The consequences of introducing an autophosphorylation site into the type I regulatory subunit of cAMP-dependent protein kinase.
The type I and type II regulatory subunits of cAMP-dependent protein kinase can be distinguished by autophosphorylation. The type II regulatory subunits have an autophosphorylation site at a proteolytically sensitive hinge region, while the type I regulatory subunits have a pseudophosphorylation site. Only holoenzyme formed with type I regulatory subunits has a high affinity binding site for MgATP. In order to determine the functional consequences of regulatory subunit phosphorylation on interaction with the catalytic subunit, an autophosphorylation site was introduced into the type I regulatory subunit using recombinant DNA techniques. When Ala97 at the hinge region of the type I regulatory subunit was replaced with Ser, the regulatory subunit became a good substrate for the catalytic subunit. Stoichiometric phosphorylation occurred exclusively at Ser97. Radioactivity was incorporated primarily into the recombinant regulatory subunit when catalytic subunit and [gamma-32P]ATP were added to the total bacterial extract. Phosphorylation of the mutant regulatory subunit also occurred readily following polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose. Phosphorylation occurred as an intramolecular event in the absence of cAMP indicating that the hinge region of the regulatory subunit occupies the substrate recognition site of the catalytic subunit in the holoenzyme complex. Holoenzyme formed with both the wild type and mutant regulatory subunits was susceptible to dissociation in the presence of high salt; however, only the native holoenzyme was stabilized by MgATP. In contrast to the wild type holoenzyme, the affinity of the mutant holoenzyme for cAMP was not reduced in the presence of MgATP. Holoenzyme formation also was not facilitated by MgATP.
Durgerian
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Department of Chemistry, University of California, San Diego, La Jolla 92093.
Taylor
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013698:Templates, Genetic
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; C015482:protein kinase modulator; D011494:Protein Kinases