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Akt-phosphorylated UFL1 UFMylates ArpC4 to promote metastasis

Nature Structural & Molecular Biology volume 32, pages 1528–1541 (2025)Cite this article

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Abstract

The role of modification by ubiquitin-fold modifier (‘UFMylation’) in regulating metastasis has remained enigmatic. Cell migration, a critical step in metastasis, is driven by actin polymerization mediated by actin-related proteins 2 and 3 (Arp2/3) at the leading edge of lamellipodia. Here, we report that UFM1-specific E3 ligase 1 (UFL1) interacts with and catalyzes the UFMylation of ArpC4, a core subunit of the Arp2/3 complex. Akt has a key role in this process, which involves phosphorylating UFL1 at T426, thereby enhancing its interaction with ArpC4 and inducing ArpC4 UFMylation. Through ArpC4 UFMylation and potentially other targets, UFL1 facilitates lamellipodia formation and promotes cell migration, invasion and metastasis, making UFL1 an attractive therapeutic target for cancer.

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Fig. 1: UFL1 promotes cell migration, invasion and metastasis.
Fig. 2: UFL1 promotes lamellipodia formation.
Fig. 3: UFL1 catalyzes ArpC4 UFMylation.
Fig. 4: ArpC4 UFMylation is crucial for cell migration, invasion and metastasis.
Fig. 5: UFMylation of ArpC4 at K130 promotes Arp2/3 activation.
Fig. 6: UFL1 is phosphorylated by Akt.
Fig. 7: Akt-mediated phosphorylation of UFL1 at T426 facilitates its interaction with ArpC4 to induce ArpC4 UFMylation.
Fig. 8: T426 phosphorylation is essential for UFL1 to promote cell migration, invasion and metastasis.

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Data availability

The protein structures used in this study were obtained from the PDB under accession codes 5IAA, 1K8K and 6W18. The data supporting the findings of this study are available within the article and Supplementary Information. Source data are provided with this paper.

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Acknowledgements

We thank Y. Yin of the Mass Spectrometry System at the National Facility for Protein Science (Shanghai Advanced Research Institute, Chinese Academy of Science) for mass spectrometry sample preparation, data collection and data analysis. We also thank X. Yao for helpful discussion. This work was supported by grants from the National Natural Science Foundation of China (32270811 and 32470749, to Y.M.; 32200614, to K.Z.), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB0940302, to Y.M.), the Center for Advanced Interdisciplinary Science and Biomedicine of IHM (QYPY20220006, to Y.M.) and the Youth Science and Technology Talents Support Program (2024) by Anhui Association for Science and Technology (RCTJ202408, to K.Z.). This work was also supported by the advanced computing resources provided by the Supercomputing Center of the USTC. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Kailiang Zhao, Hao Hu, Debao Fang, Mingran Xie.

Authors and Affiliations

  1. Department of Thoracic Surgery, The First Affiliated Hospital of USTC, National Key Laboratory of Immune Response and Immunotherapy, Center for Advanced Interdisciplinary Science and Biomedicine of IHM, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Kailiang Zhao, Hao Hu, Debao Fang, Mingran Xie, Suyun Tang, Mingsheng Wu, Xiaorui Guo, Ning Yu, Bao Yao & Yide Mei

  2. School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Kailiang Zhao & Yide Mei

  3. MOE Key Laboratory for Membraneless Organelles & Cellular Dynamics, School of Life Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, China

    Jiasheng Chen, Shan Zhang, Wenli Jiang & Chao Wang

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Contributions

K.Z. and Y.M. designed the research. K.Z., H.H., D.F., M.X., J.C., S.Z., S.T., M.W., X.G., N.Y., B.Y. and W.J. performed the research. K.Z., H.H., D.F., M.X., J.C., S.Z., S.T., M.W., X.G., N.Y., B.Y., W.J., C.W. and Y.M. analyzed the data. K.Z. and Y.M. wrote the paper.

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Correspondence to Yide Mei.

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Extended data

Extended Data Fig. 1 UFL1 promotes cell migration, invasion, and metastasis.

