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Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics

A Publisher Correction to this article was published on 26 February 2020

This article has been updated

Abstract

Neddylation is the post-translational protein modification most closely related to ubiquitination. Whereas the ubiquitin-like protein NEDD8 is well studied for its role in activating cullin−RING E3 ubiquitin ligases, little is known about other substrates. We developed serial NEDD8-ubiquitin substrate profiling (sNUSP), a method that employs NEDD8 R74K knock-in HEK293 cells, allowing discrimination of endogenous NEDD8- and ubiquitin-modification sites by MS after Lys-C digestion and K-εGG-peptide enrichment. Using sNUSP, we identified 607 neddylation sites dynamically regulated by the neddylation inhibitor MLN4924 and the de-neddylating enzyme NEDP1, implying that many non-cullin proteins are neddylated. Among the candidates, we characterized lysine 112 of the actin regulator cofilin as a novel neddylation event. Global inhibition of neddylation in developing neurons leads to cytoskeletal defects, altered actin dynamics and neurite growth impairments, whereas site-specific neddylation of cofilin at K112 regulates neurite outgrowth, suggesting that cofilin neddylation contributes to the regulation of neuronal actin organization.

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Fig. 1: sNUSP by K-εGG MS.
Fig. 2: sNUSP from Nedd8R74K-knock-in cells in response to MLN4924 and NEDP1 deletion.
Fig. 3: NEDDylated versus ubiquitinated cell proteome derived from sNUSP.
Fig. 4: Validation of COF1 and ADF as NEDD8 substrates regulated by MLN4924 and NEDP1.
Fig. 5: Validation of COF1 as a NEDD8 and NEDP1 substrate in cells and brain tissue.
Fig. 6: Neddylation modulates the actin cytoskeleton and controls neurite outgrowth in neurons.
Fig. 7: Non-neddylatable cofilinK112R impairs dendrite development of cortical neurons in vivo.

Data availability

MS raw data files were deposited at UCSD MassIVE with the data set identifier MSV000084683. Source data for Figs. 2 and 47 and Extended Data Figs. 1, 3 and 58 are available with the paper online. All other data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

PTMscan studies were performed at Genentech under a limited-use license from Cell Signaling Technology. We thank A. Attorresi, C. Pollak and S. Romano for helping with confocal and live-imaging studies. We also thank J. Cupp and members of the FACS lab for assistance. This work was supported by the German Science Foundation Collaborative Research Centre (CRC) 870 (W.W.) and by the Network Fund of the Helmholtz Association (AMPro project, to W.W.). This work was also supported by the DFG SPP1365 grant (D.R.), the Max Planck Society (D.R. and F.D.S.), the Volkswagen Stiftung (D.R.), the Agencia Nacional de Promoción Científica y Tecnológica, Argentina (D.R. and F.D.S.), the Alexander von Humboldt Foundation (F.D.S.) and the fondo para la convergencia estructural del mercosur-FOCEM (D.R.).

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Authors and Affiliations

Authors

Contributions

A.M.V. designed the study, performed experiments, and analyzed results. L.P., T.H., E.V., and D.S.K. designed, performed and analyzed proteomic experiments. R.B. and S.G. performed and analyzed cultured neuron and in utero electroporation experiments. M.D.B. and F.D.S. set up and performed super-resolution microscopy. P.Y. performed consensus site analysis. A.H. designed CRISPR constructs. M.A. performed and analyzed imaging experiments. C.C.H. helped analyze cytoskeleton experiments. W.W. directed the generation of Nedp1–/– mice. M.S. and D.R. directed the study. A.M.V., D.S.K., D.R. and M.S. wrote the manuscript. All authors read and edited the manuscript.

Corresponding authors

Correspondence to Donald S. Kirkpatrick, Damian Refojo or Morgan Sheng.

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Competing interests

A.M.V., L.P., E.V., T.H., M.A., A.H., C.C.H., D.S.K. and M.S. are employees and shareholders of Genentech, Inc., a member of the Roche group. The authors declare no additional competing financial interests.

