Lysine 63-linked polyubiquitination is required for EGF receptor degradation

Abstract

Ubiquitination mediates endocytosis and endosomal sorting of various signaling receptors, transporters, and channels. However, the relative importance of mono-versus polyubiquitination and the role of specific types of polyubiquitin linkages in endocytic trafficking remain controversial. We used mass spectrometrybased targeted proteomics to show that activated epidermal growth factor receptor (EGFR) is ubiquitinated by one to two short (two to three ubiquitins) polyubiquitin chains mainly linked via lysine 63 (K63) or conjugated with a single monoubiquitin. Multimonoubiquitinated EGFR species were not found. To directly test whether K63 polyubiquitination is necessary for endocytosis and post-endocytic sorting of EGFR, a chimeric protein, in which the K63 linkage-specific deubiquitination enzyme AMSH [associated molecule with the Src homology 3 domain of signal transducing adaptor molecule (STAM)] was fused to the carboxyl terminus of EGFR, was generated. MS analysis of EGFR-AMSH ubiquitination demonstrated that the fraction of K63 linkages was substantially reduced, whereas relative amounts of monoubiquitin and K48 linkages increased, compared with that of wild-type EGFR. EGFR-AMSH was efficiently internalized into early endosomes, but, importantly, the rates of ligand-induced sorting to late endosomes and degradation of EGFR-AMSH were dramatically decreased. The slow degradation of EGFR-AMSH resulted in the sustained signaling activity of this chimeric receptor. Ubiquitination patterns, rate of endosomal sorting, and signaling kinetics of EGFR fused with the catalytically inactive mutant of AMSH were reversed to normal. Altogether, the data are consistent with the model whereby short K63-linked polyubiquitin chains but not multimonoubiquitin provide an increased avidity for EGFR interactions with ubiquitin adaptors, thus allowing rapid sorting of activated EGFR to the lysosomal degradation pathway.

Deubiquitylation of Protein Cargo Is Not an Essential Step in Exosome Formation

“…Combined with our observations that all classes of polyubiquitin chains (K6, K11, K27, K29, K33, K48, and K63) are detected on cargo from urinary exosomes with variable frequencies, these data support the growing body of evidence that multimonoubiquitylation and polyubiquitylation are necessary to overcome the low-affinity binding of ubiquitin to ubiquitin interaction motifs on adapter proteins and facilitate rapid internalization and endosomal sorting of particular classes of membrane proteins e.g. (27,28). Furthermore, the high abundance of K63-linked ubiquitin chains we detected on urinary exosome cargo is in line with studies in yeast and cultured mammalian cells that K63-linkage is a better signal than monoubiquitylation during endosomal sorting and may even be important for MVB biogenesis (29).…”

Proteasome‐independent functions of lysine‐63 polyubiquitination in plants

“…The presence of K63-linked chains is absolutely essential for the proper intracellular sorting of endocytosed plasma membrane proteins. Cells expressing the mutant Ub K63R as a sole source of Ub, or translational fusion of the cargo to a K63-specific DUB, results in mistargeting or defects in protein degradation in the vacuole/lysosome Paiva et al, 2009;Huang et al, 2013). Ubiquitinated proteins are recognized in endosomes by the ESCRT complex (Hurley & Ren, 2009).…”

Proteomic Analysis of the Epidermal Growth Factor Receptor (EGFR) Interactome and Post-translational Modifications Associated with Receptor Endocytosis in Response to EGF and Stress

“…4B). The K 48 linkage has been associated mainly with receptor signaling rather than degradation (14,26,53). Ubiquitination at EGFR K 875 increased only 2.5-fold after EGF treatment at 37°C (relative to Cont), whereas other sites such as K 867 had ϳ20-fold increases.…”