Alix/AIP1 antagonizes epidermal growth factor receptor downregulation by the Cbl-SETA/CIN85 complex

Abstract

The assembly of the Cbl-SETA/CIN85-endophilin complex at the C terminus of the epidermal growth factor receptor (EGFR) following ligand activation mediates its internalization and ubiquitination. We found that the SETA/CIN85-interacting protein Alix/AIP1, which also binds endophilins, modulates this complex. Alix was found to associate indirectly with EGFR, regardless of its activation state, and with DeltaEGFR, which signals at low intensity and does not bind Cbls or SETA/CIN85. In agreement with this, Alix interaction did not occur via SETA/CIN85. However, SETA/CIN85 and Alix were capable of mutually promoting their interaction with the EGFR. Increasing the level of Alix weakened the interaction between SETA/CIN85 and Cbl and reduced the tyrosine phosphorylation of c-Cbl and the level of ubiquitination of EGFR, SETA/CIN85, and Cbls. This antagonism of the Cbl-SETA/CIN85 complex by Alix was reflected in its diminution of EGFR internalization. In agreement with this, small interfering RNA-mediated knockdown of Alix promoted EGFR internalization and downregulation. It has been suggested that SETA/CIN85 promotes receptor internalization by recruiting endophilins. However, Alix was also capable of increasing the level of endophilin associated with EGFR, implying that this is not sufficient to promote receptor internalization. We propose that Alix inhibits EGFR internalization by attenuating the interaction between Cbl and SETA/CIN85 and by inhibiting Cbl-mediated ubiquitination of the EGFR.

FIG. 1.

Alix binds to both EGFR and ΔEGFR. (A) Flag-tagged Alix was transfected into U87MG glioma cells. IP of endogenous EGFR revealed receptor-associated Alix within the precipitate (lane 2). (B) Alix and ΔEGFR (lanes 1 and 3) or EGFR (lane 2) were transfected into HEK293 cells, which were then serum starved for 18 h and treated with 100 ng of EGF/ml for 5 min before lysis. IPs were performed with an antibody against EGFR (Ab-1; Oncogene Science; lanes 2 and 3) or with EGFR preclearing of the ΔEGFR lysate with the same EGFR antibody, followed by IP with MAb 806 to specifically isolate the ΔEGFR (lane 1). Alix was recovered along with both receptors (lanes 1 to 3). However, less Alix was associated with ΔEGFR. (C) CHO-EGFR cells were either transfected with Flag-tagged Alix (lane 1) or not and subjected to EGFR IP followed by EGFR or Alix WB. Although a stronger Alix band is obtained in the EGFR IP when Alix is transfected, the endogenous Alix is also recovered, and the levels of endogenous Alix are equivalent regardless of whether cells were serum starved for 18 h and either not treated further (lane 2) or challenged with 50 ng of EGF/ml for 10 min (lane 3; ns, not starved). (D) Fat rich and low-fat fractions of mouse brain homogenates were subjected to IP with the anti-EGFR antibody, and the recovered material was analyzed by Alix and EGFR WB. Alix was recovered in EGFR IPs from both fractions and was also observed in the lysates. EGFR was detected in the lysates at longer exposures (not shown).

FIG. 2.

Alix binding to EGFR and ΔEGFR is independent of receptor activation. Alix was transfected into HEK293 cells together with EGFR (A) or ΔEGFR (B). In panel B endogenous EGFR was removed from ΔEGFR lysates as described for Fig. 1. After 24 h cells were serum starved and received no further treatment (lane 1), were treated with 100 ng of EGF/ml for 5 min (lane 2), or were treated with 10 μM EGFR inhibitor AG1478 (lane 3) or 20 μM src kinase inhibitor PP2 (lane 4) for 1 h in the absence of EGF stimulation to further reduce background EGFR phosphorylation levels. No treatment had an effect on the amount of Alix associated with EGFR or ΔEGFR, indicating that the interaction between Alix and receptors was constitutive. P-TYR, phosphotyrosine.

FIG. 3.

Alix binding to ΔEGFR is independent of the receptor's tyrosine phosphorylation or its kinase activity. Alix was transfected into HEK293 cells together with EGFR (lane 10), ΔEGFR (lane 1), a kinase-dead ΔEGFR receptor (DK; lane 2), and several ΔEGFR tyrosine-to-phenylalanine exchange receptors (DYs; lanes 3 to 9; a cartoon of the EGFR C terminus and details of DYs are at the right). Cells transfected with EGFR were serum starved for 18 h and exposed to 100 ng of EGF/ml for 5 min. EGFR and ΔEGFR IPs were performed as described for Fig. 1. No alteration had a major effect on the Alix-ΔEGFR interaction, indicating that it is constitutive and independent of ΔEGFR phosphorylation or kinase activity. Minor differences could be observed. For example, DY4 was associated with an increased amount of Alix compared to the other constructs (lane 6). This offers the possibility that structural differences in the C terminus of ΔEGFR impact Alix binding. EGFR bound more Alix than any of the ΔEGFR proteins. P-TYR, phosphotyrosine.

FIG. 4.

