Tumor invasion requires efficient cell migration, which is achieved by the generation of persiste... more Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 an...
Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), ... more Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 wild-type (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621...
STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE).... more STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca(2+) concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.
... W: A muscle protein in search of a function 137 L Walt Ream, William R. Vorachek and Phillip ... more ... W: A muscle protein in search of a function 137 L Walt Ream, William R. Vorachek and Phillip D. Whanger Chapter 13 The 15 kDa selenoprotein (SeplS): functional studies and a role in cancer etiology 147 Vadim N. Gladyshev, Alan M Diamond and Dolph L. Hatfield Page 11. ...
Tumor invasion requires efficient cell migration, which is achieved by the generation of persiste... more Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 an...
Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), ... more Stromal interaction molecule 1 (STIM1) plays a pivotal role in store-operated Ca2+ entry (SOCE), an essential mechanism in cellular calcium signaling and in maintaining cellular calcium balance. Because O-GlcNAcylation plays pivotal roles in various cellular function, we examined the effect of fluctuation in STIM1 O-GlcNAcylation on SOCE activity. We found that both increase and decrease in STIM1 O-GlcNAcylation impaired SOCE activity. To determine the molecular basis, we established STIM1-knockout HEK293 (STIM1-KO-HEK) cells using the CRISPR/Cas9 system and transfected STIM1 wild-type (STIM1-KO-WT-HEK), S621A (STIM1-KO-S621A-HEK), or T626A (STIM1-KO-T626A-HEK) cells. Using these cells, we examined the possible O-GlcNAcylation sites of STIM1 to determine whether the sites were O-GlcNAcylated. Co-immunoprecipitation analysis revealed that Ser621 and Thr626 were O-GlcNAcylated and that Thr626 was O-GlcNAcylated in the steady state but Ser621 was not. The SOCE activity in STIM1-KO-S621...
STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE).... more STIM1 (stromal interaction molecule 1) is a key regulator of store-operated calcium entry (SOCE). Upon depletion of Ca(2+) concentration within the endoplasmic reticulum (ER), STIM1 relocalizes at ER-plasma membrane junctions, activating store-operated calcium channels (SOCs). Although the molecular details for STIM1-SOC binding is known, the regulation of SOCE remains largely unknown. A detailed list of phosphorylated residues within the STIM1 sequence has been reported. However, the molecular pathways controlling this phosphorylation and its function are still under study. Using phosphospecific antibodies, we demonstrate that ERK1/2 mediates STIM1 phosphorylation at Ser575, Ser608 and Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.
... W: A muscle protein in search of a function 137 L Walt Ream, William R. Vorachek and Phillip ... more ... W: A muscle protein in search of a function 137 L Walt Ream, William R. Vorachek and Phillip D. Whanger Chapter 13 The 15 kDa selenoprotein (SeplS): functional studies and a role in cancer etiology 147 Vadim N. Gladyshev, Alan M Diamond and Dolph L. Hatfield Page 11. ...
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Papers by Francisco Javier Martin-Romero
Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type
STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.
Ser621 during Ca(2+) store depletion, and that Ca(2+) entry and store refilling restore phosphorylation to basal levels. This phosphorylation occurs in parallel to the dissociation from end-binding protein 1 (EB1), a regulator of growing microtubule ends. Although Ser to Ala mutation of residues 575, 608 and 621 showed a constitutive binding to EB1 even after Ca(2+) store depletion, Ser to Glu mutation of these residues (to mimic the phosphorylation profile attained after store depletion) triggered full dissociation from EB1. Given that wild-type
STIM1 and STIM1(S575E/S608E/S621E) activate SOCE similarly, a model is proposed to explain how ERK1/2-mediated phosphorylation of STIM1 regulates SOCE. This regulation is based on the phosphorylation of STIM1 to trigger dissociation from EB1 during Ca(2+) store depletion, an event that is fully reversed by Ca(2+) entry and store refilling.