Biophysical studies on interactions and assembly of full-size E3 ubiquitin ligase: suppressor of cytokine signaling 2 (SOCS2)-elongin BC-cullin 5-ring box protein 2 (RBX2).

Abstract

The multisubunit 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, among their other functions, serve as substrate receptors of CRL5 complexes. A member of the CRL family, SOCS2-EloBC-Cul5-Rbx2 (CRL5(SOCS2)), binds phosphorylated growth hormone receptor as its main substrate. Here, we demonstrate that the components of CRL5(SOCS2) can be specifically pulled from K562 human cell lysates using beads decorated with phosphorylated growth hormone receptor peptides. Subsequently, SOCS2-EloBC and full-length Cul5-Rbx2, recombinantly expressed in Escherichia coli and in Sf21 insect cells, respectively, were used to reconstitute neddylated and unneddylated CRL5(SOCS2) complexes in vitro. Finally, diverse biophysical methods were employed to study the assembly and interactions within the complexes. Unlike other E3 ligases, CRL5(SOCS2) was found to exist in a monomeric state as confirmed by size exclusion chromatography with inline multiangle static light scattering and native MS. Affinities of the protein-protein interactions within the multisubunit complex were measured by isothermal titration calorimetry. A structural model for full-size neddylated and unneddylated CRL5(SOCS2) complexes is supported by traveling wave ion mobility mass spectrometry data.

• Authors: Emil Bulatov, Esther M Martin, Sneha Chatterjee, Axel Knebel, Satoko Shimamura, Albert Konijnenberg, Clare Johnson, Nico Zinn, Paola Grandi, Frank Sobott, Alessio Ciulli
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 290 Issue 7 Pg. 4178-91 (Feb 13 2015) ISSN: 1083-351X [Electronic] United States
• PMID: 25505247 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Chemical References

• CUL5 protein, human
• Cullin Proteins
• Elongin
• NEDD8 Protein
• NEDD8 protein, human
• SOCS2 protein, human
• Suppressor of Cytokine Signaling Proteins
• Transcription Factors
• Ubiquitins
• RNF7 protein, human
• Ubiquitin-Protein Ligases

Topics

• Cullin Proteins (chemistry, genetics, metabolism)
• Elongin
• Humans
• K562 Cells
• Mass Spectrometry
• Models, Molecular
• NEDD8 Protein
• Protein Binding
• Protein Conformation
• Suppressor of Cytokine Signaling Proteins (chemistry, genetics, metabolism)
• Transcription Factors (chemistry, genetics, metabolism)
• Ubiquitin-Protein Ligases (chemistry, genetics, metabolism)
• Ubiquitination
• Ubiquitins (chemistry, genetics, metabolism)