Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Takumi Kawasaki,Takehiko Ueyama,Ingo Lange,Stefan Feske,Naoaki Saito

doi:10.1074/jbc.m109.022996

Takumi Kawasaki, Takehiko Ueyama + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m109.022996

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Abstract

Ca(2+) signals through store-operated Ca(2+) (SOC) channels, activated by the depletion of Ca(2+) from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca(2+) sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca(2+) entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCbeta both resulted in increased Ca(2+) influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.

Highlights

Tel.: 81-78-803-5961; Fax: 81-78-8035971; E-mail: naosaito@kobe-u.ac.jp. 3 The abbreviations used are: store-operated Ca2؉ (SOC), store-operated Ca2ϩ; store-operated Ca2؉ entry (SOCE), store-operated Ca2ϩ entry; ER, endoplasmic reticulum; CRAC, Ca2ϩ release-activated Ca2ϩ; PKC, protein kinase C; DAG, diacylglycerol; cPKC, conventional PKC; nPKC, novel PKC; aPKC, atypical PKC; STIM, stromal interaction molecule; aa, amino acid(s); GST, glutathione S-transferase; shRNA, short hairpin RNA; GFP, green fluorescent protein; TG, thapsigargin; PS, phosphatidylserine; ing Ca2ϩ influx from the extracellular space
The PKC family consists of at least 10 subtypes and is divided into three subfamilies based on primary structure: conventional PKC, novel PKC, and atypical PKC [5, 6]. cPKC is activated by Ca2ϩ and DAG, nPKC is activated by DAG but not Ca2ϩ, whereas aPKC is independent of both DAG and Ca2ϩ
To avoid the suppressive effect on SOCE caused by overexpression of wild-type Orai1 [30, 31], the transfected cells were maintained for 7 days before experiments, because we found that longer cultivation of cells after transfection decreased the suppressive effect of Orai1 on SOCE

Summary

Introduction

Ing Ca2ϩ influx from the extracellular space. Ca2ϩ entry through SOC channels was proposed in the 1980s [2] and has been characterized extensively in patch clamp experiments. Results from in vitro and in vivo phosphorylation experiments show that Orai1 phosphorylation in HEK293 cells is mediated by PKC␤I and is dependent on Ca2ϩ influx.

Results

Conclusion