EGF receptor ubiquitination is not necessary for its internalization

Abstract

Ubiquitination of the EGF receptor (EGFR) has been implicated in EGF-induced receptor internalization, lysosomal degradation, and down-regulation. Mutation of EGFR ubiquitination sites identified by mass spectrometry yielded receptor mutants that are weakly ubiquitinated and not down-regulated by EGF. However, these EGFR mutants were normally internalized. To examine whether this internalization was mediated by the residual ubiquitination, systematic mutagenesis of lysine residues in the kinase domain of the EGFR was performed to generate a receptor mutant that is not ubiquitinated. Mutations of a number of lysines inhibited kinase activity of the EGFR, thus leading to the inhibition of receptor internalization. However, a mutant lacking 15 lysine residues (15KR), which was negligibly ubiquitinated and normally phosphorylated, was internalized at a rate similar to that of the wild-type EGFR. As in the case of the wild-type EGFR, internalization of the 15KR mutant depended on the presence of clathrin, Grb2 adaptor, and Cbl ubiquitin ligase. These data imply that EGFR ubiquitination is not necessary for its internalization by clathrin-coated pits. Interestingly, the reconstitution of two major ubiquitination sites in the 16KR receptor mutant, which had impaired kinase activity and slow internalization kinetics, resulted in a partial rescue of ubiquitination and a complete rescue of receptor internalization. This result suggests that ubiquitination of the kinase-impaired receptor can mediate its internalization by the clathrin pathway. Altogether these data emphasize the robustness of the EGFR internalization process, which can be controlled by multiple kinase- and ubiquitination-dependent and -independent mechanisms.

Fig. 1.

Mutations of lysine residues in the kinase domain of the EGFR and their effect on receptor phosphorylation. (A) Mutated lysine residues were mapped on a previously reported structure of the EGFR kinase domain (1M14). Lysines previously identified as ubiquitination sites by mass spectrometry are shown in red. Lysines mutations that did not affect receptor tyrosine phosphorylation are shown in brown. Lysines mutations, which resulted in a moderate or severe inhibition of receptor phosphorylation, are shown in yellow or green, respectively. (B) Wild-type (WT) and mutant receptors were transiently expressed in PAE cells. The cells were untreated or treated with 20 ng/ml EGF for 2 min at 37°C. EGFR immunoprecipitates were probed with phosphotyrosine (PY20) and EGFR (1005) antibodies. Examples of several representative experiments are shown. (C) The amounts of phosphotyrosine in the EGFR immunoprecipitates normalized to the total EGFR were averaged from two experiments performed as in B and expressed as a percentage of that phosphorylation of the WT EGFR.

Fig. 2.

Ubiquitination and tyrosine phosphorylation of EGFR mutants stably expressed in PAE cells. (A) PAE cells stably expressing wild-type (WT) or mutant EGFR were untreated or treated with 20 ng/ml EGF for 2 min at 37°C. The EGFR immunoprecipitates were sequentially probed for ubiquitin, total phosphotyrosine (PY20), phosphotyrosine 1068 (pY1068), and total EGFR. Examples of three representative experiments are shown. Cell clone numbers are indicated after the names of the mutants. (B) The mean amounts of ubiquitin and phosphotyrosine in the EGFR immunoprecipitates were normalized to total EGFR (±SD) from three to four experiments performed as in A and expressed as a percentage of the ubiquitination and phosphorylation of the WT EGFR.

Fig. 3.

Internalization of ¹²⁵I-EGF by EGFR mutants. (A) WT and 15KR mutant-expressing PAE cells (clone 1) were incubated with 1 ng/ml ¹²⁵I-EGF for the indicated times at 37°C, and the ratio of internalized and surface ¹²⁵I-EGF was determined and plotted against time. (B) Rate constants (K_e) (±SD) of 1 ng/ml ¹²⁵I-EGF internalization were measured in cells stably expressing WT and 15KR receptors (clones 1 and 10). (C) PAE cells expressing 16KR (clone 2) and 16KR/2RK (clone 9) were incubated with 1 ng/ml ¹²⁵I-EGF for the indicated times, and the ratio of internalized and surface ¹²⁵I-EGF was determined and plotted against time. (D) Rate constants (K_e) (±SD) of 1 ng/ml ¹²⁵I-EGF internalization were measured in cells stably expressing WT EGFR and 16KR (clones 2 and 8) and 16KR/2RK (clones 9 and 11) mutants.

Fig. 4.

Effect of siRNA knockdown of CHC, Grb2, and Cbl. Internalization rates of WT EGFR, 15KR (clone 1), and 16KR/2RK (clone 9) mutants were measured by using 1 ng/ml ¹²⁵I-EGF in cells transfected with control siRNA (mock), CHC siRNA, Grb2 siRNA, or a mixture of c-Cbl and Cbl-b siRNAs. The data are expressed as a percentage of the internalization rates in cells treated with nontargeting control siRNA.

Fig. 5.

Mutations of ubiquitination sites slow degradation and down-regulation of EGFR. (A) Cells stably expressing WT EGFR, 15KR, 16KR, and 16KR/2RK mutants were incubated with 100 ng/ml EGF for the indicated times before lysis in the absence of orthovanadate and N-ethylmaleimide. EGFR was detected in cell lysates with antibodies 1005. (B) Cells stably expressing WT EGFR, 15KR, 16KR, and 16KR/2RK were incubated with 200 ng/ml EGF for the indicated times. The cells were then acid-washed to remove unlabeled EGF from surface receptors, and the number of surface EGF-binding sites was measured by using incubation with 50 ng/ml ¹²⁵I-EGF at 4°C for 1 h.