Abstract
Mitogen-activated protein (MAP) kinase activity is essential for tumor necrosis factor (TNF) alpha receptor 1 regulation of intestinal epithelial cell proliferation. However, the mechanism of TNF-alpha mediated activation of extracellular signal-regulated kinase (ERK)/M1AP kinase has not been established clearly. Both TNF-alpha and cell-permeable ceramide have been reported to increase the kinase activity of kinase suppressor of Ras (KSR). To determine the role of KSR in TNF-alpha-induced ERK1/ERK2 activation, we studied young adult mouse colon cells expressing a dominant-negative, kinase-inactive (ki) KSR. We report that TNF-alpha, a cell-permeable ceramide, and sphingomyelinase stimulate ERK1/ERK2 activation and increase the phosphoserine content of KSR, which are inhibited by kiKSR expression in intact cells. Furthermore, TNF-alpha-induced Raf-1 threonine phosphorylation, kinase activity toward MEK1, and association with KSR are also inhibited by kiKSR expression. Our data also show by sequential in vitro kinase assays that TNF-alpha enhances KSR phosphorylation of Raf-1 on threonine, enhancing Raf-1 kinase activity toward MAP kinase kinase. We therefore conclude that KSR is an essential upstream regulator of TNF-alpha-stimulated ERK1/ERK2 activation, most likely mediated via direct phosphorylation of Raf-1.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Cell Line
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Ceramides / pharmacology
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Colon / cytology
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Colon / enzymology*
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Enzyme Activation
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Epithelial Cells / enzymology
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Intestinal Mucosa / enzymology*
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JNK Mitogen-Activated Protein Kinases*
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MAP Kinase Kinase 1
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MAP Kinase Kinase 4
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MAP Kinase Signaling System / drug effects
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Mice
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Mitogen-Activated Protein Kinase 1 / metabolism*
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinase Kinases / metabolism
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Mitogen-Activated Protein Kinases / metabolism*
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Phosphorylation
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Phosphoserine / metabolism
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Phosphothreonine / metabolism
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Protein Kinases / metabolism
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Protein Kinases / physiology*
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Protein Serine-Threonine Kinases / metabolism
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Proto-Oncogene Proteins c-raf / metabolism
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Sphingomyelin Phosphodiesterase / pharmacology
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Tumor Necrosis Factor-alpha / antagonists & inhibitors
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Tumor Necrosis Factor-alpha / pharmacology*
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Tumor Necrosis Factor-alpha / physiology
Substances
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Ceramides
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Tumor Necrosis Factor-alpha
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Phosphothreonine
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Phosphoserine
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Protein Kinases
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KSR-1 protein kinase
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-raf
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JNK Mitogen-Activated Protein Kinases
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 1
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MAP Kinase Kinase 4
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Map2k1 protein, mouse
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Mitogen-Activated Protein Kinase Kinases
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Sphingomyelin Phosphodiesterase