An Inhibitory Segment of the Catalytic Subunit of Phosphorylase Kinase Does Not Act as a Pseudosubstrate

Cheryl Bartleson,Donald J Graves

doi:10.1074/jbc.m102308200

Abstract

The C terminus of the catalytic gamma subunit of phosphorylase kinase contains two autoinhibitory calmodulin binding domains designated PhK13 and PhK5. These peptides inhibit truncated gamma(1-300). Previous data show that PhK13 (residues 302-326) is a competitive inhibitor with respect to phosphorylase b, with a K(i) of 1.8 microm. This result suggests that PhK13 may bind to the active site of truncated gamma(1-300). Variants of PhK13 were prepared to localize the determinants for interaction with the catalytic fragment gamma(1-300). PhK13-1, containing residues 302-312, was found to be a competitive inhibitor with respect to phosphorylase b with a K(i) of 6.0 microm. PhK13 has been proposed to function as a pseudosubstrate inhibitor with Cys-308 occupying the site that normally accommodates the phosphorylatable serine in phosphorylase b. A PhK13-1 variant, C308S, was synthesized. Kinetic characterization of this peptide reveals that it does not serve as a substrate but is a competitive inhibitor. Additional variants were designed based on previous knowledge of phosphorylase kinase substrate determinants. Variants were analyzed as substrates and as inhibitors for truncated gamma(1-300). Although PhK13-1 does not appear to function as a pseudosubstrate, several specificity determinants employed in the recognition of phosphorylase b as substrate are utilized in the recognition of PhK13-1 as an inhibitor.

Highlights

The catalytic activity of many protein kinases is regulated by an autoinhibitory mechanism known as intrasteric regulation (2, 3)
Substantial evidence exists implicating this mechanism in protein kinase G, protein kinase C, myosin light chain kinase, calmodulin-dependent protein kinase I, and phosphorylase kinase (2, 3, 7, 8)
Binding of calmodulin to the autoinhibitory domain in the presence of calcium would result in a conformational change that is transmitted to the active site and results in the activation of the enzyme

Summary

Introduction

The catalytic activity of many protein kinases is regulated by an autoinhibitory mechanism known as intrasteric regulation (2, 3). Binding of calmodulin to the autoinhibitory domain in the presence of calcium would result in a conformational change that is transmitted to the active site and results in the activation of the enzyme. Dasgupta and Blumenthal (13) determined the Ki values for PhK13 and PhK5 (300 nM and 20 ␮M, respectively) with holoenzyme phosphorylase kinase These values fall into the range of Ki values obtained with other protein kinase autoinhibitory domains (2, 3). Previous data show that PhK13 is a competitive inhibitor of ␥(1-300), with respect to phosphorylase b, with a Ki of 1.8 ␮M (1) This suggests that PhK13 may act as a pseudosubstrate with cysteine 308 occupying the site normally occupied by the serine of the phosphorylatable substrate.

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