| In 1962,Ferruccio Ritossa observed that when the Drosophila exposed to 37?,its salivary gland can synthesis a kind of protein.With research,this kind of protein have a wide spread of function from protecting cells to maintaining the stability of internal environment,so those proteins were named heat shock protein(HSP)and those genes expression and physical protective phenomenon were called heat shock reaction(HSR).Heat shock response,the most primitive and conservative reaction of all the stress response,can induce the synthesis of HSP,as a "molecular chaperone" to maintain the proper folded state of proteins,protect cells against stress injury.including heat shock,hypoxia,ischemia-reperfusion injury,endotoxin,heavy metals,ethanol,enhance the tolerance to damage of the cell,maintain the normal function of cells and the metabolism,improve the survival rate of the cells,with a strong protective effect.In the stress response,HSP genes are expressed firstly,the other genes have been temporarily suppressed.In the high temperature,sodium arsenite,prostaglandin-Al induced HSR,the expression of HSP can inhibit the pro-inflammatory cytokines,which is in order to prevent excessive inflammatory response by inhibiting the activation of some transcription factor in cell damage.In mammalian cells,extracellular stimuli often triggers various signaling pathways.Among these signaling pathways,the most prominent ones are mitogen-activated protein kinases(MAPKs),including extracellular signal-regulated kinase(ERK),c-Jun N-terminal kinase(JNK),and p38 MAPK activation,involving phosphorylation on tyrosine and threonine residues,which leads to the phosphorylation of downstream effector proteins that carry out the necessary changes to respond to the stimulus.It has been found that MAPKs signaling pathways involve in infection,inflammation,immune response,cells apoptosis and tumor pathological process,cell growth and cell differentiation by regulating the expression of target gene.The magnitude and duration of MAPK activity can lead to complications such as autoimmune disease,septic shock,hypertension,and cancer.so it is important to tightly regulate MAPK activity in order to prevent potentially harmful consequences of extended MAPK function.At least ten MKPs have been identified in mammalian cells so far,with MKP1 being the archetype of the MKP family.Structurally,all MKPs have a highly conserved C-terminal catalytic domain and a less conserved N-terminal region that engages the cognate MAPKs.MKP-1 also termed dual specificity phosphatases 1(DUSP1),CL 100,VHV1,3CH134,it was the first protein phosphatase found to be specific for the MAP kinases.Electively inactivates all three MAPK families by dephosphorylating them at catalytic tyrosine and threonine residues,but it acts preferentially upon p38 and JNK.MKP-1 is encoded by immediate-early genes,is rapidly induced by many of the same stimuli that activate MAPKs,for example growth factors and stress,it has been proposed that MKP-1 function as a feedback control mechanism for MAPK signaling.so it is important to tightly regulate the expression of MKP-1 gene.The regulation of gene expression in a variety of levels,including the transcriptional level,post-transcriptional level and translational level.So far scientists pay attentions to the regulation on post-transcriptional more and more,given its ability to rapidly,precisely control gene expression.Several transcription factors(including Sp1,Sp3,and AP-1)were shown to influence the MKP-1 gene transcription in response to growth factors and stress stimuli.However,little is known about the post-transcriptional regulation of MKP-1 expression,despite the fact that MKP-1 is an early response gene.It has been found that the post-transcriptional regulation mainly includes mRNA localization,transcript stability,and the translational efficiency.Related cis-regulatory elements are mainly found in the 5'UTR(untranslated regions)and 3'UTR of transcripts.And regulatory function of 3'UTR has been intensively analyzed.Therefore,exploring entirety the mRNA 3'UTR function and regulation mechanism will contribute to better reveal the mysteries of life activities.Several studies found that RNA binding proteins are an important regulator in the post-transcription regulation which interact with mRNA 3'UTR.RNA binding proteins are a class of highly conserved and widespread protein during evolution,they specifically bind to the target RNA through its RNA binding domain,which directly or indirectly involved the pre-mRNA shearing,editing,transporting,stability,degradation and translation process.Human antigen R(human antigen R),which is an RNA binding protein,is only expressed in most of the cells generally of human embryonic lethal abnormal vision(ELAV)family.Also it can bind and stabilize the AREs in 3'UTR to stabilize mRNA stability and enhance the expression of the protein with the cis-elements together.ELAV initially was discovered to be essential for the development of the drosophila melanogaster nervous system,and it contains HuR,HuB,HuC and HuD.They mainly regulate target gene expression by post-transcriptional regulation of gene in cells.HuR,one of them,is widely expressed in mammalian cells,the others are expressed only in the nervous system.HuR is an important RNA binding protein which widely expressed in several tissues of body.By their stability and(or)the regulation of the target mRNA translational efficiency to affect the level of target gene expression in a cell,Thus participate in the regulation of cell life activities.Recent studies have revealed:HuR protein is an important regulatory factors in every