Autoinhibition and Isoform-specific Dominant Negative Inhibition of the Type II cGMP-dependent Protein Kinase

Merritt K Taylor,Rehan Ahmed,Michael Begley,Michael D Uhler

doi:10.1074/jbc.m202060200

Merritt K Taylor, Rehan Ahmed + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m202060200

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Abstract

In the absence of cyclic nucleotides, the cAMP-dependent protein kinase and cGMP-dependent protein kinases (cGKs) suppress phosphotransfer activity at the catalytic cleft by competitive inhibition of substrate binding with a pseudosubstrate sequence within the holoenzyme. The magnitude of inhibition can be diminished by autophosphorylation near this pseudosubstrate sequence. Activation of type I cGK (cGKI) and type II cGK (cGKII) are differentially regulated by their cyclic nucleotide-binding sites. To address the possibility that the distinct activation mechanisms of cGKII and cGKI result from differences in the autophosphorylation of the inhibitory domain, we investigated the effects of autophosphorylation on the kinetics of activation. Unlike the type I cGKs (cGKIalpha and Ibeta), cGKII autophosphorylation did not alter the basal activity, nor the sensitivity of the enzyme to cyclic nucleotide activation. To determine residues responsible for autoinhibition of cGKII, Ala was substituted for basic residues (Lys(122), Arg(118), and Arg(119)) or a hydrophobic residue (Val(125)) within the putative pseudosubstrate domain of cGKII. The integrity of these residues was essential for full cGKII autoinhibition. Furthermore, a cGKII truncation mutant containing this autoinhibitory region demonstrated a nanomolar IC(50) toward a constitutively active form of cGKII. Finally, we present evidence that the dominant negative properties of this truncation mutant are specific to cGKII when compared with cAMP-dependent protein kinase Calpha and cGKIbeta. These findings extend the known differences in the activation mechanisms among cGK isoforms and allow the design of an isoform-specific cGKII inhibitor.

Highlights

The cyclic nucleotide-dependent protein kinases possess a regulatory domain that suppresses activity at the catalytic site through competitive inhibition of a pseudosubstrate sequence within the regulatory domain [1]
Our results showed that cGKII did undergo significant autophosphorylation in the presence of cAMP and cGMP
CGKII autoinhibition was distinct from other cyclic nucleotide-dependent protein kinases in that autophosphorylation did not lead to activation of the cGKII kinase

Summary

Introduction

The cyclic nucleotide-dependent protein kinases possess a regulatory domain that suppresses activity at the catalytic site through competitive inhibition of a pseudosubstrate sequence within the regulatory domain [1]. Autophosphorylation of cAK lowers the Ka for cAMP and decreases the rate of association between the type II form of the R and C subunits [6, 7] This is due in part to electrostatic repulsion between the acidic residues within the catalytic cleft and the negatively charged phosphates incorporated into the autophosphorylated regulatory domain. Both splice variants of the type I isoform of cGK (I␣ and I␤) share several autoinhibitory characteristics with cAK. The truncated regulatory domain of cGKII showed dominant negative properties toward wild type cGKII in the presence of lower concentrations of 8-Br-cGMP. Given the lack of specificity of many inhibitors structurally analogous to ATP or cyclic nucleotides [15], these findings offer an isoformspecific, dominant negative inhibitor of cGKII

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