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Cbl promotes clustering of endocytic adaptor proteins

Nature Structural & Molecular Biology volume 12, pages 972–979 (2005)Cite this article

Abstract

The ubiquitin ligases c-Cbl and Cbl-b play a crucial role in receptor downregulation by mediating multiple monoubiquitination of receptors and promoting their sorting for lysosomal degradation. Their function is modulated through interactions with regulatory proteins including CIN85 and PIX, which recognize a proline-arginine motif in Cbl and thus promote or inhibit receptor endocytosis. We report the structures of SH3 domains of CIN85 and β-PIX in complex with a proline-arginine peptide from Cbl-b. Both structures reveal a heterotrimeric complex containing two SH3 domains held together by a single peptide. Trimerization also occurs in solution and is facilitated by the pseudo-symmetrical peptide sequence. Moreover, ternary complexes of CIN85 and Cbl are formed in vivo and are important for the ability of Cbl to promote epidermal growth factor receptor (EGFR) downregulation. These results provide molecular explanations for a novel mechanism by which Cbl controls receptor downregulation.

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Figure 1: Domain structure of β-PIX and CIN85 and sequence alignment of SH3 domains.
Figure 2: Overall structure of the β-PIX–Cbl-b and CIN85A–Cbl-b heterotrimeric complexes and details of the protein-peptide contacts.
Figure 3: Isothermal titration calorimetry measurements of complex formation.
Figure 4: c-Cbl and Cbl-b mediate ternary complex formation with CIN85 in vivo.
Figure 5: Comparison of the ternary β-PIX–Cbl-b complex with a SuperSH3 domain.

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Acknowledgements

The β-PIX-SH3 plasmid was a gift of E. Manser (Institute of Molecular and Cell Biology, Singapore). We are grateful to S. Howell and L. Haire (National Institute for Medical Research, London) for mass spectrometry; P. Fletcher (National Institute for Medical Research, London) and W. Mawby (University of Bristol, Bristol, UK) for peptide synthesis; J. Nicholson and R. Kehoe at Daresbury Laboratory for beamline assistance; A. Albert for help in data collection and M. Ortiz for help with CIN85A structure refinement. Research on CIN85 was supported by grant SAF2003-03860 of the Ministerio de Ciencia y Tecnología, Spain, and N.C. received a fellowship from the Ramón Areces Foundation. D.J. and K.R. are funded by the Medical Research Council, UK. Work by Y.L.D, D.H. and I.D. is supported by grants from Deutsche Forschungsgemeinschaft, Germany.

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Author notes

  1. Daniela Jozic, Nayra Cárdenes and Yonathan Lissanu Deribe: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Protein Structure, National Institute for Medical Research, The Ridgeway, London, NW7 1AA, UK

    Daniela Jozic & Katrin Rittinger

  2. Signal Transduction Group, Structural Biology and Biocomputing Programme, Centro Nacional de Investigaciones Oncológicas (CNIO), Melchor Fernández Almagro 3, Madrid, E-28029, Spain

    Nayra Cárdenes, Gabriel Moncalián & Jerónimo Bravo

  3. Institute of Biochemistry II, Goethe University Medical School, Theodor-Stern-Kai 7, Frankfurt a/M, 60590, Germany

    Yonathan Lissanu Deribe, Daniela Hoeller & Ivan Dikic

  4. Abteilung Biomolekulare Mechanismen, Max-Planck-Institut fuer Medizinische Forschung, Jahnstrasse 29, Heidelberg, 69120, Germany

    Yvonne Groemping

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Correspondence to Ivan Dikic, Katrin Rittinger or Jerónimo Bravo.

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Jozic, D., Cárdenes, N., Deribe, Y. et al. Cbl promotes clustering of endocytic adaptor proteins. Nat Struct Mol Biol 12, 972–979 (2005). https://doi.org/10.1038/nsmb1000

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