Abstract
The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. Recently, sphingolipids metabolites, ceramide, sphingosine and sphingosine-1-phosphate, have been implicated as second messengers in cell growth regulation and differentiation. In this paper, we examined the possibility that interaction of the B subunit with membrane GM1 leads to alterations in metabolism of glycosphingolipids and that increased levels of sphingolipids metabolites may mediate the biological effects of the B subunit. While the B subunit did not induce a change in the level of ceramide or sphingosine, the level of sphingosine-1-phosphate was rapidly and transiently increased. The B subunit also transiently activated cytosolic sphingosine kinase activity, which catalyzes the phosphorylation of the primary hydroxyl group of sphingosine to produce sphingosine-1-phosphate. To determine whether the increase in sphingosine-1-phosphate level plays a role in B subunit-induced mitogenicity, we used a competitive inhibitor of sphingosine kinase, D,L-threo-dihydrosphingosine. D,L-thereo-Dihydrosphingosine not only inhibited B subunit-induced DNA synthesis by 26%, it also reduced its ability to stimulate DNA-binding activity of the transcription factor AP-1. This sphingosine kinase inhibitor also inhibited B subunit-induced increases in the activity of cell cycle-regulated, cyclin-dependent serine/threonine kinases, cdk2 and p34cdc2. These findings suggest that sphingosine-1-phosphate may play a role in the signal transduction pathways activated by binding of the B subunit to endogenous ganglioside GM1.
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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3T3 Cells / cytology
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3T3 Cells / drug effects
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3T3 Cells / metabolism
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Animals
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Antibodies / pharmacology
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Binding, Competitive
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CDC2 Protein Kinase / drug effects
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CDC2 Protein Kinase / metabolism
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CDC2-CDC28 Kinases*
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Cell Division / drug effects
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Cell Division / physiology*
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Ceramides / metabolism
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Cholera Toxin / metabolism
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Cholera Toxin / pharmacology
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases / drug effects
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Cyclin-Dependent Kinases / metabolism
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DNA / biosynthesis
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DNA / drug effects
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Enzyme Activation / drug effects
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Enzyme Inhibitors / pharmacology
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Epidermal Growth Factor / pharmacology
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G(M1) Ganglioside / immunology
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G(M1) Ganglioside / metabolism*
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Lysophospholipids*
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Mice
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Mitogens / metabolism
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Mitogens / pharmacology
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Peptide Fragments / metabolism
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Peptide Fragments / pharmacology
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Phosphotransferases (Alcohol Group Acceptor) / drug effects
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Phosphotransferases (Alcohol Group Acceptor) / metabolism
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Platelet-Derived Growth Factor / pharmacology
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Protein Serine-Threonine Kinases / drug effects
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Protein Serine-Threonine Kinases / metabolism
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Sphingolipids / metabolism*
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Sphingosine / analogs & derivatives
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Sphingosine / metabolism
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Sphingosine / pharmacology
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Tetradecanoylphorbol Acetate / pharmacology
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Transcription Factor AP-1 / metabolism
Substances
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Antibodies
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Ceramides
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Enzyme Inhibitors
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Lysophospholipids
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Mitogens
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Peptide Fragments
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Platelet-Derived Growth Factor
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Sphingolipids
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Transcription Factor AP-1
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cholera toxin B subunit (50-75)
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sphingosine 1-phosphate
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G(M1) Ganglioside
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Epidermal Growth Factor
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DNA
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Cholera Toxin
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Phosphotransferases (Alcohol Group Acceptor)
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sphingosine kinase
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Protein Serine-Threonine Kinases
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CDC2 Protein Kinase
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CDC2-CDC28 Kinases
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Cdk2 protein, mouse
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases
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Sphingosine
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Tetradecanoylphorbol Acetate
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safingol