The Ubiquitin Isopeptidase UBPY Regulates Endosomal Ubiquitin Dynamics and Is Essential for Receptor Down-regulation

Paula E Row,Ian A Prior,John Mccullough,Michael J Clague,Sylvie Urbé

doi:10.1074/jbc.m512615200

Abstract

UBPY is a ubiquitin-specific protease that can deubiquitinate monoubiquitinated receptor tyrosine kinases, as well as process Lys-48- and Lys-63-linked polyubiquitin to lower denomination forms in vitro. Catalytically inactive UBPY localizes to endosomes, which accumulate ubiquitinated proteins. We have explored the sequelae of short interfering RNA-mediated knockdown of UBPY. Global levels of ubiquitinated protein increase and ubiquitin accumulates on endosomes, although free ubiquitin levels are unchanged. UBPY-depleted cells have more and larger multivesicular endosomal structures that are frequently associated through extended contact areas, characterized by regularly spaced, electron-dense, bridging profiles. Degradation of acutely stimulated receptor tyrosine kinases, epidermal growth factor receptor and Met, is strongly inhibited in UBPY knockdown cells suggesting that UBPY function is essential for growth factor receptor down-regulation. In contrast, stability of the UBPY binding partner STAM is dramatically compromised in UBPY knockdown cells. The cellular functions of UBPY are complex but clearly distinct from those of the Lys-63-ubiquitin-specific protease, AMSH, with which it shares a binding site on the SH3 domain of STAM.

Highlights

Classical studies elucidated polyubiquitin chains linked through an internal lysine (Lys-48) as a proteasomal degradation signal [11,12,13]
Substrate Specificity of ubiquitin-specific processing protease Y (UBPY)—We have adapted an in vitro assay for deubiquitinating enzymes (DUBs) activity that monitors the processing of polyubiquitin chains to lower denomination forms [22, 30]
associated molecule with the SH3-domain of signal transducing adapter molecule (STAM) (AMSH) showed specificity for Lys-63-linked, whereas UBPY showed specificity for Lys-48-linked polyubiquitin [22]. This observation came with the caveat that the Lys-63-linked chains available to us at that time were derived from mutant ubiquitin in which all lysines, with the exception of Lys-63, had been converted to arginines. We have repeated these experiments with polyubiquitin chains derived from wild-type ubiquitin and find that in contrast to AMSH, UBPY shows little discrimination between Lys-48 and Lys-63-linked chains (Fig. 1)

Summary

Introduction

Classical studies elucidated polyubiquitin chains linked through an internal lysine (Lys-48) as a proteasomal degradation signal [11,12,13]. In this paper we show that knockdown of UBPY by RNA interference has multiple cellular effects that include the accumulation of ubiquitinated proteins on endosomes, an increase in both number and size of multivesicular endosomes, and a block in RTK degradation.

Results

Conclusion