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: 14.46). 06/2012; 47(3):434-43. DOI: 10.1016/j.molcel.2012.05.015
Source: PubMed

ABSTRACT 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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Folding and maturation of most protein kinases require chaperone assistance. In higher eukaryotes, CDC37 is the predominant cochaperone that facilitates the transfer of kinase clients to HSP90. Kinase recognition is thought to occur through the N-terminal domain which has thus far eluded structure determination. Client processing also requires the phosphorylation of the N-terminal tail at Ser13 by protein kinase CK2 (casein kinase 2). How phosphorylation alters the molecular properties of CDC37 is not understood. We show that the phosphorylation at Ser13 induces a large shift towards a more compact structure based on ANS fluorescence while modestly increasing secondary structure. Moreover, this transition requires interactions of the N-terminal domain and the remainder of CDC37. The stabilizing property of the phosphorylation event can be recreated in trans by a (phospho Ser13) peptide derived from the N-terminal tail. However, the phosphorylation induced transition is not dependent on the transferred phosphate group, but rather the loss of serine-like properties at position 13. The complete absence of the N-terminal tail results in reduced secondary structure and unresponsiveness to subsequent addition of peptides. The N-terminal tail may therefore serve as an intramolecular chaperone that ensures that CDC37 assumes one of two readily interconvertible states in a manner that impacts the interaction of the client binding N-domain and the MC-domains, involved in dimerization and HSP90 binding.
    Biochemistry 01/2015; DOI:10.1021/bi501129g · 3.19 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human Hsp90 isoforms are molecular chaperones that are often up-regulated in malignances and represent a primary target for Hsp90 inhibitors undergoing clinical evaluation. Hsp90α is a stress-inducible isoform of Hsp90 that plays a significant role in apoptosis and metastasis. Though Hsp90α is secreted into the extracellular space under metastatic conditions, its role in cancer biology is poorly understood. We report that Hsp90α associates with the Aha1 co-chaperone and found this complex to localize in secretory vesicles and at the leading edge of migrating cells. Knockdown of Hsp90α resulted in a defect in cell migration. The functional role of Hsp90α/Aha1 was studied by treating the cells with various novobiocin-based Hsp90 C-terminal inhibitors. These inhibitors disrupted the Hsp90α/Aha1 complex, caused a cytoplasmic redistribution of Hsp90α and Aha1, and decreased cell migration. Structure-function studies determined that disruption of Hsp90α/Aha1 association and inhibition of cell migration correlated with the presence of a benzamide side chain, as an acetamide substituted analog was less effective. Our results show that disruption of Hsp90α/Aha1 interactions with novobiocin-based Hsp90 C-terminal inhibitors may limit the metastatic potential of tumors.
    ACS Chemical Biology 11/2014; 10(2). DOI:10.1021/cb5008713 · 5.36 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In eukaryotes, the molecular chaperones Hsp90 and Hsp70 are connected via the co-chaperone Sti1/Hop, which allows transfer of clients. Here, we show that the basic functions of yeast Sti1 and human Hop are conserved. These include the simultaneous binding of Hsp90 and Hsp70, the inhibition of the ATPase activity of Hsp90, and the ability to support client activation in vivo. Importantly, we reveal that both Hop and Sti1 are subject to inhibitory phosphorylation, although the sites modified and the influence of regulatory phosphorylation is species specific. Phospho-mimetic variants have a reduced ability to activate clients in vivo and different affinity for Hsp70. Hop is more tightly regulated, as phosphorylation affects also the interaction with Hsp90 and induces structural rearrangements in the core part of the protein.
    EMBO Reports 12/2014; 16(2). DOI:10.15252/embr.201439198 · 7.86 Impact Factor


Available from
May 27, 2014