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Adaptor Proteins in Regulation of Receptor Endocytosis
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
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. , p. 63
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1366
Keywords [en]
Cell and molecular biology
Keywords [sv]
Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-4477ISBN: 91-554-6011-9 (print)OAI: oai:DiVA.org:uu-4477DiVA, id: diva2:164916
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
List of papers
1. Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors
Open this publication in new window or tab >>Cbl-CIN85-endophilin complex mediates ligand-induced downregulation of EGF receptors
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2002 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 416, no 6877, p. 183-187Article 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.

Keywords
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:nbn:se:uu:diva-90464 (URN)10.1038/416183a (DOI)11894095 (PubMedID)
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: 2017-12-14Bibliographically approved
2. Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors.
Open this publication in new window or tab >>Identification of a novel proline-arginine motif involved in CIN85-dependent clustering of Cbl and down-regulation of epidermal growth factor receptors.
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2003 In: J Biol Chem, Vol. 278, no 41, p. 39735-46Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91985 (URN)
Available from: 2004-09-14 Created: 2004-09-14Bibliographically approved
3. Dab2 links CIN85 with clathrin-mediated receptor internalization.
Open this publication in new window or tab >>Dab2 links CIN85 with clathrin-mediated receptor internalization.
2003 In: FEBS Lett, Vol. 554, no 1-2, p. 81-7Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91986 (URN)
Available from: 2004-09-14 Created: 2004-09-14Bibliographically approved
4. CIN85 associates with multiple effectors controlling intracellular trafficking of EGF receptors.
Open this publication in new window or tab >>CIN85 associates with multiple effectors controlling intracellular trafficking of EGF receptors.
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2004 In: Mol Biol Cell, Vol. 15, no 7, p. 3155-66Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-91987 (URN)
Available from: 2004-09-14 Created: 2004-09-14Bibliographically approved
5. Suppressors of T-cell Receptor Signaling Sts-1 and Sts-2 Bindto Cbl and Inhibit Endocytosis of Receptor Tyrosine Kinases
Open this publication in new window or tab >>Suppressors of T-cell Receptor Signaling Sts-1 and Sts-2 Bindto Cbl and Inhibit Endocytosis of Receptor Tyrosine Kinases
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2004 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 279, no 31, p. 32786-32795Article in journal (Refereed) Published
Abstract [en]

The ubiquitin (Ub) ligase Cbl plays a critical role in attenuation of receptor tyrosine kinase (RTK) signaling by inducing ubiquitination of RTKs and promoting their sorting for endosomal degradation. Herein, we describe the identification of two novel Cbl-interacting proteins, p70 and Clip4 (recently assigned the names Sts-1 and Sts-2, respectively), that inhibit endocytosis of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor. Sts-1 and Sts-2 contain SH3 domains that interacted with Cbl, Ub-associated domains, which bound directly to mono-Ub or to the EGFR/Ub chimera as well as phosphoglycerate mutase domains that mediated oligomerization of Sts-1/2. Ligand-induced recruitment of Sts-1/Sts-2 into activated EGFR complexes led to inhibition of receptor internalization, reduction in the number of EGFR-containing endocytic vesicles, and subsequent block of receptor degradation followed by prolonged activation of mitogenic signaling pathways. On the other hand, interference with Sts-1/Sts-2 functions diminished ligand-induced receptor degradation, cell proliferation, and oncogenic transformation in cultured fibroblasts. We suggest that Sts-1 and Sts-2 represent a novel class of Ub-binding proteins that regulate RTK endocytosis and control growth factor-induced cellular functions.

Place, publisher, year, edition, pages
The American Society for Biochemistry and Molecular Biology, Inc., 2004
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-91988 (URN)10.1074/jbc.M403759200 (DOI)15159412 (PubMedID)
Available from: 2004-09-14 Created: 2004-09-14 Last updated: 2017-12-14Bibliographically approved

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