Abstract
The family of seven-in-absentia (SIAH) ubiquitin E3 ligases functions in the control of numerous key signaling pathways. These enzymes belong to the RING (really interesting new gene) group of E3 ligases and mediate the attachment of ubiquitin chains to substrates, which then leads to their proteasomal degradation. Here, we describe a protocol that allows measuring SIAH-mediated ubiquitination and degradation of its client proteins as exemplified by acetyl transferases using simple overexpression experiments. The impact of SIAH expression on the relative amounts of target proteins and their mRNAs can be quantified by Western blotting and quantitative PCR (qPCR) as described here.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schmitz ML, Grishina I (2012) Regulation of the tumor suppressor PML by sequential post-translational modifications. Front Oncol 2:204
Scott JD, Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Science 326:1220–1224
Lorenz S, Cantor AJ, Rape M, Kuriyan J (2013) Macromolecular juggling by ubiquitylation enzymes. BMC Biol 11:65
Walczak H, Iwai K, Dikic I (2012) Generation and physiological roles of linear ubiquitin chains. BMC Biol 10:23
Lauwers E, Jacob C, Andre B (2009) K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway. J Cell Biol 185:493–502
Kleiger G, Mayor T (2014) Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Biol 24:352–359
Hoeller D, Dikic I (2009) Targeting the ubiquitin system in cancer therapy. Nature 458:438–444
Lill JR, Wertz IE (2014) Toward understanding ubiquitin-modifying enzymes: from pharmacological targeting to proteomics. Trends Pharmacol Sci 35:187–207
Richardson PG, Sonneveld P, Schuster MW, Irwin D, Stadtmauer EA, Facon T, Harousseau JL, Ben-Yehuda D, Lonial S, Goldschmidt H, Reece D, San-Miguel JF, Blade J, Boccadoro M, Cavenagh J, Dalton WS, Boral AL, Esseltine DL, Porter JB, Schenkein D, Anderson KC (2005) Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med 352:2487–2498
Popovic D, Vucic D, Dikic I (2014) Ubiquitination in disease pathogenesis and treatment. Nat Med 20:1242–1253
Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78:399–434
Budhidarmo R, Nakatani Y, Day CL (2012) RINGs hold the key to ubiquitin transfer. Trends Biochem Sci 37:58–65
House CM, Moller A, Bowtell DD (2009) Siah proteins: novel drug targets in the Ras and hypoxia pathways. Cancer Res 69:8835–8838
Krämer OH, Stauber RH, Bug G, Hartkamp J, Knauer SK (2013) SIAH proteins: critical roles in leukemogenesis. Leukemia 27:792–802
Qi J, Kim H, Scortegagna M, Ronai ZA (2013) Regulators and effectors of Siah ubiquitin ligases. Cell Biochem Biophys 67:15–24
Calzado MA, de la Vega L, Moller A, Bowtell DD, Schmitz ML (2009) An inducible autoregulatory loop between HIPK2 and Siah2 at the apex of the hypoxic response. Nat Cell Biol 11:85–91
Grishina I, Debus K, Garcia-Limones C, Schneider C, Shresta A, Garcia C, Calzado MA, Schmitz ML (2012) SIAH-mediated ubiquitination and degradation of acetyl-transferases regulate the p53 response and protein acetylation. Biochim Biophys Acta 1823:2287–2296
Khurana A, Nakayama K, Williams S, Davis RJ, Mustelin T, Ronai Z (2006) Regulation of the ring finger E3 ligase Siah2 by p38 MAPK. J Biol Chem 281:35316–35326
Depaux A, Regnier-Ricard F, Germani A, Varin-Blank N (2007) A crosstalk between hSiah2 and Pias E3-ligases modulates Pias-dependent activation. Oncogene 26:6665–6676
Famulski JK, Trivedi N, Howell D, Yang Y, Tong Y, Gilbertson R, Solecki DJ (2010) Siah regulation of Pard3A controls neuronal cell adhesion during germinal zone exit. Science 330:1834–1838
Kramer OH, Muller S, Buchwald M, Reichardt S, Heinzel T (2008) Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RARalpha. FASEB J 22:1369–1379
Moller A, House CM, Wong CS, Scanlon DB, Liu MC, Ronai Z, Bowtell DD (2009) Inhibition of Siah ubiquitin ligase function. Oncogene 28:289–296
Nakayama K, Gazdoiu S, Abraham R, Pan ZQ, Ronai Z (2007) Hypoxia-induced assembly of prolyl hydroxylase PHD3 into complexes: implications for its activity and susceptibility for degradation by the E3 ligase Siah2. Biochem J 401:217–226
Eisenberg E, Levanon EY (2013) Human housekeeping genes, revisited. Trends Genet 29:569–574
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta delta C(T)) method. Methods 25:402–408
Acknowledgements
The work from M.L.S. is supported by grants from the Deutsche Forschungsgemeinschaft (SFB 1213) and Deutsche Krebshilfe (111447). The work of M.K. is supported from the Deutsche Forschungsgemeinschaft (Kr1143/5-3 and Kr1143/7-3). Both laboratories are further supported by SFB/TRR81, SFB1021, and the Excellence Cluster Cardio-Pulmonary System (ECCPS).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Hagenbucher, J., Stekman, H., Rodriguez-Gil, A., Kracht, M., Schmitz, M.L. (2017). Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases. In: Krämer, O. (eds) HDAC/HAT Function Assessment and Inhibitor Development. Methods in Molecular Biology, vol 1510. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6527-4_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6527-4_22
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6525-0
Online ISBN: 978-1-4939-6527-4
eBook Packages: Springer Protocols