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Peer review information Katarzyna Marcinkiewicz was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 NEDD8R74K knock-in (KI) and NEDP1 knockout (KO) strategy for sNUSP.

a, Representative immunoblotting of total cell lysates of Nedp1WT and Nedp1KO CRISPR HEK293 cell clones (KO #1: sgRNA 1 + 4; KO #2: sgRNA 2 + 5), with anti-NEDD8, anti-NEDP1, anti-tubulin, and anti-actin antibodies. b, Rescue of neddylation patterns in Nedp1KO cells by re-expression of NEDP1WT. Nedp1WT and Nedp1KO HEK293 cell clones were transiently transfected with pcDNA3.1EMPTY, NEDP1WT-HA, NEDP1C163A-HA, or CSN5WT-FLAG constructs, and treated with DMSO or 2 μM MLN4924 for 24 h, as indicated. Representative immunoblotting of total cell lysates with anti-NEDD8, anti-NEDP1, anti-tubulin, anti-actin, and anti-GAPDH antibodies. c, Analysis of neddylation and ubiquitination patterns of CRISPR Nedd8WT or R74K Nedp1WT or KO HEK293 cell clones. Representative immunoblotting of total cell lysates from indicated cell clones, treated with DMSO or 3 μM MLN4924 for 24 h, with anti-NEDD8, anti-ubiquitin (FK2), anti-tubulin, anti-actin, and anti-GAPDH antibodies. Uncropped blot images are provided as source data. Experiments were repeated at least three times with similar results. Molecular mass is indicated in kilodaltons.

Source data

Extended Data Fig. 2 MLN4924- and NEDP1-regulated NEDD8R74K K-εGG proteins from sNUSP.

ac, Tables show all significantly MLN4924- and NEDP1-regulated NEDD8R74K K-εGG proteins for key comparisons of DMSO vs. MLN4924 in Nedp1WT cells (a), DMSO vs. MLN4924 in Nedp1KO cells (b), and Nedp1KO (DMSO) vs. Nedp1WT (DMSO) cells (c) with log2 fold change >1.0 and –log10 adjusted P value >1.3 (P < 0.05). Experiment was repeated twice.

Extended Data Fig. 3 Analysis of neddylation substrates identified by sNUSP.

a, Cullin NEDD8R74K-specific and ubiquitin-specific K-εGG sites detected in sNUSP experiments. Asterisks mark known and experimentally confirmed canonical neddylation sites of cullins. b, Regulation of NEDD8R74K and ubiquitin K-εGG sites of CUL3 by MLN4924 (downregulation in red) and NEDP1 loss (downregulation in blue). c, Regulation of cullin neddylation by MLN4924 and NEDP1 at the protein level. d, Gene Ontology (GO) term enrichment analysis of neddylated proteins identified in NEDD8R74K K-εGG MS experiments (PANTHER, http://www.pantherdb.org/, overrepresentation test, P values defined by Fisher’s exact test). Protein class categories with positive fold enrichment and P values <0.01 are shown, and the number of hits compared to the category size is indicated after each GO term. e, Number of NEDD8R74K-modified (pink) and ubiquitin-modified (blue) K-εGG sites per protein. On average, 2.03 neddylation sites (1,327 sites/609 proteins) compared to 3.85 ubiquitination sites (12,599 sites/3,270 proteins) per protein; large majority of NEDD8 substrates with only one or two neddylation sites per protein (491 of 609 proteins), and one to five ubiquitination sites per protein for ubiquitinated substrates (2,658 of 3,270 proteins). Data for graphs in d,e are available as Source data.

Source data

Extended Data Fig. 4 Analysis of neddylation and ubiquitination events on substrate proteins.

a–g, Regulation of NEDD8R74K and ubiquitin K-εGG sites of (a) UBC12, (b) UBA3, (c) NEDD8, (d) PARP1, (e) CAND1, (f) LMNA and (g) LMNB1 by MLN4924 (downregulation in red) and Nedp1KO (upregulation in red).

