**4.1. MAPK pathway**

The close relationship between SOCE and mitogen-activated protein kinases (MAPKs) was revealed by Machaca and Haun [35], when they investigated the inactivation of SOCE in Xenopus oocyte maturation. SOCE is an active pathway in almost all eukaryotic cells, but during M-phase of cell cycle it becomes inactivated [36–38]. Machaca and Haun demonstrated that SOCE inactivation at germinal vesicle breakdown of Xenopus oocytes coincided with an increase in levels of MAPK and maturation-promoting factor (MPF), but they also demonstrated that MPF triggered SOCE inactivation by inhibiting the coupling between store depletion and SOCE activation, and not by blocking Ca2+ influx through SOCE channels [35].

In cells at interphase, some evidence supports a role for SOCE on ERK1/2 activation. In this regard, it has been proposed that SOCE activates extracellular signal-regulated kinases 1/2 (ERK1/2) in parotid acinar cells [39] and melanoma cells [40]. This proposal fits well with SOCE as an upstream regulator of ERK1/2. However, this proposal does not seem to be applicable to all cell lines, since ERK1/2 can be activated in the absence of extracellular Ca2+ and therefore in the absence of Ca2+ influx in HEK293 cells [20], the Ishikawa adenocarcinoma cell line [18], osteosarcoma U2OS cells [34], and prostate PC3 cells [19]. In addition, STIM1 knockdown did not modify phosphorylation of MEK1/2-ERK1/2 in gastric cancer cells [41], and STIM1 and ORAI1 knockdown did not inhibit the activation of ERK1/2 in response to EGF [42]. Moreover, ERK1/2 is fully activatable in STIM1-KO PC3 cells [19], with no active SOCE, demonstrating that SOCE is dispensable for ERK1/2 activation.

On the contrary, increasing evidence demonstrates that SOCE is a target for ERK1/2 activity, and that ERK1/2 is an upstream regulator of STIM1 and SOCE (reviewed in [43]). Pozo-Guisado et al. reported that STIM1 is phosphorylated by ERK1/2 at residues Ser575, Ser608, and Ser621 [25]. This phosphorylation is required for the full activation of STIM1 and for triggering the dissociation of STIM1 from microtubules [17]. Accordingly, phospho-STIM1 is enriched at the leading edge of migrating cells, that is, in the vicinity of receptor tyrosine kinases [34], where phospho-STIM1 acts in cooperation with ORAI1 to regulate the Ca2+ influx that rules cell migration. Consequently, phospho-STIM1 is an effector of ERK1/2 and an essential mediator for the activation of Ca2+ influx upon stimulation of cells with IGF-1 [20], or EGF [18, 19, 34].

Other MAPKs, such as p38 MAPK, have been shown to regulate SOCE, although by an indirect mechanism. Transforming growth factor beta (TGFβ) regulates megakaryocyte maturation and platelet formation by upregulating the expression of the serum-glucocorticoid inducible kinase SGK1 [44, 45], which is p38 MAPK-dependent [46]. SGK1 stimulates nuclear translocation of transcription factor NF-κB, which upregulates ORAI1 expression, increasing SOCE. This increase was demonstrated to be sensitive to p38 MAPK inhibition, SGK1 inhibition, and NF-κB inhibition, demonstrating the role of p38 MAPK in the upregulation of SOCE [47]. Another proposal was reported by Sundivakkam et al. [48], who reported that p38 MAPK directly phosphorylates STIM1. In this report, it was shown that pharmacological inhibition of p38 MAPK increased SOCE and that p38β knockdown prevented STIM1 phosphorylation and potentiated SOCE. However, this report did not identify the phosphorylated Ser/Thr residue(s), since the findings were based on the use of a phospho-Ser antibody [48], but not a site-specific phospho-specific antibody.

#### **4.2. cAMP and PKA**

of plasma membrane ruffling [34]. This membrane ruffling is the reorganization of the cortical cytoskeleton required for the formation of filopodia and lamellipodia, and STIM1-KO (knockout) and ORAI1-KO cells, engineered by CRISPR/Cas9 genome editing, showed defective membrane ruffling and largely diminished cell migration [34], demonstrating that Ca2+

Given the importance of Ca2+ in many signaling pathways, the impact of STIM1, ORAI, and SOCE on cell signaling is also remarkable. Increasing evidence prove the significant role of this Ca2+ entry pathway in cell physiology and tissue homeostasis, and we focus here on the role of STIM and ORAI proteins on modulators of signaling pathways, such as kinases, phosphatases, and transcription factors. We also describe recent findings that unravel how STIM1

