Expression and characterization of mutant forms of the type I regulatory subunit of cAMP-dependent protein kinase. The effect of defective cAMP binding on holoenzyme activation.
The mouse wild type and four mutant regulatory type I (RI) subunits were expressed in Escherichia coli and subjected to kinetic analyses. The defective RI subunits had point mutations in either cAMP-binding site A (G200/E), site B (G324/D, R332/H), or in both binding sites. In addition, a truncated form of RI which lacked the entire cAMP-binding site B was generated. All of the mutant RI subunits which bound [3H]cAMP demonstrated more rapid rates of cAMP dissociation compared to the wild type RI subunit. Dissociation profiles showed only a single dissociation component, suggesting that a single nonmutated binding site was functional. The mutant RI subunits associated with purified native catalytic subunit to form chromatographically separable holoenzyme complexes in which catalytic activity was suppressed. Each of these holoenzymes could be activated but showed varying degrees of cAMP responsiveness with apparent Ka values ranging from 40 nM to greater than 5 microM. The extent to which the mutated cAMP-binding sites were defective was also shown by the resistance of the respective holoenzymes to activation by cAMP analogs selective for the mutated binding sites. Kinetic results support the conclusions that 1) Gly-200 of cAMP-binding site A and Gly-324 or Arg-332 of site B are essential to normal conformation and function, 2) activation of type I cAMP-dependent protein kinase requires that only one of the cAMP-binding sites be functional, 3) mutational inactivation of site B (slow exchange) has a much more drastic effect than that of site A on increasing the Ka of the holoenzyme for cAMP, as well as in altering the rate of cAMP dissociation from the remaining site of the free RI subunit. The strong dependence of one cAMP-binding site on the integrity of the other site suggests a tight association between the two sites.
Woodford
T A
TA
Howard Hughes Medical Institute, Vanderbilt University, Nashville, Tennessee 37232.
Correll
L A
LA
McKnight
G S
GS
Corbin
J D
JD
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D000242:Cyclic AMP / Q000502:physiology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; C015482:protein kinase modulator; D000242:Cyclic AMP; D011494:Protein Kinases