Abstract
The signalling cascade including Raf, mitogen-activated protein kinase (MAPK) kinase and extracellular-signal-regulated kinase (ERK) is important in many facets of cellular regulation1,2,3. Raf is activated through both Ras-dependent and Ras-independent mechanisms4,5,6, but the regulatory mechanisms of Raf activation remain unclear7,8,9. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified10,11,12,13 and characterized as negative regulators of growth-factor-induced ERK activation14,15,16,17,18,19,20,21,22,23,24,25. But the molecular functions of mammalian Sproutys have not been clarified. Here we show that mammalian Sprouty4 suppresses vascular epithelial growth factor (VEGF)-induced, Ras-independent activation of Raf1 but does not affect epidermal growth factor (EGF)-induced, Ras-dependent activation of Raf1. Sprouty4 binds to Raf1 through its carboxy-terminal cysteine-rich domain, and this binding is necessary for the inhibitory activity of Sprouty4. In addition, Sprouty4 mutants of the amino-terminal region containing the conserved tyrosine residue, which is necessary for suppressing fibroblast growth factor signalling19,25, still inhibit the VEGF-induced ERK pathway. Our results show that receptor tyrosine kinases use distinct pathways for Raf and ERK activation and that Sprouty4 differentially regulates these pathways.
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Acknowledgements
We thank E. Nishida for the ERK2 plasmid; S. Ohno for the PKC plasmids; T. Sato and Y. Kaziro for the Ras plasmids; G. Christofori for the antibody against Sprouty2; A. E. Koromilas, Y. Hiromi, S. Kondo, M. Iwanami, M. Ohtubo, A. Takeda and E. Mekada for comments; N. Arifuku for manuscript preparation; and Y. Kawabata and H. Ohgusu for technical assistance. Part of this work was supported by grants from the Ministry of Science, Education, Culture and Sports of Japan, the Japan Research Foundation for Clinical Pharmacology and Human Frontier International Program Organization (to A.Y.), the Fukuoka Cancer Society and the TAKEDA Science Foundation (to A.S.).
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Supplementary Figures
Figure S1, Suppression of VEGF-induced, but not EGF-induced Raf1 kinase activity by Sprouty4. (PDF 351 kb)
Figure S2, KDR activates ERK through the PLCã-PKC pathway but not through the Grb2/Ras pathway.
Figure S3, Subcellular localization of Sprouty4 mutants in HeLa cells visualized by immuno-fluorescence microscopy.
Figure S4, S5, Binding of Raf1 to WT and deletion mutants of Sprouty and Spred.
Figure S6, In vitro binding of Sprouty4 and Raf1.
Figure S7, Oligomer formation of WT and RBM-deletion mutant (ΔRBM) Sprouty4.
Figure S8, ΔRBM-Sprouty4 reverted the inhibitory effect of WT-Sprouty4.
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Sasaki, A., Taketomi, T., Kato, R. et al. Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding to Raf1. Nat Cell Biol 5, 427–432 (2003). https://doi.org/10.1038/ncb978
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DOI: https://doi.org/10.1038/ncb978
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