Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases

Article metrics

Abstract

Rab5 is a small GTPase involved in the control of intracellular trafficking, both at the level of receptor endocytosis and endosomal dynamics1. The finding that Rab5 can be activated by receptor tyrosine kinases (RTK)2 raised the question of whether it also participates in effector pathways emanating from these receptors. Here we show that Rab5 is indispensable for a form of RTK-induced actin remodelling, called circular ruffling. Three independent signals, originating from Rab5, phosphatidylinositol-3-OH kinase and Rac, respectively, are simultaneously required for the induction of circular ruffles. Rab5 signals to the actin cytoskeleton through RN-tre, a previously identified Rab5-specific GTPase-activating protein (GAP)3. Here we demonstrate that RN-tre has the dual function of Rab5-GAP and Rab5 effector. We also show that RN-tre is critical for macropinocytosis, a process previously connected to the formation of circular ruffles4,5. Finally, RN-tre interacts with both F-actin and actinin-4, an F-actin bundling protein. We propose that RN-tre establishes a three-pronged connection with Rab5, F-actin and actinin-4. This may aid crosslinking of actin fibres into actin networks at the plasma membrane. Thus, we have shown that Rab5 is a signalling GTPase and have elucidated the major molecular elements of its downstream pathway.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Rab5 and circular ruffles.
Figure 2: Biological effects of RN-tre and its mutants.
Figure 3: Interaction between RN-tre and ACTN4.
Figure 4: Role of RN-tre and ACTN4 in circular ruffling.

References

  1. 1

    Zerial, M. & McBride, H. Rab proteins as membrane organizers. Nature Rev. Mol. Cell Biol. 2, 107–117 (2001)

  2. 2

    Barbieri, M. A. et al. Epidermal growth factor and membrane trafficking. EGF receptor activation of endocytosis requires Rab5a. J. Cell Biol. 151, 539–550 (2000)

  3. 3

    Lanzetti, L. et al. The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5. Nature 408, 374–377 (2000)

  4. 4

    Swanson, J. & Watts, C. Macropinocytosis. Trends Cell Biol. 5, 424–428 (1995)

  5. 5

    Hall, A. Rho GTPases and the actin cytoskeleton. Science 279, 509–514 (1998)

  6. 6

    Bar-Sagi, D. & Hall, A. Ras and Rho GTPases: a family reunion. Cell 103, 227–238 (2000)

  7. 7

    Suetsugu, S., Yamazaki, D., Kurisu, S. & Takenawa, T. Differential roles of WAVE1 and WAVE2 in dorsal and peripheral ruffle formation for fibroblast cell migration. Dev. Cell 5, 595–609 (2003)

  8. 8

    Eriksson, A., Siegbahn, A., Westermark, B., Heldin, C. H. & Claesson-Welsh, L. PDGF alpha- and beta-receptors activate unique and common signal transduction pathways. EMBO J. 11, 543–550 (1992)

  9. 9

    Bar-Sagi, D. & Feramisco, J. R. Induction of membrane ruffling and fluid-phase pinocytosis in quiescent fibroblasts by ras proteins. Science 233, 1061–1068 (1986)

  10. 10

    Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D. & Hall, A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410 (1992)

  11. 11

    Dharmawardhane, S., Sanders, L. C., Martin, S. S., Daniels, R. H. & Bokoch, G. M. Localization of p21-activated kinase 1 (PAK1) to pinocytic vesicles and cortical actin structures in stimulated cells. J. Cell Biol. 138, 1265–1278 (1997)

  12. 12

    Scita, G. et al. EPS8 and E3B1 transduce signals from Ras to Rac. Nature 401, 290–293 (1999)

  13. 13

    Horiuchi, H. et al. A novel Rab5 GDP/GTP exchange factor complexed to Rabaptin-5 links nucleotide exchange to effector recruitment and function. Cell 90, 1149–1159 (1997)

  14. 14

    Tall, G. G., Barbieri, M. A., Stahl, P. D. & Horazdovsky, B. F. Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1. Dev. Cell 1, 73–82 (2001)

  15. 15

    Spaargaren, M. & Bos, J. L. Rab5 induces Rac-independent lamellipodia formation and cell migration. Mol. Biol. Cell 10, 3239–3250 (1999)

