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RPEL-family rhoGAPs link Rac/Cdc42 GTP loading to G-actin availability

Nature Cell Biology volume 21, pages 845–855 (2019)Cite this article

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Abstract

RPEL proteins, which contain the G-actin-binding RPEL motif, coordinate cytoskeletal processes with actin dynamics. We show that the ArhGAP12- and ArhGAP32-family GTPase-activating proteins (GAPs) are RPEL proteins. We determine the structure of the ArhGAP12/G-actin complex, and show that G-actin contacts the RPEL motif and GAP domain sequences. G-actin inhibits ArhGAP12 GAP activity, and this requires the G-actin contacts identified in the structure. In B16 melanoma cells, ArhGAP12 suppresses basal Rac and Cdc42 activity, F-actin assembly, invadopodia formation and experimental metastasis. In this setting, ArhGAP12 mutants defective for G-actin binding exhibit more effective downregulation of Rac GTP loading following HGF stimulation and enhanced inhibition of Rac-dependent processes, including invadopodia formation. Potentiation or disruption of the G-actin/ArhGAP12 interaction, by treatment with the actin-binding drugs latrunculin B or cytochalasin D, has corresponding effects on Rac GTP loading. The interaction of G-actin with RPEL-family rhoGAPs thus provides a negative feedback loop that couples Rac activity to actin dynamics.

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Fig. 1: Two families of rhoGAPs contain an RPEL motif.
Fig. 2: ArhGAP12 interaction with G-actin requires the RPEL motif.
Fig. 3: Structural analysis of the ArhGAP12/G-actin complex.
Fig. 4: G-actin inhibits ArhGAP12 GAP activity by occluding rho protein binding.
Fig. 5: ArhGAP12 controls GTP loading on Rac and Cdc42 in melanoma cells.
Fig. 6: ArhGAP12 regulates Rac-dependent processes in cells.
Fig. 7: G-actin regulates Rac activity in melanoma cells.

Data availability

The ArhGAP12/G-actin structure has been deposited in the Protein Data Bank (PDB; https://www.rcsb.org) with the primary accession code 6GVC. Structures of MRTF-A RPEL2/G-actin and ArhGAP15 that were re-analysed in this study were obtained from PDB under the accession codes 2V52 and 3BYI, respectively.

Previously published RNA sequencing data that were re-analysed here are available under the accession code GSE45888.

The human melanoma survival data were derived from the TCGA Research Network (http://cancergenome.nih.gov). The dataset derived from this resource that supports the findings of this study is available in OncoLnc (http://www.oncolnc.org).

The source data for Figs. 1c, 2a,d, 3a,f, 4a–c, 5a–d, 6a–d, 7a–c,e,f,h and Supplementary Figs. 1e,f,i, 2f, 3c,d, 4b–d,f,–h, 5a–d,g–i, 6a–e have been provided as Supplementary Table 1. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Krause, M. & Gautreau, A. Steering cell migration: lamellipodium dynamics and the regulation of directional persistence. Nat. Rev. Mol. Cell Biol. 15, 577–590 (2014).

    Article  CAS  Google Scholar 

  2. Przybyla, L., Muncie, J. M. & Weaver, V. M. Mechanical control of epithelial-to-mesenchymal transitions in development and cancer. Annu. Rev. Cell Dev. Biol. 32, 527–554 (2016).

    Article  CAS  Google Scholar 

  3. Skau, C. T. & Waterman, C. M. Specification of architecture and function of actin structures by actin nucleation factors. Annu. Rev. Biophys. 44, 285–310 (2015).

    Article  CAS  Google Scholar 

  4. Dominguez, R. & Holmes, K. C. Actin structure and function. Annu. Rev. Biophys. 40, 169–186 (2011).

    Article  CAS  Google Scholar 

  5. Lawson, C. D. & Ridley, A. J. Rho GTPase signaling complexes in cell migration and invasion. J. Cell Biol. 217, 447–457 (2018).

    Article  CAS  Google Scholar 

  6. Hodge, R. G. & Ridley, A. J. Regulating Rho GTPases and their regulators. Nat. Rev. Mol. Cell Biol. 17, 496–510 (2016).

    Article  CAS  Google Scholar 

  7. Cook, D. R., Rossman, K. L. & Der, C. J. Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease. Oncogene 33, 4021–4035 (2014).

    Article  CAS  Google Scholar 

  8. Laurin, M. & Cote, J. F. Insights into the biological functions of dock family guanine nucleotide exchange factors. Genes Dev. 28, 533–547 (2014).

    Article  CAS  Google Scholar 

  9. Tcherkezian, J. & Lamarche-Vane, N. Current knowledge of the large RhoGAP family of proteins. Biol. Cell 99, 67–86 (2007).

