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Abstract
Addition of sphingosine 1-phosphate induces proliferation of quiescent Swiss 3T3 fibroblasts by unknown mechanisms. To identify the pathways involved, the ability of sphingosine 1-phosphate to activate mitogen-activated protein (MAP) kinase was studied. Sphingosine 1-phosphate rapidly activated the Raf/MAP kinase kinase (MKK)/MAP kinase pathway, and the concentration dependence for MAP kinase activation correlated with that for induction of DNA synthesis. Both MKK1 and MKK2 were activated by sphingosine 1-phosphate, assessed by specific immune complex kinase assays. Prior treatment of the Swiss 3T3 cells with pertussis toxin inhibited 70-80% of the sphingosine 1-phosphate-stimulated MAP kinase activity. Thus, one of the direct or indirect targets of exogenous sphingosine 1-phosphate appears to be a G(i)/G(o) protein.
Highlights
From the +Howard Hughes Medical Institute, Departments of Medicine and Pharmacology and the Markey Center for Cell Signaling, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 and the Wepartment of Biochemistry and Molecular Biology, Georgetown University Medical Center, Washington, D
Sphingosine i-Phosphate Activates mitogenactivated protein (MAP) Kinase in Swiss 3T3 Cells-To determine whether exogenous sphingosine I-phosphate can stimulate MAP kinase activity in Swiss 3T3 fibroblasts, quiescent Swiss 3T3 fibroblasts were treated with a mitogenic concentration of sphingosine I-phosphate (5 /-tM) or PDGF (65 ng/ml) for 1-15 min, and p42mapk activities were assayed by a specific immune complex assay [25]
Our results showed that sphingosine I-phosphate added to Swiss 3T3 cells rapidly activates the protein kinase cascade sequentiall y involving Raf-I, MAP kina e kinase, and MAP kinase
Summary
Medical Institute, Box 577, Hospital West, University of Virginia Health Sciences Center, Charlottesville, VA 22908. MAP kinases are activated by dual tyrosine and threonine phosphorylations occurring in a TXY motif near the conserved A(S)PE and phosphorylate (S/ T)P motifs in their target proteins. A 42-kDa MAP kinase (p42mapkIERK2) and a 44-kDa MAP kinase (p44mapkIERK2) are the two best characterized MAP kinases, collectively referred to here as MAP kinase Both enzymes are activated by two remarkably specific dual specificity MAP kinase kinases (MKKs or MEKs) [14,15,16,17]. It is critically important to identify the downstream components involved To this end, the potential involvement of MAP kinase in signaling for sphingosine I-phosphate in Swiss 3T3 fibroblasts was studied. We report that exogenous sphingosine I-phosphate rapidly stimulates the activation of the MAP kinase pathway by a pertussis toxin-sensitive mechanism
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