  16. 16

    Benmerah, A., Bayrou, M., Cerf-Bensussan, N. & Dautry-Varsat, A. Inhibition of clathrin-coated pit assembly by an Eps15 mutant. J. Cell Sci. 112, 1303–1311 (1999)

  17. 17

    Joneson, T., White, M. A., Wigler, M. H. & Bar-Sagi, D. Stimulation of membrane ruffling and MAP kinase activation by distinct effectors of RAS. Science 271, 810–812 (1996)

  18. 18

    Wennstrom, S. et al. Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Curr. Biol. 4, 385–393 (1994)

  19. 19

    Honda, K. et al. Actinin-4, a novel actin-bundling protein associated with cell motility and cancer invasion. J. Cell Biol. 140, 1383–1393 (1998)

  20. 20

    Djinovic-Carugo, K., Young, P., Gautel, M. & Saraste, M. Structure of the alpha-actinin rod: molecular basis for cross-linking of actin filaments. Cell 98, 537–546 (1999)

  21. 21

    Araki, N., Hatae, T., Yamada, T. & Hirohashi, S. Actinin-4 is preferentially involved in circular ruffling and macropinocytosis in mouse macrophages: analysis by fluorescence ratio imaging. J. Cell Sci. 113, 3329–3340 (2000)

  22. 22

    Tocque, B. et al. Ras-GTPase activating protein (GAP): a putative effector for Ras. Cell. Signal. 9, 153–158 (1997)

  23. 23

    Greenwood, J. A., Theibert, A. B., Prestwich, G. D. & Murphy-Ullrich, J. E. Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin. J. Cell Biol. 150, 627–642 (2000)

  24. 24

    Corgan, A. M., Singleton, C., Santoso, C. B. & Greenwood, J. A. Phosphoinositides differentially regulate alpha-actinin flexibility and function. Biochem. J. 378, 1067–1072 (2004)

  25. 25

    Fraley, T. S. et al. Phosphoinositide binding inhibits alpha-actinin bundling activity. J. Biol. Chem. 278, 24039–24045 (2003)

  26. 26

    Krueger, E. W., Orth, J. D., Cao, H. & McNiven, M. A. A dynamin-cortactin-Arp2/3 complex mediates actin reorganization in growth factor-stimulated cells. Mol. Biol. Cell 14, 1085–1096 (2003)

  27. 27

    Schafer, D. A. et al. Dynamin2 and cortactin regulate actin assembly and filament organization. Curr. Biol. 12, 1852–1857 (2002)

  28. 28

    Fazioli, F. et al. Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. EMBO J. 12, 3799–3808 (1993)

  29. 29

    Seastone, D. J. et al. The WASp-like protein scar regulates macropinocytosis, phagocytosis and endosomal membrane flow in Dictyostelium. J. Cell Sci. 114, 2673–2683 (2001)

Download references

Acknowledgements

This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro and Human Science Frontier Program to P.P.D.F. and G.S., and by grants from the Association for International Cancer Research, European Community (VI Framework), Italian Ministry for University and Research and Consiglio Nazionale delle Ricerche to P.P.D.F. We thank E. Frittoli for technical assistance. L.L. was supported in part by a fellowship from the Associazione Sviluppo Piemonte.

Author information

Correspondence to Pier Paolo Di Fiore.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure S1

Active Ras and Rac induce lamellipodia, but not circular ruffles. (PDF 2509 kb)

Supplementary Figure S2

Coated pit-mediated endocytosis and PDGF-induced circular ruffling are independent events. (PDF 3610 kb)

Supplementary Figure S3

Effects of Rab5 and various GTPases and their effectors on the actin cytoskeleton. (PDF 4816 kb)

Supplementary Figure S4

Involvement of RN-tre in circular ruffling and macropinocytosis. (PDF 17070 kb)

Supplementary Figure Legends (DOC 33 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lanzetti, L., Palamidessi, A., Areces, L. et al. Rab5 is a signalling GTPase involved in actin remodelling by receptor tyrosine kinases. Nature 429, 309–314 (2004) doi:10.1038/nature02542

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.