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EphrinA1-induced cytoskeletal re-organization requires FAK and p130cas

Nature Cell Biology volume 4, pages 565–573 (2002)Cite this article

Abstract

Ephrins and Eph receptors are involved in axon guidance and cellular morphogenesis. An interaction between ephrin and Eph receptors elicits neuronal growth-cone collapse through cytoskeletal disassembly. When NIH3T3 cells were plated onto an ephrinA1-coated surface, the cells both adhered and spread. Adhesion and spreading proceeded concomitantly with changes in both the actin and microtubule cytoskeleton. EphA2, focal adhesion kinase (FAK) and p130cas were identified as the major ephrin-dependent phosphotyrosyl proteins during the ephrin-induced morphological changes. Mouse embryonic fibroblasts (MEFs) derived from FAK−/− and p130cas−/− mice had severe defects in ephrinA1-induced cell spreading, which were reversed after re-expression of FAK or p130cas, respectively. Expression of a constitutively active EphA2 induced NIH3T3 cells to undergo identical, but ligand-independent, morphological changes. These data show that ephrinA1 can induce cell adhesion and actin cytoskeletal changes in fibroblasts in a FAK- and p130cas-dependent manner, through activation of the EphA2 receptor. The finding that ephrin–Eph signalling can result in actin cytoskeletal assembly, rather than disassembly, has many implications for ephrin–Eph responses in other cell types.

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Figure 1: EphrinA1 induces cell adhesion and spreading.
Figure 2: Localization of F-actin, tubulin and phosphotyrosine in NIH3T3 cells plated onto ephrinA1 or fibronectin.
Figure 3: Localization of F-actin, Paxillin and Vinculin in NIH3T3 cells plated onto ephrinA1 or fibronectin.
Figure 4: EphrinA1 induces increased tyrosine phosphorylation of FAK, p130cas, Paxillin and EphA2.
Figure 5: Immunolocalization of FAK and p130cas after plating NIH3T3 cells onto ephrinA1.
Figure 6: FAK−/− MEFs exhibit severe deficiencies in spreading on ephrinA1-coated surfaces.
Figure 7: p130cas−/− MEFs exhibit no spreading on ephrinA1-coated surfaces.
Figure 8: A constitutively active EphA2 mimics the actin re-organization induced by ephrinA1.
Figure 9: RhoA is required for ephrinA1-dependent spreading of NIH3T3 cells.

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Acknowledgements

Funding for parts of this work was provided by a Human Frontiers Science Program Organisation long-term fellowship and an Amgen Inc. fellowship (to N.C.) as well as National Institutes of Health grants from the National Cancer Institute, CA141915, CA39780 and CA82863 (to T.H.). We are indebted to J. Meisenhelder, S. Simon, H. Mondala and M. Rosenthal for considerable help and expert technical assistance. We also thank C. Vaziri for numerous helpful discussions and R. Lindberg, J. Ruiz, L. Robertson, D. Schlaepfer, N. Somia, W. Jiang and B. Seed for generously donating their time, reagents and expertise. We are especially grateful to R.Gage, B. Summers and L. Kitabayashi for access to and help with confocal microscopy, and J. Simon for invaluable help with Adobe Photoshop. T.H. is a Frank and Elise Schilling American Cancer Society Research Professor.

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Authors and Affiliations

  1. Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, 92037, CA, USA

    Nigel Carter & Tony Hunter

  2. Department of Haematology and Oncology, Graduate School of Medicine, University of Tokyo, Hongo, 113-8655, Tokyo, Japan

    Tetsuya Nakamoto & Hisamaru Hirai

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Correspondence to Nigel Carter.

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Supplementary Figures

Figure S1. Plating NIH3T3 cells on to ephrinA1 induces cell adhesion and spreading in a large percentage of cells and this is mimicked by expression of an activated EphA2 receptor. (PDF 228 kb)

Figure S2. Profiling ephrinA1-responsive EphA receptors in NIH3T3 cells. Serum starved NIH3T3 cells were treated with 2µg/ml ephrinA1-Fc or buffer control for 30 minutes.

Figure S3. Adherence of NIH3T3 and PC3 cells to culture dishes coated with differing concentrations of EphrinA1.

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Carter, N., Nakamoto, T., Hirai, H. et al. EphrinA1-induced cytoskeletal re-organization requires FAK and p130cas. Nat Cell Biol 4, 565–573 (2002). https://doi.org/10.1038/ncb823

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