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

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 inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.

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