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Stimulus-dependent phosphorylation of profilin-1 in angiogenesis

Nature Cell Biology volume 14, pages 1046–1056 (2012)Cite this article

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Abstract

Angiogenesis, the formation of new blood vessels, is fundamental to development and post-injury tissue repair. Vascular endothelial growth factor (VEGF)-A guides and enhances endothelial cell migration to initiate angiogenesis. Profilin-1 (Pfn-1) is an actin-binding protein that enhances actin filament formation and cell migration, but stimulus-dependent regulation of Pfn-1 has not been observed. Here, we show that VEGF-A-inducible phosphorylation of Pfn-1 at Tyr 129 is critical for endothelial cell migration and angiogenesis. Chemotactic activation of VEGF receptor kinase-2 (VEGFR2) and Src induces Pfn-1 phosphorylation in the cell leading edge, promoting Pfn-1 binding to actin and actin polymerization. Conditional endothelial knock-in of phosphorylation-deficient Pfn1Y129F in mice reveals that Pfn-1 phosphorylation is critical for angiogenesis in response to wounding and ischaemic injury, but not for developmental angiogenesis. Thus, VEGFR2/Src-mediated phosphorylation of Pfn-1 bypasses canonical, multistep intracellular signalling events to initiate endothelial cell migration and angiogenesis, and might serve as a selective therapeutic target for anti-angiogenic therapy.

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Figure 1: VEGF-A induces phosphorylation of Pfn-1 at Tyr 129 during endothelial cell migration.
Figure 2: Phosphorylation of Pfn-1 at Tyr 129 promotes binding to actin and actin polymerization.
Figure 3: Phosphorylation of Pfn-1 at Tyr 129 by VEGFR2 and Src.
Figure 4: Phosphorylation of Pfn-1 at Tyr 129 is spatially restricted to the leading edge of migrating endothelial cells, and is required for VEGF-A-induced endothelial cell migration.
Figure 5: Phosphorylation of Pfn-1 at Tyr 129 is expressed preferentially in infarcted cardiac blood vessels.
Figure 6: Knock-in of phospho-deficient Pfn1Y129F / Y129F in mouse endothelial cells reduces VEGF-A-induced cell migration.
Figure 7: Phosphorylation of Pfn-1 at Tyr 129 is required for angiogenesis during post-injury tissue repair but not for developmental angiogenesis.

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Acknowledgements

We are grateful to A. Levine and T. Egelhoff for helpful suggestions, X. West, A. Vasanji and J. Boerckel for technical assistance in punch-wound and microCT studies, and J. Drazba for image analysis. Cdh5-Cre/ERT2 mice were provided by R. Adams, Max Planck Institute, Germany. This work was supported by National Institutes of Health grants P01 HL029582, P01 HL076491 and R21 HL094841 (to P.L.F.).

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Authors and Affiliations

  1. Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

    Yi Fan, Abul Arif, Jie Jia, Sandeepa M. Eswarappa & Paul L. Fox

  2. Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

    Yanqing Gong & Arie Horowitz

  3. Mass Spectrometry Laboratory for Protein Sequencing, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

    Belinda Willard

  4. Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, Ohio 44195, USA

    Linda M. Graham

  5. Department of Integrated Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio 44272, USA

    Marc S. Penn

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Contributions

Y.F. designed, performed and analysed all experiments, produced figures, and wrote the initial draft of the paper. A.A. generated and analysed the data in Figs 1c–e, 2e and Supplementary Fig. S4d. Y.Q. designed and performed immunohistochemistry analysis in Fig. 5a. J.J. contributed to the analysis of the BIAcore in Fig. 2d. S.M.E. performed phylogenic analysis of Pfn-1. B.W. identified the Pfn-1 phosphorylation site by mass spectrometry as shown in Fig. 1b and Supplementary Fig. S2. A.H., L.M.G. and M.S.P. helped write and edit the final draft of the manuscript. P.L.F designed, supervised, and analysed all experiments and wrote the final draft of the manuscript.

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Correspondence to Paul L. Fox.

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Fan, Y., Arif, A., Gong, Y. et al. Stimulus-dependent phosphorylation of profilin-1 in angiogenesis. Nat Cell Biol 14, 1046–1056 (2012). https://doi.org/10.1038/ncb2580

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