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SUMOylation of the GTPase Rac1 is required for optimal cell migration

Nature Cell Biology volume 12, pages 1078–1085 (2010)Cite this article

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Abstract

The Rho-like GTPase, Rac1, induces cytoskeletal rearrangements required for cell migration. Rac activation is regulated through a number of mechanisms, including control of nucleotide exchange and hydrolysis, regulation of subcellular localization or modulation of protein-expression levels1,2,3. Here, we identify that the small ubiquitin-like modifier (SUMO) E3-ligase, PIAS3, interacts with Rac1 and is required for increased Rac activation and optimal cell migration in response to hepatocyte growth factor (HGF) signalling. We demonstrate that Rac1 can be conjugated to SUMO-1 in response to hepatocyte growth factor treatment and that SUMOylation is enhanced by PIAS3. Furthermore, we identify non-consensus sites within the polybasic region of Rac1 as the main location for SUMO conjugation. We demonstrate that PIAS3-mediated SUMOylation of Rac1 controls the levels of Rac1–GTP and the ability of Rac1 to stimulate lamellipodia, cell migration and invasion. The finding that a Ras superfamily member can be SUMOylated provides an insight into the regulation of these critical mediators of cell behaviour. Our data reveal a role for SUMO in the regulation of cell migration and invasion.

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Figure 1: PIAS3 is a Rac1-binding protein required for optimal Rac activation and cell migration in response to HGF treatment.
Figure 2: PIAS3 regulates Rac1 activation in the cytoplasm.
Figure 3: Rac1 is SUMOylated in vitro and in vivo.
Figure 4: Rac1 is SUMOylated in the polybasic region and SUMOylation affects its GTP levels.
Figure 5: SUMOylation of the polybasic region is required for optimal cell migration and invasion.

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References

  1. Heasman, S. J. & Ridley, A. J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat. Rev. Mol. Cell Biol. 9, 690–701 (2008).

    Article  CAS  Google Scholar 

  2. Bustelo, X. R., Sauzeau, V. & Berenjeno, I. M. GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. Bioessays 29, 356–370 (2007).

    Article  CAS  Google Scholar 

  3. Ellenbroek, S. I. & Collard, J. G. Rho GTPases: functions and association with cancer. Clin. Exp. Metastasis 24, 657–672 (2007).

    Article  CAS  Google Scholar 

  4. Royal, I., Lamarche-Vane, N., Lamorte, L., Kaibuchi, K. & Park, M. Activation of cdc42, rac, PAK and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol. Biol. Cell 11, 1709–1725 (2000).

    Article  CAS  Google Scholar 

  5. Gentile, A., Trusolino, L. & Comoglio, P. M. The Met tyrosine kinase receptor in development and cancer. Cancer Metastasis Rev. 27, 85–94 (2008).

    Article  CAS  Google Scholar 

  6. Hays, J. L. & Watowich, S. J. Oligomerization-induced modulation of TPR-MET tyrosine kinase activity. J. Biol. Chem. 278, 27456–27463 (2003).

    Article  CAS  Google Scholar 

  7. Zhang, S. et al. Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J. Biol. Chem. 270, 23934–23936 (1995).

    Article  CAS  Google Scholar 

  8. Ridley, A. J., Comoglio, P. M. & Hall, A. Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac and Rho in MDCK cells. Mol. Cell Biol. 15, 1110–1122 (1995).

    Article  CAS  Google Scholar 

  9. Kotaja, N., Karvonen, U., Janne, O. A. & Palvimo, J. J. PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases. Mol. Cell Biol. 22, 5222–34 (2002).

    Article  CAS  Google Scholar 

  10. Palvimo, J. J. PIAS proteins as regulators of small ubiquitin-related modifier (SUMO) modifications and transcription. Biochem. Soc. Trans. 35, 1405–1408 (2007).

    Article  CAS  Google Scholar 

  11. Lanning, C. C., Daddona, J. L., Ruiz-Velasco, R., Shafer, S. H. & Williams, C. L. The Rac1 C-terminal polybasic region regulates the nuclear localization and protein degradation of Rac1. J. Biol. Chem. 279, 44197–44210 (2004).

