The Stability Regulation Of The TRIB2 Protein Via Nutritional Stress And Its Molecular Mechanism Research

Background and Objectives:The TRIBs(Tribbles)protein family has a pseudokinase domain and mainly serves as a scaffolding protein.There are three homologous gene subtypes of TRIB1,2,and 3 in mammalian cells,which binds to a variety of signaling molecules including E3 ubiquitin ligase COP1(Constitutive photomorphogenic Protein1),thereby exerting corresponding biological functions.Structurally,TRIBs are mainly composed of three parts: the amino terminus(N terminus),the intermediate pseudokinase-like domain,and the carboxy terminus(C terminus).The N-terminal contains a domain rich in proline,glutamic acid,serine and threonine(P \ E \ S \ T),which is called "PEST" domain.It is generally believed that proteins with PEST domains are unstable,Previous studies have shown that in mammalian adipocytes,limiting the concentration of glucose in the cell culture medium can enhance the stability of TRIB3.TRIB3 combines with COP1 to degrade acetyl-Co A carboxylase(ACC),limiting fatty acid synthesis;in the liver cells,the overexpression of TRIB1 can reduce fatty acid oxidation and triglyceride synthesis,and the expression level of TRIB1 is inversely correlated with glucose concentration.Therefore,both TRIB1 and TRIB3 play an important role in the process of cellular energy metabolism.Other studies have shown that the expression level of TRIB3 in adipocytes is significantly increased under glucose starvation conditions in vivo and in vitro,but it is still unclear whether this phenomenon also exists in other cells.It is worth noting that TRIB2 in liver cancer cells is also unstable under basic culture conditions.Research suggests that TRIB2 is often used as an oncogene in certain melanoma,liver cancer,acute leukemia and other malignant tumor cells,and is closely related to the occurrence and development of tumors.As a member of the TRIB family,does TRIB2 play a role in the energy metabolism of cells? What role does it play? How does it affect the biological function of tumors? There is still a lack of research.In order to explore whether TRIB2 plays a role in cellular energy metabolism,we selected melanoma cells with high expression of TRIB2 protein as a research model,using glucose starvation,amino acid starvation,and 2-deoxy-D-glucose,treatment of cells under various nutritional stress conditions,it was found that the expression of TRIB2 protein was significantly reduced under the above nutritional starvation state,and the decrease in TRIB2 protein expression was mediated by the proteasome,which revealed for the first time that TRIB2 protein stability received cell nutrition signals regulation.Based on other previous reports that TRIB2 containing PEST domain and TRIB2 protein stability is affected by COP1,β-Tr CP(β-transducin repeat-containing protein)and Smurf1(SMAD Specific E3 Ubiquitin Protein Ligase 1),but these studies were carried out under basic cell culture conditions,therefore,we studied whether the TRIB2 PEST domain and these three E3 ubiquitin ligases in glucose starvation mediate the degradation of TRIB2 protein.After excluding the PEST domain and the effects of COP1,Smurf1 E3 ubiquitin ligase,by TRIB2 amino acid sequence analysis,we found that there is a potential β-Tr CP binding sequence DSGX(2-4)S(including Aspartic acid(D)can also be phosphorylated S or T,X can be any amino acid,and both S need to be modified by phosphorylation),because the S or T in this motif needs to be phosphorylated only under the premise of modification can it be recognized by β-Tr CP E3 ligase,also known as phosphodegron.Therefore,we further investigated whether β-Tr CP mediates the degradation effect of TRIB2 under glucose starvation,and whether AMPK(AMP-activated protein kinase)activated under glucose starvation is involved in phosphorylating TRIB2 phosphodegron to reveal the molecular mechanism of TRIB2 protein degradation under glucose starvation.More and more studies have shown that TRIB2 as an oncogene is increased in certain malignant tumor cells such as