Testing the Effects of SIAH Ubiquitin E3 Ligases on Lysine Acetyl Transferases

  • Jan Hagenbucher
  • Hilda Stekman
  • Alfonso Rodriguez-Gil
  • Michael Kracht
  • M. Lienhard Schmitz
Part of the Methods in Molecular Biology book series (MIMB, volume 1510)


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.

Key words

Ubiquitin E3 ligases Proteasome Protein degradation Transfection Western blot qPCR 



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).


  1. 1.
    Schmitz ML, Grishina I (2012) Regulation of the tumor suppressor PML by sequential post-translational modifications. Front Oncol 2:204CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Scott JD, Pawson T (2009) Cell signaling in space and time: where proteins come together and when they’re apart. Science 326:1220–1224CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Lorenz S, Cantor AJ, Rape M, Kuriyan J (2013) Macromolecular juggling by ubiquitylation enzymes. BMC Biol 11:65CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Walczak H, Iwai K, Dikic I (2012) Generation and physiological roles of linear ubiquitin chains. BMC Biol 10:23CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    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 185:493–502CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Kleiger G, Mayor T (2014) Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Biol 24:352–359CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Hoeller D, Dikic I (2009) Targeting the ubiquitin system in cancer therapy. Nature 458:438–444CrossRefPubMedGoogle Scholar
  8. 8.
    Lill JR, Wertz IE (2014) Toward understanding ubiquitin-modifying enzymes: from pharmacological targeting to proteomics. Trends Pharmacol Sci 35:187–207CrossRefPubMedGoogle Scholar
  9. 9.
    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 352:2487–2498CrossRefPubMedGoogle Scholar
  10. 10.
    Popovic D, Vucic D, Dikic I (2014) Ubiquitination in disease pathogenesis and treatment. Nat Med 20:1242–1253CrossRefPubMedGoogle Scholar
  11. 11.
    Deshaies RJ, Joazeiro CA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78:399–434CrossRefPubMedGoogle Scholar
  12. 12.
    Budhidarmo R, Nakatani Y, Day CL (2012) RINGs hold the key to ubiquitin transfer. Trends Biochem Sci 37:58–65CrossRefPubMedGoogle Scholar
  13. 13.
    House CM, Moller A, Bowtell DD (2009) Siah proteins: novel drug targets in the Ras and hypoxia pathways. Cancer Res 69:8835–8838CrossRefPubMedGoogle Scholar
  14. 14.
    Krämer OH, Stauber RH, Bug G, Hartkamp J, Knauer SK (2013) SIAH proteins: critical roles in leukemogenesis. Leukemia 27:792–802CrossRefPubMedGoogle Scholar
  15. 15.
    Qi J, Kim H, Scortegagna M, Ronai ZA (2013) Regulators and effectors of Siah ubiquitin ligases. Cell Biochem Biophys 67:15–24CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    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 11:85–91CrossRefPubMedGoogle Scholar
  17. 17.
    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 1823:2287–2296CrossRefPubMedGoogle Scholar
  18. 18.
    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 281:35316–35326CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Depaux A, Regnier-Ricard F, Germani A, Varin-Blank N (2007) A crosstalk between hSiah2 and Pias E3-ligases modulates Pias-dependent activation. Oncogene 26:6665–6676CrossRefPubMedGoogle Scholar
  20. 20.
    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 330:1834–1838CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    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 22:1369–1379CrossRefPubMedGoogle Scholar
  22. 22.
    Moller A, House CM, Wong CS, Scanlon DB, Liu MC, Ronai Z, Bowtell DD (2009) Inhibition of Siah ubiquitin ligase function. Oncogene 28:289–296CrossRefPubMedGoogle Scholar
  23. 23.
    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 401:217–226CrossRefPubMedGoogle Scholar
  24. 24.
    Eisenberg E, Levanon EY (2013) Human housekeeping genes, revisited. Trends Genet 29:569–574CrossRefPubMedGoogle Scholar
  25. 25.
    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 25:402–408CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Jan Hagenbucher
    • 1
  • Hilda Stekman
    • 1
  • Alfonso Rodriguez-Gil
    • 1
  • Michael Kracht
    • 2
  • M. Lienhard Schmitz
    • 1
  1. 1.Medical Faculty, Institute of BiochemistryJustus-Liebig-UniversityGiessenGermany
  2. 2.Rudolf-Buchheim-Institute of PharmacologyJustus-Liebig-University GiessenGiessenGermany

Personalised recommendations