(a–c) A549 cells expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#2 were subjected to wound-healing (b) and transwell migration (c) assays. The successful knockdown of UFL1 was verified by western blotting (a). Scale bar in b: 200 μm; c: 100 μm. (d, e) U2OS cells expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#2 were subjected to transwell migration assays (e). The successful knockdown of UFL1 was verified by western blotting (d). Scale bar in e: 100 μm. (f, g) A549 cells expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#2 were subjected to single cell tracking (f) and transwell invasion (g) assays. Cell migration speed, persistence, and mean-squared displacement (MSD) in single cell tracking assay were also analyzed using an Excel macro described by Gorelik and Gautreau (f). Scale bar in f: 25 μm; g: 100 μm. (h) U2OS cells expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#2 were subjected to transwell invasion assays. Scale bar: 100 μm. (i–k) H1299 expressing control sgRNA, UFL1 sgRNA#1, or UFL1 sgRNA#2 were subjected to transwell migration (j) and transwell invasion (k) assays. Cell lysates were also analyzed by western blotting (i). Scale bar in j and k: 100 μm. All experiments were repeated three times independently with similar results. The shown blots and images are representative of three independent experiments. Data in (b, c, e, g, h, j, k) are mean ± SD (n = 3). Data in (f) are mean ± SEM (n = 10, 8 and 10, respectively). Statistical analysis was performed using one-way ANOVA (b, c, e–h, j, k).

Source data

Extended Data Fig. 2 UFL1 promotes lamellipodia formation.

(a–d) A549 cells with or without ectopic Flag-UFL1 expression were serum-starved for 6 h before they were stimulated with or without 10% FBS or 100 ng/ml EGF for 30 min, followed by immunostaining with either anti-UFL1 (a) or anti-Flag (c) antibody, along with FITC-phalloidin. The plot profiles (a, c) and Pearson’s correlation coefficient (b, d) were quantified. The arrows indicate the leading edge of migrating cells. Scale bar: 20 μm. (e–h) H1299 cells (e, f) or A549 cells (g, h) expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#2 were serum-starved for 6 h before they were scratched. Cells were then treated with 100 ng/ml EGF (e, f) or 10% FBS (g, h) for 30 min, followed by immunofluorescence assay. (e, g) The lamellipodia protrusions driven by branched actin networks were shown by immunostaining with anti-Cortactin antibody. White arrows indicate lamellipodia region. (f, h) The percentage of lamellipodia-positive cells were calculated. Scale bar: 20 μm. (i) A549 cells expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#2 were subjected to cell spreading assay. Scale bars: 50 μm. (j) H1299 cells expressing control shRNA, UFL1 shRNA#1, or UFL1 shRNA#1 plus shRNA-resistant Flag-UFL1 were subjected to cell spreading assay. Scale bars: 50 μm. All experiments were repeated three times independently with similar results. The shown images are representative of three independent experiments. Data in (b, d, f, h, i, j) are mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA (f, h–j).

Source data

Extended Data Fig. 3 ArpC4 is a bona fide substrate of UFMylation.

(a) Lysates from HEK293T cells expressing Flag-UFL1 alone or together with the indicated HA-tagged each single subunit of the Arp2/3 complex were immunoprecipitated with anti-Flag antibody, followed by western blot analysis. (b) Lysates from HEK293T cells expressing HA-UFL1 alone or HA-UFL1 plus Flag-tagged Arp3, Cortactin, Coronin, and Cofilin as indicated were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. (c) Lysates from HEK293T cells expressing HA-UFL1 alone or HA-UFL1 plus Flag-tagged N-WASP were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. (d) Myc-tagged UBA5 and UFC1, HA-tagged UFL1, UFBP1, and UFM1-ΔC2 were expressed in HEK293T cells with Flag-tagged ArpC4 as indicated. Denatured cell lysates were subjected to pull-down with anti-Flag antibody, followed by western blot analysis. (e) ArpC4, UFC1, UFL1, and UFBP1, together with UFM1-ΔC2 or UFM1-ΔC3, were expressed in HEK293T cells. Denatured cell lysates were subjected to pull-down with Ni-NTA resins, followed by western blot analysis. (f) Flag-tagged ArpC4 and the UFMylation components were expressed in HEK293T cells with or without HA-tagged UFSP2. Denatured cell lysates were subjected to pull-down with anti-Flag antibody, followed by western blot analysis. (g) His-tagged wild-type ArpC4 or its KO mutant (all lysine residues were replaced with arginine) was expressed in HEK293T cells with the UFMylation components as indicated. Denatured cell lysates were subjected to pull-down with Ni-NTA resins, followed by western blot analysis. (h) UFSP2-KO H1299 cells expression control or Flag-UFM1-ΔC2 were infected with lentiviruses expressing either control shRNA or ArpC4 shRNA as indicated. Denatured cell lysates were subjected to pull-down with anti-Flag antibody, followed by western blot analysis. (i) Coomassie blue staining of recombinant His-tagged UBA5, UFC1, UFM1-ΔC2, UFL1/UFBP1, and MBP-tagged ArpC4. All experiments were repeated three times independently with similar results. The shown blots/gels are representative of three independent experiments.

