Identification of cancer-associated missense mutations in hace1 that impair cell growth control and Rac1 ubiquitylation

Emilie Andrio,Raymond Ruimy,Romain Lotte,Jacqueline Cherfils,Anne Doye,Mads Daugaard,Frédéric Bost,Daniel Hamaoui,Emmanuel Lemichez,Poul H Sorensen,Amel Mettouchi

doi:10.1038/srep44779

Abstract

The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation.

Highlights

The abnormal expression of mammalian HECT family E3 ubiquitin-protein ligases is involved in age-related diseases notably cancer but little is known about missense mutations in these proteins and how they affect their function[1,2,3]
To gain insight into the function of the non-catalytic ANK and MID domains of HACE1 and their role in cancer, we reasoned that missense mutations identified in cancer samples in these domains may reflect defects in their functions leading to impaired control of cell proliferation
Our data identify amino acids outside the catalytic HECT domain that are essential for the control of cell proliferation by HACE1, some of which are critical for the regulation of Rac[1] by ubiquitylation

Summary

Introduction

The abnormal expression of mammalian HECT family E3 ubiquitin-protein ligases is involved in age-related diseases notably cancer but little is known about missense mutations in these proteins and how they affect their function[1,2,3]. HECT-domain and Ankyrin-repeat Containing E3 ubiquitin protein ligase 1 (HACE1) is an E3 ubiquitin ligase with an important role in cancer that was first unveiled by drawing a genetic link between hace[1] loss of expression and sporadic Wilms’ tumors[4]. This was followed by the report that mice knockout for hace[1] developed spontaneously cancers originating from the three germ layers during aging, pointing to a protective function of HACE1 in cell homeostasis[5]. The role of the ANK and MID domains in the control of Rac[1] ubiquitylation has remained poorly characterized

Methods

Results

Conclusion