SUMMARY Many critical protein kinases rely on the Hsp90 chaperone machinery for stability and function. After initially forming a ternary complex with kinase client and the co-chaperone p50Cdc37, Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50Cdc37 and recruitment of the ATPase activating co-chaperone AHA1, but the molecular regulation of this complex process at the cellular level is poorly understood. We demonstrate that a series of tyrosine phosphorylation events, involving both p50Cdc37 and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50Cdc37 phosphorylation on Y4 and Y298 disrupts client-p50Cdc37 association, while Hsp90 phosphorylation on Y197 dissociates p50Cdc37 from Hsp90. Hsp90 phosphorylation on Y313 promotes recruitment of AHA1 which stimulates Hsp90 ATPase activity, furthering the chaperoning process. Finally, at completion of the chaperone cycle, Hsp90 Y627 phosphorylation induces dissociation of the client and remaining co-chaperones.

Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA.

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Recent studies have identified development of resistance to tyrosine kinase inhibition (TKI) as a significant roadblock to effective treatment. One mechanism of resistance recently appreciated involves 'oncogene switching', or the re-activation of signaling pathways by one or more redundant upstream activators. In breast cancer models, ErbB TKIs such as gefitinib have been shown to lose the ability to modulate ErbB-driven signaling pathways over time, even though ErbB inhibition is maintained. Although incomplete ErbB inhibition has been proposed to underlie this phenomenon, our findings suggest that oncogene switching can also re-activate downstream signaling pathways in breast cancer cells, even when ErbB inhibition is complete. We find that ErbB TKI-induced Src activation mediates downstream signaling rebound in SKBR3 cells, and we show that combination of Src and ErbB inhibitors is more effective and longlasting than is either TKI alone. Finally, the Hsp90 inhibitor 17-AAG, by simultaneously and durably inhibiting multiple signaling activators including ErbB and Src kinases, does not permit oncogene switching and results in a more prolonged and robust inhibition of downstream signaling pathways in breast cancer cells than do individual TKIs. These data support the continued clinical evaluation of Hsp90 inhibitors in breast cancer.