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 regulation. Raf is activated through both Ras-dependent and Ras-independent mechanisms, but the regulatory mechanisms of Raf activation remain unclear. Two families of membrane-bound molecules, Sprouty and Sprouty-related EVH1-domain-containing protein (Spred) have been identified and characterized as negative regulators of growth-factor-induced ERK activation. 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 signalling, 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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Cell Membrane / drug effects
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Cell Membrane / metabolism*
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Endothelial Growth Factors / metabolism
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Endothelial Growth Factors / pharmacology
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Epidermal Growth Factor / metabolism
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Epidermal Growth Factor / pharmacology
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Eukaryotic Cells / cytology
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Eukaryotic Cells / drug effects
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Eukaryotic Cells / metabolism*
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Fibroblast Growth Factors / metabolism
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Fibroblast Growth Factors / pharmacology
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Growth Substances / metabolism*
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Humans
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Immunohistochemistry
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Intercellular Signaling Peptides and Proteins / metabolism
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Intercellular Signaling Peptides and Proteins / pharmacology
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Intracellular Signaling Peptides and Proteins
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Lymphokines / metabolism
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Lymphokines / pharmacology
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MAP Kinase Signaling System / drug effects
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MAP Kinase Signaling System / genetics*
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Mitogen-Activated Protein Kinases / genetics
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Mitogen-Activated Protein Kinases / metabolism
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Mutation / genetics
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Nerve Tissue Proteins / metabolism*
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Protein Binding / drug effects
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Protein Binding / genetics
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Protein Structure, Tertiary / drug effects
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Protein Structure, Tertiary / genetics
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Proto-Oncogene Proteins c-raf / genetics
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Proto-Oncogene Proteins c-raf / metabolism*
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Receptor Protein-Tyrosine Kinases / drug effects
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Receptor Protein-Tyrosine Kinases / metabolism*
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Signal Transduction / drug effects
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Signal Transduction / genetics
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
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ras Proteins / genetics
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ras Proteins / metabolism
Substances
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Endothelial Growth Factors
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Growth Substances
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Intercellular Signaling Peptides and Proteins
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Intracellular Signaling Peptides and Proteins
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Lymphokines
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Nerve Tissue Proteins
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SPRY4 protein, human
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Vascular Endothelial Growth Factor A
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Vascular Endothelial Growth Factors
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Fibroblast Growth Factors
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Epidermal Growth Factor
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Receptor Protein-Tyrosine Kinases
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Proto-Oncogene Proteins c-raf
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Mitogen-Activated Protein Kinases
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ras Proteins