Date on Master's Thesis/Doctoral Dissertation

12-2008

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Pharmacology and Toxicology

Committee Chair

Gozal, Evelyne

Author's Keywords

Cell survival; Hsp90; Geldanamycin

Subject

Protein kinases; Protein binding

Abstract

The chaperone heat shock protein 90 (Hsp90) regulates physiologically and pathological cellular processes, by binding and stabilizing kinases involved in basal cellular functions and in cellular responses to stress, respectively. I hypothesize that Hsp90 binding to kinases Akt and Raf-l and to co-chaperone Cdc37 and Akt-dependent phosphorylation 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 role in cell survival. Indeed, GA cytotoxicity is attributed to disruption of Hsp90 binding, although the role of this drug's benzoquinone in its cytotoxicity was never studied. This study used GA, the antioxidant precursor N-acetyl cysteine (NAC), and the classical quinone menadione (MEN), to shows that oxidative stress and disruption of Hsp90 binding contribute to GA cytotoxicity. In addition, Hsp90 binding promotes survival by regulating protein degradation. Proteasomal inhibition prevented MEN-induced protein degradation, but failed to inhibit GA-induced protein degradation. Thus, GA induces cytotoxicity by early disruption of Hsp90 binding, followed by oxidative stress~induced non~proteasomal protein degradation. Additional factors, such as Akt~dependent phosphorylation of Hsp90, may promote survival by regulating Hsp90 binding. Akt phosphorylates Hsp90 in vitro and in PC~12 cells expressing active Akt or exposed to 6h SH, concomitant with increased Akt phosphorylation. Proteomic analysis of Hsp90 immunoprecipitates identified additional Hsp90~binding proteins that may be recruited to and released from the Hsp90 complex in response to 6h SH. Most identified Hsp90 binding proteins dissociate in response to Akt inhibition by the Akt inhibitor, Akti 112, suggesting that Akt phosphorylation regulates the protein associations of the Hsp90 complexes. Survival studies with Akti 112 demonstrate that basal Akt phosphorylation, but not the 6h SH~induced increase in Akt phosphorylation is critical to survival. However, constitutive Akt phosphorylation is not sufficient to prevent death at 24h SH, suggesting additional factors are required for survival to SH. In summary, Akt~dependent phosphorylation of Hsp90 regulates protein binding and PC~ 12 cell survival.

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