Mechanisms of activation and autoinhibition for the type II cGMP -dependent protein kinase: Molecular determinants and inhibitor design.

cAMP and cGMP dependent protein kinases (cAKs and cGKs) are involved in a wide variety of neuronal signal transduction pathways. However, there is controversy over the nature and magnitude of the contribution of the two cGK isoforms to some neurophysiological phenomena. In part, this ambiguity is due to the lack of an inhibitor with high specificity for each kinase. This study investigates the mechanisms of autoregulation of cGK isoforms with the goal of exploiting differences to develop an isoform-specific cGK inhibitor. Autoregulation of several cAK and cGK isoforms is mediated by cyclic nucleotide dependent regulation of an autoinhibitory pseudosubstrate sequence within a regulatory portion of the holoenzyme, allowing for low activity of the kinases in the absence of cyclic nucleotide. Site-directed mutagenesis of critical residues in each of the two cyclic nucleotide binding sites demonstrated that the amino-terminal binding site was essential for cyclic nucleotide dependent activation of cGKII. Additionally, mutagenesis was used to determine the inhibitory properties of residues that share homology with the determinants for potent endogenous pseudosubstrate inhibitors of cAK. These mutants demonstrated that cGKII autoinhibition is uniquely dependent upon residues peripheral to a pseudosubstrate sequence. Further, a cGKII truncation mutant that contained the region that possessed these potentially inhibitory residues inhibited a constitutively active cGKII in the nanomolar range. This inhibitory potency was dramatically decreased (>100-fold) when these residues were mutated or when the truncation mutant was bound to cGMP. These studies demonstrate that the interactions between the regulatory and catalytic domains in cGKII have a high affinity that is dependent upon residues both within and outside the pseudosubstrate sequence, and that cGMP binding abrogates this affinity. Preliminary studies indicate that the inhibitory properties of this truncation mutant are cGKII-specific. The effects of cyclic nucleotide blockade by mutagenesis and determination of residues essential for autoinhibition can be used for the design of cyclic nucleotide independent; high-affinity, isoform-specific cGK inhibitors.Ph.D.BiochemistryBiological SciencesNeurosciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/126623/2/3016968.pd