    Article  CAS  Google Scholar 

  10. Amin, E. et al. Deciphering the molecular and functional basis of RHOGAP family proteins: a systematic approach toward selective inactivation of Rho family proteins. J. Biol. Chem. 291, 20353–20371 (2016).

    Article  CAS  Google Scholar 

  11. Miralles, F., Posern, G., Zaromytidou, A. I. & Treisman, R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113, 329–342 (2003).

    Article  CAS  Google Scholar 

  12. Vartiainen, M. K., Guettler, S., Larijani, B. & Treisman, R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science 316, 1749–1752 (2007).

    Article  CAS  Google Scholar 

  13. Mouilleron, S., Guettler, S., Langer, C. A., Treisman, R. & McDonald, N. Q. Molecular basis for G-actin binding to RPEL motifs from the serum response factor coactivator MAL. EMBO J. 27, 3198–3208 (2008).

    Article  CAS  Google Scholar 

  14. Wiezlak, M. et al. G-actin regulates the shuttling and PP1 binding of the RPEL protein Phactr1 to control actomyosin assembly. J. Cell Sci. 125, 5860–5872 (2012).

    Article  CAS  Google Scholar 

  15. Huet, G. et al. Actin-regulated feedback loop based on Phactr4, PP1 and cofilin maintains the actin monomer pool. J. Cell Sci. 126, 497–507 (2013).

    Article  CAS  Google Scholar 

  16. Esnault, C. et al. Rho-actin signaling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts. Genes Dev. 28, 943–958 (2014).

    Article  CAS  Google Scholar 

  17. Cen, B. et al. Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes. Mol. Cell. Biol. 23, 6597–6608 (2003).

    Article  CAS  Google Scholar 

  18. Allen, P. B., Greenfield, A. T., Svenningsson, P., Haspeslagh, D. C. & Greengard, P. Phactrs 1-4: a family of protein phosphatase 1 and actin regulatory proteins. Proc. Natl Acad. Sci. USA 101, 7187–7192 (2004).

    Article  CAS  Google Scholar 

  19. Sagara, J. et al. Scapinin, a putative protein phosphatase-1 regulatory subunit associated with the nuclear nonchromatin structure. J. Biol. Chem. 278, 45611–45619 (2003).

    Article  CAS  Google Scholar 

  20. Pawłowski, R., Eeva Kaisa Rajakylä, E. K., Vartiainen, M. K. & Treisman, R. An actin-regulated importin α/β-dependent extended bipartite NLS directs nuclear import of MRTF-A. EMBO J. 29, 3448–3458 (2010).

    Article  Google Scholar 

  21. Hirano, H. & Matsuura, Y. Sensing actin dynamics: structural basis for G-actin-sensitive nuclear import of MAL. Biochem. Biophys. Res. Commun. 414, 373–378 (2011).

    Article  CAS  Google Scholar 

  22. Furukawa, Y. et al. Isolation of a novel human gene, ARHGAP9, encoding a Rho-GTPase activating protein. Biochem. Biophys. Res. Commun. 284, 643–649 (2001).

    Article  CAS  Google Scholar 

  23. Gentile, A. et al. Met-driven invasive growth involves transcriptional regulation of Arhgap12. Oncogene 27, 5590–5598 (2008).

    Article  CAS  Google Scholar 

  24. Seoh, M. L., Ng, C. H., Yong, J., Lim, L. & Leung, T. ArhGAP15, a novel human RacGAP protein with GTPase binding property. FEBS Lett. 539, 131–137 (2003).

    Article  CAS  Google Scholar 

  25. Zhao, C. et al. GC-GAP, a Rho family GTPase-activating protein that interacts with signaling adapters Gab1 and Gab2. J. Biol. Chem. 278, 34641–34653 (2003).

    Article  CAS  Google Scholar 

  26. Matsuda, M. et al. Identification of adherens junction-associated GTPase activating proteins by the fluorescence localization-based expression cloning. Exp. Cell Res. 314, 939–949 (2008).

    Article  CAS  Google Scholar 

  27. Monastyrskaya, K. et al. miR-199a-5p regulates urothelial permeability and may play a role in bladder pain syndrome. Am. J. Pathol. 182, 431–448 (2013).

    Article  CAS  Google Scholar 

  28. Rudnicki, A. et al. Next-generation sequencing of small RNAs from inner ear sensory epithelium identifies microRNAs and defines regulatory pathways. BMC Genom. 15, 484 (2014).

    Article  Google Scholar 

  29. Lecat, S., Matthes, H. W., Pepperkok, R., Simpson, J. C. & Galzi, J. L. A fluorescent live imaging screening assay based on translocation criteria identifies novel cytoplasmic proteins implicated in G protein-coupled receptor signaling pathways. Mol. Cell. Proteomics 14, 1385–1399 (2015).