Extended Data Fig. 5 Validation of cofilin as NEDD8 and NEDP1 substrate.

a, Structure of cofilin (green) and actin (purple and cyan) (modified from PDB 5YU8) with the cofilinK112 neddylation site (dark blue). b, Lysine (K) to arginine (R) mutagenesis of neddylated lysine residues identified by NEDD8R74K-specific K-εGG MS confirms K112 as the main neddylation site of cofilin. Nedp1KO HEK293 cells were transiently transfected with the different COF1BIO-HA constructs and treated with DMSO or 3 μM MLN4924 for 24 h, as indicated. Representative immunoblotting of denaturing biotin-streptavidin pulldown samples (COF1) and lysates for endogenous NEDD8 with anti-NEDD8 antibodies and COF1BIO-HA with anti-HA antibodies. Note, the neddylated COF1 band is absent in all samples with K112R mutations. c, Increased cofilin neddylation in Nedp1KO cells. Nedp1KO and Nedp1WT HEK293 cells were transiently transfected with COF1WT-BIO-HA constructs and treated with DMSO or 2 μM MLN4924 for 24 h, as indicated. Representative immunoblotting of denaturing biotin-streptavidin pulldown samples (COF1) and lysates for endogenous NEDD8 with anti-NEDD8 antibodies and COF1BIO-HA with anti-HA antibodies. Uncropped blot images for b and c are available as Source data. Experiments in b,c were repeated at least three times with similar results. Molecular mass is indicated in kilodaltons.

Source data

Extended Data Fig. 6 Regulation of the neuronal actin cytoskeleton by neddylation.

a, Neddylation-deficient neurons form enlarged axonal growth cones. Mouse hippocampal neurons treated with DMSO or 1 μM MLN4924 at DIV2 for 24 h, fixed at DIV3 and stained for microtubules (tyr-tubulin) and F-actin (phalloidin). Measurement of axonal growth cone area and actin/microtubule ratio in stage 3 neurons (unpaired, two-tailed t tests, growth cone area, t(22) = 3.25, **P < 0.01 (P = 0.0037); actin/microtubule ratio, t(22) = 2.413, *P < 0.05 (P = 0.0246), n = 11 control, 13 MLN4924 neurons, from one culture). Scale bar, 2 μm. b, Neddylation inhibition increases actin dynamics and retrograde flow. Spinning disk confocal microscopy (every 2 s for 5 min) of axon growth cones of DIV2 rat hippocampal neurons, nucleofected with LifeAct-GFP and treated with DMSO or 1 μM MLN4924 for 3−5 h. Measurement of relative changes in LifeAct-GFP pixel intensity (unpaired, two-tailed t test, t(72) = 2.511, *P < 0.05 (P = 0.0143), n = 38 DMSO and 36 MLN4924 growth cones, from four neuron cultures) and retrograde flow (unpaired, two-tailed t test, t(8) = 3.304, *P < 0.05 (P = 0.0108), each data point represents a single growth cone in which the retrograde flow was measured and averaged in kymographs of three ROIs, each for six events, n = 5 growth cones per condition, from two cultures). Scale bars, 10 μm. Related to Supplementary Video 2. Data for graphs are available as Source data. Data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 7 Neddylation regulates neurite outgrowth in developing neurons.

a, Representative images of time lapse imaging (DIV1–5) of mouse hippocampal neurons, separately nucleofected with ControlIRES-mRFP (red) or UBC12C111S-IRES-GFP (green) and mixed before plating. Quantification of neurons in developmental stages 1–3 at DIV1 and 3 (two-way ANOVA, treatment x stage interaction, F(6,219) = 66.42, ***P < 0.001; Tukey’s multiple comparisons test, comparing stages 1, 2 and 3 for control vs. UBC12C111S, *P < 0.05, ***P < 0.001; n = 12−27 fields of view per group, from two cultures). Arrow marks growing axon, arrowhead marks UBC12C111S-expressing cell. Scale bar, 25 μm. b, Representative images of rat hippocampal neurons, nucleofected with pCAGGS-GFP, treated with DMSO or 0.5 μM MLN4924 at DIV1 and 2, imaged at DIV3 (24 h after last treatment). Quantification of total neurite outgrowth per cell (unpaired, two-tailed t test, t(6) = 8.002, ***P < 0.001 (P = 0.0002); n = 4 areas with >1,000 neurons each, from two cultures). Scale bar, 100 μm. Data for graphs are available as Source data. Data are presented as mean ± s.e.m.

