InsP3R-associated cGMP Kinase Substrate Determines Inositol 1,4,5-Trisphosphate Receptor Susceptibility to Phosphoregulation by Cyclic Nucleotide-dependent Kinases

Wataru Masuda,Matthew J Betzenhauser,David I Yule

doi:10.1074/jbc.m110.168989

Wataru Masuda, Matthew J Betzenhauser + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m110.168989

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Abstract

Ca(2+) release through inositol 1,4,5-trisphosphate receptors (InsP(3)R) can be modulated by numerous factors, including input from other signal transduction cascades. These events shape the spatio-temporal characteristics of the Ca(2+) signal and provide fidelity essential for the appropriate activation of effectors. In this study, we investigate the regulation of Ca(2+) release via InsP(3)R following activation of cyclic nucleotide-dependent kinases in the presence and absence of expression of a binding partner InsP(3)R-associated cGMP kinase substrate (IRAG). cGMP-dependent kinase (PKG) phosphorylation of only the S2+ InsP(3)R-1 subtype resulted in enhanced Ca(2+) release in the absence of IRAG expression. In contrast, IRAG bound to each InsP(3)R subtype, and phosphorylation of IRAG by PKG attenuated Ca(2+) release through all InsP(3)R subtypes. Surprisingly, simply the expression of IRAG attenuated phosphorylation and inhibited the enhanced Ca(2+) release through InsP(3)R-1 following cAMP-dependent protein kinase (PKA) activation. In contrast, IRAG expression did not influence the PKA-enhanced activity of the InsP(3)R-2. Phosphorylation of IRAG resulted in reduced Ca(2+) release through all InsP(3)R subtypes during concurrent activation of PKA and PKG, indicating that IRAG modulation is dominant under these conditions. These studies yield mechanistic insight into how cells with various complements of proteins integrate and prioritize signals from ubiquitous signaling pathways.

Highlights

A primary locus for modifying the characteristics of the intracellular Ca2ϩ signal is through regulating the activity of the InsP3R2 family of Ca2ϩ release channels
In order to establish if InsP3R-associated PKG substrate (IRAG) can potentially modulate other InsP3R family members and splice variants, experiments were first performed to ascertain whether IRAG physically interacts with all subtypes of InsP3R and is capable of forming a tertiary complex with PKG1␤
IRAG Does Not Impact protein kinase (PKA) Regulation of InsP3R-2 Unless Phosphorylated by PKG—we addressed whether IRAG

Summary

Introduction

A primary locus for modifying the characteristics of the intracellular Ca2ϩ signal is through regulating the activity of the InsP3R2 family of Ca2ϩ release channels. Outside of the conserved NH2-terminal InsP3 binding pocket and the COOH-terminal channel domain, the primary sequence of the individual proteins is quite divergent, allowing for potential InsP3R subtype-specific regulation of Ca2ϩ release. This regulation, along with the particular complement of InsP3R expressed, is thought to make a significant contribution to defining the particular Ca2ϩ signals observed in individual cell types. The alternatively spliced S2Ϫ InsP3R-1 variant, the major InsP3R-1 splice variant expressed outside the neural system, resulting in 40 amino acids excised between phosphorylation sites, was not subject to regulation by PKG [20] These data indicate that all PKA consensus sequences are not necessarily PKG substrates. It is not known if IRAG interacts with all subtypes of InsP3R or if its expression is ubiquitous, this mechanism may reconcile earlier observations of decreased Ca2ϩ release following PKG and PKA activation in various tissues [22, 24, 28]

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