Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry

Eulalia Pozo-Guisado, David G. Campbell, Maria Deak, Alberto Alvarez-Barrientos, Nicholas A. Morrice, Ignacio S. Alvarez, Dario R. Alessi, Francisco Javier Martin-Romero

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    Abstract

    Store-operated calcium entry (SOCE) is an important Ca2+ entry pathway that regulates many cell functions. Upon store depletion, STIM1, a transmembrane protein located in the endoplasmic reticulum (ER), aggregates and relocates close to the plasma membrane (PM) where it activates store-operated calcium channels (SOCs). Although STIM1 was early defined as a phosphoprotein, the contribution of the phosphorylation has been elusive. In the present work, STIM1 was found to be a target of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in vitro, and we have defined the ERK1/2-phosphorylated sites on the STIM1 sequence. Using HEK293 cells stably transfected for the expression of tagged STIM1, we found that alanine substitution mutants of ERK1/2 target sites reduced SOCE significantly, suggesting that phosphorylation of these residues are required to fully accomplish SOCE. Indeed, the ERK1/2 inhibitors PD184352 and PD0325901 decreased SOCE in transfected cells. Conversely, 12-O-tetradecanoylphorbol-13-acetate, which activates ERK1/2, enhanced SOCE in cells expressing wild-type tagged STIM1, but did not potentiate Ca2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca2+ influx without disturbing the aggregation of STIM1 upon store depletion and without affecting the relocalization in ER-PM punctae. However, our results suggest that STIM1 phosphorylation at ERK1/2 target sites can modulate SOCE by altering STIM1 binding to SOCs, because a significant decrease in FRET efficiency was observed between alanine substitution mutants of STIM1-GFP and ORAI1-CFP.

    Original languageEnglish
    Pages (from-to)3084-3093
    Number of pages10
    JournalJournal of Cell Science
    Volume123
    Issue number18
    Early online date24 Aug 2010
    DOIs
    Publication statusPublished - 15 Sep 2010

    Keywords

    • STIM1
    • Calcium
    • Store-operated calcium entry
    • SOCE
    • Phosphorylation
    • ERK1/2

    Cite this

    Pozo-Guisado, E., Campbell, D. G., Deak, M., Alvarez-Barrientos, A., Morrice, N. A., Alvarez, I. S., Alessi, D. R., & Martin-Romero, F. J. (2010). Phosphorylation of STIM1 at ERK1/2 target sites modulates store-operated calcium entry. Journal of Cell Science, 123(18), 3084-3093. https://doi.org/10.1242/jcs.067215