The role of Hsp90 in PC-12 cell survival.

Cell survival;Hsp90;Geldanamycin

Christina Blume Clark

University:University of Louisville

Department:Pharmacology and Toxicology

关键词: Cell survival;    Hsp90;    Geldanamycin;   

Others  :  https://ir.library.louisville.edu/cgi/viewcontent.cgi?article=1256&context=etd

美国|英语

来源: The Universite of Louisville's Institutional Repository

【 摘 要 】

The chaperone heat shock protein 90 (Hsp90) regulates physiologically andpathological cellular processes, by binding and stabilizing kinases involved in basalcellular functions and in cellular responses to stress, respectively. I hypothesize thatHsp90 binding to kinases Akt and Raf-l and to co-chaperone Cdc37 and Akt-dependentphosphorylation of Hsp90 regulate PC-12 cell survival.Hsp90 binding was inhibited using the classical Hsp90 inhibitor, Geldanamycin(GA). Disruption of Hsp90 binding by GA correlated with similar cell death at normoxia(RA) and at 0.1 % O2 sustained hypoxia (SH), suggesting that Hsp90 binding plays a rolein cell survival.Indeed, GA cytotoxicity is attributed to disruption of Hsp90 binding, although therole of this drug's benzoquinone in its cytotoxicity was never studied. This study usedGA, the antioxidant precursor N-acetyl cysteine (NAC), and the classical quinonemenadione (MEN), to shows that oxidative stress and disruption of Hsp90 bindingcontribute to GA cytotoxicity. In addition, Hsp90 binding promotes survival byregulating protein degradation. Proteasomal inhibition prevented MEN-induced proteindegradation, but failed to inhibit GA-induced protein degradation. Thus, GA induces cytotoxicity by early disruption of Hsp90 binding, followed by oxidative stress~inducednon~proteasomal protein degradation.Additional factors, such as Akt~dependent phosphorylation of Hsp90, maypromote survival by regulating Hsp90 binding. Akt phosphorylates Hsp90 in vitro and inPC~12 cells expressing active Akt or exposed to 6h SH, concomitant with increased Aktphosphorylation. Proteomic analysis of Hsp90 immunoprecipitates identified additionalHsp90~binding proteins that may be recruited to and released from the Hsp90 complex inresponse to 6h SH. Most identified Hsp90 binding proteins dissociate in response to Aktinhibition by the Akt inhibitor, Akti 112, suggesting that Akt phosphorylation regulates theprotein associations of the Hsp90 complexes. Survival studies with Akti 112 demonstratethat basal Akt phosphorylation, but not the 6h SH~induced increase in Aktphosphorylation is critical to survival. However, constitutive Akt phosphorylation is notsufficient to prevent death at 24h SH, suggesting additional factors are required forsurvival to SH.In summary, Akt~dependent phosphorylation of Hsp90 regulates protein bindingand PC~ 12 cell survival.