Source data

Extended Data Fig. 4 Mass spectrometric identification of lysine residues of ArpC4 modified by UFM1.

(a–g) Recombinant MBP-ArpC4 and the UFMylation components were subjected to in vitro UFMylation assay. The products were separated by SDS-PAGE, followed by commassie blue staining. The band of UFMylated ArpC4 was cut and delivered to mass spectrometry analysis. Lys35 (a), Lys44 (b), Lys60 (c), Lys77 (d), Lys84 (e), Lys107 (f), and Lys166 (g) of ArpC4 were identified as the potential UFMylation sites.

Extended Data Fig. 5 ArpC4 UFMylation plays an important role in cell migration, invasion, and metastasis.

(a–c) Control or ArpC4 knockdown A549 cells were infected with lentiviruses expressing Flag-tagged ArpC4 or its 5KR mutant. Cells were subjected to wound-healing (a) and transwell migration (c) assays. Cell lysates were also analyzed by western blot (b). Scale bar in a: 200 μm; c: 100 μm. (d–f) The analysis of cell migration speed (d), persistence (e), and mean-squared displacement (MSD) (f) of Fig. 4d. (g) Control or ArpC4 knockdown A549 cells were infected with lentiviruses expressing Flag-tagged ArpC4 or its 5KR mutant. Cells were subjected to transwell invasion assays. Scale bar: 100 μm. (h) H1299 cells expressing either control shRNA or ArpC4 shRNA were infected with lentiviruses expressing Flag-tagged wild-type ArpC4 or its 5KR mutant. Cells were serum-starved for 6 h before they were scratched. Cells were then treated with 10% FBS for 30 min, followed by immunofluorescence assay. The percentage of lamellipodia-positive cells were calculated. (i) H&E staining of lung lobes from the NCG mice intravenously injected with A549 cells expressing control shRNA, ArpC4 shRNA, ArpC4 shRNA plus wild-type ArpC4, or ArpC4 shRNA plus 5KR mutant (each also expressing luciferase). Scale bar: 200 μm. (j) H1299 cells expressing either control or Flag-UFL1 were infected with lentiviruses expressing control shRNA, ArpC4 shRNA, ArpC4 shRNA plus wild-type ArpC4, or ArpC4 shRNA plus 5KR mutant as indicated. Cells lysates were subjected to western blot analysis. All experiments were repeated three times independently with similar results. The shown blots and images are representative of three independent experiments. Data in (a, c, g, h) are mean ± SD (n = 3). Data in (d–f) are mean ± SEM (n = 18, 14, 16 and 14, respectively). Statistical analysis was performed using one-way ANOVA (a, c, d, g, h).

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Extended Data Fig. 6 K130 is crucial for mediating the functional effects of ArpC4 UFMylation on cell migration and invasion.

(a–g) A549 cells expressing ArpC4 shRNA were reconstituted with the indicated Flag-ArpC4 proteins. Cells were subjected to wound-healing (a and b), transwell migration (d and e), and transwell invasion assay (f and g). Cell lysates were analyzed by western blotting (c). Scale bar in a: 200 μm; in d and f: 100 μm. (h–j) H1299 cells expressing either control or Flag-UFL1 were infected with lentiviruses expressing control shRNA, ArpC4 shRNA, ArpC4 shRNA plus wild-type ArpC4, or ArpC4 shRNA plus ArpC4 mutants as indicated. Cells were subjected to transwell migration (h) and transwell invasion (j) assays. Cell lysates were analyzed by western blotting (i). Scale bars in h and j: 100 μm. (k) H1299 cells expressing control shRNA, UFL1 shRNA-#1, and UFL1 shRNA-#2 were serum-starved for 6 h before they were treated with or without 10% FBS for 30 min. Cell lysates were incubated with glutathione beads immobilized recombinant GST-PAK1-PBD. The input and GST-PAK1-PBD bound Rac1 were analyzed by western blotting. GST-PAK1-PBD bound Rac1 was considered as active Rac1. (l) Cell lysates from H1299 cells expressing control shRNA or UFL1 shRNA were immunoprecipitated with control IgG or anti-Arp2 antibody, followed by western blot analysis. The phosphorylation of Arp2 was detected using anti-phospho-Arp2 (T237/T238) antibody. (m) Cell lysates from H1299 cells expressing control shRNA or UFL1 shRNA were immunoprecipitated with control IgG or anti-ArpC1 antibody, followed by western blot analysis. The phosphorylation of ArpC1 was detected using pan phospho-serine/threonine antibody. All experiments were repeated three times independently with similar results. The shown blots and images are representative of three independent experiments. Data in (b, e, g, h, j) are mean ± SD (n = 3). Statistical analysis was performed using one-way ANOVA (b, e, g, h, j).

