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

Abstract

SummaryStore-operated calcium entry (SOCE) is an important Ca 2+ 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-signalregulated 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 Ca 2+ influx in cells expressing serine to alanine mutations in ERK1/2 target sites of STIM1. Alanine substitution mutations decreased Ca 2+ 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.

Phosphorylation of residues inside the SNARE complex suppresses secretory vesicle fusion

“…Together, our proteomics analysis identifies several putative substrates of PKCB such as MUNC18 (Brochetta et al , 2014; Genc et al , 2014), SNAP23 (Polgár et al , 2003; Lorentz et al , 2012), STIM1 (Pozo‐Guisado et al , 2010), and MYH9 (Ludowyke et al , 2006) (Appendix Table S1) that may enhance mast cell secretion as a positive arm in the incoherent feed‐forward regulation (Fig 4F). Surprisingly, we also discovered that VAMP8 has an evolutionarily conserved phosphorylation motif in the center of the SNARE fusion complex that acts in parallel as a negative regulatory arm in the same circuit to prevent fusion (Fig 4G).…”

STIM1/Orai1-mediated SOCE: current perspectives and potential roles in cardiac function and pathology

“…is required for thapsigargin-induced activation of SOCE by enhancing STIM1-Orai1 coupling (92). In neonatal cardiomyocytes we have shown that activation of SOCE by thapsigargin also increases STIM1 phosphorylation (153).…”

“…The K domain is predicted to contain a flexible central amphipathic ␣-helix that is flanked by globular positively charged regions in a dumbbell-like structure (148). The region between the ID and K domains appears to be the major regulatory region and contains most of the currently identified phosphorylation sites (62,92,109,143). Although it is now well recognized that STIM1 is a phosphoprotein (31,62), the molecular pathways responsible for controlling STIM1 phosphorylation are not well defined and the physiological function of many of the phosphorylation sites remain to be identified.…”

Alternative splicing converts STIM2 from an activator to an inhibitor of store-operated calcium channels

“…Several mechanisms that affect the magnitude of SOCE have been identified, such as transcriptional regulation , posttranslational modifications Hawkins et al, 2010;Pozo-Guisado et al, 2010), and accessory proteins Store-operated calcium entry (SOCE) regulates a wide variety of essential cellular functions. SOCE is mediated by STIM1 and STIM2, which sense depletion of ER Ca 2+ stores and activate Orai channels in the plasma membrane.…”