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

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.

Deubiquitylases From Genes to Organism

“…The body of data around USP8 is more complex. Its depletion has more severe effects on the organization of the endocytic pathway that combine to inhibit EGFR downregulation (25,177,218,219). One contributing factor to such defective EGFR trafficking is that USP8 controls the stability of ESCRT-0 components, Hrs and STAM in cells and in conditional knockout mice (185,219).…”

“…Its depletion has more severe effects on the organization of the endocytic pathway that combine to inhibit EGFR downregulation (25,177,218,219). One contributing factor to such defective EGFR trafficking is that USP8 controls the stability of ESCRT-0 components, Hrs and STAM in cells and in conditional knockout mice (185,219). However, for Frizzled receptor (Fz) and Smoothened (Smo), key components of Wingless (Wnt) and Hedgehog signaling pathways, respectively, USP8 plays a negative regulatory role with regard to receptor degradation more akin to that originally proposed for AMSH (153,180,281).…”

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation

“…Although some investigators have noted an increase in total cellular EGFR when USP8 is depleted (24,28), others have determined that USP8 depletion leads to an increase in EGFR lysosomal degradation, accompanied by a decrease in EGFR protein levels in both cellular and mouse conditional knock-out models (23,36,37). USP8 has a role not only in protein degradation but also in protein trafficking.…”

“…Its highest expression is in the CA1 and dentate gyrus of the hippocampus, as well as the preoptic nucleus and the paraventricular nucleus in the diencephalon (22). Within the cell, USP8 has been found in the cytoplasm and at the endosome (23)(24)(25). USP8 degrades Lys-48-, Lys-63-, and Lys-6-linked ubiquitin chains (26,27).…”

“…USP8 has a role not only in protein degradation but also in protein trafficking. It has been shown that USP8 binds to the Src homology 3 domain of signal transducing adaptor molecule (STAM) (38) and stabilizes it (24). STAM is a component of the ESCRT0 complex.…”