HECTD3 Mediates an HSP90-Dependent Degradation Pathway for Protein Kinase Clients

Zhaobo Li,Lihong Zhou,Chrisostomos Prodromou,Velibor Savic,Laurence H Pearl

doi:10.1016/j.celrep.2017.05.078

Zhaobo Li, Lihong Zhou + Show 3 more

Open Access

https://doi.org/10.1016/j.celrep.2017.05.078

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Abstract

SummaryInhibition of the ATPase cycle of the HSP90 chaperone promotes ubiquitylation and proteasomal degradation of its client proteins, which include many oncogenic protein kinases. This provides the rationale for HSP90 inhibitors as cancer therapeutics. However, the mechanism by which HSP90 ATPase inhibition triggers ubiquitylation is not understood, and the E3 ubiquitin ligases involved are largely unknown. Using a siRNA screen, we have identified components of two independent degradation pathways for the HSP90 client kinase CRAF. The first requires CUL5, Elongin B, and Elongin C, while the second requires the E3 ligase HECTD3, which is also involved in the degradation of MASTL and LKB1. HECTD3 associates with HSP90 and CRAF in cells via its N-terminal DOC domain, which is mutationally disrupted in tumor cells with activated MAP kinase signaling. Our data implicate HECTD3 as a tumor suppressor modulating the activity of this important oncogenic signaling pathway.

Highlights

The HSP90 molecular chaperone is responsible for the stabilization and biological activity of a diverse set of ‘‘clients,’’ including clinically important proteins such as nuclear hormone receptors and a broad range of protein kinases (Taipale et al, 2010)
A Fluorescence-Based Assay for Client Protein Degradation The proto-oncogene kinases BRAF and CRAF are well-documented HSP90 client proteins that have previously been shown to be ubiquitylated and degraded in tumor cell lines treated with the HSP90 inhibitor AUY922 (Sharp et al, 2007)
AUY922, like other HSP90 inhibitors, strongly inhibits cell growth and promotes apoptosis in tumor cell lines such as HT29 and HCT116, which are addicted to mitogen-activated protein kinase (MAPK) signaling mediated by RAF kinases, potentially confounding reliable measurement of protein levels

Summary

Introduction

The HSP90 molecular chaperone is responsible for the stabilization and biological activity of a diverse set of ‘‘clients,’’ including clinically important proteins such as nuclear hormone receptors and a broad range of protein kinases (Taipale et al, 2010). The involvement of the HSP90 (heat shock protein 90) system in the cellular stabilization of oncogenic protein kinases such as ErbB2, BRaf-V600E, FGFR-G719S, BCR-ABL, and EML4-ALK has marked it as a prime target for drug discovery, and a number of potent HSP90 inhibitors are at various stages of clinical trial in a range of tumor types (Neckers and Workman, 2012) These compounds act as competitive inhibitors of ATP binding to the N-terminal domain of the chaperone molecule, blocking the ATPase-coupled conformational cycle that is essential for HSP90s activity (Ali et al, 2006; Panaretou et al, 1998; Prodromou et al, 2000). Whether this proceeds through the same pathway as the HSP90-inhibitor-triggered degradation is uncertain

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