Epidermal growth factor receptor signaling intensity determines intracellular protein interactions, ubiquitination, and internalization

Abstract

Ligand activation of the epidermal growth factor receptor (EGFR) causes the binding of Cbls, which leads to EGFR polyubiquitination and internalization through endophilin complexes that contain the adaptor protein SH3-domain encoding, expressed in tumorigenic astrocytes/Cbl-interacting protein of 85 kDa/regulator of ubiquitous kinase (SETA/CIN85/Ruk). In cells grown at high density, high levels of SETA interfered in the recruitment of Casitas B-lineage (Cbl) proteins to the EGFR and reduced its polyubiquitination, suggesting that SETA has a regulatory function in the formation of the EGFR-Cbl-endophilin complex and in EGFR down-regulation. In a situation where there is EGFR signaling but no internalization or down-regulation, as is the case with the EGFR with exons 2-7 deleted (DeltaEGFR) oncogene, these proteins were absent altogether. By using mAb 806, which recognizes an EGFR-activation state and preferentially immunoprecipitates DeltaEGFR, we show that DeltaEGFR did not interact with Cbls, SETA, or endophilin A1, providing a mechanistic explanation for its lack of internalization. As would be expected by the absence of Cbl proteins in the DeltaEGFR complex, the mutant receptor was also not polyubiquitinated. The intracellular C terminus and tyrosine autophosphorylation pattern of DeltaEGFR are similar to wild-type EGFR, but it signals at a lower intensity as determined by levels of EGFR phosphotyrosine. To test the implication that the lack of interaction with the Cbl-SETA-endophilin complex is because of differences in signal intensity, EGFR-expressing cells were treated with tyrphostin AG1478 EGFR inhibitor. Attenuation of wild-type EGFR signal to levels similar to that found in DeltaEGFR resulted in the dissociation of SETA and Cbl proteins and a concomitant attenuation of receptor internalization.

Fig. 1.

SETA regulates the Cbl–EGFR interaction. (A) In HEK293 cells, EGFR IP (lanes 1 and 2) identified SETA as part of the EGFR complex (lane 2). Antipolyubiquitin IPs contained SETA (lane 3). (B) In HEK293, cells were allowed to reach confluency, serum-starved (ss), and were then exposed to 100 ng/ml EGF for 5 min (EGF). c-Cbl proteins and SETA could be seen to move to the EGFR on EGF stimulation (lanes 1–4). Cotransfection of SETA abrogated the increased association of c-Cbl with the EGFR after stimulation (lanes 5 and 6). (C) In subconfluent HEK 293 cells, analysis of EGFR IP showed that the presence of c-Cbl and the levels of EGFR polyubiquitination, detected as a high-molecular-weight smear, were unaffected by the presence of SETA. (D). In contrast, in confluent HEK293 cells, analysis of EGFR IPs showed that SETA and Cbl proteins were present, and that SETA reduced the amount of EGFR-associated Cbls (compare lanes 2–4 with lanes 6–8), as well as EGFR polyubiquitination.

Fig. 2.

ΔEGFR does not interact with c-Cbl, Cbl-b, SETA, or endophilin. (A) HEK293 cells were transfected with c-Cbl and EGFR (lane 1) or ΔEGFR (lanes 2–4), serum-starved overnight, and incubated with 100 ng/ml EGF for 5 min. EGFR IP (Ab-1, Oncogene Science) revealed one band corresponding to wild-type EGFR (lane 1), or two bands corresponding to EGFR and ΔEGFR when cells were transfected with ΔEGFR (lane 2). When a mAb 806 IP was prepared from cells transfected with ΔEGFR, two bands were obtained, with the higher, less-intense band corresponding to wild-type EGFR (lane 3). When anti-EGFR preclearing preceded mAb806 IP, only one band corresponding to ΔEGFR was obtained (lane 4). c-Cbl was found in IPs when EGFR, but not ΔEGFR, was present (lanes 1–4). (B) Similar experiments with HEK293 cells transfected with Cbl-b showed Cbl-b in wild-type but not ΔEGFR-specific IPs (lanes 1–3). (C) Similar studies in cells grown in the continuous presence of serum demonstrated that SETA was found in IPs from HEK293 cells that contained wild-type EGFR (lanes 2 and 3), but not ΔEGFR alone (lane 1). A phosphotyrosine blot verified that receptors were activated. Please note that IPs in lanes 1 and 3 were from the same lysates, and so SETA protein was expressed in the sample from which lane 1 was prepared. (D) Although expression levels of endophilin A1 were higher when it was cotransfected with ΔEGFR than with EGFR (compare lanes 1 and 2), it was not present in ΔEGFR-specific IP (lane 2), but was present in EGFR IP (lane 1) as expected. Differences in endophilin A1 levels may relate to its higher turnover when EGFR is cotransfected.

Fig. 3.

ΔEGFR is not polyubiquitinated. ΔEGFR and c-Cbl (lanes 1, 5, 7, and 11), Cbl-b (lanes 2, 6, 8, and 12), AIP1 (lanes 3 and 9), and SETA (lanes 4 and 10), with or without HA-tagged polyubiquitin (HA-Ub), were cotransfected into HEK293 cells. ΔEGFR-specific IPs as described in Fig. 2, and the lysates from which they were derived, were subjected to HA Western blotting. c-Cbl or Cbl-b increased polyubiquitination of proteins in the lysates, which was detected as an HA-reactive high-molecular-weight smear when ub-HA was cotransfected (compare lanes 11 and 12 with lanes 9 and 10 with lanes 7 and 8). No smear could be detected in the corresponding ΔEGFR immunoprecipitates (lanes 5 and 6). SETA or its binding partner AIP1, which is Flag-tagged, did not mediate polyubiquitination of any proteins on their own and serve as controls.

Fig. 4.

Inhibition of EGFR reduces phosphorylation, association with SETA and Cbl, and polyubiquitination. CHO cells were treated with AG1478, and, if transfected with EGFR, were also stimulated with EGF for 5 min. EGFR or ΔEGFR-specific IPs were prepared as appropriate and showed that, with increased dosage of AG1478, the level of phosphotyrosine on EGFR was reduced, as was its binding to SETA and Cbls. In addition, polyubiquitination of the EGFR, visible as an HA-positive smear, was suppressed by AG1478. (Lower) Quantification of the phosphotyrosine signal relative to the EGFR signal is shown, with the values for untreated EGFR being set arbitrarily to 1 in each case. The phosphorylation level of ΔEGFR is similar to that of EGFR from cells treated with 4 nM AG1478. This concentration is the start of the threshold at which levels of SETA and Cbl proteins in EGFR complexes declined.

Fig. 5.

Inhibition of EGFR by AG1478 reduces its internalization. (A) The percentage EGFR internalization was measured in CHO cells, transfected with EGFR, and treated with AG1478, using radiolabeled EGF. Treatment with 5–10 nM AG1478 results in a decline in the amount of EGFR internalization. (B) The relative remaining EGFR on the surface of CHO cells, transfected with EGFR and treated with the indicated concentration of AG1478, was measured after exposure to EGF for the times indicated, using radiolabeled EGF. The values are expressed as a percentage of control cells that were not exposed to EGF. Treatment with AG1478 inhibited the decline in EGFR that was present at the cell surface over time.