Source data

Extended Data Fig. 8 General neddylation inhibition and specific blockade of NEDD8 conjugation to K112 reduce cofilin phosphorylation in neurons.

a, Neddylation inhibition does not change neuronal cofilin protein levels. Representative immunoblotting of total cell lysates of cultured rat cortical neurons, treated with DMSO, 1 μM MLN4924 or 1 μM MG132 for 2, 4, 6 and 24 h at DIV14–15, with anti-cofilin, anti-tubulin, anti-actin, anti-GAPDH, anti-NEDD8 and anti-ubiquitin (FK2) antibodies. Cofilin levels were measured and normalized to tubulin (Kruskal-Wallis tests; 2 h, *P = 0.0107, Dunn’s multiple comparison test, *P < 0.05 (DMSO vs. MG132); 4 h, P = 0.1965; 6 h, P = 0.5403; 24 h, P = 0.5474; n = 4 samples per treatment for 2, 4 and 6 h, n = 8 wells per treatment for 24 h, from two cultures). b, MLN4924 treatment decreases cofilin Ser3 phosphorylation. Immunoblotting of total cell lysates of cultured rat hippocampal neurons, treated with DMSO or 1 μM MLN4924 for 24, 12 or 8 h from DIV1–2 (representative blot) and DIV5–6, with anti-P-cofilin (Ser3), anti-cofilin, anti-tubulin, anti-actin, and anti-GAPDH antibodies. Quantification of P-cofilin/total cofilin levels (one-way ANOVA, F(5,35) = 14.3, ***P < 0.001; Sidak’s multiple comparisons (DMSO vs. MLN4924), *P < 0.05, ***P < 0.001; n = 6 DMSO and n = 7 or 8 MLN4924, from three neuron cultures). c, Reduced phosphorylation of non-neddylatable cofilinK112R. Immunoblotting of total cell lysates of cultured rat hippocampal neurons, nucleofected with cofilinBIO-HA WT or K112R constructs from DIV0–7, with anti-P-cofilin (Ser3), anti-cofilin, anti-tubulin, and anti-actin antibodies. Quantification of P-cofilin/total cofilin levels (unpaired, two-tailed t test, t(6) = 4.622, **P < 0.01 (P = 0.0036), n = 4 samples per group, from two cultures). Uncropped blot images and data for graphs are available as Source data. Molecular mass is indicated in kilodalton. Data are presented as mean ± s.e.m.

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Supplementary information

Supplementary Information

Supplementary Notes, Supplementary Figs. 1–8 and Supplementary Table 3.

Reporting Summary

Supplementary Table 1

Quantitative sNUSP NEDD8R74K-specific K-εGG mass spectrometry data

Supplementary Table 2

Quantitative sNUSP ubiquitin K-εGG mass spectrometry data

Supplementary Video 1

Transient formation of ectopic lamellipodia structures by neddylation inhibition. Differential interference contrast (DIC) time lapse imaging of a DIV2 mouse hippocampal neuron before, during, and after 1 μM MLN4924 treatment. Experiment was repeated twice with similar results and with 10–15 cells imaged per experiment.

Supplementary Video 2

Increased actin dynamics and retrograde flow in axonal growth cones of MLN4924-treated neurons. Rat hippocampal neurons, nucleofected with LifeAct-GFP before plating and treated with DMSO or 1 μM MLN4924 at DIV2 (~43 h after plating). Axonal growth cones imaged 3–5 h later on a spinning disk confocal microscope (every 2 s for 5 min). Scale bar, 10 μm. Experiment was repeated four times with similar results. Related to Extended Data Fig. 6b.

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Unprocessed western blots

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Vogl, A.M., Phu, L., Becerra, R. et al. Global site-specific neddylation profiling reveals that NEDDylated cofilin regulates actin dynamics. Nat Struct Mol Biol 27, 210–220 (2020). https://doi.org/10.1038/s41594-019-0370-3

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