life activities,such as,cell division,cell senescence,immune cell activation and differentiation of muscle.Its dysfunction is closely related to the development of inflammation,cancer and other diseases.The stability of mRNA which is one of important mechanisms for regulating gene expression in eukaryotic cells and belong to post-transcriptional gene regulation,can affect the efficiency of protein translation.HuR gene is located on human chromosome 19p13.2 region,and encodes into mature full-length mRNA about 996nt.Its protein molecular weight is approximately 36 kDa.HuR protein contains three RNA recognition motif(RRM)domains,the N-terminal RRM1 and RRM2 molecules involved in the binding with the target mRNA.HuR binding protein can specifically target mRNA 3'untranslated sequences(UTR)of the element ARE(AU rich element)by increasing the stability and(or)translation of the target mRNA and the efficiency of expression of the target gene upregulated in cells level.However,recent studies indicate that HuR protein can also negatively regulate expression of target genes in the cells,such as HuR can specifically bind to p27Kip1 mRNA 5'UTR sequence of the internal ribosome entry site(IRES),inhibition p27Kip1 protein translation.It is necessary for HuR activity to have nuclear-cytoplasmic shuttling.Under normal circumstances,HuR is mainly in the nucleus of cells.But in variety of conditions such as inflammatory cytokine stimulation,hypoxia,radiation therapy,chemotherapy and immunotherapy and other stimuli,it will be transported from the nucleus to cytoplasm through combining RRMs with mRNA 3'UTR AREs and other trans-acting factors,also HuR combine with a variety related factors mRNA involved in tumor growth,apoptosis resistance,resistance to chemotherapy,angiogenesis and lymphangiogenesis,and chang the spatial structure of RNA to inhibit deadenylation and recruit mRNA to the exonuclease or block recognition AREs in nucleic acid endonuclease activity,which in turn enhance mRNA stability,improving protein expression,ultimately promote tumor progression.The function of HuR protein in the cell is affected by phosphorylation of p38 MAPK.Activation of p38 MAPK catalyzes the phosphorylation of HuR Thr118 to quickly shift HuR from the nucleus to the cytoplasm to improve the stability of targeted mRNA.When the cells are stimulated H2O2,CHK2 kinase was activated to catalyze the phosphorylation of HuR Ser88,Ser100 and Thr118,thus affecting combination it with SIRT1 mRNA molecules.kinase it has also been reported to that CHK2 can catalytic the phosphorylation of HuR Thr118 to increase the stability of it in the cell.In addition,phosphorylation of HuR Ser202 by CDK1 kinases was also prompted to interact with 14-3-3proteins,which remain in the nucleus.PKCa catalyzes the phosphorylation of HuR Ser221 protein modification,which is crucial for the cells when they are stimulated by the ATP,HuR shift from the nucleus to the cytoplasm.In summary,we believe that it is helpful to reveal the mechanism of posttranscriptional regulation of gene expression MKP-1 to promote us learning more about the mechanism of MAPKs signal pathway,through the preliminary research on functions of MKP-1 mRNA 3'UTR and their interaction combination protein HuR.In the this study,we first examined the stability of MKP-1 mRNA and MKP-1 protein in heat shock(43 0C,5%CO2).We found that the stability of both MKP-1 mRNA and MKP-1 protein were raised.Based on the above experiment result,we further identified that HuR can interact with MKP-1 mRNA through use of biotin RNA-pull down technology.We first synthesized biotin-labeled RNA probes of MKP-1 mRNA 3'UTR by vitro transcription method,and incubated the probes with MEF cell lysates in heat shock or control.Then Western blotting detected the HuR protein while the control can't.To verify the factor of p38 signaling pathways in this process,we found MKP-1 mRNA significantly lowered after p38 interference by the real-time quantitative PCR.Finally,we have made further research on the biological function of HuR.The results show that HuR significantly phosphorylated in heat shock and up regulation of MKP-1 protein expression too.Suggesting that HuR can regulate the MKP-1 mRNA possibly through p38 signaling pathway.Meanwhile,we detected the half-life of MKP-1 mRNA and protein in p38 gene knockout MEF cells and normal respectively.We found that the half-life of MKP-1 mRNA and protein in p38 gene knockout MEF cells was significantly shorter compared to the normal group.In conclusion,we found that HuR can positive regulation of MKP-1 mRNA in heat shock and in the process,after phosphorylation of HuRT118 threonine by p38,they became stress bodies which moved from the nucleus to cytoplasm.In this study,we achieved the following results:1.We detected the half-life of MKP-1 mRNA and protein after heat shock,we found that the half-life of MKP-1 mRNA and protein was significantly extended.2.We detected the half-life of MKP-1 mRNA and protein after heat shock in p38 gene knockout MEF cells and normal respectively.we found that the half-life of MKP-1 mRNA and protein in p38 gene knockout MEF cells was significantly shorter compared to the normal group.3.After p38 suppression,the expression of MKP-1 mRNA was significantly inhibited.4.The half-life of MKP-1 mRNA and protein was prolonged after transfection HuR.5.We identified that HuR can interact with MKP-1 mRNA through use of biotin RNA-pull down technology.6.After heat shock the phosphorylation of HuRT118 threonine was significantly.7.The intracellular localization of HuR by immunofluorescence. |
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