Dynamic tyrosine phosphorylation modulates cycling of the HSP90-P50(CDC37)-AHA1 chaperone machine

Urologic Oncology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Molecular cell (Impact Factor: 14.02). 06/2012; 47(3):434-43. DOI: 10.1016/j.molcel.2012.05.015
Source: PubMed


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 cochaperone p50(Cdc37), Hsp90 proceeds through a cycle of conformational changes facilitated by ATP binding and hydrolysis. Progression through the chaperone cycle requires release of p50(Cdc37) and recruitment of the ATPase activating cochaperone 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 p50(Cdc37) and Hsp90, are minimally sufficient to provide directionality to the chaperone cycle. p50(Cdc37) phosphorylation on Y4 and Y298 disrupts client-p50(Cdc37) association, while Hsp90 phosphorylation on Y197 dissociates p50(Cdc37) 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 cochaperones.

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Available from: Chrisostomos Prodromou, Sep 30, 2015
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    • "It is noteworthy that isolated hAha1 and the mutant proteins had no contaminating ATPase activity (Figure S4D). We previously demonstrated that hAha1 co-exists in an hHsp90-kinase client complex (Xu et al., 2012). Therefore, we assessed the impact of Y223F and Y223E mutations on hAha1 interaction with kinase clients. "
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    ABSTRACT: The ability of Heat Shock Protein 90 (Hsp90) to hydrolyze ATP is essential for its chaperone function. The co-chaperone Aha1 stimulates Hsp90 ATPase activity, tailoring the chaperone function to specific "client" proteins. The intracellular signaling mechanisms directly regulating Aha1 association with Hsp90 remain unknown. Here, we show that c-Abl kinase phosphorylates Y223 in human Aha1 (hAha1), promoting its interaction with Hsp90. This, consequently, results in an increased Hsp90 ATPase activity, enhances Hsp90 interaction with kinase clients, and compromises the chaperoning of non-kinase clients such as glucocorticoid receptor and CFTR. Suggesting a regulatory paradigm, we also find that Y223 phosphorylation leads to ubiquitination and degradation of hAha1 in the proteasome. Finally, pharmacologic inhibition of c-Abl prevents hAha1 interaction with Hsp90, thereby hypersensitizing cancer cells to Hsp90 inhibitors both in vitro and ex vivo. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 07/2015; 12(6). DOI:10.1016/j.celrep.2015.07.004 · 8.36 Impact Factor
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    • "By contrast, indirect analysis based on the inability of PP5 phosphatase to bind the Hsp90-heme-regulated inhibitor of protein synthesis (HRI) has shown that chaperone phosphorylation enhances HRI activity (Shao et al. 2002). Phosphorylation of tyrosine 197 in human Hsp90α (Y186 in TgHsp90, Table 1) dissociates cdc37 from Hsp90 (Xu et al. 2012). This study also showed that mutation of Y627 (Y604 in T. gondii, Table 1) favours the release of client Cdk4 and co-chaperones (AHA1 and PP5). "
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    ABSTRACT: SUMMARY Hsp90 is a widely distributed and highly conserved molecular chaperone that is ubiquitously expressed throughout nature, being one of the most abundant proteins within non-stressed cells. This chaperone is up-regulated following stressful events and has been involved in many cellular processes. In Toxoplasma gondii, Hsp90 could be linked with many essential processes of the parasite such as host cell invasion, replication and tachyzoite-bradyzoite interconversion. A Protein-Protein Interaction (PPI) network approach of TgHsp90 has allowed inferring how these processes may be altered. In addition, data mining of T. gondii phosphoproteome and acetylome has allowed the generation of the phosphorylation and acetylation map of TgHsp90. This review focuses on the potential roles of TgHsp90 in parasite biology and the analysis of experimental data in comparison with its counterparts in yeast and humans.
    Parasitology 02/2014; 141(9):1-10. DOI:10.1017/S0031182014000055 · 2.56 Impact Factor
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    • "Although SMT3-MYC overexpression increased yHsp90 interaction with yAha1, association of the cochaperone with Hsp90 was not detectable in cells treated with GA prior to lysis (Figure S5B). Finally, we showed earlier (Figure 3D) that yHsp90-E33A (which binds ATP but does not hydrolyze it) is SUMOylated markedly more than wild-type yHsp90, and the equivalent mutation in hHsp90a-E47A promotes increased steady-state interaction with Aha1 in cells (Xu et al., 2012). However , GA-affinity bead pull-down of hHsp90a-E47A was less efficient than that of the wild-type protein, and Aha1 was not present in either pull-down (Figure S5C), consistent with the hypothesis that Hsp90 inhibitors and Aha1 cannot interact concurrently with N domain SUMOylated Hsp90. "
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    ABSTRACT: The stability and activity of numerous signaling proteins in both normal and cancer cells depends on the dimeric molecular chaperone heat shock protein 90 (Hsp90). Hsp90's function is coupled to ATP binding and hydrolysis and requires a series of conformational changes that are regulated by cochaperones and numerous posttranslational modifications (PTMs). SUMOylation is one of the least-understood Hsp90 PTMs. Here, we show that asymmetric SUMOylation of a conserved lysine residue in the N domain of both yeast (K178) and human (K191) Hsp90 facilitates both recruitment of the adenosine triphosphatase (ATPase)-activating cochaperone Aha1 and, unexpectedly, the binding of Hsp90 inhibitors, suggesting that these drugs associate preferentially with Hsp90 proteins that are actively engaged in the chaperone cycle. Importantly, cellular transformation is accompanied by elevated steady-state N domain SUMOylation, and increased Hsp90 SUMOylation sensitizes yeast and mammalian cells to Hsp90 inhibitors, providing a mechanism to explain the sensitivity of cancer cells to these drugs.
    Molecular cell 01/2014; 53(2):317-29. DOI:10.1016/j.molcel.2013.12.007 · 14.02 Impact Factor
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