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Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
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2002 (English)In: Nature, ISSN 0028-0836, Vol. 416, no 6877, 183-187 p.Article in journal (Refereed) Published
Abstract [en]

Cbl is a multi-adaptor protein involved in ligand-induced downregulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction. Here we demonstrate that Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K; ref. 6) and endophilins (regulatory components of clathrin-coated vesicles) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 was constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl was increased on EGF stimulation. Inhibition of these interactions was sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases.

Place, publisher, year, edition, pages
2002. Vol. 416, no 6877, 183-187 p.
Keyword [en]
Adaptor Proteins; Signal Transducing, Animals, CHO Cells, Carrier Proteins/genetics/*metabolism, Cell Line, Cricetinae, Down-Regulation/*drug effects, Endocytosis/drug effects, Epidermal Growth Factor/*pharmacology, Genes; Reporter/genetics, Humans, Ligands, Macromolecular Substances, Precipitin Tests, Protein Binding/drug effects, Proto-Oncogene Proteins/*metabolism, Proto-Oncogene Proteins c-cbl, Receptor; Epidermal Growth Factor/*metabolism, Ubiquitin-Protein Ligases
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-90464DOI: 10.1038/416183aPubMedID: 11894095OAI: oai:DiVA.org:uu-90464DiVA: diva2:162827
Note

Erratum in Nature 2002 May 2 ;417(6884):102.

Doi: 10.1038/417102b

Available from: 2003-04-28 Created: 2003-04-28 Last updated: 2013-06-12Bibliographically approved
In thesis
1. Cbl in Regulation of Growth Factor Receptor Endocytosis and Actin Dynamics
Open this publication in new window or tab >>Cbl in Regulation of Growth Factor Receptor Endocytosis and Actin Dynamics
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins belonging to the Cbl family are multidomain scaffolds that participate in numerous processes, assembling signaling complexes and mediating attachment of ubiquitin to receptor and non-receptor tyrosine kinases.

We characterized a novel role for Cbl and Cbl-b in ligand-dependent internalization of growth factor receptors. Upon stimulation with epidermal growth factor (EGF), Cbl proteins associate with EGF receptor, become phosphorylated, and bind to the three SH3 domains of CIN85, which brings endophilins to the complex with active receptors. Endophilins can induce internalization of the plasma membrane, contributing to formation of clathrin-coated pits. We identified a minimal binding domain for CIN85 in the carboxyl termini of Cbl/Cbl-b and observed constitutive association between CIN85, Cbl/Cbl-b and oncogenically stimulated receptor tyrosine kinases. In addition to functioning as a ubiquitin ligase, Cbl forms a complex with CIN85 and endophilin, which is required for efficient endocytosis and downregulation of membrane receptors.

In EGF stimulated cells, we observed inducible modification of CIN85 and related CMS proteins by attachment of a single ubiquitin molecule. Monoubiquitination of CIN85 was mediated by the RING finger and dependent on the carboxyl terminal part of Cbl/Cbl-b, and demanded an intact carboxyl terminus of CIN85. Prolonged stimulation with EGF induced concomitant degradation of EGF receptors, Cbl, and monoubiquitinated forms of CIN85 in lysosomes.

Cbl regulates cytoskeletal processes in a variety of cell systems. We identified SH3P2, a protein with SH3 domain and ankyrin repeats, as a Cbl partner and described its phosphorylation by Src and its distribution in fibroblasts and osteoclasts. SH3P2 formed inducible complexes with Cbl and actin in spread cells and colocalized with dynamic actin structures.

Our data contribute to better understanding of the role of Cbl in downregulation of receptor tyrosine kinases as well as in controlling actin rearrangement.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2003. 58 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1243
Keyword
Cell and molecular biology, RTK, Cbl, endocytosis, ubiquitination, actin, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Cellbiology
Identifiers
urn:nbn:se:uu:diva-3443 (URN)91-554-5583-2 (ISBN)
Public defence
2003-05-22, B42, Biomedical Center (BMC), Uppsala, 13:15
Opponent
Supervisors
Available from: 2003-04-28 Created: 2003-04-28 Last updated: 2013-06-10Bibliographically approved
2. Adaptor Proteins in Regulation of Receptor Endocytosis
Open this publication in new window or tab >>Adaptor Proteins in Regulation of Receptor Endocytosis
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Ligand-induced endocytosis of receptor tyrosine kinases (RTKs) is a dynamic process governed by numerous protein-protein and protein-lipid interactions. This is a major mechanism of signal termination and is also frequently impaired in cancer. The Cbl family of ubiquitin ligases has been shown to play a key role in downregulation of RTKs, by directing their ligand-induced ubiquitination and subsequent lysosomal degradation. My thesis work has led to the identification of novel, ubiquitin-ligase independent, functions of Cbl in receptor endocytosis. We demonstrated that the adaptor protein CIN85 links Cbl with epidermal growth factor receptor (EGFR) internalization. The three SH3 domains of CIN85 interact with Cbl/Cbl-b in a phosphotyrosine dependent manner, whereas its proline-rich region constitutively binds endophilins, known regulators of plasma membrane invagination. The SH3 domains of CIN85 recognize an atypical proline-arginine (PxxxPR) motif present in Cbl and Cbl-b. Moreover, we showed that numerous endocytic regulatory proteins, among them ASAP1 and Dab2, interact with CIN85 via their PxxxPR motifs. The SH3 domains of CIN85 are able to cluster and exchange its effectors at subsequent stages of EGFR endocytosis, thus participating in the control of receptor internalization, recycling and degradation in the lysosome. We proposed that CIN85 functions as a scaffold molecule implicated in control of multiple steps in downregulation of RTKs.

Furthermore, we identified two novel Cbl- and ubiquitin-interacting adaptor proteins named Sts-1 and Sts-2 (Suppressors of T-cell receptor signaling). Ligand-induced and Cbl-mediated recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization and subsequent block of receptor degradation followed by prolonged mitogenic signaling pathways. Our results indicate that Sts-1 and Sts-2 represent a new class of negative regulators of Cbl functions in receptor endocytosis.

In conclusion, this thesis describes novel mechanisms by which Cbl, coupled to its effectors, orchestrates trafficking of RTKs. Detailed understanding of how these processes are controlled under physiological as well as under pathological conditions may be important for future therapeutic approaches.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 63 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1366
Keyword
Cell and molecular biology, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-4477 (URN)91-554-6011-9 (ISBN)
Public defence
2004-10-08, B42, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-09-14 Created: 2004-09-14 Last updated: 2013-06-12Bibliographically approved

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