Activation Of The Stat1 Thermally Induced Activation And Its Study On The Mechanism Of Hsp90 Gene Regulation

Heat shock protein 90 (Hsp90) is one of the most popular proteins in eukaryotic cell.It can refold the misfolded proteins produced during heat shock and facilitate thedegradation of denatured proteins. Hsp90 also helps the maturation, transportation andactivity regulation of some hormone receptors, transcription factors, kinases and nitrogenmonoxide synthase. Thus Hsp90 plays a wide and important role in the cells, and itsexpression regulation study is of physical importance.There are two closely related cytoplasm isoforms of Hsp90 in mammalian cells,termed Hsp90αand Hsp90β, encoded by hsp90αand hsp90βgenes independently mappedto human chromosome 14 and chromosome 6 respectively, and regulated independently.Statl is a tumor suppressor in the signal transducers and activators of transcription(STAT) family. Statl was known to be phosphorylated in response to stress, IFN-γ, UV,TNF-αtreatment. However, the kinase that phosphorylated Statl-Tyr701 and Ser727 underheat shock treatment was perplexed, as a variety of kinases were known to be active, whilethey were each in the downstream of a specific intracellular signal transduction pathways.In this study, we provide the first evidence for the existence of two non-classical, heatshock-associated signaling pathways involving Jak2 and PKCε, which are activated underheat shock and converged on the phosphorylation of Statl. We present the first evidence ofthat the two SWI/SNF chromatin remodeling complexes work coordinately in regulatingthe responsiveness of its target gene, in which a Statl dependent switching from hBrm toBrgl is critical for the heat shock response.1. The signaling pathway involved in heat shock induced activation of Statl.As the phosphorylation on Tyr701 and Ser727 is prerequisite for Statl activation, weexamined and showed that both Tyr701 and Ser727 of the Statl were phosphorylated uponheat shock while the total amount of Statl was kept constant. We showed that ectopicStatl-M cells exhibited a G2/M arrest even before heat-shock. After 24h of recovery, 2/3 of the Statl-M cells were accumulated in G2/M that was unable to resume the normal cellcycle. Geldanamycin (GA), an inhibitor of Hsp90 was used to treat the cells before heatshock; we showed that the heat shock induced phosphorylation on both Statl-Tyr701 andSer727 was significantly inhibited. GA could completely abolished heat shock inducedactivation of Jak2 and the heat shock-induced membrane translocation of PKCεandHsp90β, which indicating Hsp90 was required for heat shock induced activation of Jak2and PKCε.Co-immunoprecipitation assays showed that Statl existed in complex with Jak2 andin the complex with PKCεduring heat shock. In heat shock Jurkat cells, phosphorylatedStatl is a prerequisite for the efficient expression of hsp90βgene, while thephosphorylation of Statl required Hsp90βto activate its upstream kinases (Jak2 andPKCε).2. Opposite effects of Statl on the regulation of hsp90 genes under heat shockGA inhibited the heat shock induced activity of the hsp90βpromoter whereas itenhanced that of the hsp90αpromoter. It was found that under heat shock conditionDN-Jak2 enhanced the promoter activity of hsp90αgene, while it significantly reducedthat of hsp90βgene. Upon heat shock, expression of PKCεRNAi construct enhanced heatshock-induced activity of the hsp90αpromoter but inhibited that of the hsp90βpromoter.Statl displayed opposite effects on the induction of hsp90 genes by heat shock. Expressionof wild-type Statl suppressed hsp90αbut enhanced hsp90βtranscription. In contrast,phosphorylation-deficient mutants of Statl (Statl^Y701F, Statl^S727A, or Stat^Y/F,S/A)significantly increased heat shock induction of the hsp90αgene but severely suppressedthat of hsp90βgene.The results indicate heat shock induced expression of hsp90βgene is regulated by apositive feedback loop involving Hsp90β-dependent activation of Statl, which in turnensure appropriate functions of Statl in a heat shock response. We found that Statlregulates the heat shock induction of hsp90αgene in diverse effects. The status and efficacy of Statl bound at the GAS (interferon Gamma Activated Sequence) of its targetgene are pivotal in determining the impact of Statl under heat shock.In this study, we provide the first evidence regarding heat shock induced expression ofhsp90βgene is regulated by a positive feedback loop involving Hsp90β-dependentactivation of Statl, which in turn ensure appropriate functions of Statl in a heat shockresponse. We found that Statl regulates the heat shock induction of hsp90αgene in diverseeffects.