Abstract
Basic fibroblast growth factor (FGF) stimulates the proliferation, differentiation, and motility of multiple cell types. Signal transduction by FGF is mediated by high affinity FGF receptors that have autophosphorylating tyrosine kinase activity and also elicit the release of low molecular weight signaling molecules, including inositol 1,4,5-trisphosphate, diacylglycerol, and arachidonate. We have shown previously that basic FGF-stimulated, phospholipase A2 (PLA2)-mediated arachidonate release regulates endothelial cell (EC) motility (Sa, G., and Fox, P.L. (1994) J. Biol. Chem. 269, 3219-3225). Here we identify the phospholipase responsible for basic FGF-mediated arachidonate release as cytosolic PLA2 (cPLA2) by demonstrating in EC lysates a requirement for micromolar Ca2+, dithiothreitol insensitivity, and inactivation by anti-cPLA2 antiserum. The role of cPLA2 is also indicated by the observed mechanisms of activation which show a requirement for p42 mitogen-activated protein kinase activity, cPLA2 phosphorylation, and cPLA2 translocation from cytosol to membranes. Phosphorylation of cPLA2, arachidonate release from prelabeled intact cells, and cell motility all have similar concentration dependencies on basic FGF. Since arachidonate release is required for basic FGF-stimulated motility of EC, our results show that p42 mitogen-activated protein kinase activation of cPLA2 may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF.
Highlights
From the Wepartment of Cell Biology, Cleveland Clinic Research Institute, Cleveland, Ohio 44195 and the "!~Molecular Biology Division, Rhfme-Poulenc-Rorer Central Research, Collegeville, Pennsylvania 19426
Since arachidonate release is required for basic fibroblast growth factor (FGF)-stimulated motility of endothelial cell (EC), our results show that p42 mitogen-activated protein kinase activation of cP~ may be a regulatory event in stimulation of cellular release of this important eicosanoid precursor during cellular responses to basic FGF
A mechanism consistent with these observations is that the phospholipase A2 (PLA2) activity present in membranes is due to cytosolic PLA2 (cPLA2) and that the amount of activity is lower than that in the cytosol due to limited translocation in the absence of agonist
Summary
2360-2366, 1995 Printed in U.S.A. Activation of Cytosolic Phospholipase A2 by Basic Fibroblast Growth Factor via a p42 Mitogen-activated Protein Kinasedependent Phosphorylation Pathway in Endothelial Cells*. The cellular effects of basic FGF are transduced by its interaction with any one of four members of a family of high affinity, cell surface FGF receptors [2] These receptors are "single-pass" transmembrane proteins with a kinase activity that induces phosphorylation of tyrosine residues in the receptor itself, as well as in phospholipase-Cy1 [3], a 34-kDa lipocortin-like protein [4], cortactin [5], and She [6]. In this report we show that the PLA2 activated by basic FGF is cPLA2 , that the activation occurs by a MAP kinase-dependent phosphorylation pathway, and that this activity is responsible for the release of arachidonate in stimulated EC
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