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Extended Data Fig. 7 Akt phosphorylates UFL1 at T426.

(a) H1299 cells expressing control or UFL1-TurboID were serum-starved for 6 h before they were treated with or without 10% FBS for 30 min in the presence of 200 μM D-biotin. Biotinylated proteins were pulled-down using streptavidin beads (Thermo Fisher Scientific). The input and biotinylated proteins were analyzed by western blotting. (b) H1299 cells expressing control or Flag-UFL1 were starved for 6 h and treated with MK2206 or PD98059 for 2 h, followed by 10% FBS stimulation for 30 min. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody and western blot analysis. (c) Prediction of potential phosphorylation sites on UFL1 using Group-based Prediction System (GPS). (d) Dot blot analysis of the generated phospho-UFL1-T426 antibody against the indicated UFL1 peptides with or without T426 phosphorylation, which demonstrated that the p-UFL1(T426) antibody specifically recognize the pT426 epitope. (e) Flag-tagged UFL1 alone or together with HA-Akt1 was expressed in HEK293T cells. The cells were treated with or without MK2206 for 2 h before they were subjected to immunoprecipitation with anti-Flag antibody, followed by western blot analysis. (f) Recombinant GST-UFL1 or UFL1-T426A were incubated with purified Flag-Akt1 in the kinase buffer for 30 min at 30 °C. The phosphorylation of UFL1 was detected by western blot using anti-p-UFL1 (T426) antibody. All experiments were repeated three times independently with similar results. The shown blots are representative of three independent experiments.

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Extended Data Fig. 8 T426 phosphorylation is essential for UFL1 to promote cell migration, invasion, and metastasis.

(a–c) UFL1 knockdown A549 cells were reconstituted with the indicated Flag-UFL1 proteins. Cells were subjected to wound-healing (a) and transwell migration (c) assay. Cell lysates were also analyzed by western blot (b). Scale bar in a: 200 μm; c: 100 μm. (d–f) The analysis of cell migration speed (d), persistence (e), and mean-squared displacement (MSD) (f) of Fig. 8d. (g) UFL1 knockdown A549 cells were reconstituted with the indicated Flag-UFL1 proteins. Cells were subjected transwell invasion assay. Scale bar: 100 μm; (h) The percentage of lamellipodia-positive cells in Fig. 8f. (i, j) UFL1 knockdown A549 cells were reconstituted with the indicated Flag-UFL1 proteins. Cells were serum-starved for 6 h before they were scratched. Cells were then treated with 10% FBS for 30 min, followed by immunofluorescence assay. (i) The lamellipodia protrusions driven by branched actin networks were shown by immunostaining with anti-Cortactin antibody. White arrows indicate lamellipodia region. (j) The percentage of lamellipodia-positive cells were calculated. Scale bar: 20 μm. (k) H&E staining of lung lobes from the NCG mice intravenously injected with A549 cells expressing control shRNA, UFL1 shRNA, UFL1 shRNA plus wild-type UFL1, UFL shRNA plus UFL1-T426A, or UFL shRNA plus UFL1-T426D (each also expressing luciferase). Scale bar: 200 μm. All experiments were repeated three times independently with similar results. The shown blots and images are representative of three independent experiments. Data in (a, c, g, h, j) are mean ± SD (n = 3). Data in (d–f) are mean ± SEM (n = 15 for each group). Statistical analysis was performed using one-way ANOVA (a, c, d, g, h, j).

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Extended Data Fig. 9 Analysis of UFL1 phosphorylation in clinical lung cancer samples.

(a, b) UFL1 and phospho-UFL1 (T426) levels in lung cancer tissues (T) and matched adjacent normal tissues (N) (a). The expression levels of UFL1 in (a) and (Fig. 8i) were quantified and normalized to β-actin (b). (c, d) Levels of UFL1, phospho-UFL1 (T426), and phospho-Akt (S473) in lung cancer tissues (c). UFL1 levels in (c) and (Fig. 8k) were quantified and normalized to β-actin (d). All experiments were repeated three times independently with similar results. The shown blots are representative of three independent experiments. Data in (b) were analyzed by a paired Student’s t test (two-tailed) (n = 33). Data in (d) are mean ± SD (n = 22, 13 and 11, respectively), analyzed by one-way ANOVA.

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Zhao, K., Hu, H., Fang, D. et al. Akt-phosphorylated UFL1 UFMylates ArpC4 to promote metastasis. Nat Struct Mol Biol 32, 1528–1541 (2025). https://doi.org/10.1038/s41594-025-01576-8

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