The Molecular Mechanism Of Orphan Receptor TR3 Phosphorylation Induced By C-Jun NH2-terminal Protein Kinase (JNK)

c-Jun N-terminal kinase(JNK) family is one of members of mitogen-activated protein kinase(MAPK) superfamily.The JNK signal pathway can be activated by cytokines,growth factors,stresses,and so on.Many studies suggest that the JNK signal pathway plays a pivotal role in cell differentiation,apoptosis,stress,the initiation and progress of considerable human diseases.Orphan receptor TR3,a member of nuclear receptor superfamily,is a product of immediate early gene which is expressed rapidly after induced by several proliferation factors,such as EGF,FGF,NGF,PDGF,phorbol ester,and different apoptosis inducers.It is similar in structure with the members of steroid/retinoid receptor superfamily that all have a DNA binding region of two zinc fingers and a ligand binding region.As a transcription factor,TR3 is involved in proliferation, differentiation and apoptosis of cells.In our previous study,we found that JNK activator anisomycin can induce TR3 phosphorylation.After constructing various TR3 deletion mutants(TR3ΔC574,TR3ΔN98,TR3ΔN50,TR3ΔN74,TR3ΔN84,TR3ΔN92),we identified the JNK phosphorylation site located in the region of aa 93-98 in TR3(93-AT S*P AS-98), around which a classic JNK phosphorylation motif-S*P exists.In the present study, we further demonstrate that anisomycin induced TR3 phosphorylation through JNK1 rather than p38 and ERK signals,which is mediated by its upstream factors MAPK kinase 4 and MAPK kinase 7.We also mutated the serine into araine at aa 95 site,and confirmed that the precise JNK phosphorylation site is serine95.Furthermore,we demonstrate that TR3 phosphorylation by JNK coincided with its ubiquitination and degradation,resulting in the loss of its mitogenic activity in HEK293T cells.Finally, we showed that JNK-induced phosphorylation blocked the DNA binding property of TR3 and hence diminished its transactivation activity.In conclusion,we,for the first time,have not only determined the precise phosphorylation site on TR3 by JNK,but also indicated the correlation between phosphorylation and ubiquitination,and some signal pathways for TR3 regulated by JNK through phosphorylation and ubiquitination.Our study provides the basis for further studies of interaction between TR3 and JNK in the development of carcinoma and for the screening new drugs to cure TR3-dependent tumors.