The Effects And Molecular Mechanisms Of Endoplasmic Reticulum Stress Signals On The Stability Of TRIB2

Backgroud and Objectives:TRIB2 is a homologue of the drosophila pseudokinase “Tribbles” and is highly conserved evolutionarily.Structurally,TRIB2 protein is mainly composed of three domains,including the N-terminal “PEST” domain,the carboxy-terminal(C-terminal)domain and the central pseudokinase domain.The N-terminal “PEST” domain is rich in proline(P),glutamate(E),serine(S),threonine(T).It is generally believed that “PEST”domain is an unstable domain of protein,and protein degradation is caused by phosphorylation of this domain,resulting in protein instability.Lacking of kinase activity,the pseudokinase domain behaves as a scaffold mediating protein-protein interaction.The c-terminal domain contains two conservative motifs: the HPW [F/L] motif mediating its interaction with MEK1 and the DQXVP(D/E)motif mediating ints interaction with E3 ubiquitin ligase COP1,respectively.TRIB2 is overexpressed in several kinds of malignant tumors,and is widely recognized as an oncogene.TRIB2 was found to be an unstable protein by previous studies.Its protein stability is regulated by several E3 ubiquitin ligases,including COP1,Smurf1,and β-Tr CP.Our previous study revealed that stability of TRIB2 is responsive to glucose starvation,raising the possibility that TRIB2 is a endoplasmic reticulum(ER)stress responsive protein.To this end,we utilized two melanoma cell lines with high expression of TRIB2 to observe the protein expression level of TRIB2 under ER stress.After establishing TRIB2 is indeed an ER stress responsive molecule,we investigated the molecular mechanisms regulating TRIB2 degradation under ER stress conditions,involving TRIB2 structure,phosphorylation,and its E3 ubiquitin liase.Our study sheds new light on designing new strategy for targeted therapy of clinical melanoma.Methods:(1)Western Blot was carried out to study the protein expression level of TRIB2 in melanoma cells under endoplasmic reticulum stress conditions such as protein glycation inhibition,glucose starvation and oxidative stress.(2)Melanoma cells were treated with proteasome inhibitor MG132,Cullin neddylation inhibitor MLN4924,and ER stress inducer,and the expression level of TRIB2 protein was detected by Western Blot.(3)Site-directed DNA mutagenesis was carried out to construct COP1-binding deficient TRIB2 mutant,Co-immunoprecipitation(Co-IP)between mutant TRIB2 and COP1 was carried out to confirm the loss of binding between the two proteins,and protein stability of COP1-binding deficient TRIB2 mutant in response to ER stress was observed by Western blot.In the meantime,lentivirally mediated sh RNA silencing of COP1 was used to study the expression level of TRIB2 under ER stress.(4)Overlapping PCR was carried out to contruct truncated TRIB2 lacking “PEST”domain,and site-directed DNA mutation of the serine/threonine of “PEST” domain-into alanines to construct un-phosphorylatable “PEST” domain of TRIB2 to investigate whether “PEST” domain or its phosphorylation mediated the degradation of TRIB2 under ER stress.(5)Protein phosphatase treatment was used to study whether phosphorylation of TRIB2 caused the slow mobility of TRIB2 on Western blot.Co-immunoprecipitation(CoIP)between p38 and TRIB2 was carried out to study if they interact each other.p38 knockout cells established by Cas9 nuclease and p38 MAPK inhibitor SB203580 were used to study whether p38 regulates the phosphorylation and stability of TRIB2 under ER stress.(6)The effect of TRIB2 on the proliferation of melanoma cells in vitro was studied by CCK8 cell proliferation assay and tumor clone formation assay.Results:(1)The protein expression level of TRIB2 in melanoma cells decreased significantly under ER stress.(2)The proteasome inhibitor MG132 and Cullin Neddylation inhibitor MLN4924 increased the protein expression of TRIB2 in melanoma cells under basal culture conditions,and MLN4924 reversed the protein degradation of TRIB2 induced by ER stress.(3)COP1 and TRIB2 could be immunoprecipitated.TRIB2 protein was stabilized in COP1-silenced melanoma cells under ER stress.and the COP1-binding deficient mutant of TRIB2 was stable upon ER stress induction.(4)TRIB2 lacking “PEST” domain was stable under the induction of ER stress.(5)The serine/threonine residues were phosphorylated.Mutations of all the 9serine/threonine residues or 4 serine/threonine residues in front of proline into alanine were able to stabilized the TRIB2 upon ER stress,indicating one or some proline-directed protein kinase(s)mediated the phosphorylation of “PEST” domain and regulated the stability of TRIB2 protein.But p38,a typical proline-directed kinase,which is activated upon ER stress,fails to regulate the protein degradation of TRIB2 induced by ER stress despite that p38 interacts with TRIB2.(6)TRIB2 protein enhances the proliferation of melanoma cells.Conclusions:In this study,we demonstrated for the first time that the expression level of TRIB2 protein in melanoma cells decreased significantly under ER stress,and further revealed that the degradation of TRIB2 is mediated by ubiquitin-proteasome system and is dependent on Cullin-Ring E3 ubiquitin ligases.The C-terminus of TRIB2 mediates the binding of TRIB2 with the Cullin-Ring E3 ubiquitin ligase COP1.TRIB2 mutant losing COP1-binding is stable under ER stress,indicating that COP1 is the E3 ubiquitin ligase that mediates the degradation of TRIB2 protein induced by ER stress.The N-terminal “PEST” domain of TRIB2 and its phosphorylation also participate in regulating the protein degradation of TRIB2 induced by ER stress.But stress-responsive p38 protein kinase is not involved in the regulation of TRIB2 protein stability under ER stress conditions.