Molecular Mechanisms Of STIP In Regulating Mdm2-p53Pathway

STIP (septin and tuftelin-interacting proteins) protein is a kind of nucleoproteinscontaining multiple structural domains including G-patch, coiled-coil and six Try-Try repeats.STIP is highly homologous from worms to human. Overexpression of STIP canself-assembled into rod-liked macromolecular structure in nucleus. Knockdown of STIP byRNAi results in the stagnation of early embryonic development in16cells in C.elegans. Thedeadly phenotype can be rescued by re-expression of human or Drosophila stip gene, thissuggests that STIP has very important conservative function in biological evolution. However,its molecular function has not fully elucidated as yet.As an important transcription factor, tumor suppressor p53regulate the expression ofmore than150genes. In the face of the stress signals such as cancer gene activation and DNAdamage, p53play its important transcription activation function, then lead to a series of stressreactions such as cell cycle arrest, apoptosis, and DNA damage repair. On the other hand, theactivation, expression and location of p53are regulated by posttranslational modification.Among then, the intracellular level of p53is tightly controlled by the ubiquitin-proteasomepathway. E3ubiquitin ligase Mdm2is one of the most important molecules which areinvolved in the p53ubiquitination modification. The N-terminal of Mdm2interacted with theN-terminal Zn finger domain and the C-terminal RING domain of p53and connected theubiquitin chains to p53, by that p53can be identified and degraded through proteasomepathway. Mdm2also can connect single ubiquitin to p53, which can induce p53nuclearoutput. However, Mdm2is the target molecule of p53. Its transcription level is directregulated by p53, which formed Mdm2-p53feedback loop.Our studies show the expression levels of, STIP has certain correlation with cellularMdm2and p53. With the same as exogenous STIP, endogenous STIP localized in nucleus andassembled into stiposome. In order to define whether STIP participate in the regulation ofMdm2-p53pathway, we constructed Flag-Mdm2and Flag-p53plasmid. We found thatoverexpression of STIP strongly raise Mdm2and p53protein levels in U2OS, H1299,HCT116(p53wt) and HCT116(p53-/-), whereas downregulation of STIP decreased Mdm2andp53levels in dose-dependent manner. To define that STIP influence Mdm2and p53in theprotein level or mRNA level, we detected the mRNA of Mdm2and p53in overexpression ordownregulation of STIP by real-time PCR assay respectively. It revealed that STIP had nosignificant influence on mRNA of p53and mdm2. Furthermore, when cells were treated withproteasome inhibitor MG132, STIP lost the influence on Mdm2and p53, which suggests that STIP may regulate the protein level of Mdm2and p53through the ubiquitin-proteasomepathway. Our findings revealed that overexpression of STIP reduced the ubiquitination ofMdm2and p53, extended the half-life of Mdm2and p53, whereas downregulation of STIPincrease the ubiquitination of Mdm2and p53, shorten the half-life of Mdm2and p53, whichsuggest STIP stabilize the Mdm2and p53by regulating the ubiquitination of Mdm2and p53.Considering that the opposite biological function of Mdm2and p53, we used clone-formationassay and MTT assay to reveal STIP’s biological function after stabilized Mdm2and p53,then find that STIP can promote the growth of tumor cells. The clarification of molecularmechanism of STIP regulate Mdm2-p53pathway and uncover of STIP’s biological functionsbreak the ground in this field, provide a theoretical basis for further systematic research ofSTIP and rich related theory of Mdm2-p53pathways. The relationship between STIP andMdm2-p53provides new targets for early diagnosis and drug treatment of human tumor.