Store-operated calcium entry is dispensable for the activation of ERK1/2 pathway in prostate cancer cells

Cell Signal. 2017 Dec:40:44-52. doi: 10.1016/j.cellsig.2017.08.010. Epub 2017 Aug 31.

Abstract

STIM1, the endoplasmic reticulum Ca2+ sensor that modulates the activity of plasma membrane Ca2+ channels, becomes phosphorylated at ERK1/2 target sites during Ca2+ store depletion triggered by thapsigargin or epidermal growth factor (EGF). This ERK1/2-dependent phosphorylation regulates STIM1 localization and dissociation from microtubules, and it is known that enhances the binding to ORAI1, a store-operated Ca2+ entry (SOCE) channel, leading to the activation of this Ca2+ influx pathway. However, there remained some evidence of a role for SOCE in the activation of ERK1/2, and here we assessed the contribution of SOCE to ERK1/2 activation by generating a STIM1-deficient cell line by CRISPR/Cas9 genome editing of the STIM1 locus in prostate cancer PC3 cells. The genomic modification consisted of a 16 base-pair insertion in exon 5 of both alleles, therefore abrogating STIM1 synthesis. STIM1-KO cells did show a striking decrease in Ca2+ influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca2+ entry in PC3 cells during stimulation with EGF. Moreover, identical levels of total ERK1/2 were found in STIM1-KO cells and the parental cell line, and ERK1/2 activation was fully activated in KO cells, both in the presence and in the absence of extracellular Ca2+, a result that supports that STIM1 and SOCE are not required for ERK1/2 activation. This activation was sensitive to Src kinase inhibition, but not to CAMKII nor PKC inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.

Keywords: CRISPR; Calcium; ERK1/2; SOCE; STIM1; Src.

MeSH terms

  • CRISPR-Cas Systems / genetics
  • Calcium / metabolism*
  • Calcium Channels / genetics*
  • Calcium Channels / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
  • Cell Line, Tumor
  • Cell Membrane / genetics
  • Cell Membrane / metabolism
  • Epidermal Growth Factor / genetics
  • Epidermal Growth Factor / metabolism
  • Gene Knockout Techniques
  • Humans
  • Intracellular Calcium-Sensing Proteins
  • MAP Kinase Signaling System / genetics
  • Male
  • Membrane Proteins / genetics*
  • Microtubules / genetics
  • Microtubules / metabolism
  • Prostatic Neoplasms / genetics*
  • Prostatic Neoplasms / pathology
  • Stromal Interaction Molecule 1 / genetics*
  • Stromal Interaction Molecule 1 / metabolism
  • src-Family Kinases / antagonists & inhibitors

Substances

  • Calcium Channels
  • Intracellular Calcium-Sensing Proteins
  • Membrane Proteins
  • SARAF protein, human
  • Stromal Interaction Molecule 1
  • Epidermal Growth Factor
  • src-Family Kinases
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Calcium