    Article  CAS  Google Scholar 

  12. Michaelson, D. et al. Rac1 accumulates in the nucleus during the G2 phase of the cell cycle and promotes cell division. J. Cell Biol. 181, 485–496 (2008).

    Article  CAS  Google Scholar 

  13. Yamashina, K., Yamamoto, H., Chayama, K., Nakajima, K. & Kikuchi, A. Suppression of STAT3 activity by Duplin, which is a negative regulator of the Wnt signal. J. Biochem. 139, 305–314 (2006).

    Article  CAS  Google Scholar 

  14. Hay, R. T. SUMO: a history of modification. Mol. Cell 18, 1–12 (2005).

    Article  CAS  Google Scholar 

  15. Schmidt, D. & Muller, S. Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity. Proc. Natl Acad. Sci. USA 99, 2872–2877 (2002).

    Article  CAS  Google Scholar 

  16. Mukhopadhyay, D. & Dasso, M. Modification in reverse: the SUMO proteases. Trends Biochem. Sci. 32, 286–95 (2007).

    Article  CAS  Google Scholar 

  17. Suzuki, T. et al. A new 30-kDa ubiquitin-related SUMO-1 hydrolase from bovine brain. J. Biol. Chem. 274, 31131–31134 (1999).

    Article  CAS  Google Scholar 

  18. Martin, S. F., Hattersley, N., Samuel, I. D., Hay, R. T. & Tatham, M. H. A fluorescence-resonance-energy-transfer-based protease activity assay and its use to monitor paralog-specific small ubiquitin-like modifier processing. Anal. Biochem. 363, 83–90 (2007).

    Article  CAS  Google Scholar 

  19. Tatham, M. H. et al. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J. Biol. Chem. 276, 35368–35374 (2001).

    Article  CAS  Google Scholar 

  20. Tatham, M. H., Rodriguez, M. S., Xirodimas, D. P. & Hay, R. T. Detection of protein SUMOylation in vivo. Nat. Protoc. 4, 1363–1371 (2009).

    Article  CAS  Google Scholar 

  21. Noren, N. K., Niessen, C. M., Gumbiner, B. M. & Burridge, K. Cadherin engagement regulates Rho family GTPases. J. Biol. Chem. 276, 33305–33308 (2001).

    Article  CAS  Google Scholar 

  22. Kamitani, T. et al. Identification of three major sentrinization sites in PML. J. Biol. Chem. 273, 26675–26682 (1998).

    Article  CAS  Google Scholar 

  23. Hoege, C., Pfander, B., Moldovan, G. L., Pyrowolakis, G. & Jentsch, S. RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419, 135–141 (2002).

    Article  CAS  Google Scholar 

  24. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–72 (2008).

    Article  CAS  Google Scholar 

  25. ten Klooster, J. P., Jaffer, Z. M., Chernoff, J. & Hordijk, P. L. Targeting and activation of Rac1 are mediated by the exchange factor β-Pix. J. Cell Biol. 172, 759–769 (2006).

    Article  CAS  Google Scholar 

  26. Tolias, K. F., Couvillon, A. D., Cantley, L. C. & Carpenter, C. L. Characterization of a Rac1- and RhoGDI-associated lipid kinase signaling complex. Mol. Cell Biol. 18, 762–770 (1998).

    Article  CAS  Google Scholar 

  27. Tolias, K. F. et al. Type Iα phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. Curr. Biol. 10, 153–156 (2000).

    Article  CAS  Google Scholar 

  28. van Hennik, P. B. et al. The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity. J. Biol. Chem. 278, 39166–39175 (2003).

    Article  CAS  Google Scholar 

  29. Williams, C. L. The polybasic region of Ras and Rho family small GTPases: a regulator of protein interactions and membrane association and a site of nuclear localization signal sequences. Cell Signal 15, 1071–1080 (2003).

    Article  CAS  Google Scholar 

  30. Vidali, L., Chen, F., Cicchetti, G., Ohta, Y. & Kwiatkowski, D. J. Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor. Mol. Biol. Cell 17, 2377–2390 (2006).

