three forms: c-cbl, CBLb and CBL-3) MDM2, MDMX and SCF Fbxo11

Transcription

1 Supplementary information S1 (table) Reported Neddylation Substrates Target protein Biological function of target Effect of neddylation E2 E3 Regulation of neddylation Cullins RING ubiquitin E3 ligases Activation of ubiquitin UBC12, RBX1/RBX2 Enhanced by DCNLs, transfer activity UBE2F Tfb3 (budding yeast) and ubiquitylation substrates, reversed by the CSN complex SMURF1 HECT Ubiquitin E3 ligase Activation of ubiquitin transfer activity TGFβRII TGF-β signal transduction, inhibition of proliferation TGFβRII stabilization, thus promoting the antiproliferative effect of TGF-β Neddylated lysines (method) Human CUL1: Lys720 Human CUL2: Lys689 Human CUL3: Lys712 Human CUL4A: Lys705 Human CUL4B: Lys859 Human CUL5: Lys724 UBC12 SMURF1 Not known Lys324, Lys495, Lys545, Lys558, Lys559, and Lys667 (mass spectrometry) UBC12 c-cbl c- CBL- mediated neddylation depends on TGFβRII kinase activity; reversed by DEN1 Lys556, Lys567 (mass spectrometry and mutagenesis) Addressed criteria* Reference s 1, 2, 3, 4, 5 1 1, 2, 4, 5 2 1, 2, 4, 5 3 EGFR Growth factor signal transduction Enhanced ubiquitylation and subsequent lysosomal degradation TP53 Transcription factor Inhibition of transcription activity, cytoplasmic localization TP73 Transcription factor Inhibition of transcription activity, cytoplasmic localization Not studied Not studied CBL (all three forms: c-cbl, CBLb and CBL-3) MDM2, MDMX and SCF Fbxo11 Stimulated by EGF Reversed by DEN1; inhibited by TIP60. NUB1 decreases TP53 neddylation Not studied MDM2 Reversed by DEN1. Only full length TP73 but not the N- terminally truncated isoform is neddylated Multiple lysines in tyrosine kinase domain Lys370, Lys372 and Lys373 1, 5 4 1, 2, 4, Not known 1, 2, 5 11

2 E2F (E2F-1, -2, -3, -4 and DP1) IKKγ (also known as NEMO) BCA3 (also known as AKIP1) Transcription factor, regulating the G1/S phase cell cycle transition Regulatory subunit of the IKK complex Regulator of NF-κB transcription Down- regulation of transcriptional activity; destabilization Inhibition of NF-κB signalling Inhibition of NF-κB transcription Mainly UBE2F MDM2 RING E3 ligase Stabilization Not studied MDM2 (autoneddylation) Not identified Reversed by DEN1; inhibited by TIP60 Reversed by DEN1. SET7/9-mediated methylation promotes neddylation Not studied TRIM40 Neddylation is higher in normal gastric epithelium than in gastric cancer tissue Not studied Not studied Reversed by DEN1; inhibited by ostrogen Not known 1, 5 5,12 Mainly Lys residues in the DNA-binding domain (Lys117, Lys120, Lys125, Lys182, Lys183 and Lys 185) 1, 3, 5 13,14 Not identified 1, 5 15 All 11 Lys residues 1, 2, 5 16 BRAP2 RING E3 regulating the Ras- MAPK pathway; associated with inflammatory dysfunction; suppressor of NF- κb signalling No effect for inhibition of NF-κB Not studied Not studied Not studied Lys HIF1α /HIF2α VHL HIF transcription factor component; survival signalling under hypoxia Substrate receptor of a CRL2 E3 ligase; regulating hypoxia signalling through degradation of HIF1α; positive regulator of fibronectin-mediated extracellular matrix assembly Stabilization in both normoxia and hypoxia Prevents CUL2 and fibronectin binding; might trigger the switch from CUL2 to fibronectin Not studied Not studied Not studied PAS-B domain (truncation) Not studied Not studied Not studied All three available Lys residues (Lys159, Lys171 and Lys196) with Lys159 as the major acceptor 1, 2, , 2 19,20

