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 RI␣ 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 RI␣. An additional conserved carboxylate, Glu 101 in RI␣ and the equivalent, Glu 99 in RII␣ were mutated to Ala. Replacement of Glu 101 had no effect while rRII(E99A) was very defective. RI␣ and RII␣ 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. Asp 170 , which contributes directly to the hydrogen bonding network in cAMPbinding site A, thus contributes also to holoenzyme stability.Cyclic AMP-dependent protein kinase (cAPK) 1 exists as an inactive tetramer (R 2 C 2 ) that dissociates in the presence of cAMP to release two active catalytic (C) subunits and a dimeric regulatory (R) subunit saturated with four molecules of cAMP (R 2 -cAMP 4 ). The two classes of cAPK, type I and type II, differ primarily in their regulatory subunits (1). The two known families of physiological inhibitors of cAPK, the regulatory subunits (RI and RII) and the heat-stable protein kinase inhibitor (PKI) employ a common mechanism for the inhibition of C. Each inhibitor has an autoinhibitory domain that resembles the substrate consensus sequence, "RRXS/T⌿," where X is any amino acid, and the Pϩ1 site is a hydrophobic group (⌿). In RII the P-site is a Ser, while in RI and PKI, this site is an Ala (2-4).Although binding of this autoinhibitor region to the active site of C is essential for inhibition, this interaction alone is not sufficient for high affinity binding. The R-subunits, as well as PKI, bind to C with K d values that lie in the subnanomolar range (5-7), while peptides containing only the autoinhibitor site bind with micromolar affinity (8, 9). Furthermore, mutant forms of RI and RII with Ala substitutions at the P-2 and P-3 arginines can still form stable holoenzyme complexes in the absence of cAMP (10, 11), indicating that there are additional regions of interaction between R and C that lie beyond the autoinhibitor site. These sites are referred to here as peripheral sites.As shown in Fig. 1, the R-subunit has a well defined domain structure consisting of an N-terminal dimerization domain, followed by the autoinhibitory region and two tandem cAMPbinding domains, A and B. Proteolytic cleavage of the RIIsubunit at a site just prior to the P-3 Arg yielded a monomeric R-subunit fra...

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 TPKI and corresponding to Lys213, Lys217, and Lys189 in murine Ca, 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 TPKI, 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.

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