melanoma,liver cancer,acute leukemia,etc.,which is closely related to the occurrence and development of tumors.Although TRIB2 has been studied as an oncogene in hepatocarcinoma,acute leukemia and other tumorigenesis,its function and mechanism in melanoma cells are still poorly studied,and the relationship between TRIB2 and melanoma cell energy metabolism is even no more report has been reported.This study aims to reveal the relationship between TRIB2 and cell energy metabolism in melanoma cells and its molecular mechanism,and provide an experimental basis for exploring new melanoma treatment strategies.Methods:(1)Treatment of melanoma cells in vitro with various nutritional stress conditions and observing the expression level of TRIB2 by Western Blot;(2)Using proteasome or Cullin neddylation inhibitor to investigate whether the decreased expression of TRIB2 under nutrient starvation is mediated by proteasome and Cullin-dependent E3 ligase;(3)Overlapping PCR technique was used to construct TRIB2 mutant with PEST domain truncated,by which to study whether the "PEST" domain mediates the degradation of TRIB2 under starvation;(4)Using lentiviral sh RNA to silence COP1 expression or COP1 binding defective TRIB2 mutant to study the expression level and stability of TRIB2 under nutritional stress conditions;(5)Using Co-Immunoprecipitation,GST fusion protein-Pull-Down Assay,or phosphodegron Peptide-Pull-Down assay to investigate whether there is a physical interaction between Trib2 and E3 ubiquitin ligase β-Tr CP;(6)Using lentiviral Cullin1 or β-Tr CP sh RNA to study the regulatory effect of Cullin1-dependent E3 ubiquitin ligase β-Tr CP on the stability of Trib2 protein;(7)Using In vivo ubiquitination assay to study whether Trib2 is a substrate for E3 ubiquitin ligase β-Tr CP;(8)Using site-directed DNA mutagenesis to mutate the serine / threonine in the potential phosphodegron consensus on Trib2 c DNA to alanine(A),failing to accept phosphorylation modification,then to study its binding capacity to E3 ubiquitin ligaseβ-Tr CP and its protein stability under nutritional stress;(9)A variety of AMPK activators were used to study the effect of AMPK activation on TRIB2 expression,and to compare the stability of TRIB2over-expressed in both wild type mouse embryonic fibroblasts(MEF)and AMPK null MEF to reveal the regulatory effect of AMPK on the degradation of TRIB2 under nutritional stress;(10)Using GST fusion protein pull-down experiment to study whether AMPK binds to TRIB2;(11)Using In vitro kinase assay to simulate the phosphorylation environment outside the cell to investigate whether AMPK can phosphorylate bacterially expressed Trib2 protein without post-translational modification;(12)Tumor cell clone formation experiments were used to study the effect of TRIB2 on melanoma cell clone formation in vitro.Results:(1)Under nutritional stress conditions such as glucose starvation,amino acid starvation,and 2-DG treatment,TRIB2 protein expression in melanoma cells decreased significantly.(2)Proteasome inhibitors and neddylation inhibitors enhanced the expression level of TRIB2 protein in melanoma cells under basic culture conditions.The neddylation inhibitor reversed glucose starvation-induced degradation of TRIB2 protein in melanoma cells.(3)Degradation rate of TRIB2 without the "PEST" domain under glucose starvation was not significantly different from that of wild-type TRIB2.(4)In the basic culture condition,although the expression level of TRIB2 in COP1 silent melanoma cells was significantly increased,under glucose starvation,the TRIB2 protein in COP1 silent cells was still degraded,and COP1 binds to the defective TRIB2 mutant Under glucose starvation,its protein degradation rate was not significantly different from that of wild-type TRIB2.(5)Both β-Tr CP and TRIB2 can not only co-immunoprecipitate,but also the GST-TRIB2 fusion protein successfully pulls down the endogenous β-Tr CP,TRIB2 phosphodegron pre-phosphorylated synthetic short peptide successfully pulls down the endogenous β-Tr CP,and the same short peptide without phosphorylation treatment cannot pull down β-Tr CP.