The close relationship between SOCE and mitogen-activated protein kinases (MAPKs) was revealed by Machaca and Haun [35], when they investigated the inactivation of SOCE in Xenopus oocyte maturation. SOCE is an active pathway in almost all eukaryotic cells, but during M-phase of cell cycle it becomes inactivated [36–38]. Machaca and Haun demonstrated that SOCE inactivation at germinal vesicle breakdown of Xenopus oocytes coincided with an increase in levels of MAPK and maturation-promoting factor (MPF), but they also demonstrated that MPF triggered SOCE inactivation by inhibiting the coupling between store depletion and SOCE activation, and not by blocking Ca2+ influx through SOCE channels [35]. In cells at interphase, some evidence supports a role for SOCE on ERK1/2 activation. In this regard, it has been proposed that SOCE activates extracellular signal-regulated kinases 1/2 (ERK1/2) in parotid acinar cells [39] and melanoma cells [40]. This proposal fits well with SOCE as an upstream regulator of ERK1/2. However, this proposal does not seem to be applicable to all cell lines, since ERK1/2 can be activated in the absence of extracellular Ca2+ and therefore in the absence of Ca2+ influx in HEK293 cells [20], the Ishikawa adenocarcinoma cell line [18], osteosarcoma U2OS cells [34], and prostate PC3 cells [19]. In addition, STIM1 knockdown did not modify phosphorylation of MEK1/2-ERK1/2 in gastric cancer cells [41], and STIM1 and ORAI1 knockdown did not inhibit the activation of ERK1/2 in response to EGF [42]. Moreover, ERK1/2 is fully activatable in STIM1-KO PC3 cells [19], with no active

influx through STIM1-activated ORAI1 is essential for these events.

**4. The role of STIM1 and ORAI1 on cell signaling**

and ORAI1 are modulated by posttranslational modifications.

SOCE, demonstrating that SOCE is dispensable for ERK1/2 activation.

On the contrary, increasing evidence demonstrates that SOCE is a target for ERK1/2 activity, and that ERK1/2 is an upstream regulator of STIM1 and SOCE (reviewed in [43]). Pozo-Guisado et al. reported that STIM1 is phosphorylated by ERK1/2 at residues Ser575, Ser608, and Ser621 [25]. This phosphorylation is required for the full activation of STIM1 and for triggering the dissociation of STIM1 from microtubules [17]. Accordingly, phospho-STIM1 is enriched at the leading edge of migrating cells, that is, in the vicinity of receptor tyrosine

**4.1. MAPK pathway**

8 Calcium and Signal Transduction

The crosstalk between SOCE and cAMP-activated pathways has been investigated thoroughly since the molecular description of STIM1 and ORAI1. For instance, the Ca2+/CaM-stimulated adenylyl cyclase 8 (AC8) was found to be activated by SOCE and co-localized with STIM1 and ORAI1 in lipid rafts [49]. Interestingly, other authors found that lowering the concentration of free Ca2+ within the ER led to recruitment of adenylyl cyclases, enhancing the production of cAMP with the subsequent PKA activation, being this action independent of the [Ca2+]i [50]. Because activation of STIM1 and translocation to ER-PM junctions were required for coupling ER-Ca2+ depletion and adenylyl cyclase activity, without altering [Ca2+]i, those authors proposed the occurrence of a pathway termed store-operated cAMP signaling (SOcAMPS), a pathway that was later confirmed for other AC isoforms, such as AC3 [51]. More recently, it was confirmed that other Ca2+ channels, including TRPC1, were also involved in the activation of ACs [52]. It has been reported that STIM1 interacts with the plasma membrane adenylyl cyclase 6 to regulate melanogenesis [53], and this interaction is mediated by the Ser/Pro-rich C-terminal region of STIM1. These reports, together with the finding that ORAI1- and SOCEdeficient fibroblasts showed impaired cAMP production and cAMP-dependent signaling [54], strongly support the direct relationship between Ca2+ depletion at ER stores, STIM1 activation, and enhanced production of cAMP.

As for other pathways, the regulation between STIM1 and PKA seems to be reciprocal. In addition to the CRAC channel ORAI1, the plasma membrane-resident STIM1 activates storeindependent arachidonic acid regulated Ca2+ (ARC) channels, and this activation depends on the phosphorylation of STIM1 at Thr389 by PKA, which requires the scaffold protein AKAP79 [55]. This phosphorylation triggers a structural change in the SOAR region of STIM1 (also known as CAD) being essential for the selective activation of ARC channels [56].