    Article  CAS  Google Scholar 

  31. Bossis, G. & Melchior, F. SUMO: regulating the regulator. Cell Div. 1, 13 (2006).

    Article  Google Scholar 

  32. Rooney, C. et al. The Rac activator STEF (Tiam2) regulates cell migration by microtubule-mediated focal adhesion disassembly. EMBO Rep. 11, 292–298 (2010).

    Article  CAS  Google Scholar 

  33. Woodcock, S. A., Jones, R. C., Edmondson, R. D. & Malliri, A. A modified tandem affinity purification technique identifies that 14-3-3 proteins interact with Tiam1, an interaction which controls Tiam1 stability. J. Proteome Res. 8, 5629–5641 (2009).

    Article  CAS  Google Scholar 

  34. Woodcock, S. A. et al. SRC-induced disassembly of adherens junctions requires localized phosphorylation and degradation of the rac activator tiam1. Mol. Cell 33, 639–653 (2009).

    Article  CAS  Google Scholar 

  35. Tatham, M. H., Rodriguez, M. S., Xirodimas, D. P. & Hay, R. T. Detection of protein SUMOylation in vivo. Nat. Protoc. 4, 1363–71 (2009).

    Article  CAS  Google Scholar 

  36. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V. & Mann, M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nat. Protoc. 1, 2856–2860 (2006).

    Article  CAS  Google Scholar 

  37. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  Google Scholar 

  38. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).

    Article  CAS  Google Scholar 

  39. Kersey, P. J. et al. The international protein index: an integrated database for proteomics experiments. Proteomics 4, 1985–1988 (2004).

    Article  CAS  Google Scholar 

  40. Maiolica, A. et al. Structural analysis of multiprotein complexes by cross-linking, mass spectrometry, and database searching. Mol. Cell Proteomics 6, 2200–2211 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This study was supported by CRUK grant C147/A6058 and an EMBO Long-Term fellowship to S.C.L. M.H.T. and E.G.J. were supported by CRUK. We thank N. Maurice (Glasgow, UK) for mass spectrometry analysis, I. Matic (Dundee, UK) for help with SUMO–Rac1 branched-peptide data interpretation, H. Yokosawa, L. Trusolino and L. Vidali for reagents, N. Mack for help with the calcium-switch experiments, I. Arozarena and C. Wellbrock for help with invasion assays, and members of the Cell Signalling Group, A. Hurlstone, N. Divecha, J. Pérez-Martín and C. Wilkinson, for critical reading of the manuscript and helpful discussions.

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Author notes

  1. Richard C. Jones

    Present address: Current address: MS Bioworks, 3950 Varsity Drive, Ann Arbor, MI 48108, USA.,

  2. Ricky D. Edmondson

    Present address: Current address: Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.,

Authors and Affiliations

  1. Cell Signalling Group, Cancer Research UK Paterson Institute for Cancer Research, The University of Manchester, Manchester, M20 4BX, UK

    Sonia Castillo-Lluva & Angeliki Malliri

  2. Wellcome Trust Centre for Gene Regulation and Expression, Sir James Black Centre College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK

    Michael H. Tatham, Ellis G. Jaffray & Ronald T. Hay

  3. National Center for Toxicological Research (NCTR), Food and Drug Administration (FDA), Jefferson, 72079, AR, USA

    Richard C. Jones & Ricky D. Edmondson

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Contributions

S.C.L. co-wrote the manuscript and designed and executed all the experiments apart from the mass spectrometry of the sites of SUMO modification, which was performed by M.H.T. R.C.J. and R.D.E. performed the mass spectrometry analysis of the TAP–Rac1 purification. E.G.J. purified all the components required for the in vitro SUMOylation and helped with the in vitro SUMOylation experiments. R.T.H. provided expertise and help with the SUMOylation experiments. A.M. provided team leadership, project management and wrote the manuscript.

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Correspondence to Angeliki Malliri.

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Castillo-Lluva, S., Tatham, M., Jones, R. et al. SUMOylation of the GTPase Rac1 is required for optimal cell migration. Nat Cell Biol 12, 1078–1085 (2010). https://doi.org/10.1038/ncb2112

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