G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

Ana Ruiz-Gomez,Britt Mellström,Daniel Tornero,Esperanza Morato,Magali Savignac,Helena Holguín,Koldo Aurrekoetxea,Paz González,Carmen González-García,Valentín Ceña,Federico Mayor,Jose R Naranjo

doi:10.1074/jbc.m607166200

Abstract

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

Highlights

G protein-coupled receptor kinases (GRKs)3 are a family of proteins that share the ability to selectively phosphorylate the
In this study we describe the interaction between the neuronal calcium sensor Downstream regulatory element antagonist modulator (DREAM)/KChIP3 and GRK2 or GRK6 and analyzed the consequences of this interaction for different cellular functions of DREAM
We show that the phosphorylation state of DREAM at Ser-95 is regulated by GRK2 and calcineurin and that this is important for the regulation by DREAM of Kv4 channel cell surface expression

Summary

Introduction

G protein-coupled receptor kinases (GRKs) are a family of proteins that share the ability to selectively phosphorylate the. A common property of all three GRK subfamilies is their ability to interact with Ca2ϩ binding proteins of the EF-hand class, which reduces their kinase activity and provides a mechanism to terminate the receptor desensitization (for reviews, S-transferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonic acid. DREAM, named KChIP3 [20], interacts with potassium channels of the Kv4 class directing their trafficking to and inside the plasma membrane [24] and regulating in a Ca2ϩ-dependent manner the gating properties of the channel [20]. In the cytosol DREAM binds to the C-terminal region of the presenilins [26] and blocks the release of Ca2ϩ from the endoplasmic reticulum and the apoptosis induced by presenilin mutants associated to Alzheimer disease [27]

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