The MIT Domain of UBPY Constitutes a CHMP Binding and Endosomal Localization Signal Required for Efficient Epidermal Growth Factor Receptor Degradation

Paula E Row,Han Liu,Sebastian Hayes,Rebecca Welchman,Panagoula Charalabous,Kay Hofmann,Michael J Clague,Christopher M Sanderson,Sylvie Urbé

doi:10.1074/jbc.m704009200

Abstract

We have identified and characterized a Microtubule Interacting and Transport (MIT) domain at the N terminus of the deubiquitinating enzyme UBPY/USP8. In common with other MIT-containing proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7. We also show that in common with AMSH, UBPY deubiquitinating enzyme activity can be stimulated by STAM but is unresponsive to its cognate CHMPs. The UBPY MIT domain is dispensable for its catalytic activity but is essential for its localization to endosomes. This is functionally significant as an MIT-deleted UBPY mutant is unable to rescue its binding partner STAM from proteasomal degradation or reverse a block to epidermal growth factor receptor degradation imposed by small interfering RNA-mediated depletion of UBPY.

Highlights

Lysosomal degradation rates determine the levels of cell surface receptor tyrosine kinases, an important parameter in the control of cell growth [1, 2]
Tel.: 49-151-794-5432; Fax: 44-151-794-4434; E-mail: urbe@ liv.ac.uk. 3 The abbreviations used are: MVB, multivesicular body; VPS, vacuolar protein-sorting; ESCRT, endosomal sorting complex required for transport; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; STAM, signal-transducing adaptor molecule; deubiquitinating enzymes (DUBs), deubiquitinating enzyme; siRNA, small interfering RNA; EGF, epidermal growth factor; Microtubule Interacting and Transport (MIT), microtubule-interacting and transport domain; GFP, green fluorescent protein; GST, glutathione S-transferase; HEK, human embryonic kidney; PBS, phosphate-buffered saline
UBPY Contains an MIT Domain—Fig. 1A shows an alignment of the N-terminal region of UBPY with VPS4 and other MIT domain-containing proteins, suggesting that like the other STAM binding DUB, AMSH, UBPY contains an MIT domain

Summary

Introduction

Lysosomal degradation rates determine the levels of cell surface receptor tyrosine kinases, an important parameter in the control of cell growth [1, 2]. In common with other MITcontaining proteins such as AMSH and VPS4, UBPY can interact with CHMP proteins, which are known to regulate endosomal sorting of ubiquitinated receptors. Comparison of binding preferences for the 11 members of the human CHMP family between the UBPY MIT domain and another ubiquitin isopeptidase, AMSH, reveals common interactions with CHMP1A and CHMP1B but a distinct selectivity of AMSH for CHMP3/VPS24, a core subunit of the ESCRT-III complex, and UBPY for CHMP7.

Results

Conclusion