Abstract
Formation of a stable lamellipodium at the front of migrating cells requires localization of Rac activation to the leading edge. Restriction of α4 integrin phosphorylation to the leading edge limits the interaction of α4 with paxillin to the sides and rear of a migrating cell. The α4–paxillin complex inhibits stable lamellipodia, thus confining lamellipod formation to the cell anterior. Here we report that binding of paxillin to the α4 integrin subunit inhibits adhesion-dependent lamellipodium formation by blocking Rac activation. The paxillin LD4 domain mediates this reduction in Rac activity by recruiting an ADP-ribosylation factor GTPase-activating protein (Arf-GAP) that decreases Arf activity, thereby inhibiting Rac. Finally, the localized formation of the α4–paxillin–Arf-GAP complex mediates the polarization of Rac activity and promotes directional cell migration. These findings establish a mechanism for the spatial localization of Rac activity to enhance cell migration.
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References
Lauffenburger, D. A. & Horowitz, A. F. Cell migration: a physically integrated molecular process. Cell 84, 359–369 (1996).
Ridley, A. J. et al. Cell migration: integrating signals from front to back. Science 302, 1704–1709 (2003).
Funamoto, S., Meili, R., Lee, S., Parry, L. & Firtel, R. A. Spatial and temporal regulation of 3-phosphoinositides by PI 3-kinase and PTEN mediates chemotaxis. Cell 109, 611–623 (2002).
Iijima, M. & Devreotes, P. Tumor suppressor PTEN mediates sensing of chemoattractant gradients. Cell 109, 599–610 (2002).
Etienne-Manneville, S. & Hall, A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell 106, 489–498 (2001).
Hall, A. Rho GTPases and the actin cytoskeleton. Science 23, 509–514 (1998).
Nobes, C. D. & Hall, A. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J. Cell Biol. 144, 1235–1244 (1999).
Kraynov, V. S. et al. Localized Rac activation dynamics visualized in living cells. Science 290, 333–337 (2000).
Rodriguez, O. C. et al. Conserved microtubule-actin interactions in cell movement and morphogenesis. Nature Cell Biol. 5, 599–609 (2003).
Srinivasan, S. et al. Rac and Cdc42 play distinct roles in regulating PI(3,4,5)P3 and polarity during neutrophil chemotaxis. J. Cell Biol. 160, 375–385 (2003).
Yang, J. T., Rayburn, H. & Hynes, R. O. Cell adhesion events by alpha4 integrins are essential in placental and cardiac development. Development 121, 549–560 (1995).
Hemler, M. E. VLA proteins in the integrin family: Structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 8, 365–400 (1990).
Arroyo, A. G., Yang, J. T., Rayburn, H. & Hynes, R. O. Differential requirements for α4 integrins in hematopoiesis. Cell 85, 997–1008 (1996).
Liu, S. et al. Binding of paxillin to α4 integrins modifies integrin-dependent biological responses. Nature 402, 676–681 (1999).
Han, J. et al. Phosphorylation of the integrin α4 cytoplasmic domain regulates paxillin binding. J. Biol. Chem. 276, 40903–40909 (2001).
Han, J., Rose, D. M., Woodside, D. G., Goldfinger, L. E. & Ginsberg, M. H. Integrin α4β1-dependent T cell migration requires both phosphorylation and dephosphorylation of the α4 cytoplasmic domain to regulate the reversible binding of paxillin. J. Biol. Chem. 278, 34845–34853 (2003).
Goldfinger, L. E., Han, J., Kiosses, W. B., Howe, A. K. & Ginsberg, M. H. Spatial restriction of α4 integrin phosphorylation regulates lamellipodial stability and α4β1-dependent cell migration. J. Cell Biol. 162, 731–741 (2003).
Tumbarello, D. A., Brown, M. C. & Turner, C. E. The paxillin LD motifs. FEBS Lett. 513, 114–118 (2002).
Turner, C. E. Paxillin interactions. J. Cell Sci. 113, 4139–4140 (2000).
Turner, C. E. Paxillin and focal adhesion signalling. Nature Cell Biol. 2, E231–E236 (2000).
Kiyokawa, E., Hashimoto, Y., Kurata, T., Sugimura, H. & Matsuda, M. Evidence that DOCK180 up-regulates signals from the CrkII-p130Cas complex. J. Biol. Chem. 273, 24479–24484 (1998).
Hagel, M. et al. The adaptor protein paxillin is essential for normal development in the mouse and is critical transducer of fibronectin signaling. Mol. Cell. Biol. 22, 901–915 (2002).
Wade, R., Bohl, J. & Vande, P. S. Paxillin null embryonic stem cells are impaired in cell spreading and tyrosine phosphorylation of focal adhesion kinase. Oncogene 21, 96–107 (2002).
Nishiya, N., Tachibana, K., Shibanuma, M., Mashimo, J. I. & Nose, K. Hic-5-reduced cell spreading on fibronectin: competitive effects between paxillin and Hic-5 through interaction with focal adhesion kinase. Mol. Cell. Biol. 21, 5332–5345 (2001).