3 APP (amyloid precursor protein) intracellular domain (AICD) Component of a transcription factor Inhibition of transcription activity Not studied Not studied Not studied All 7 Lys residues (Lys612, Lys624, Lys , Lys676 and Lys688); Lys676 and Lys688 are sites in AICD 1 21 Parkin RING-in-between-RING E3 ligase; regulator of mitophagy; mutated in Parkinson s disease Increased E3 ligase activity PINK1 Protein kinase, activator of Parkin Stabilization of its 55 kda fragment Histone H4 Core nucleosome component; Polyneddylation recruits multiple post-translational RNF168 and facilitates modifications regulate repair chromatin state and DNA repair drice D. melanogaster homologue Inhibition of caspase of capsase 7; effector caspase activity in apoptosis signalling RPL11 Ribosomal protein Stabilization and nucleolar localization; non-neddylated form activates TP53 by binding to MDM2 RPS14 Ribosomal protein Stabilization and nucleolar localization; neddylated form activates TP53 by binding to MDM2 Not studied Not studied Reversed by DEN1 Multiple Lys residues, along the entire protein. Lys76 was detected by mass spectrometry from Parkinson s disease patients brain samples. 1, 2, 5 22,23 Not studied Not studied Reversed by DEN1 Not studied 1 22 UBC12 RNF111 Triggered by DNA damage Not studied Drosophila IAP1 Reversed by DEN1 N-terminal Lys residues All 9 surface exposed Lys residues Not studied MDM2 Reversed by DEN1 All 16 Lys residues 1, 2, 4, , 2, , 2, Not studied MDM2 Reversed by DEN1 Not studied 1, 2, 5 31

4 HuR RCAN1 mrna stabilization; cell dedifferentiation; proliferation Inhibitor of calcineurin signaling HuR stabilization; nuclear localization Enhanced stability; increased binding to calcineurin Not studied MDM2 Reversed by DEN1 Lys283, Lys313 and Lys326 Not studied Not studied Endogenous neddylation observed in mouse embryonic brain but not adult; reduced by oxidative stress Lys96, Lys104 and Lys170 1, , 2, 5 33 *The criteria for a genuine neddylation target are: 1, covalent attachment of NEDD8 through its C- terminal glycine to a Lys residue in the target protein; 2, detectable neddylation under homeostatic conditions and at endogenous levels of NEDD8 and substrate expression; 3, neddylation of the proposed target is sensitive to MLN4924 treatment under conditions that block cullin neddylation but not ubiquitylation, and NEDD8 is thus activated by NAE. Ideally, studies of a neddylation target should further define: 4, the specific NEDD8 E2 and E3 enzyme(s); 5, the regulation and biological consequences of neddylation. References 1. Bennett, E. J., Rush, J., Gygi, S. P. & Harper, J. W. Dynamics of cullin- RING ubiquitin ligase network revealed by systematic quantitative proteomics. Cell 143, (2010). 2. Xie, P. et al. The covalent modifier Nedd8 is critical for the activation of Smurf1 ubiquitin ligase in tumorigenesis. Nat Commun 5, 3733 (2014). 3. Zuo, W. et al. c- Cbl- mediated neddylation antagonizes ubiquitination and degradation of the TGF- β type II receptor. Mol Cell 49, (2013). 4. Oved, S. et al. Conjugation to Nedd8 instigates ubiquitylation and down- regulation of activated receptor tyrosine kinases. J. Biol. Chem. 281, (2006). 5. Xirodimas, D. P., Saville, M. K., Bourdon, J.- C., Hay, R. T. & Lane, D. P. Mdm2- mediated NEDD8 conjugation of p53 inhibits its transcriptional activity. Cell 118, (2004). 6. Dohmesen, C., Koeppel, M. & Dobbelstein, M. Specific inhibition of Mdm2- mediated neddylation by Tip60. Cell Cycle 7, (2008). 7. Singh, R. K., Iyappan, S. & Scheffner, M. Hetero- oligomerization with MdmX rescues the ubiquitin/nedd8 ligase activity of RING finger mutants of Mdm2. J. Biol. Chem. 282, (2007).