(6)Lentivirus-mediated sh RNA successfully silenced the expression of Cullin1 or β-Tr CP in melanoma cells,resulting in enhanced TRIB2 protein stability under glucose starvation.(7)In vivo ubiquitination experiments have shown that β-Tr CP promotes the ubiquitination of TRIB2 under glucose starvation.(8)Phosphodegron sequence mutant TRIB2-3A(TRIB2 T226,S227,S231 mutated to alanine A)compared with wild-type TRIB2,the efficiency of immunoprecipitation with β-Tr CP was significantly reduced,and GST-TRIB2-3A fusion protein was pulled down β-Tr CP capacity is also significantly reduced compared to the wild-type TRIB2-GST fusion protein.In vivo ubiquitination experiment,the extent of TRIB2-3A mutants receiving β-Tr CP ubiquitination under glucose starvation conditions was also significantly reduced compared to wild-type TRIB2.(9)The kinase AMPK is activated under glucose starvation conditions,accompanied by a decrease in the stability of TRIB2.Under glucose starvation,TRIB2 overexpressed in wild-type MEF cells was rapidly degraded,while the stability of TRIB2 protein overexpressed in AMPK gene knockout MEF cells was significantly improved,indicating that AMPK promotes TRIB2 under glucose starvation degradation.(10)GST-TRIB2 fusion protein successfully pulls down endogenous AMPK.(11)In vitro kinase assay shows that AMPK can phosphorylate expressed in E.coli in vitro,without post-translationally modified TRIB2 protein but phosphorylate TRIB2-3A protein not.(12)TRIB2 expression enhances the colony formation of melanoma cells in vitro.Conclusions:This study reveals for the first time that TRIB2 protein is rapidly degraded under the stimulation of many nutritional stress signals.Cullin neddylation inhibitors can reverse this degradation,indicating that the degradation of TRIB2 is mediated by Culllin-dependent E3 ubiquitin ligase and proteasome.Unlike other proteins with PEST domains,the PEST domain of TRIB2 has no regulatory effect on the degradation of TRIB2 protein under glucose starvation.Cullin neddylation inhibitors reverse the degradation of TRIB2 induced by glucose starvation,which basically excludes the possibility that the HECT domain family E3 ubiquitin ligase Smurf1 mediates the degradation of TRIB2.However,COP1 silent cells still maintain the ability of TRIB2 degradation under glucose starvation and the degradation rate of COP1 binding-deficient TRIB2 mutants under glucose starvation is not significantly different from that of wild-type TRIB2,indicating that COP1 is not in the main E3 ubiquitin ligase that leads to TRIB2 degradation.The following experimental results show that the synergistic effect of protein kinase AMPK and E3 ubiquitin ligaseβ-Tr CP leads to the production of TRIB2 phosphodegron is the main molecular mechanism leading to the degradation of TRIB2 under glucose starvation: 1)TRIB2,β-Tr CP co-precipitation,GST-TRIB2 fusion protein or TRIB2 phosphodegron short peptide successfully pull down β-Tr CP,these results indicate that there is physical binding between TRIB2 and β-Tr CP.2)In the state of glucose starvation,the silence of Cullin1 or β-Tr CP stabilizes TRIB2,and β-Tr CP overexpression promotes the ubiquitination of TRIB2 in vivo.3)Not only the ability of TRIB2-3A mutant to co-precipitate with β-Tr CP decreases,but its GST fusion protein’s ability to pull downβ-Tr CP is also significantly reduced,and the ability of TRIB2-3A mutant to accept ubiquitination under glucose starvation conditions obviously weakened.4)The GST-TRIB2 fusion protein successfully pulls down AMPK and AMPK can phosphorylate wild-type TRIB2 in vitro and lose phosphorylation ability to the 3A mutant.Overexpression of TRIB2 in MEFs knocked out by AMPK under glucose starvation conditions is degraded and wild-type is blocked MEF degrades rapidly.These experimental results indicate that AMPK may be a protein kinase that mediates the phosphorylation of TRIB2 phosphodegron.This study provides new ideas for exploring the clinical treatment of melanoma with high expression of TRIB2,and has important theoretical value and clinical application value.