West, K. A. et al. The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL). J. Cell Biol. 154, 161–176 (2001).
Zhao, Z. S., Manser, E., Loo, T. H. & Lim, L. Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly. Mol. Cell. Biol. 20, 6354–6363 (2000).
Turner, C. E. et al. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: a role in cytoskeletal remodeling. J. Cell Biol. 145, 851–863 (1999).
Song, J., Khachikian, Z., Radhakrishna, H. & Donaldson, J. G. Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading. J. Cell Sci. 111, 2257–2267 (1998).
Franco, M. et al. EFA6, a sec7 domain-containing exchange factor for ARF6, coordinates membrane recycling and actin cytoskeleton organization. EMBO J. 18, 1480–1491 (1999).
Santy, L. C. & Casanova, J. E. Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell Biol. 154, 599–610 (2001).
Liu, S. et al. A fragment of Paxillin binds the alpha 4 integrin cytoplasmic domain (Tail) and selectively inhibits α4-mediated cell migration. J. Biol. Chem. 277, 20887–20894 (2002).
Premont, R. T. et al. β2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein. Proc. Natl Acad. Sci. USA 95, 14082–14087 (1998).
Boshans, R. L., Szanto, S., Van Aelst, L. & D'Souza-Schorey, C. ADP-ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA. Mol. Cell. Biol. 20, 3685–3694 (2000).
Radhakrishna, H., Al Awar, O., Khachikian, Z. & Donaldson, J. G. ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements. J. Cell Sci. 112, 855–866 (1999).
Price, L. S., Leng, J., Schwartz, M. A. & Bokoch, G. M. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol. Biol. Cell 9, 1863–1871 (1998).
Brown, M. C., West, K. A. & Turner, C. E. Paxillin-dependent paxillin kinase linker and p21-activated kinase localization to focal adhesions involves a multistep activation pathway. Mol. Biol. Cell 13, 1550–1565 (2002).
Michiels, F. et al. Regulated membrane localization of Tiam1, mediated by the NH2-terminal pleckstrin homology domain, is required for Rac-dependent membrane ruffling and C-Jun NH2-terminal kinase activation. J. Cell Biol. 137, 387–398 (1997).
Tarricone, C. et al. The structural basis of Arfaptin-mediated cross-talk between Rac and Arf signalling pathways. Nature 411, 215–219 (2001).
Honda, A. et al. Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell 99, 521–532 (1999).
Krauss, M. et al. ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Iγ. J. Cell Biol. 162, 113–124 (2003).
Han, J. et al. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science 279, 558–560 (1998).
Yoshii, S. et al. alphaPIX nucleotide exchange factor is activated by interaction with phosphatidylinositol 3-kinase. Oncogene 18, 5680–5690 (1999).
Fleming, I. N., Gray, A. & Downes, C. P. Regulation of the Rac1-specific exchange factor Tiam1 involves both phosphoinositide 3-kinase-dependent and -independent components. Biochem. J. 351, 173–182 (2000).
Chan, B. M. C. et al. Distinct cellular functions mediated by different VLA integrin α subunit cytoplasmic domains. Cell 68, 1051–1060 (1992).
Kil, S. H., Krull, C. E., Cann, G., Clegg, D. & Bronner-Fraser, M. The α4 subunit of integrin is important for neural crest cell migration. Dev. Biol. 202, 29–42 (1998).
Sengbusch, J. K., He, W., Pinco, K. A. & Yang, J. T. Dual functions of α4β1 integrin in epicardial development: initial migration and long-term attachment. J. Cell Biol. 157, 873–882 (2002).
Tzima, E. et al. Activation of Rac1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression. EMBO J. 21, 6791–6800 (2002).
Dell'Angelica, E. C. et al. GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex. J. Cell Biol. 149, 81–94 (2000).
Arias-Salgado, E. G. et al. Src kinase activation by direct interaction with the integrin beta cytoplasmic domain. Proc. Natl Acad. Sci. USA 100, 13298–13302 (2003).
Yano, H. et al. Roles played by a subset of integrin signaling molecules in cadherin-based cell–cell adhesion. J. Cell Biol. 166, 283–295 (2004).
Acknowledgements
We thank Dr Miguel A. Del Pozo, Dr Larry E. Goldfinger and Dr Sergio Lizano for valuable discussions, and Dr Christopher E. Turner for critical review of the manuscript. This work was supported by grants from the National Institutes of Health. N.N. was supported by The Naito Foundation and then by a postdoctoral fellowship from the American Heart Association. J.H. was an advanced Fellow of the National Multiple Sclerosis Society.
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Nishiya, N., Kiosses, W., Han, J. et al. An α4 integrin–paxillin–Arf-GAP complex restricts Rac activation to the leading edge of migrating cells. Nat Cell Biol 7, 343–352 (2005). https://doi.org/10.1038/ncb1234
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DOI: https://doi.org/10.1038/ncb1234
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