SH2D4A, A New Signaling Molecule, Regulates Cell Proliferation And Apoptosis

IntroductionSH2 domain containing 4A(SH2D4A) is a novel protein of the SH2 signaling protein family that only contains a single SH2 domain.We cloned the SH2D4A gene from the human chromosome 8p22 region,and indicated SH2D4A protein is located in the cytoplasm and is expressed ubiquitously.The SH2 domain is the prototype for protein-protein interaction modules,binds to a phosphotyrosine,and mediates the formation of multiprotein complexes in protein tyrosine kinase pathways.SH2 domains are typically found in multidomain signaling enzymes or in adaptor proteins,where they mediate protein-protein interactions and regulate associated catalytic subunits.The lack of additional domains in SH2D4A suggests that it is unlikely to function in either of these roles.However,only a few reports of SH2D4A have been published so far,and its detailed biological function is still poorly understood.To investigate the biological function of SH2D4A,we constructed SH2D4A expression vector and its SH2 domain-deleted expression vector,and detected the effect of SH2D4A on cell proliferation and apoptosis using MTT assay and Flow-cytometric analyses and protein kinase C(PKC) activity assay.The data showed that SH2 domain is the functional domain of SH2D4A,and SH2D4A inhibited cell proliferation and promoted cell apoptosis.Furthermore,SH2D4A suppressed the insulin-like growth factorâ…¡(IGFâ…¡)-induced Ca²⁺release and PKC activation.Therefore,these findings suggested that SH2D4A inhibited cell proliferation and promoted cell apoptosis by suppression of PKC signaling pathway.We further performed a yeast two-hybrid screen using SH2D4A as the bait and found SH2D4A directly bind to estrogen receptorα(ERα).Co-immunoprecipitation and GST pull-down assays further confirmed their interaction.We also demonstrated an inhibitory effect of SH2D4A on estrogen-induced cell proliferation.We showed that this effect was due to suppression of a non-genomic action of ERαvia an ERα/phospholipase C-γ(PLC-γ)/PKC signaling pathway.In addition,we confirmed a new relationship among SH2D4A,ERα,and PLC-γ.SH2D4A may be useful for the development of a new anti-cancer drug acting as an ER signaling modulator.Our findings provide insight into the biological function of SH2D4A.Materials and MethodsMaterials1,Human embryonic kidney cell line(HEK293)2,Reagents for construction expression vector3,Reagents for Microculture tetrazolium(MTT) assay.4,Reagents for Flow-cytometric analyses.5,Reagents for PKC activity assay.6,Fluorescent probe Fluo-3/AM7,Reagents for Western blot8,Reagents for Yeast two-hybrid screen and assay9,Reagents for Co-immunoprecipitation10,Reagents for GST pull-down assay11,Equipments for experiments:fluorospectrophotometer,UV spectrophotometer, microplate spectrophotometer,flowcytometry,gel-formatter,oscillatory incubator et al.12,Database and software:genome data base,protein analytic data base,analytic software of imaging and scanning,Primer3,SPSS version 13.0 et al.Methods1,To investigate the biological function of SH2 domain in SH2D4A,we constructed SH2 domain-deleted expression vector pSH2D4A-Delet by enzymatic digestion,ligation and transform technique.2,The MTT assay was performed to test the effect of SH2D4A in cell rpoliferation.3,Flow-cytometric analyses was used to observe the influence of in cell cycle,cell proliferation and cell apoptosis.4,The IGFâ…¡or estrogen-induced activation of PKC was measured by the PepTag Protein Kinase Assay.5,Changes in Ca²⁺ concentration were measured by the Ca²⁺-sensitive fluorescent probe Fluo-3/AM.6,Through constructed pAD/ERα,yeast two-hybrid screen and assay were performed to investigate the interaction between SH2D4A and ERa.7,After constructed pSH2D4A-HA,co-immunoprecipitation was performed to investigate the relationship among SH2D4A,ERαand PLC-γ.8,After constructed pGST/SH2D4A and pGST/ERα,GST pull-down was performed to confirmed the directly interaction between SH2D4A and ERα.Results1,Constructions of expression vectors,such as pAD/ERα,pSH2D4A-HA, pGST/SH2D4A and pGST/ERα,provided the bases for investigating the biological function of SH2D4A.2,SH2D4A was found to inhibite cell proliferation and promote apoptosis. SH2D4A also suppressed IGFâ…¡-induced cell proliferation and its anti-apoptosis effect. But SH2D4A only arrested estrogen-induced cell proliferation and didn't influence its anti-apoptosis effect.3,The activity of PKC was measured by PepTag Protein Kinase Assay.SH2D4A was observed to inhibit the activation of PKC,and suppressed the IGFâ…¡or estrogen-induced activation of PKC4,The concentration of intracellular Ca²⁺ was tested using a fluorescent probe. SH2D4A arrested the IGFâ…¡or estrogen-induced release of Ca²⁺. 5,We found the interaction between ERαand SH2D4A in yeast two-hybrid screen, and verified this interaction via the yeast two-hybrid assay.SH2D4A interacted with ERαin yeast cell.6,We further investigated the interaction between SH2D4A and ERαin vivo using co-immunoprecipitation assay.The data confirmed the interaction of SH2D4A with ERαin eukaryotic cells.7,After expression-inducion and purification of fusion protein,GST pull-down assays were used to determine the interaction between SH2D4A and ERαin vitro.The interaction between SH2D4A and ERαwas direct and independent on additional proteins in the cells.8,In the co-immunoprecipitation assay,PLC-γinteracted with ERαin bidirectional reactions,and this interaction was suppressed by SH2D4A.Conclusion1,SH2D4A regulated cell proliferation and apoptosis by suppression of PKC signaling pathway.2,SH2D4A directly interacted with ERα;PLC-γ,interacted with ERα;SH2D4A competed with PLC-γfor binding to ERαand regulated cell proliferation via an ERα/PLC-γ/PKC signal pathway.