SETA/CIN85 and Alix mutually enhance their binding to EGFR. (A) To determine whether SETA/CIN85 and Alix, which independently interact with the EGFR, can modulate each other's interaction, Alix and an mutant Alix with reduced ability to bind SETA/CIN85 were transfected together with EGFR and lacZ or SETA/CIN85 into HEK293 cells grown in the continuous presence of serum. EGFR immunoprecipitations revealed that SETA/CIN85 increased the amount of Alix that is associated with the EGFR 1.7-fold (compare lanes 1 and 4) but not the amount of Alix-Δ717-784, which does not bind SETA/CIN85 (compare lanes 2 and 5). (B) Cotransfection of full-length Alix or Alix-Δ717-784 together with EGFR and lacZ or SETA/CIN85 followed by EGFR IP demonstrated that Alix was capable of increasing the amount of SETA/CIN85 bound to wild-type EGFR 1.7-fold (lane 2) while the non-SETA/CIN85-binding Alix-Δ717-784 was not (lane 3).

FIG. 5.

Alix antagonizes SETA/CIN85's association with c-Cbl and Cbl-b. (A) SETA/CIN85, Cbls, and lacZ or Alix were transfected into HEK293 cells and either Cbls were immunoprecipitated by their HA tags or Alix and a mutant Alix were immunoprecipitated via their Flag tags. Alix strongly decreased the amount of SETA/CIN85 associated with c-Cbl (compare lanes 1 and 2) or Cbl-b (compare lanes 4 and 5). The amount of SETA/CIN85 associated with Alix, on the other hand, remained unaffected by Cbl proteins (compare lanes 3, 6, and 7). Alix-Δ717-784 did not precipitate any SETA/CIN85 and served as a negative control (lane 8). (B) In the converse experiment, SETA/CIN85, c-Cbl, and lacZ, Alix, or Alix-Δ717-784 were transfected into HEK293 cells and SETA/CIN85 immunoprecipitates were prepared. HA WB (detecting c-Cbl) showed that Alix reduced the amount of c-Cbl associated with SETA/CIN85 while Alix-Δ717-784 did not (compare lane 2 to 1 and 3). (C) Various amounts of the Alix plasmid were transfected along with c-Cbl and SETA/CIN85. The amount of c-Cbl recovered in SETA/CIN85 immunoprecipitates was measured and showed a stepwise reduction to 98 (lane 2), 79 (lane 3), or 64% (lane 4) of the amount in the control (lane 1; by densitometry). The amounts of Alix protein in lanes 3 and 4 were 1.8 and 2.1 times, respectively, the amount observed in lane 2 (by densitometry).

FIG. 6.

Alix reduces protein ubiquitination and attenuates Cbl activity. (A) EGFR, SETA/CIN85, c-Cbl, and Cbl-b were transfected together with Flag-tagged monoubiquitin and either lacZ (lanes 1, 3, 5, and 7) or Alix (lanes 2, 4, 6, and 8) into HEK293 cells as indicated. Cells were serum starved and stimulated with 50 ng of EGF/ml for 5 min before lysis and IP with the following specific antibodies: EGFR, Labvision Ab-11 (lanes 1 and 2); SETA/CIN85, a polyclonal antibody (lanes 3 and 4), c-Cbl and Cbl-b, their HA tags (lanes 5 to 8). Although there was no impact of Alix transfection on the overall level of the high-molecular-weight Flag-tagged ubiquitinated smear in cell lysates from which the immunoprecipitates were prepared (bottom), there was an effect on certain proteins. Specifically, the level of ubiquitination detectable in EGFR immunoprecipitates, seen as a high-molecular-weight smear, was reduced, while the EGFR protein was unaffected (lanes 1 and 2). Similarly, the level of SETA/CIN85 ubiquitination, seen as a faint band running underneath Alix in the Flag WB or above SETA in the SETA WB, was reduced by the presence of Alix while the level of SETA/CIN85 was not altered (lanes 3 and 4). Lanes 1 through 4 measured ubiquitination mediated by endogenous E3 ubiquitin ligases. Alix was also able to reduce the activity and monoubiquitination of c-Cbl and Cbl-b E3 ubiquitin ligases, as demonstrated by the reduction in the intensity of the Flag-tagged high-molecular-weight smear and the individual Cbl protein bands (lanes 5 through 8). (B) EGFR, c-Cbl, Alix, or GFP control vector were transfected into HEK293 cells as indicated. Cells were serum starved and challenged with 50 ng of EGF/ml for 5 min before lysis, and lysates were subjected to EGFR or Cbl IP and WB with the indicated antibodies. The tyrosine phosphorylation of the EGFR was not altered by Alix, as determined by densitometry of the EGFR, EGFR phosphotyrosine (P-Tyr), and PY1045 bands (lanes 1 to 4), nor was the amount of c-Cbl recovered in EGFR IPs (determined by densitometry of the Cbl and EGFR bands in the EGFR IP; lanes 3 and 4). In contrast tyrosine phosphorylation of c-Cbl was dependent on the presence of EGFR and was attenuated strongly by the presence of Alix (middle). Analysis of lysates is shown at the bottom and demonstrates relative expression levels. Note that expression of c-Cbl reduced the level of EGFR as expected.

