Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase

suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2)

Emil Bulatov, Esther M. Martin, Sneha Chatterjee, Axel Knebel, Satoko Shimamura, Albert Konijnenberg, Clare Johnson, Nico Zinn, Paola Grandi, Frank Sobott, Alessio Ciulli (Lead / Corresponding author)

    Research output: Contribution to journalArticle

    12 Citations (Scopus)

    Abstract

    The multi-subunit Cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes and cancer, and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, amongst their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) binds phosphorylated growth hormone receptor (GHR) as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated GHR peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in E. coli and in Sf21 insect cells, respectively, were used to reconstitute CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike many other E3 ligases, the CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multi-angle static light scattering (SEC-MALS) and native mass spectrometry (MS). Affinities of the protein-protein interactions within the multi-subunit complex were measured by isothermal titration calorimetry (ITC). A structural model for the full-size CRL5SOCS2 is supported by travelling wave ion-mobility mass spectrometry (TWIM-MS) data.

    Original languageEnglish
    Pages (from-to)4178-4191
    Number of pages14
    JournalJournal of Biological Chemistry
    Volume290
    Issue number7
    DOIs
    Publication statusPublished - 13 Feb 2015

    Fingerprint

    Ubiquitin-Protein Ligases
    Suppressor of Cytokine Signaling Proteins
    Cullin Proteins
    Cytokines
    Somatotropin Receptors
    Mass spectrometry
    Mass Spectrometry
    Proteins
    Substrates
    Sf9 Cells
    Calorimetry
    K562 Cells
    Size exclusion chromatography
    Ubiquitination
    Structural Models
    Medical problems
    Titration
    Light scattering
    Escherichia coli
    Gel Chromatography

    Cite this

    Bulatov, Emil ; Martin, Esther M. ; Chatterjee, Sneha ; Knebel, Axel ; Shimamura, Satoko ; Konijnenberg, Albert ; Johnson, Clare ; Zinn, Nico ; Grandi, Paola ; Sobott, Frank ; Ciulli, Alessio. / Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase : suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2). In: Journal of Biological Chemistry. 2015 ; Vol. 290, No. 7. pp. 4178-4191.
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    title = "Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2)",
    abstract = "The multi-subunit Cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes and cancer, and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, amongst their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) binds phosphorylated growth hormone receptor (GHR) as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated GHR peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in E. coli and in Sf21 insect cells, respectively, were used to reconstitute CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike many other E3 ligases, the CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multi-angle static light scattering (SEC-MALS) and native mass spectrometry (MS). Affinities of the protein-protein interactions within the multi-subunit complex were measured by isothermal titration calorimetry (ITC). A structural model for the full-size CRL5SOCS2 is supported by travelling wave ion-mobility mass spectrometry (TWIM-MS) data.",
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    Bulatov, E, Martin, EM, Chatterjee, S, Knebel, A, Shimamura, S, Konijnenberg, A, Johnson, C, Zinn, N, Grandi, P, Sobott, F & Ciulli, A 2015, 'Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2)', Journal of Biological Chemistry, vol. 290, no. 7, pp. 4178-4191. https://doi.org/10.1074/jbc.M114.616664

    Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase : suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2). / Bulatov, Emil; Martin, Esther M.; Chatterjee, Sneha; Knebel, Axel; Shimamura, Satoko; Konijnenberg, Albert; Johnson, Clare; Zinn, Nico; Grandi, Paola; Sobott, Frank; Ciulli, Alessio (Lead / Corresponding author).

    In: Journal of Biological Chemistry, Vol. 290, No. 7, 13.02.2015, p. 4178-4191.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase

    T2 - suppressor of cytokine signaling 2 (SOCS2):ElonginBC:Cullin5:RING-box protein 2 (Rbx2)

    AU - Bulatov, Emil

    AU - Martin, Esther M.

    AU - Chatterjee, Sneha

    AU - Knebel, Axel

    AU - Shimamura, Satoko

    AU - Konijnenberg, Albert

    AU - Johnson, Clare

    AU - Zinn, Nico

    AU - Grandi, Paola

    AU - Sobott, Frank

    AU - Ciulli, Alessio

    N1 - Copyright © 2014, The American Society for Biochemistry and Molecular Biology.

    PY - 2015/2/13

    Y1 - 2015/2/13

    N2 - The multi-subunit Cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes and cancer, and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, amongst their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) binds phosphorylated growth hormone receptor (GHR) as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated GHR peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in E. coli and in Sf21 insect cells, respectively, were used to reconstitute CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike many other E3 ligases, the CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multi-angle static light scattering (SEC-MALS) and native mass spectrometry (MS). Affinities of the protein-protein interactions within the multi-subunit complex were measured by isothermal titration calorimetry (ITC). A structural model for the full-size CRL5SOCS2 is supported by travelling wave ion-mobility mass spectrometry (TWIM-MS) data.

    AB - The multi-subunit Cullin RING E3 ubiquitin ligases (CRLs) target post-translationally modified substrates for ubiquitination and proteasomal degradation. The suppressors of cytokine signaling (SOCS) proteins play important roles in inflammatory processes, diabetes and cancer, and therefore represent attractive targets for therapeutic intervention. The SOCS proteins, amongst their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5SOCS2) binds phosphorylated growth hormone receptor (GHR) as its main substrate. Here, we demonstrate that the components of CRL5SOCS2 can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated GHR peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in E. coli and in Sf21 insect cells, respectively, were used to reconstitute CRL5SOCS2 complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike many other E3 ligases, the CRL5SOCS2 was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multi-angle static light scattering (SEC-MALS) and native mass spectrometry (MS). Affinities of the protein-protein interactions within the multi-subunit complex were measured by isothermal titration calorimetry (ITC). A structural model for the full-size CRL5SOCS2 is supported by travelling wave ion-mobility mass spectrometry (TWIM-MS) data.

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    EP - 4191

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    JF - Journal of Biological Chemistry

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