Identification of electrostatic interaction sites between the regulatory and catalytic subunits of cyclic AMP-dependent protein kinase.
Two classes of molecules inhibit the catalytic subunit (C) of the cyclic AMP-dependent protein kinase (cAPK), the heat-stable protein kinase inhibitors (PKIs) and the regulatory (R) subunits. Basic sites on C, previously identified as important for R/C interaction in yeast TPK1 and corresponding to Lys213, Lys217, and Lys189 in murine C alpha, were replaced with either Ala or Thr and characterized for their kinetic properties and ability to interact with RI and PKI. rC(K213A) and rC(K217A) were both defective in forming holoenzyme with RI but were inhibited readily with PKI. This contrasts with rC(R133A), which is defective in binding PKI but not RI (Wen & Taylor, 1994). Thus, the C-subunit employs two distinct electrostatic surfaces to achieve high-affinity binding with these two types of inhibitory molecules even though all inhibitors share a common consensus site that occupies the active site cleft. Unlike TPK1, mutation of Lys189 had no effect. The mutant C subunits that were defective in binding RI, rC(K213A) and rC(K217A), were then paired with three RI mutants, rRI(D140A), rRI(E143A), and rRI(D258A), shown previously to be defective in recognition of C. Although the mutations at Asp140 and Asp258 in RI were additive with respect to the C mutations. rC(K213A) and rRI(E143A) were compensatory, thus identifying a specific electrostatic interaction site between RI and C. The results are discussed in terms of the RI and C crystal structures and the sequence homology between the yeast and mammalian enzymes.
Gibson
R M
RM
Department of Chemistry and Biochemistry. University of California, San Diego, La Jolla 92093-0654, USA.
Ji-Buechler
Y
Y
Taylor
S S
SS
D000409:Alanine; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004563:Electrochemistry; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry; D012045:Regulatory Sequences, Nucleic Acid; D013329:Structure-Activity Relationship
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine; D000409:Alanine