    Article  CAS  Google Scholar 

  30. Schlam, D. et al. Phosphoinositide 3-kinase enables phagocytosis of large particles by terminating actin assembly through Rac/Cdc42 GTPase-activating proteins. Nat. Commun. 6, 8623 (2015).

    Article  CAS  Google Scholar 

  31. Ba, W. et al. ARHGAP12 functions as a developmental brake on excitatory synapse function. Cell Rep. 14, 1355–1368 (2016).

    Article  CAS  Google Scholar 

  32. Nakamura, T. et al. PX-RICS mediates ER-to-Golgi transport of the N-cadherin/β-catenin complex. Genes Dev. 22, 1244–1256 (2008).

    Article  CAS  Google Scholar 

  33. Mouilleron, S., Wiezlak, M., O’Reilly, N., Treisman, R. & McDonald, N. Q. Structures of the Phactr1 RPEL domain and RPEL motif complexes with G-actin reveal the molecular basis for actin binding cooperativity. Structure 20, 1960–1970 (2012).

    Article  CAS  Google Scholar 

  34. Guettler, S., Vartiainen, M. K., Miralles, F., Larijani, B. & Treisman, R. RPEL motifs link the serum response factor cofactor MAL but not myocardin to Rho signaling via actin binding. Mol. Cell. Biol. 28, 732–742 (2008).

    Article  CAS  Google Scholar 

  35. Posern, G., Sotiropoulos, A. & Treisman, R. Mutant actins demonstrate a role for unpolymerized actin in control of transcription by serum response factor. Mol. Biol. Cell 13, 4167–4178 (2002).

    Article  CAS  Google Scholar 

  36. Mouilleron, S., Langer, C. A., Guettler, S., McDonald, N. Q. & Treisman, R. Structure of a pentavalent G-actin*MRTF-A complex reveals how G-actin controls nucleocytoplasmic shuttling of a transcriptional coactivator. Sci. Signal. 4, ra40 (2011).

    Article  Google Scholar 

  37. Radu, M. et al. ArhGAP15, a Rac-specific GTPase-activating protein, plays a dual role in inhibiting small GTPase signaling. J. Biol. Chem. 288, 21117–21125 (2013).

    Article  CAS  Google Scholar 

  38. Zamboni, V. et al. Disruption of ArhGAP15 results in hyperactive Rac1, affects the architecture and function of hippocampal inhibitory neurons and causes cognitive deficits. Sci. Rep. 6, 34877 (2016).

    Article  CAS  Google Scholar 

  39. Graziano, B. R. et al. A module for Rac temporal signal integration revealed with optogenetics. J. Cell Biol. 216, 2515–2531 (2017).

    Article  CAS  Google Scholar 

  40. Kurisu, S., Suetsugu, S., Yamazaki, D., Yamaguchi, H. & Takenawa, T. Rac-WAVE2 signaling is involved in the invasive and metastatic phenotypes of murine melanoma cells. Oncogene 24, 1309–1319 (2005).

    Article  CAS  Google Scholar 

  41. Nakahara, H. et al. Involvement of Cdc42 and rac small G proteins in invadopodia formation of RPMI7951 cells. Genes Cells 8, 1019–1027 (2003).

    Article  CAS  Google Scholar 

  42. Yamaguchi, H. et al. Sphingosine-1-phosphate receptor subtype-specific positive and negative regulation of Rac and haematogenous metastasis of melanoma cells. Biochem. J. 374, 715–722 (2003).

    Article  CAS  Google Scholar 

  43. Stengel, K. & Zheng, Y. Cdc42 in oncogenic transformation, invasion, and tumorigenesis. Cell. Signal. 23, 1415–1423 (2011).

    Article  CAS  Google Scholar 

  44. Revach, O. Y., Winograd-Katz, S. E., Samuels, Y. & Geiger, B. The involvement of mutant Rac1 in the formation of invadopodia in cultured melanoma cells. Exp. Cell Res. 343, 82–88 (2016).

    Article  CAS  Google Scholar 

  45. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  Google Scholar 

  46. Eddy, R. J., Weidmann, M. D., Sharma, V. P. & Condeelis, J. S. Tumor cell invadopodia: invasive protrusions that orchestrate metastasis. Trends Cell Biol. 27, 595–607 (2017).

    Article  CAS  Google Scholar 

  47. Medjkane, S., Perez-Sanchez, C., Gaggioli, C., Sahai, E. & Treisman, R. Myocardin-related transcription factors and SRF are required for cytoskeletal dynamics and experimental metastasis. Nat. Cell Biol. 11, 257–268 (2009).

