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

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.

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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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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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