Cbl-directed monoubiquitination of CIN85 is involved in regulation of ligand-induced degradation of EGF receptors

Abstract

Addition of ubiquitin or ubiquitin chains to target proteins leads to their mono- or polyubiquitination, respectively. Whereas polyubiquitination targets proteins for degradation, monoubiquitination is thought to regulate receptor internalization and endosomal sorting. Cbl proteins are major ubiquitin ligases that promote ligand-dependent polyubiquitination and degradation of receptor tyrosine kinases. They also recruit CIN85-endophilin in the complex with activated receptors, thus controlling receptor endocytosis. Here we show that the adaptor protein CIN85 and its homologue CMS are monoubiquitinated by Cbl/Cbl-b after epidermal growth factor (EGF) stimulation. Monoubiquitination of CIN85 required direct interactions between CIN85 and Cbl, the intact RING finger domain of Cbl and a ubiquitin acceptor site present in the carboxyl terminus of CIN85. Cbl-b and monoubiquitinated CIN85 are found in the complex with polyubiquitinated EGF receptors during prolonged EGF stimulation and are degraded together in the lysosome. Dominant interfering forms of CIN85, which have been shown previously to delay EGF receptor degradation, were also impaired in their monoubiquitination. Thus, our data demonstrate that Cbl/Cbl-b can mediate polyubiquitination of cargo as well as monoubiquitination of CIN85 to control endosomal sorting and degradation of receptor tyrosine kinases.

Fig 1.

CIN85 and CMS are monoubiquitinated (MonoUbi) after EGF stimulation. (A) HEK293T cells were transiently transfected with expression vectors coding for CIN85, EGFR, Cbl-b, and FLAG-tagged ubiquitin (FLAG-Ubi). Thirty hours after transfection, cells were starved for 12 h and mock-treated (−) or stimulated with 100 ng/ml EGF (+) for 10 min. CIN85 was immunoprecipitated (IP) with anti-CIN85 (CT) antibodies, and monoubiquitination was detected with anti-FLAG (M5) antibodies. Levels of precipitated CIN85 were determined with anti-CIN85 (CT) antibodies. Transfection of pcDNA3-CIN85 into HEK293T cells led to expression of two isoforms migrating with the relative molecular mass of ≈78 and 85 kDa. (B) NIH 3T3-EGFR cells, transfected with FLAG-tagged ubiquitin, were starved for 12 h and mock-treated (−) or stimulated with 100 ng/ml EGF for 5 or 30 min. Immunoprecipitates of CIN85 were analyzed by Western blotting with antiubiquitin antibodies, anti-CIN85 (CT), and anti-EGFR (RK2) antibodies. Two CIN85 isoforms (p78 and p85) may represent previously described alternatively spliced forms of CIN85 (32, 33). (C) HEK293T cells overexpressing EGFRs and FLAG-tagged ubiquitin without or with Cbl, Cbl-b, or Nedd4, and FLAG-CIN85 or FLAG-CMS were mock-treated (−) or stimulated with 100 ng/ml EGF (+) for 10 min. CIN85 was immunoprecipitated with anti-CIN85 antibodies (Top) and CMS with anti-FLAG (M2) antibodies (Middle), and their monoubiquitination was analyzed by blotting with anti-FLAG (M5) antibodies. Levels of Cbl, Cbl-b, and Nedd4 in representative TCLs are shown (Bottom).

Fig 2.

Monoubiquitination (MonoUbi) of CIN85 is mediated by Cbl and Cbl-b. (A) HEK293T cells overexpressing EGFRs, FLAG-tagged ubiquitin, FLAG-tagged CIN85 and Cbl, or Cbl-70Z were stimulated with EGF as described for Fig. 1A. Levels of EGFR, Cbl, Cbl-70Z, CIN85, and monoubiquitinated CIN85 were determined in the TCLs. (B) HEK293T cells expressing EGFR, FLAG-tagged ubiquitin (FLAG-Ubi), CIN85 and Cbl-b, Cbl-b-N1/2, or Cbl-b-C2/3 were stimulated with EGF as described in Fig. 1A. TCLs were analyzed for the presence of monoubiquitinated CIN85 by blotting with anti-CIN85 antibodies (CT), and levels of Cbl-b and Cbl-b deletion forms are shown (Upper). wt, wild type. (C) Cbl-b-N1/2 contains the SH2 domain and RING finger domain (RF) of Cbl-b. Cbl-b-C2/3 consists of the RING finger domain, proline-rich region (PxxP), and leucine zipper (LZ) domain of Cbl-b.

Fig 3.

Monoubiquitination (MonoUbi) of the carboxyl terminus of CIN85. (A) HEK293T cells overexpressing EGFRs, Cbl-b, and FLAG-tagged ubiquitin (FLAG-Ubi) together with FLAG-tagged CIN85-3SH3 (3SH3), CIN85-PCc (PCc), or wild-type CIN85 (wt) were stimulated with EGF as described for Fig. 1A. CIN85 and CIN85 mutants were immunoprecipitated (IP) with anti-CIN85 (CT or SH3) antibodies, and a Western blot was probed with anti-EGFR (RK2), anti-Cbl-b (RF), and anti-FLAG (M5) antibodies as indicated. (B) HEK293T cells overexpressing EGFR, Cbl-b, and FLAG-tagged ubiquitin together with FLAG-tagged wild-type CIN85 (wt), CIN85-3SH3-M (3SH3-M), CIN85-Cc-lysine-less (Cc-K less), CIN85-ΔCc (ΔCc), or CIN85-ΔP (ΔP) were stimulated with EGF as described for Fig. 1A. CIN85 and CIN85 mutants were immunoprecipitated by using anti-CIN85 (SH3) antibodies, and their monoubiquitination was analyzed by Western blotting with anti-FLAG (M5) antibodies. Levels of Cbl-b in TCLs are shown (Lower).

Fig 4.

CIN85 is degraded together with Cbl-b and EGFRs. (A) HEK293T cells overexpressing EGFRs, FLAG-tagged ubiquitin (Ubi), FLAG-tagged CIN85, and Cbl-b or Cbl-G306E were starved and either mock-treated or treated for 1 h with 20 μM of the proteasome inhibitor MG132 as indicated, followed by mock treatment or EGF (100 ng/ml) stimulation for indicated times. TCLs were analyzed for EGFR, Cbl-b, CIN85, and ERK2 levels with specific antibodies. ERK2 was used as a control for equal loading, because it is not degraded in the complex with EGFRs. (B) A schematic model of a Cbl–CIN85 role in EGFR down-regulation. After ligand binding, EGFRs dimerize and autophosphorylate, leading to recruitment of Cbl to phosphorylated EGFRs. Cbl polyubiquitinates the EGFR and recruits CIN85, which also becomes monoubiquitinated by Cbl. The EGFR–Cbl–CIN85 complex is sorted along the endocytic pathway and targeted to the MVB and lysosome for degradation. Cbl thus mediates ubiquitination of both EGFRs and CIN85 in the same complex. CIN85 also has a dual role in controlling EGFR down-regulation, because it both promotes receptor internalization and participates in endosomal sorting and subsequent degradation of activated receptors.