    Article  CAS  Google Scholar 

  48. Jaiswal, M. et al. Functional cross-talk between ras and Rho pathways: a Ras-specific GTPase-activating protein (p120RasGAP) competitively inhibits the RhoGAP activity of deleted in liver cancer (DLC) tumor suppressor by masking the catalytic arginine finger. J. Biol. Chem. 289, 6839–6849 (2014).

    Article  CAS  Google Scholar 

  49. Fujii, T., Iwane, A. H., Yanagida, T. & Namba, K. Direct visualization of secondary structures of F-actin by electron cryomicroscopy. Nature 467, 724–728 (2010).

    Article  CAS  Google Scholar 

  50. Murakami, K. et al. Structural basis for actin assembly, activation of ATP hydrolysis, and delayed phosphate release. Cell 143, 275–287 (2010).

    Article  CAS  Google Scholar 

  51. Aktories, K., Lang, A. E., Schwan, C. & Mannherz, H. G. Actin as target for modification by bacterial protein toxins. FEBS J. 278, 4526–4543 (2011).

    Article  CAS  Google Scholar 

  52. Ceccarelli, D. F. et al. Non-canonical interaction of phosphoinositides with pleckstrin homology domains of Tiam1 and ArhGAP9. J. Biol. Chem. 282, 13864–13874 (2007).

    Article  CAS  Google Scholar 

  53. Kong, L. & Ge, B. X. MyD88-independent activation of a novel actin-Cdc42/Rac pathway is required for Toll-like receptor-stimulated phagocytosis. Cell Res. 18, 745–755 (2008).

    Article  CAS  Google Scholar 

  54. Winter, G., Lobley, C. M. & Prince, S. M. Decision making in xia2. Acta Crystallogr. D 69, 1260–1273 (2013).

    Article  CAS  Google Scholar 

  55. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  Google Scholar 

  56. Adams, P. D. et al. PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    Article  CAS  Google Scholar 

  57. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of coot. Acta Crystallogr. D 66, 486–501 (2010).

    Article  CAS  Google Scholar 

  58. Vaguine, A. A., Richelle, J. & Wodak, S. J. SFCHECK: a unified set of procedures for evaluating the quality of macromolecular structure-factor data and their agreement with the atomic model. Acta Crystallogr. D 55, 191–205 (1999).

    Article  CAS  Google Scholar 

  59. The PyMOL Molecular Graphics System, Version 2.1.1 (Schrödinger, LLC, 2010).

  60. Edelstein, A. D. et al. Advanced methods of microscope control using muManager software. J. Biol. Methods 1, e10 (2014).

    Article  Google Scholar 

  61. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Crick Science Technology Platforms for their support and advice during this work, especially M. Renshaw and K. Anderson (Advanced Light Microscopy), P. Chakravarty and A. Stewart (Bioinformatics and Biostatistics); C. Watkins and J. Bee (Biological Research); N. Patel and A. Alidoust (Fermentation Facility); D. Davis (Flow Cytometry); G. Clark (Genomics Equipment Park); M. Howell (High-throughput screening); N. O’Reilly (Peptide Chemistry) and P. Walker (Structural Biology). X-ray data were collected at the Diamond Light Source (ID24 beamline, mx8015). We thank M. Matsuda (Kyoto University) for the RaichuEV-Rac plasmid and M. Way and members of the R.T. and N.Q.M. groups for their helpful discussions. This work was supported by Cancer Research UK core funding until 31 March 2015. Since then, support to R.T. and N.Q.M. has been provided by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001-190 and FC001-115), the UK Medical Research Council (FC001-190 and FC001-115) and the Wellcome Trust (FC001-190 and FC001-115), and by an ERC Advanced Grant (grant no. 268690) to R.T.

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Authors and Affiliations

  1. Signalling and Transcription Group, The Francis Crick Institute, London, UK

    Jessica Diring & Richard Treisman

  2. Structural Biology Science Technology Platform, The Francis Crick Institute, London, UK

    Stephane Mouilleron

  3. Signalling and Structural Biology Group, The Francis Crick Institute, London, UK

    Neil Q. McDonald

  4. Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck College, London, UK

    Neil Q. McDonald

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  1. Jessica Diring
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Contributions

All authors designed and interpreted the experiments. J.D. conducted the biochemical and cell biology studies. S.M. determined the structure of the actin/ArhGAP12 complex and conducted the comparative structural analysis. J.D. and R.T. wrote the manuscript with input from S.M. and N.Q.M.

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Correspondence to Richard Treisman.

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Diring, J., Mouilleron, S., McDonald, N.Q. et al. RPEL-family rhoGAPs link Rac/Cdc42 GTP loading to G-actin availability. Nat Cell Biol 21, 845–855 (2019). https://doi.org/10.1038/s41556-019-0337-y

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