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The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation.

ABSTRACT: It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr(115)/Thr(425)/Thr(603), of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a 'phosphorylation switch' for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.

SUBMITTER: Lu XA

PROVIDER: S-EPMC3927929 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Targeting the Hsp90-Cdc37-client protein interaction to disrupt Hsp90 chaperone machinery.

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Project description:Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature.

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Project description:Maintaining a healthy proteome is fundamental for the survival of all organisms1. Integral to this are Hsp90 and Hsp70, molecular chaperones that together facilitate the folding, remodelling and maturation of the many 'client proteins' of Hsp902. The glucocorticoid receptor (GR) is a model client protein that is strictly dependent on Hsp90 and Hsp70 for activity3-7. Chaperoning GR involves a cycle of inactivation by Hsp70; formation of an inactive GR-Hsp90-Hsp70-Hop 'loading' complex; conversion to an active GR-Hsp90-p23 'maturation' complex; and subsequent GR release8. However, to our knowledge, a molecular understanding of this intricate chaperone cycle is lacking for any client protein. Here we report the cryo-electron microscopy structure of the GR-loading complex, in which Hsp70 loads GR onto Hsp90, uncovering the molecular basis of direct coordination by Hsp90 and Hsp70. The structure reveals two Hsp70 proteins, one of which delivers GR and the other scaffolds the Hop cochaperone. Hop interacts with all components of the complex, including GR, and poises Hsp90 for subsequent ATP hydrolysis. GR is partially unfolded and recognized through an extended binding pocket composed of Hsp90, Hsp70 and Hop, revealing the mechanism of GR loading and inactivation. Together with the GR-maturation complex structure9, we present a complete molecular mechanism of chaperone-dependent client remodelling, and establish general principles of client recognition, inhibition, transfer and activation.

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