Skip to main content
SpringerLink
Log in

Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

  • Original Article
  • Published:
Journal of Chemical Biology

Abstract

The tumor suppressor p53 has evolved a MDM2-dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg97/Lys98, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phospho-mimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10

Similar content being viewed by others

References

  1. Adams J, Chen ZP, Van Denderen BJ, Morton CJ, Parker MW, Witters LA, Stapleton D, Kemp BE (2004) Protein Sci 13:155

    Article  CAS  Google Scholar 

  2. Arva NC, Gopen TR, Talbot KE, Campbell LE, Chicas A, White DE, Bond GL, Levine AJ, Bargonetti J (2005) J Biol Chem 280:26776

    Article  CAS  Google Scholar 

  3. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Proc Natl Acad Sci U S A 98:10037

    Article  CAS  Google Scholar 

  4. Bottger V, Bottger A, Howard SF, Picksley SM, Chene P, Garcia-Echeverria C, Hochkeppel HK, Lane DP (1996) Oncogene 13:2141

    CAS  Google Scholar 

  5. Burch LR, Midgley CA, Currie RA, Lane DP, Hupp TR (2000) FEBS Lett 472:93

    Article  CAS  Google Scholar 

  6. Burch L, Shimizu H, Smith A, Patterson C, Hupp TR (2004) J Mol Biol 337:129

    Article  CAS  Google Scholar 

  7. Candeias MM, Malbert-Colas L, Powell DJ, Daskalogianni C, Maslon MM, Naski N, Bourougaa K, Calvo F, Fahraeus R (2008) Nat Cell Biol 10:1098

    Article  CAS  Google Scholar 

  8. Chen J, Marechal V, Levine AJ (1993) Mol Cell Biol 13:4107

    CAS  Google Scholar 

  9. Craig AL, Chrystal JA, Fraser JA, Sphyris N, Lin Y, Harrison BJ, Scott MT, Dornreiter I, Hupp TR (2007) Mol Cell Biol 27:3542

    Article  CAS  Google Scholar 

  10. Dornan D, Hupp TR (2001) EMBO Rep 2:139

    Article  CAS  Google Scholar 

  11. Dyson HJ, Wright PE (2002) Curr Opin Struct Biol 12:54

    Article  CAS  Google Scholar 

  12. Elenbaas B, Dobbelstein M, Roth J, Shenk T, Levine AJ (1996) Mol Med 2:439

    CAS  Google Scholar 

  13. Gannon JV, Greaves R, Iggo R, Lane DP (1990) EMBO J 9:1595

    CAS  Google Scholar 

  14. Gouet P, Courcelle E, Stuart DI, Metoz F (1999) Bioinformatics 15:305

    Article  CAS  Google Scholar 

  15. Haupt Y, Maya R, Kazaz A, Oren M (1997) Nature 387:296

    Article  CAS  Google Scholar 

  16. Kobe B, Kemp BE (1999) Nature 402:373

    Article  CAS  Google Scholar 

  17. Kobe B, Heierhorst J, Kemp BE (1997) Adv Second Messenger Phosphoprotein Res 31:29

    CAS  Google Scholar 

  18. Kostic M, Matt T, Martinez-Yamout MA, Dyson HJ, Wright PE (2006) J Mol Biol 363:433

    Article  CAS  Google Scholar 

  19. Kubbutat MH, Jones SN, Vousden KH (1997) Nature 387:299

    Article  CAS  Google Scholar 

  20. Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ, Pavletich NP (1996) Science 274:948

    Article  CAS  Google Scholar 

  21. Lai Z, Freedman DA, Levine AJ, McLendon GL (1998) Biochemistry 37:17005

    Article  CAS  Google Scholar 

  22. Linke K, Mace PD, Smith CA, Vaux DL, Silke J, Day CL (2008) Cell Death Differ 15:841

    Article  CAS  Google Scholar 

  23. Maclaine NJ, Oster B, Bundgaard B, Fraser JA, Buckner C, Lazo PA, Meek DW, Hollsberg P, Hupp TR (2008) J Biol Chem 283:28563

