Interaction of the regulatory and catalytic subunits of cAMP-dependent protein kinase. Electrostatic sites on the type Ialpha regulatory subunit.
Since a basic surface on the catalytic (C) subunit of cAMP-dependent protein kinase is important for binding to the regulatory (R) subunit, acidic residues in R were sought that might contribute to R-C interaction. Using differential labeling by a water-soluble carbodiimide (Buechler, T. A., and Taylor, S. S. (1990) Biochemistry 29, 1937-1943), seven specific carboxylates in RIalpha were identified that were protected from chemical modification in the holoenzyme; each was then replaced with Ala. Of these, rRI(E15A/E106A/D107A)), rRI(E105A), rRI(D140A), rRI(E143A), and rRI(D258A) all were defective in holoenzyme formation and define negative electrostatic surfaces on RIalpha. An additional conserved carboxylate, Glu101 in RIalpha and the equivalent, Glu99 in RIIalpha were mutated to Ala. Replacement of Glu101 had no effect while rRII(E99A) was very defective. RIalpha and RIIalpha thus differ in the molecular details of how they recognize C. Unlike wild-type RI, two additional mutants, rRI(D170A) and rRI(K242A), inhibited C-subunit stoichiometrically in the presence of cAMP and show increases in both on- and off-rates. Asp170, which contributes directly to the hydrogen bonding network in cAMP-binding site A, thus contributes also to holoenzyme stability.
Gibson
R M
RM
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654, USA.
Ji-Buechler
Y
Y
Taylor
S S
SS
D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000378:metabolism; D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009154:Mutation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D055672:Static Electricity; D013329:Structure-Activity Relationship
D054754:Cyclic AMP-Dependent Protein Kinase RIIalpha Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases