Casas-Rua Vanessa, Tomas-Martin Patricia, Lopez-Guerrero Aida M, Alvarez Ignacio S, Pozo-Guisado Eulalia, Martin-Romero Francisco Javier
Department of Biochemistry and Molecular Biology, School of Life Sciences, University of Extremadura, Badajoz, Spain.
Department of Cell Biology, School of Life Sciences, University of Extremadura, Badajoz, Spain.
Biochim Biophys Acta. 2015 Jan;1853(1):233-43. doi: 10.1016/j.bbamcr.2014.10.027. Epub 2014 Nov 4.
STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca²⁺ entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca²⁺ emptying triggered by the inhibition of the sarco(endo)plasmic Ca²⁺ -ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca²⁺, indicating that both events are upstream steps for Ca²⁺entry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca²⁺ channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.
基质相互作用分子1（STIM1）是储存性钙内流（SOCE）的关键调节因子，因此是Ca²⁺内流依赖性细胞事件的介导者。已报道，在毒胡萝卜素抑制肌浆网Ca²⁺-ATP酶引发的细胞内Ca²⁺排空过程中，细胞外调节蛋白激酶1/2（ERK1/2）靶位点的STIM1磷酸化增强了STIM1的激活。然而，这种特定磷酸化的生理功能尚不清楚。本研究探讨了STIM1磷酸化在表皮生长因子（EGF）触发的细胞信号传导中的作用。使用人子宫内膜腺癌细胞系（Ishikawa细胞），发现EGF或H-Ras的活性突变体H-Ras（G12V）可触发STIM1在Ser575、Ser608和Ser621位点的磷酸化，并且该过程对ERK1/2抑制剂PD0325901敏感。ERK1/2激活和STIM1磷酸化均在无细胞外Ca²⁺的情况下发生，表明这两个事件都是Ca²⁺内流激活的上游步骤。此外，EGF以类似于毒胡萝卜素激活STIM1的方式触发STIM1与EB1（微管正端调节因子）解离。当通过ERK1/2靶位点的丝氨酸到丙氨酸取代突变靶向STIM1磷酸化时，Ishikawa细胞的迁移受到损害。钙通道阻滞剂SKF96365也观察到这种效应。STIM1的模拟磷酸化突变将迁移恢复到与野生型STIM1相似的水平。最后，靶向STIM1磷酸化在很大程度上抑制了EGF触发的波形蛋白表达增加和E-钙黏蛋白重新定位，而STIM1-S575E/S608E/S621E使这两种上皮-间质转化标志物的表达恢复正常。