    Article  CAS  Google Scholar 

  24. Marechal V, Elenbaas B, Piette J, Nicolas JC, Levine AJ (1994) Mol Cell Biol 14:7414

    CAS  Google Scholar 

  25. McCoy MA, Gesell JJ, Senior MM, Wyss DF (2003) Proc Natl Acad Sci U S A 100:1645

    Article  CAS  Google Scholar 

  26. Momand J, Zambetti GP, Olson DC, George D, Levine AJ (1992) Cell 69:1237

    Article  CAS  Google Scholar 

  27. Neduva V, Russell RB (2005) FEBS Lett 579:3342

    Article  CAS  Google Scholar 

  28. Neduva V, Russell RB (2006) Nucleic Acids Res 34:W350

    Article  CAS  Google Scholar 

  29. Neduva V, Russell RB (2006) Curr Opin Biotechnol 17:465

    Article  CAS  Google Scholar 

  30. Nenutil R, Smardova J, Pavlova S, Hanzelkova Z, Muller P, Fabian P, Hrstka R, Janotova P, Radina M, Lane D, Coates P, Vojtesek B (2005) J Pathol 207:251

    Article  CAS  Google Scholar 

  31. Ofir-Rosenfeld Y, Boggs K, Michael D, Kastan MB, Oren M (2008) Mol Cell 32:180

    Article  CAS  Google Scholar 

  32. Passmore LA, Barford D (2004) Biochem J 379:513–525

    Article  CAS  Google Scholar 

  33. Pettersson S, Kelleher M, Pion E, Wallace M, Ball KL (2009) Role of MDM2 acid domain interactions in recognition and ubiquitination of the transcription factor IRF-2. Biochem J 418:575–585

    Article  CAS  Google Scholar 

  34. Popowicz GM, Czarna A, Rothweiler U, Sszwagierczak A, Krajewski M, Weber L, Holak TA (2007) Cell Cycle 6:2386

    CAS  Google Scholar 

  35. Poyurovsky MV, Jacq X, Ma C, Karni-Schmidt O, Parker PJ, Chalfie M, Manley JL, Prives C (2003) Mol Cell 12:875

    Article  CAS  Google Scholar 

  36. Poyurovsky MV, Priest C, Kentsis A, Borden KL, Pan ZQ, Pavletich N, Prives C (2007) EMBO J 26:90

    Article  CAS  Google Scholar 

  37. Schon O, Friedler A, Freund S, Fersht AR (2004) J Mol Biol 336:197

    Article  CAS  Google Scholar 

  38. Shimizu H, Burch LR, Smith AJ, Dornan D, Wallace M, Ball KL, Hupp TR (2002) J Biol Chem 277:28446

    Article  CAS  Google Scholar 

  39. Shimizu H, Saliba D, Wallace M, Finlan L, Langridge-Smith PR, Hupp TR (2006) Biochem J 397:355

    Article  CAS  Google Scholar 

  40. Showalter SA, Bruschweiler-Li L, Johnson E, Zhang F, Bruschweiler R (2008) J Am Chem Soc 130:6472

    Article  CAS  Google Scholar 

  41. Stevens C, Pettersson S, Wawrzynow B, Wallace M, Ball K, Zylicz A, Hupp TR (2008) FEBS J 275:4875

    Article  CAS  Google Scholar 

  42. Terzian T, Suh YA, Iwakuma T, Post SM, Neumann M, Lang GA, Van Pelt CS, Lozano G (2008) Genes Dev 22:1337–1344

    Article  CAS  Google Scholar 

  43. Thut CJ, Goodrich JA, Tjian R (1997) Genes Dev 11:1974

    Article  CAS  Google Scholar 

  44. Toledo F, Wahl GM (2007) MDM2 and MDM4: p53 regulators as targets in anticancer therapy. Int J Biochem Cell Biol 39:1476

