Heat Shock Protein 90 Chaperone Regulates the E3 Ubiquitin-Ligase Hakai Protein Stability.

Díaz-Díaz Díaz-Díaz,Casas-Pais Casas-Pais,Graña Graña,Figueroa Figueroa,Romay Romay,Concha Concha,Colombo Colombo,Roca-Lema Roca-Lema

doi:10.3390/cancers12010215

Abstract

The E3 ubiquitin-ligase Hakai binds to several tyrosine-phosphorylated Src substrates, including the hallmark of the epithelial-to-mesenchymal transition E-cadherin, and signals for degradation of its specific targets. Hakai is highly expressed in several human cancers, including colon cancer, and is considered as a drug target for cancer therapy. Here, we report a link between Hakai and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the correct folding of its client proteins, allowing them to maintain their stability and activity. Hsp90 inhibitors specifically interfere with the association with its Hsp90 client proteins, and exhibit potent anti-cancer properties. By immunoprecipitation, we present evidence that Hakai interacts with Hsp90 chaperone complex in several epithelial cells and demonstrate that is a novel Hsp90 client protein. Interestingly, by overexpressing and knocking-down experiments with Hakai, we identified Annexin A2 as a Hakai-regulated protein. Pharmacological inhibition of Hsp90 with geldanamycin results in the degradation of Hakai in a lysosome-dependent manner. Interestingly, geldanamycin-induced Hakai degradation is accompanied by an increased expression of E-cadherin and Annexin A2. We also show that geldanamycin suppresses cell motility at least in part through its action on Hakai expression. Taken together, our results identify Hakai as a novel Hsp90 client protein and shed light on the regulation of Hakai stability. Our results open the possibility to the potential use of Hsp90 inhibitors for colorectal cancer therapy through its action on Hakai client protein of Hsp90.

Highlights

heat shock protein 90 (Hsp90) (90-KDa heat shock protein) is a molecular chaperone involved in the correct folding of a selected group of proteins, named as client proteins, allowing them to maintain their proper conformation and the preservation of their activity [1]
In a proteomic study conducted for the search of interacting proteins with Hsp90, Taipale and collaborators showed that, far from expected, more than 100 proteins were E3 ubiquitin-ligases [1,9]
Given that we have previously shown a regulation of Hsp70 and Hsp90 chaperones in Hakai stably expressing MDCK epithelial cells compared to non-transformed epithelial cells [21], we decided to evaluate whether Hakai could be among the E3 ubiquitin ligases interacting proteins with these chaperones

Summary

Introduction

Hsp (90-KDa heat shock protein) is a molecular chaperone involved in the correct folding of a selected group of proteins, named as client proteins, allowing them to maintain their proper conformation and the preservation of their activity [1]. The regulation of Hsp client proteins plays a crucial role in several cellular processes, such as cell cycle control, apoptosis and cell survival, and contributes to the development of pathological conditions, such as neurodegenerative and Cancers 2020, 12, 215; doi:10.3390/cancers12010215 www.mdpi.com/journal/cancers. Many client proteins of Hsp are oncogenes, mutated or overexpressed, which are important for tumor malignant progression [3]. The activation of Hsp client proteins occurs in an ATP-dependent manner [4]. The small-molecule inhibitor geldanamycin (GA), a benzoquinone ansamycin antibiotic, occupies the ATP binding-pocket of Hsp, interfering on the association with its client proteins.

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