Advertisement

Investigating Mechanisms that Control Ubiquitin-Mediated DAF-16/FOXO Protein Turnover

  • Thomas HeimbucherEmail author
  • Coleen T. MurphyEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)

Abstract

Protein turnover of FOXO family transcription factors is regulated by the ubiquitin-proteasome system. A complex interplay of factors that covalently attach certain types of ubiquitin chains (E3-ubiquitin ligases), and enzymes that are able to remove ubiquitin conjugates (deubiquitylases), regulate the degradation of FOXO proteins by the proteasome. Here, we describe methods to characterize candidate E3-ubiquitin ligases and deubiquitylases as regulators of the FOXO ubiquitylation status. Our protocol can be utilized to purify and enrich a ubiquitylated FOXO pool from cultured cells under denaturing conditions, which inactivates cellular deubiquitylases and thereby protects ubiquitin conjugates on FOXO proteins. In addition, our method describes how ubiquitylated FOXO proteins can be renatured in a stepwise fashion to serve as substrates for in vitro deubiquitylation (DUB) assays.

Key words

FOXO DAF-16 Protein stability Ubiquitylation assay Deubiquitylation (DUB) assay Ubiquitin Proteasome 

Notes

Acknowledgments

This work was supported by the NIH NIA 1R56AG047344-01A1 (C.T.M.) and the Glenn Foundation for Medical Research. C.T.M. is the Director of the Glenn Center for Aging Research at Princeton. We thank Dr. Andrea Carrano for critically reading the manuscript.

References

  1. 1.
    Huang H, Tindall DJ (2011) Regulation of FOXO protein stability via ubiquitination and proteasome degradation. Biochim Biophys Acta 1813(11):1961–1964.  https://doi.org/10.1016/j.bbamcr.2011.01.007 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479.  https://doi.org/10.1146/annurev.biochem.67.1.425 CrossRefPubMedGoogle Scholar
  3. 3.
    Huang H, Regan KM, Wang F, Wang D, Smith DI, van Deursen JM, Tindall DJ (2005) Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation. Proc Natl Acad Sci U S A 102(5):1649–1654.  https://doi.org/10.1073/pnas.0406789102 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Yang JY, Zong CS, Xia W, Yamaguchi H, Ding Q, Xie X, Lang JY, Lai CC, Chang CJ, Huang WC, Huang H, Kuo HP, Lee DF, Li LY, Lien HC, Cheng X, Chang KJ, Hsiao CD, Tsai FJ, Tsai CH, Sahin AA, Muller WJ, Mills GB, Yu D, Hortobagyi GN, Hung MC (2008) ERK promotes tumorigenesis by inhibiting FOXO3a via MDM2-mediated degradation. Nat Cell Biol 10(2):138–148.  https://doi.org/10.1038/ncb1676 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hu MC, Lee DF, Xia W, Golfman LS, Ou-Yang F, Yang JY, Zou Y, Bao S, Hanada N, Saso H, Kobayashi R, Hung MC (2004) IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 117(2):225–237CrossRefGoogle Scholar
  6. 6.
    Fu W, Ma Q, Chen L, Li P, Zhang M, Ramamoorthy S, Nawaz Z, Shimojima T, Wang H, Yang Y, Shen Z, Zhang Y, Zhang X, Nicosia SV, Zhang Y, Pledger JW, Chen J, Bai W (2009) MDM2 acts downstream of p53 as an E3 ligase to promote FOXO ubiquitination and degradation. J Biol Chem 284(21):13987–14000.  https://doi.org/10.1074/jbc.M901758200 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    van der Horst A, de Vries-Smits AM, Brenkman AB, van Triest MH, van den Broek N, Colland F, Maurice MM, Burgering BM (2006) FOXO4 transcriptional activity is regulated by monoubiquitination and USP7/HAUSP. Nat Cell Biol 8(10):1064–1073.  https://doi.org/10.1038/ncb1469 CrossRefPubMedGoogle Scholar
  8. 8.
    Heimbucher T, Liu Z, Bossard C, McCloskey R, Carrano AC, Riedel CG, Tanasa B, Klammt C, Fonslow BR, Riera CE, Lillemeier BF, Kemphues K, Yates JR 3rd, O'Shea C, Hunter T, Dillin A (2015) The Deubiquitylase MATH-33 controls DAF-16 stability and function in metabolism and longevity. Cell Metab 22(1):151–163.  https://doi.org/10.1016/j.cmet.2015.06.002 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Zheng X, Zhai B, Koivunen P, Shin SJ, Lu G, Liu J, Geisen C, Chakraborty AA, Moslehi JJ, Smalley DM, Wei X, Chen X, Chen Z, Beres JM, Zhang J, Tsao JL, Brenner MC, Zhang Y, Fan C, DePinho RA, Paik J, Gygi SP, Kaelin WG Jr, Zhang Q (2014) Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase. Genes Dev 28(13):1429–1444.  https://doi.org/10.1101/gad.242131.114 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Li W, Gao B, Lee SM, Bennett K, Fang D (2007) RLE-1, an E3 ubiquitin ligase, regulates C. elegans aging by catalyzing DAF-16 polyubiquitination. Dev Cell 12(2):235–246.  https://doi.org/10.1016/j.devcel.2006.12.002 CrossRefPubMedGoogle Scholar
  11. 11.
    van der Knaap JA, Kumar BR, Moshkin YM, Langenberg K, Krijgsveld J, Heck AJ, Karch F, Verrijzer CP (2005) GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7. Mol Cell 17(5):695–707.  https://doi.org/10.1016/j.molcel.2005.02.013 CrossRefPubMedGoogle Scholar
  12. 12.
    Heimbucher T, Hunter T (2015) The C. elegans Ortholog of USP7 controls DAF-16 stability in insulin/IGF-1-like signaling. WormBook 4(4):e1103429.  https://doi.org/10.1080/21624054.2015.1103429 CrossRefGoogle Scholar
  13. 13.
    Treier M, Staszewski LM, Bohmann D (1994) Ubiquitin-dependent c-Jun degradation in vivo is mediated by the delta domain. Cell 78(5):787–798CrossRefGoogle Scholar
  14. 14.
    Kwon ES, Narasimhan SD, Yen K, Tissenbaum HA (2010) A new DAF-16 isoform regulates longevity. Nature 466(7305):498–502.  https://doi.org/10.1038/nature09184 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    McCloskey RJ, Kemphues KJ (2012) Deubiquitylation machinery is required for embryonic polarity in Caenorhabditis elegans. PLoS Genet 8(11):e1003092.  https://doi.org/10.1371/journal.pgen.1003092 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Wang F, Chan CH, Chen K, Guan X, Lin HK, Tong Q (2012) Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation. Oncogene 31(12):1546–1557.  https://doi.org/10.1038/onc.2011.347 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Lewis-Sigler Institute for Integrative GenomicsPrinceton UniversityPrincetonUSA
  2. 2.Paul F. Glenn Laboratories for Aging ResearchPrinceton UniversityPrincetonUSA
  3. 3.Department of Molecular BiologyPrinceton UniversityPrincetonUSA

Personalised recommendations