    Article  CAS  Google Scholar 

  45. Uhrinova S, Uhrin D, Powers H, Watt K, Zheleva D, Fischer P, McInnes C, Barlow PN (2005) J Mol Biol 350:587

    Article  CAS  Google Scholar 

  46. Uldrijan S, Pannekoek WJ, Vousden KH (2007) EMBO J 26:102

    Article  CAS  Google Scholar 

  47. Vassilev LT, Vu BT, Graves B, Carvajal D, Podlaski F, Filipovic Z, Kong N, Kammlott U, Lukacs C, Klein C, Fotouhi N, Liu EA (2004) Science 303:844

    Article  CAS  Google Scholar 

  48. Vojtesek B, Dolezalova H, Lauerova L, Svitakova M, Havlis P, Kovarik J, Midgley CA, Lane DP (1995) Oncogene 10:389

    CAS  Google Scholar 

  49. Vousden KH, Lane DP (2007) Nat Rev Mol Cell Biol 8:275

    Article  CAS  Google Scholar 

  50. Wallace M, Worrall E, Pettersson S, Hupp TR, Ball KL (2006) Mol Cell 23:251

    Article  CAS  Google Scholar 

  51. Wawrzynow B, Zylicz A, Wallace M, Hupp T, Zylicz M (2007) J Biol Chem 282:32603

    Article  CAS  Google Scholar 

  52. Wawrzynow B, Pettersson S, Zylicz A, Bramham J, Worrall E, Hupp TR, Ball KL (2009) J Biol Chem 284:11517–11530

    Google Scholar 

  53. Xirodimas D, Saville MK, Edling C, Lane DP, Lain S (2001) Oncogene 20:4972

    Article  CAS  Google Scholar 

  54. Yang Y, Ludwig RL, Jensen JP, Pierre SA, Medaglia MV, Davydov IV, Safiran YJ, Oberoi P, Kenten JH, Phillips AC, Weissman AM, Vousden KH (2005) Cancer Cell 7:547

    Article  CAS  Google Scholar 

  55. Yu GW, Rudiger S, Veprintsev D, Freund S, Fernandez-Fernandez MR, Fersht AR (2006) Proc Natl Acad Sci U S A 103:1227

    Article  CAS  Google Scholar 

  56. Yu GW, Allen MD, Andreeva A, Fersht AR, Bycroft M (2006) Protein Sci 15:384

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by Programme Grants to KLB (C377/A6355) and TRH (C483/A6354) from the Cancer Research UK. EW is funded by a Cancer Research UK PhD studentship (C483/A5547).

Author information

Author notes

  1. Liam Worrall

    Present address: Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3

Authors and Affiliations

  1. CRUK p53 Signal Transduction Group, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR

    Erin G. Worrall & Ted R. Hupp

  2. CRUK Interferon and Cell Signaling Group, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR

    Bartosz Wawrzynow & Kathryn L. Ball

  3. Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR

    Erin G. Worrall, Bartosz Wawrzynow, Kathryn L. Ball & Ted R. Hupp

  4. Institute for Translational and Chemical Biology, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR

    Liam Worrall

  5. Institute for Translational and Chemical Biology, University of Edinburgh, Edinburgh, Scotland, UK, EH9 3JR

    Malcolm Walkinshaw

Authors
  1. Erin G. Worrall
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bartosz Wawrzynow
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liam Worrall
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Malcolm Walkinshaw
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Kathryn L. Ball
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Ted R. Hupp
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ted R. Hupp.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Worrall, E.G., Wawrzynow, B., Worrall, L. et al. Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif. J Chem Biol 2, 113–129 (2009). https://doi.org/10.1007/s12154-009-0019-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12154-009-0019-5

Keywords

Search

Navigation