5 8. Abida, W. M., Nikolaev, A., Zhao, W., Zhang, W. & Gu, W. FBXO11 promotes the Neddylation of p53 and inhibits its transcriptional activity. J. Biol. Chem. 282, (2007). 9. Liu, G. & Xirodimas, D. P. NUB1 promotes cytoplasmic localization of p53 through cooperation of the NEDD8 and ubiquitin pathways. Oncogene 29, (2010). 10. Leidecker, O., Matic, I., Mahata, B., Pion, E. & Xirodimas, D. P. The ubiquitin E1 enzyme Ube1 mediates NEDD8 activation under diverse stress conditions. Cell Cycle 11, (2012). 11. Watson, I. R., Blanch, A., Lin, D. C. C., Ohh, M. & Irwin, M. S. Mdm2- mediated NEDD8 modification of TAp73 regulates its transactivation function. J. Biol. Chem. 281, (2006). 12. Watson, I. R. et al. Chemotherapy induces NEDP1- mediated destabilization of MDM2. Oncogene 29, (2010). 13. Loftus, S. J., Liu, G., Carr, S. M., Munro, S. & La Thangue, N. B. NEDDylation regulates E2F- 1- dependent transcription. EMBO Rep 13, (2012). 14. Aoki, I., Higuchi, M. & Gotoh, Y. NEDDylation controls the target specificity of E2F1 and apoptosis induction. Oncogene 32, (2013). 15. Noguchi, K. et al. TRIM40 promotes neddylation of IKKγ and is downregulated in gastrointestinal cancers. Carcinogenesis 32, (2011). 16. Gao, F., Cheng, J., Shi, T. & Yeh, E. T. H. Neddylation of a breast cancer- associated protein recruits a class III histone deacetylase that represses NFkappaB- dependent transcription. Nat. Cell Biol. 8, (2006). 17. Takashima, O. et al. Brap2 regulates temporal control of NF- κb localization mediated by inflammatory response. PLoS ONE 8, e58911 (2013). 18. Ryu, J.- H. et al. Hypoxia- inducible factor α subunit stabilization by NEDD8 conjugation is reactive oxygen species- dependent. J. Biol. Chem. 286, (2011). 19. Stickle, N. H. et al. pvhl modification by NEDD8 is required for fibronectin matrix assembly and suppression of tumor development. Mol Cell Biol 24, (2004). 20. Russell, R. C. & Ohh, M. NEDD8 acts as a molecular switch defining the functional selectivity of VHL. EMBO Rep 9, (2008). 21. Lee, M.- R., Lee, D., Shin, S. K., Kim, Y. H. & Choi, C. Y. Inhibition of APP intracellular domain (AICD) transcriptional activity via covalent conjugation with Nedd8. Biochem Biophys Res Commun 366, (2008). 22. Choo, Y. S. et al. Regulation of parkin and PINK1 by neddylation. Hum. Mol. Genet. 21, (2012). 23. Um, J. W. et al. Neddylation positively regulates the ubiquitin E3 ligase activity of parkin. J. Neurosci. Res. 90, (2012).

6 24. Ma, T. et al. RNF111- dependent neddylation activates DNA damage- induced ubiquitination. Mol Cell 49, (2013). 25. Broemer, M. et al. Systematic in vivo RNAi analysis identifies IAPs as NEDD8- E3 ligases. Mol Cell 40, (2010). 26. Xirodimas, D. P. et al. Ribosomal proteins are targets for the NEDD8 pathway. EMBO Rep 9, (2008). 27. Ebina, M. et al. Myeloma Overexpressed 2 (Myeov2) Regulates L11 Subnuclear Localization through Nedd8 Modification. PLoS ONE 8, e65285 (2013). 28. Mahata, B., Sundqvist, A. & Xirodimas, D. P. Recruitment of RPL11 at promoter sites of p53- regulated genes upon nucleolar stress through NEDD8 and in an Mdm2- dependent manner. Oncogene 31, (2012). 29. Sun, X.- X., Wang, Y.- G., Xirodimas, D. P. & Dai, M.- S. Perturbation of 60 S ribosomal biogenesis results in ribosomal protein L5- and L11- dependent p53 activation. J. Biol. Chem. 285, (2010). 30. Sundqvist, A., Liu, G., Mirsaliotis, A. & Xirodimas, D. P. Regulation of nucleolar signalling to p53 through NEDDylation of L11. EMBO Rep 10, (2009). 31. Zhang, J., Bai, D., Ma, X., Guan, J. & Zheng, X. hcinap is a novel regulator of ribosomal protein- HDM2- p53 pathway by controlling NEDDylation of ribosomal protein S14. Oncogene 33, (2012). 32. Embade, N. et al. Murine double minute 2 regulates Hu antigen R stability in human liver and colon cancer through NEDDylation. Hepatology 55, (2012). 33. Noh, E. H. et al. Covalent NEDD8 conjugation increases RCAN1 protein stability and potentiates its inhibitory action on calcineurin. PLoS ONE 7, e48315 (2012).