Ala³³⁵ is essential for high‐affinity cAMP‐binding of both sites A and B of cAMP‐dependent protein kinase type I

Abstract

A single amino acid substitution (Ala33SAsp) in cAMP binding site B of the regulatory subunit of cAMP-dependent protein kinase type I was sufficient to abolish high affinity cAMP binding for both cAMP binding sites A and B. Furthermore, the Ala33SAsp mutation increased the activation constant for cAMP of the mutant holoenzyme 30-fold and also enhanced the rate of holoenzyme formation. Thus, the substitution was responsible for the dominant negative phenotype of the enzyme. Activation of mutant holoenzyme with site-selective cAMP analogs indicated that the enzyme dissociated through binding to site A only. Our results provide evidence that Ala 335 is an essential residue for high affinity cAMP binding of both sites as well as for the functional integrity of the enzyme.B has been shown to interact with the adenine moiety since alteration of this residue changed the affinity and specificity for cGMP [13]. Recently, a sub-clone of the rat myelocytic leukemia cell line (IPC-81) has been found to harbor a heterozygously expressed point mutation [14]. This mutated clone was selected by treatment with 8-CPT-cAMP and identified as Alato-Asp at the invariant position 335 [14]. In order to study the effects of this mutation on ligand binding and holoenzyme activation we used recombinant protein expression techniques to obtain large quantities of pure recombinant Ala335Asp mutant protein. Materials and methods

Fast and Slow Cyclic Nucleotide-dissociation Sites in cAMP-dependent Protein Kinase Are Transposed in Type Iβ cGMP-dependent Protein Kinase

“…Biochemical studies of type II cAK first suggested that the more amino-terminal cyclic nucleotide-binding site is the fast site whereas the adjacent site is the slow site (9), and similar approaches were used to demonstrate the same structural order for the binding sites of type I cAK (10). Molecular modeling (11) and results of site-directed mutagenesis of the binding sites (12,13) supported these findings.…”

Dynamic unfolding of a regulatory subunit of cAMP‐dependent protein kinase by capillary electrophoresis: Impact of cAMP dissociation on protein stability

“…1b. This observation is supported by previous studies characterizing the wild-type APK protein with an apparent k off of 60 s 21 for the RIa site [31]. In addition, further experiments (data not shown) were performed using shorter effective capillary lengths (e.g., 1-30 cm) by placing the protein sample plug at closer distances to the detector window using a defined applied hydrodynamic pressure time interval prior to voltage application.…”

“…2b using the relative increase in peak area measured for the protein. The apparent K d determined by CE was 0.6 6 0.2 mM, which is about one order of magnitude weaker than reported for the wild-type APK by competitive ( 3 H)cAMP radioactive exchange with ammonium sulfate precipitation [29,31]. It is not clear whether this discrepancy is due to differences in the recombinant protein construct relative to wild-type receptor or bias in the CE method, since it is assumed that cAMP has been fully stripped from the protein during electromigration.…”

Active Site Mutations Define the Pathway for the Cooperative Activation of cAMP-Dependent Protein Kinase

“…Site B is preferentially accessible in the intact holoenzyme based on kinetic analysis (Øgreid & Døskeland, 1981a,b). Other studies with cAMP-binding site mutants (Zorn et al, 1995), deletion mutants Saraswat et al, 1988), and cAMP analogs , demonstrated that binding of cAMP to Site A is the critical step for dissociating the R-C complex. On the basis of the results described here, a clear model for the stepwise activation of the holoenzyme can be proposed (Figure 8).…”