective. The NH2 terminus of the fragment was Ser-252, indicating that it encompassed the entire predicted B domain. Although the [3H]cAMP and [3H]cGMP exchange rates of the isolated B domain were increased relative to the B domain in intact RI alpha, the [3H]cAMP exchange rate was comparable to that of the B domain of full-length RI alpha containing an unoccupied A domain. A plasmid encoding only the isolated B domain was overexpressed in Escherichia coli, and a monomeric form of the B domain was purified that had identical properties to the proteolytically generated fragment, indicating that all of the elements for the high-affinity cAMP-binding B domain are contained within the 128 amino acid carboxyl terminus of the R subunit. Prolonged induction of the B domain in E. coli or storage of the purified protein resulted in the formation of a dimer that could be reverted to the monomer by incubation in 2-mercaptoethanol. Dimerization caused an approximate fivefold increase in the rate of cyclic nucleotide exchange relative to the monomer. The results show that an isolated cAMP-binding domain can function independently of any other domain structures of the R subunit.
Shabb
J B
JB
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks 58202-9037, USA.
Poteet
C E
CE
Kapphahn
M A
MA
Muhonen
W M
WM
Baker
N E
NE
Corbin
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP