Elsevier

Experimental Cell Research

Volume 300, Issue 2, 1 November 2004, Pages 388-395
Experimental Cell Research

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

https://doi.org/10.1016/j.yexcr.2004.07.003Get rights and content

Abstract

Mutation of the binding site for Cbl (Tyr1045) in the EGF receptor (EGFR) results in impaired ubiquitination but does not affect EGFR internalization. However, the Y1045F mutation resulted in strongly decreased degradation of the EGFR, as well as efficient recycling of EGFR to the plasma membrane. Significantly, more wild-type EGFR than Y1045F EGFR was found localizing to multivesicular late endosomes. Ubiquitination of the EGFR was in HeLa cells inhibited both upon overexpressing the N-terminal part of Cbl and upon overexpressing a double mutant Grb2 incapable of interacting with Cbl and thereby being incapable of indirectly recruiting Cbl to the EGFR. Collectively, these data suggest that the ubiquitination resulting from direct binding of Cbl to pTyr1045 of the EGFR is critical for lysosomal sorting of the EGFR in contrast to ubiquitination resulting from Grb2-mediated binding of Cbl to the EGFR.

Introduction

Binding of EGF activates the EGF receptor (EGFR) due to ligand-induced dimerization and kinase activation. This results in autophosphorylation of the EGFR and phosphorylation of other intracellular substrates [1]. Signaling additionally orchestrates endocytosis and lysosomal degradation of the EGFR by as yet incompletely understood mechanisms. The EGFR has been demonstrated to be ubiquitinated upon activation [2], and the ubiquitin ligase responsible for the ubiquitination has been identified as Cbl [3], [4], [5], [6], [7], [8]. It has further been demonstrated that Cbl positively regulates clathrin-dependent endocytosis of the EGFR [7], [9], [10], [11], [12], [13], [14], [15] and that ubiquitination of the EGFR by Cbl is required for directing the EGFR to internal vesicles of late endosomes and for lysosomal degradation of the receptor to occur [6], [16], [17].

Recently, it was demonstrated that the EGFR appears to be multiply monoubiquitinated and not polyubiquitinated upon activation [13]. It has further been demonstrated that the EGFR can be ubiquitinated both by direct interaction of Cbl with the EGFR through pTyr1045 as well as by indirect interaction of Cbl with the EGFR through binding of the complex of Cbl and Grb2 to Grb2 binding sites in the EGFR tail [6], [11]. Interestingly, it has also been demonstrated that Grb2 is critical in regulating the internalization of the EGFR through clathrin-coated pits [18]. Our recently published data have confirmed that Grb2-mediated Cbl-induced ubiquitination of the EGFR is required for the EGFR to enter clathrin-coated pits [19]. It has recently been published that even though the Y1045F mutant of EGFR is reduced in EGF-induced ubiquitination, it is efficiently endocytosed from clathrin-coated pits [14]. In the present work, we have studied the endocytosis and subsequent intracellular sorting of the Y1045F EGFR in more detail. We have confirmed that the Y1045F EGFR is inefficiently ubiquitinated but efficiently endocytosed. We have further demonstrated that the Y1045F EGFR is very inefficiently degraded, but efficiently recycled, compared to wild-type EGFR (wt EGFR). Accordingly, wt EGFR is by immuno-EM frequently found in multivesicular, late endosomes, while the Y1045F mutant more frequently is observed in early endosomes and at the plasma membrane, consistent with a rapid recycling pattern. Our data therefore suggest that direct interaction of Cbl with pTyr1045 results in ubiquitination of the EGFR, qualitatively different from the ubiquitination resulting from indirect binding of Cbl through Grb2. We propose that while indirect binding of Cbl to the EGFR via Grb2 is required for a ubiquitination pattern allowing endocytosis through clathrin-coated pits, direct binding of Cbl to the EGFR is required for a ubiquitination pattern allowing sorting to lysosomes

Section snippets

Materials

Human recombinant EGF was from Bachem AG (Budendorf, Switzerland). 125I-EGF was from Amersham Biosciences (Buckinghamshire, UK). Rabbit anti-conjugated ubiquitin antibody was from Sigma-Aldrich (St. Louis, MO). Mouse anti-phosphorylated EGFR (pY1173) antibody was from Upstate (Lake Placid, NY), and sheep anti-EGFR antibody was from Fitzgerald (Concord, MA). Mouse anti-EGFR antibody (Ab-3) was from NeoMarkers, Lab Vision Corporation (Fremont, CA). Mouse anti-EGFR (sc-120) and rabbit anti-EGF

The EGFR mutant Y1045F is inefficiently ubiquitinated upon ligand binding

EGF-induced ubiquitination of wt EGFR and Y1045F EGFR was compared by Western blotting upon immunoprecipitation of the EGFR (Fig. 1). Consistent with previously published results [18], we found that ubiquitination of Y1045F EGFR was impaired but not abolished upon incubation with EGF. The reduced ubiquitination was expected, given the reported finding that Cbl binds to pTyr1045 in the EGFR [6]. Residual ubiquitination was further consistent with the reported findings that Cbl can also interact

Discussion

The EGFR is endocytosed and sorted to lysosomes upon incubation of cells with EGF, and in both these processes, recruitment of Cbl to the EGFR has been demonstrated to play a role [3], [4], [7], [12], [16], [18], [19], [26]. Both direct binding of Cbl to the EGFR and indirect binding of Cbl to the EGFR through Grb2 has been reported to result in ubiquitination of the EGFR [6], [11]. Further, only indirect binding of Cbl to the EGFR through Grb2 is considered important for recruitment of the

Acknowledgments

Robin M. Scaife, Alan Hall, and Dirk Bohmann are acknowledged for gifts of valuable reagents and Randi Idsoe for technical assistance. A. Sorkin was supported by NCI, while E. Stang and I.H. Madshus were supported by The Norwegian Research Council, The Norwegian Cancer Society, Medinnova, NOVO Nordic Foundation, Anders Jahre's Foundation for the Promotion of Science, Torsted's Legacy, Blix Legacy, and Bruun's Legacy.

References (29)

  • Z. Galcheva-Gargova et al.

    The epidermal growth factor receptor is covalently linked to ubiquitin

    Oncogene

    (1995)
  • G. Levkowitz et al.

    c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor

    Genes Dev.

    (1998)
  • C.A. Joazeiro et al.

    The tyrosine kinase negative regulator c-Cbl as a RING-type. E2-dependent ubiquitin-protein ligase

    Science

    (1999)
  • P. Soubeyran et al.

    Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors

    Nature

    (2002)
  • Cited by (146)

    • Receptor-kinase EGFR-MAPK adaptor proteins mediate the epithelial response to Candida albicans via the cytolytic peptide toxin, candidalysin

      2022, Journal of Biological Chemistry
      Citation Excerpt :

      However, Shp2 is known to negatively regulate IL-6 release in macrophages in response to TLR ligands (59); thus, we anticipate that Shp2 has a similar function during OEC infections. The c-Cbl adaptor is important in EGFR trafficking and regulation (60, 61); notably, ubiquitination of EGFR by c-Cbl is implicated in clathrin-independent endocytosis of EGFR and downstream signaling (62). Additionally, though the p38 MAPK pathway is pivotal in EGFR endocytosis (63), little is known about the role of p38 in EGFR cellular localization in response to infection.

    • Early Endosomal Compartments

      2022, Encyclopedia of Cell Biology: Volume 1-6, Second Edition
    • ESCRT-dependent cargo sorting at multivesicular endosomes

      2018, Seminars in Cell and Developmental Biology
      Citation Excerpt :

      Post-translational modification of integral membrane proteins by ubiquitin serves as a key sorting signal for endocytosis and targeting to MVEs. In particular, members of the Cbl family of E3 ubiquitin ligases have been tied to the internalization and ESCRT-dependent degradation of dozens of activated cell surface receptors [11,38–40]. Although the addition of a single ubiquitin moiety has been shown to be sufficient to enable ESCRT-mediated protein sorting at MVEs [41], many cargoes including EGF receptor undergo polyubiquitin modification (predominantly via K63-linkages), which may enhance their rate of sequestration within ILVs [18,42–44].

    View all citing articles on Scopus
    View full text