HRAS reduces store-operated calcium entry (SOCE) in RAS-less MEF cells

JULIETA MANSILLA RICARTTI; LUTZ BIRNBAUMER; SEBASTIAN SUSPERREGUY

Congreso; LXVII Reunion Anual de la Sociedad Argentina de Investigacion Clinca (SAIC); 2022

The store-operated calcium entry(SOCE) is the major Ca2+ entry pathway in nonexcitable cells, that is activated as a cellular response to refill the Ca2+ stores whenreticulum endoplasmic (ER)-Ca2+ store are depleted. The depletionof ER Ca2+ results in STIM1 translocation to ER-plasma membranejunctions where they bind and activate Orai1, the pore subunit ofthe Ca2+ release-activated Ca2+ (CRAC) channel. Ras GTPasesare molecular switches localized at plasma membrane that cyclebetween an inactive GDP-bound state and an activate GTP- boundstate that engages effector proteins to regulate multiple signal transduction pathways. In humans, three RAS genes encode four distinctisoforms: HRAS, NRAS, and the two splice variants of KRAS gene,KRAS4a and KRAS4b, containing exons 4a and 4b, respectively.Our work has focused on elucidating whether RAS has a role in theSOCE mechanism. Experimental approach: To determine whetherthe protooncogene RAS is involved in the mechanism of SOCE, weused a unique cell model of mouse embryonic fibroblast devoid ofRAS proteins (RAS-less MEFs). First, we analyzed the Ras-lessMEF and RAS-less clones generated by stable transfection of human cDNAs of HRAS or KRAS4A (RAS-less MEF HRAS+, RASless MEF KRAS4A+) by Tg-evoked SOCE using the radiometric fura2 assay. Next, we transfected the clone of MEF devoid of RASproteins with EYFP-tagged H-,N- or KRAS and analyzed the changeof SOCE. Finally, we used pharmacological inhibitors of MEK-ERK,PI3K and PKC to investigate the intracellular signaling pathwaysinvolved in the effects of RAS Key results: We found Tg-evokedSOCE was reduced in RAS-less MEFs by more than 50%, whereas in the clone that stable express HRAS (RAS-less MEF HRAS+) SOCE was reduced by almost 90%. Moreover, HRAS transfected into RAS-less MEFs reduced Tg-induced SOCE in a dose-dependent manner. PKC blockades with GF109203X increased SOCEin SOCE RAS-less MEF BRAF by almost 30%, whereas blockadeof MEK/ERK with UO126 or PI3-K with LY294002 had no effect.Finally, HRAS transfected still reduced SOCE in RAS-less MEFswhen the intracellular signaling pathways MEK/ERK or PI3-K wereblocked, whereas HRAS transfected did not reduce SOCE whenPKC was blocked. Conclusions and implications: In conclusion, theresults suggest HRAS reduces SOCE without using MEK/ERK andPI3-K, the major intracellular signaling pathways activated by GTPbound RAS