FIG. 7.

Alix increases the amount of endophilin associated with the EGFR in an activity-independent manner. (A) Flag-tagged Alix and human CIN85 were cotransfected with HA-tagged endophilin A1 into HEK293 cells, which were serum starved and stimulated for 5 min with 100 ng of EGF/ml. Alix and SETA/CIN85 were immunoprecipitated from cell lysates via their Flag epitopes, and in both cases endophilin was also recovered, with the Alix IP containing more. Although a slightly lower level of SETA/CIN85 was expressed in the lysates, the signal intensity of the CIN85 protein in the Flag IP was similar to that of Alix, suggesting similar levels of protein, as they were both detected with the same anti-Flag antibody. Both SETA/CIN85 and Alix were recovered in the HA-endophilin IP, and again less SETA/CIN85 was recovered. (B) In vitro confrontation of various radiolabeled Alix proteins made by in vitro transcription and translation with bacterially made GST proteins showed that GST-SETA binds Alix and Alix-784Stop (a truncation of Alix at position 784) efficiently but shows no binding to Alix-Δ717-784 and low-efficiency binding to Alix-R745Gthat is equivalent to that observed for lacZ. Note that in this experiment Alix was relatively underloaded. (C) Transfection of Alix and Alix-R745G increased the level of endophilin that was recovered in EGFR immunoprecipitates beyond the increase mediated by stimulation with EGF.

FIG. 8.

Alix reduces EGFR internalization and antagonizes the promotion of EGFR internalization by Cbl. (A) The percentage of EGFR internalization in CHO cells transfected with EGFR, c-Cbl, and Alix was measured by using radiolabeled ¹²⁵I-EGF. Expression of Alix results in a decline in the amount of c-Cbl-mediated EGFR internalization. (B) The relative remaining EGFR on the surfaces of CHO cells, transfected with EGFR, c-Cbl, and Alix, was measured after exposure to EGF for the times indicated to radiolabeled EGF. The values are expressed as percentages of control cells that were not exposed to EGF. Expression of Alix inhibited the c-Cbl-induced decline in EGFR present at the cell surface.

FIG. 9.

Knockdown of Alix promotes the internalization and degradation of the EGFR. (A) HeLa and CHO-EGFR cells were incubated with various amounts of a 20 μM Alix siRNA duplex solution using 1 μl of LP2000 for 72 h. Subsequent immunoblotting using an anti-Alix antibody revealed a complete and specific knockdown of endogenous protein after application of 1 μl of siRNA solution. Erk2 levels verified loading of equal amounts of protein. (B) Receptor internalization in HeLa cells was measured by flow cytometry. Alix knockdown caused a significant increase in EGFR removal from the cell membrane compared to control cells. The insert shows confirmation of downregulation of Alix in the cells used in this analysis. (C) The amount of EGFR remaining on the cell surface was determined by receptor labeling with ¹²⁵I-EGF ligand and subsequent measurement in a gamma counter. Analysis revealed a significant acceleration of receptor downregulation in CHO-EGFR cells permanently overexpressing EGFR.

FIG. 10.

Model of different SETA/CIN85 complexes associated with EGFR. (A and B) The data demonstrate that Alix associates with both inactive and active EGFR (A), while previous work has shown that Cbl and SETA/CIN85 do not bind to the inactive receptor, but that EGFR activation leads to phosphorylation of the EGFR and the binding of Cbl and SETA/CIN85, which results in SETA/CIN85, Cbl, and EGFR ubiquitination and EGFR internalization (B). (C) When levels of SETA/CIN85 are increased, additional Alix is recruited to the EGFR via direct interaction with SETA/CIN85. Note that for the sake of clarity we have omitted the complex multimeric clusters that SETA/CIN85 forms with Cbls, both by multimerization via SETA/CIN85's C-terminal coiled coil and via the binding of multiple Cbls by a single SETA/CIN85 molecule, which is likely to afford many Alix binding sites. (D) Conversely increasing the amount of Alix allows it to compete with Cbls for SETA/CIN85 binding as well as inhibit the activity of the Cbls, consequently reducing the levels of EGFR, SETA/CIN85, and Cbl ubiquitination and EGFR internalization. (E) Reduction in the level of Alix stimulates the rate of internalization of active EGFR, presumably by removing the weak inhibitory effect that endogenous levels of Alix mediate. Endophilins bind constitutively to SETA/CIN85 and Alix. Binding domains are indicated by small target symbols on proteins, and are labeled as follows: RING, RING finger domain of Cbls; PxxP, P-X-X-P motifs in Cbls; PTB, phosphotyrosine binding domain of Cbls, a variant SH2 domain; CT, C termini of Cbls, which are modified by phosphorylation and where SETA/CIN85 binds; SH3 1, 2, and 3, the three SH3 domains of SETA/CIN85; PRCT, proline-rich C terminus in SETA/CIN85 and Alix; Ub, ubiquitin; Y-P, phosphorylated tyrosine in the C terminus of EGFR; endo, endophilin.