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 spectrometry-based 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.

Fig. 1.

Stoichiometry of EGFR ubiquitination. (A) PAE/wtEGFR cells were treated with or without 20 ng/mL EGF for 5 min at 37 °C. EGFR was immunoprecipitated, immunoprecipitates were resolved by SDS/PAGE, and the gels were stained with Coomassie blue. A representative gel is shown, with red lines indicating regions R1, R2, and R3 containing EGFR that were excised and analyzed by AQUA-based targeted proteomics as described in Materials and Methods. (B) Quantification of the number of Ub per EGFR molecule (Ub/EGFR), number of Ub-conjugated sites in EGFR (Ub’n sites/EGFR), and number of Ub per chain conjugated to EGFR (Ub/chain) in gel regions R1, R2 and R3 using equations in SI Appendix. Graph bars represent mean values (±SEM; n = 5). The cartoon illustration below depicts ubiquitinated EGFR kinase domain and summarizes the stoichiometry of EGFR ubiquitination in each of these gel regions calculated on the basis of AQUA analysis. 4%, 11%, and 85% (mean values; n = 5) of total EGFRs are present in R1, R2, and R3, respectively. All receptors present is R1 and R2 are considered to be EGF-activated, whereas in R3, ∼15% of total EGFR are EGF-activated (EGF-occupied before cell solubilization). (C) Representative tandem mass spectra of Ub peptides demonstrating K48-chain and K63-chain ubiquitination. Peaks matching expected singly and doubly charged (++) b and y ions are labeled. Insets show representative mass chromatograms for the unlabeled (L) and isotope-labeled internal standard peptide (H) from the LC-SRM.

Fig. 2.

AMSH attachment to EGFR results in impaired K63 ubiquitination of the receptor. (A) Schematic representation of EGFR-AMSH chimeric proteins. (B) PAE cells expressing wtEGFR, EGFR-AMSH (clone 10), or EGFR-AMSH* (clone 27) were treated or not with 20 ng/mL EGF for 5 min at 37 °C and lysed, and EGFRs were immunoprecipitated. Immunoprecipitates were resolved by SDS/PAGE and probed with Ub, phosphotyrosine, and EGFR antibodies. Note that Ab1005 poorly recognize activated EGFR. (C) PAE cells expressing wtEGFR, EGFR-AMSH (clone 10), or EGFR-AMSH* (clone 27) were treated with 20 ng/mL EGF for 5 min at 37 °C and lysed, and EGFRs were immunoprecipitated. Absolute amounts (femtomole quantities) of total Ub and K48 and K63 polyUb-chain linkages were measured by SRM-based MS. The data are presented as percentages of polyUb linkages to the total amount of Ub in the combined three gel regions R1, R2, and R3 of EGFR staining that was excised similarly to Fig.1A.

Fig. 3.

Internalization of EGFR-AMSH chimeric proteins. (A) Internalization rate constants (k_e) of [¹²⁵I]EGF (1 ng/mL) were measured in several single cell clones of cells stably expressing wtEGFR, EGFR-AMSH, or EGFR-AMSH*. *P < 0.05 (EGFR-AMSH* relative to EGFR-AMSH). (B) Cells stably expressing similar levels of wtEGFR, EGFR-AMSH, or EGFR-AMSH* were incubated with EGF-Rh (100 ng/mL) for 10 min at 37 °C. After fixation, the cells were stained with antibody to EEA.1 followed by secondary Cy5-conjugated antibody. A z stack of confocal images were acquired although 561 nm (EGF-Rh, red) and 640 nm (EEA.1, green) channels. Confocal sections through the middle of the cell are shown. “Yellow” signifies the overlap of rhodamine and Cy5 fluorescence. (Scale bars: 10 μm.) (C) The relative amount of EGF-Rh in EEA.1-containing endosomes was calculated from two independent experiments performed as in A). Data represent values averaged from 10 cells (± SEM).

Fig. 4.

Slow degradation and lysosomal targeting of EGFR-AMSH. (A) Several individual single-cell clones of cells expressing wtEGFR, EGFR-AMSH, and EGFR-AMSH* were serum-starved and incubated with EGF (100 ng/mL) for the indicated times before lysis in the absence of OV and NEM. EGFR was detected with antibodies 1005. (B) The amount of EGFR immunoreactivity was quantitated from five to six experiments for each variant, and the mean values (± SEM) for each type of EGFR/mutant-expressing cells were plotted against time. (C) Cells were preincubated with leupeptin and then incubated with EGF-FITC (100 ng/mL) for 2 h at 37 °C, including with LysoTrackerRed for the last 30 min of this incubation with EGF-FITC. After fixation, a z stack of images were acquired although 561-nm (LysoTrackerRed) and 488-nm (EGF-FITC) channels. Confocal sections through the middle of the cell are shown. “Yellow” signifies the overlap of red and green fluorescence. Arrows indicate examples of colocalization of EGF-FITC and LysoTracker. (Scale bars: 10 μm.) (D) The percentage of EGF-FITC located in vesicles containing LysoTrackerRed relative to the total cell-associated EGF-FITC was calculated from two experiments performed as in C. Each data represents value averaged from 10 cells (± SEM). *P < 0.05 (EGFR-AMSH relative to wtEGFR and EGFR-AMSH*).

Fig. 5.

Signaling by EGFR-AMSH chimeric proteins. (A) Cells expressing wtEGFR, EGFR-AMSH, or EGFR-AMSH* were serum-starved, treated with 10 ng/mL EGF for 5 min at 37 °C, washed, and further incubated for the indicated times (Chase Time) without EGF. The cells were lysed in the presence of OV and NEM. The cell lysates were probed for active EGFR (antibody pY1068), total EGFR, phosphorylated ERK1/2, and total ERK1/2. The experiment is representative of three independent experiments. (B) Mean amounts of active ERK1/2 normalized to total ERK1/2 from three experiments (± SEM) plotted against chase time are presented on the graphs.