Abstract
The eight SOCS (Suppressor of Cytokine Signaling) proteins encoded in the human genome all contain a C-terminal domain, the SOCS box, that allows them to function as E3 ubiquitin ligases and thereby catalyze the ubiquitination of components of the JAK/STAT signaling pathway. This activity is key to their function as cytokine signaling inhibitors as, once ubiquitinated, signaling molecules are degraded by the proteasome which allows the cell to return to its basal (unstimulated) state. SOCS based E3s are a subset of the CRL (Cullin-Ring-Ligase) family of ubiquitin ligases with the SOCS protein acting as the substrate recruitment module and interacting specifically with Cullin5, the E3 scaffold. Included here are protocols for the expression and purification of SOCS-based E3 complexes and their use in in vitro ubiquitination assays to characterize potential substrates. We have currently verified two components of the JAK/STAT pathway as substrates for ubiquitination using this method.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Hilton DJ, Richardson RT, Alexander WS, Viney EM, Willson TA, Sprigg NS, Starr R, Nicholson SE, Metcalf D, Nicola NA (1998) Twenty proteins containing a C-terminal SOCS box form five structural classes. Proc Natl Acad Sci U S A 95:114–119
Starr R, Willson TA, Viney EM, Murray LJ, Rayner JR, Jenkins BJ, Gonda TJ, Alexander WS, Metcalf D, Nicola NA, Hilton DJ (1997) A family of cytokine-inducible inhibitors of signalling. Nature 387:917–921
Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N, Taniguchi T, Fujita T, Kanakura Y, Komiya S, Yoshimura A (1997) A new protein containing an SH2 domain that inhibits JAK kinases. Nature 387:921–924
Naka T, Narazaki M, Hirata M, Matsumoto T, Minamoto S, Aono A, Nishimoto N, Kajita T, Taga T, Yoshizaki K, Akira S, Kishimoto T (1997) Structure and function of a new STAT-induced STAT inhibitor. Nature 387:924–929
Zhang JG, Farley A, Nicholson SE, Willson TA, Zugaro LM, Simpson RJ, Moritz RL, Cary D, Richardson R, Hausmann G, Kile BJ, Kent SB, Alexander WS, Metcalf D, Hilton DJ, Nicola NA, Baca M (1999) The conserved SOCS box motif in Suppressor of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc Natl Acad Sci U S A 96:2071–2076
Dye BT, Schulman BA (2007) Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins. Annu Rev Biophys Biomol Struct 36:131–150
Hochstrasser M (2006) Lingering mysteries of ubiquitin-chain assembly. Cell 124:27–34
Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533
Zheng N, Schulman BA, Song L, Miller JJ, Jeffrey PD, Wang P, Chu C, Koepp DM, Elledge SJ, Pagano M, Conaway RC, Conaway JW, Harper JW, Pavletich NP (2002) Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416:703–709
Petroski MD, Deshaies RJ (2005) Function and regulation of cullin-RING ubiquitin ligases. Nat Rev Mol Cell Biol 6:9–20
Kamura T, Maenaka K, Kotoshiba S, Matsumoto M, Kohda D, Conaway RC, Conaway JW, Nakayama KI (2004) VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases. Genes Dev 18:3055–3065
Reynolds PJ, Simms JR, Duronio RJ (2008) Identifying determinants of cullin binding specificity among the three functionally different Drosophila melanogaster Roc proteins via domain swapping. PLoS One 3:e2918
Babon JJ, Sabo JK, Soetopo A, Yao S, Bailey MF, Zhang JG, Nicola NA, Norton RS (2008) The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase. J Mol Biol 381:928–940
Babon JJ, Sabo JK, Zhang JG, Nicola NA, Norton RS (2009) The SOCS box encodes a hierarchy of affinities for Cullin5: implications for ubiquitin ligase formation and cytokine signalling suppression. J Mol Biol 387:162–174
Kile BT, Schulman BA, Alexander WS, Nicola NA, Martin HM, Hilton DJ (2002) The SOCS box: a tale of destruction and degradation. Trends Biochem Sci 27:235–241
Krebs DL, Hilton DJ (2001) SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19:378–387
Hilton DJ (1999) Negative regulators of cytokine signal transduction. Cell Mol Life Sci 55:1568–1577
Li T, Pavletich NP, Schulman BA, Zheng N (2005) High-level expression and purification of recombinant SCF ubiquitin ligases. Methods Enzymol 398:125–142
Acknowledgments
This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. This research was supported by an NHMRC Program Grant (461219), NIH Grant (CA022556), NHMRC CDA (JJB, 516777), and NHMRC Project Grant (1011804).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Babon, J.J., Laktyushin, A., Kershaw, N.J. (2013). In Vitro Ubiquitination of Cytokine Signaling Components. In: Nicholson, S., Nicola, N. (eds) JAK-STAT Signalling. Methods in Molecular Biology, vol 967. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-242-1_19
Download citation
DOI: https://doi.org/10.1007/978-1-62703-242-1_19
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-241-4
Online ISBN: 978-1-62703-242-1
eBook Packages: Springer Protocols