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Cbl-directed monoubiquitination of CIN85 is involved in regulation of ligand-induced degradation 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.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
2002 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 99, no 19, 12191-12196 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2002. Vol. 99, no 19, 12191-12196 p.
Keyword [en]
Adaptor Proteins; Signal Transducing, Animals, Carrier Proteins/chemistry/genetics/*metabolism, Cell Line, Cytoskeletal Proteins, Epidermal Growth Factor/pharmacology, Humans, Ligands, Models; Biological, Mutagenesis; Site-Directed, Oncogene Protein v-cbl, Receptor; Epidermal Growth Factor/genetics/*metabolism, Recombinant Proteins/chemistry/genetics/metabolism, Retroviridae Proteins; Oncogenic/genetics/*metabolism, Ubiquitin/metabolism
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-91755DOI: 10.1073/pnas.192462299PubMedID: 12218189OAI: oai:DiVA.org:uu-91755DiVA: diva2:164588
Note

De två första författarna delar första författarskapet.

Available from: 2004-04-23 Created: 2004-04-23 Last updated: 2017-12-14Bibliographically approved
In thesis
1. Ubiquitination and Receptor Endocytosis
Open this publication in new window or tab >>Ubiquitination and Receptor Endocytosis
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Protein ubiquitination is an evolutionary conserved mechanism that controls a wide variety of cellular functions. Polyubiquitinated proteins are generally degraded in the proteasome, whereas monoubiquitination controls various other cellular processes, including endocytosis and endosomal sorting.

Termination of signaling by activated receptor tyrosine kinases (RTKs) largely occurs via their endocytosis and subsequent lysosomal degradation, processes accompanied by receptor ubiquitination. Cbl family proteins are major ubiquitin ligases that promote RTK ubiquitination and downregulation. We showed that epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptors are monoubiquitinated at multiple sites following their ligand-induced activation and that a single ubiquitin is sufficient for both receptor internalization and degradation. Cbl also controls EGF receptor (EGFR) downregulation by binding to CIN85, which recruits endophilins to EGFR/Cbl complexes. In the complex with activated EGFRs, Cbl directs monoubiquitination of CIN85, and the entire complex is targeted for degradation in the lysosome. We propose that multiple monoubiquitination of activated receptors and associated protein complexes ensures proper receptor sorting towards the lysosome. Importantly, the functions of Cbl are also negatively controlled in order to maintain cellular homestasis. Sprouty2 blocks EGFR downregulation by sequestering Cbl from activated EGFRs. We showed that Sprouty2 also associates with CIN85 and that this binding is required for efficient inhibition of EGFR ubiquitination and endocytosis.

Cbl is also implicated in other aspects of RTK signaling, including organization of the actin cytoskeleton. We found that growth factor receptor signals promote lamellipodia formation in neuronal cells via a complex containing Cbl, the adaptor protein ArgBP2 and Pyk2. The lamellipodia formation required intact lipid rafts and the recruitment of Crk and PI(3)K to tyrosine phosphorylated Cbl.

In conclusion, our findings contribute to a better understanding of monoubiquitin signals in downregulation of RTKs and point at a role of Cbl in the regulation of cytoskeleton dynamics.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2004. 79 p.
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1351
Keyword
Cell and molecular biology, Ubiquitination, receptor tyrosine kinase, endocytosis, Cbl, CIN85, EGFR, Cell- och molekylärbiologi
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-4259 (URN)91-554-5965-X (ISBN)
Public defence
2004-05-14, B22, Biomedical Center (BMC), Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2004-04-23 Created: 2004-04-23Bibliographically approved
2. 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

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