Title
Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration
Author
Gaubius Instituut TNO
Schalkwijk, C.G.
van der Heijden, M.A.G.
Bunt, G.
Maas, R.
Tertoolen, L.G.J.
van Bergen Henegouwen, P.M.P.
Verkleij, A.J.
van den Bosch, H.
Boonstra, J.
Publication year
1996
Abstract
The 85 kDa cytosolic phospholipase A2 (cPLA2) preferentially catalyses the hydrolysis of arachidonic acid from the sn-2 position of phospholipids. cPLA2 can be activated by extracellular stimuli such as thrombin, platelet-derived growth factor and epidermal growth factor (EGF). A full activation of cPLA2 requires an increase of intracellular Ca2+ concentration and phosphorylation on Ser-505 by mitogen-activated protein (MAP) kinase. Because EGF can provoke an increase in intracellular [Ca2+] ([Ca2+](i)) and activation of MAP kinase, we investigated the role of these pathways in EGF-induced activation of cPLA2. Characterization of two cell lines expressing different numbers of EGF receptors (HERc13 and HER14) revealed that both were activating MAP kinase in response to EGF, but only HER14 responded with an increase in [Ca2+](i). In this study we used both cell lines as a tool to clarify the role of each pathway in cPLA2 activation. We show that EGF stimulates cPLA2 activity in both cell lines in vitro as measured in cytosolic fractions, but only in HER14 in vivo as measured by 3H release from cells prelabelled with [3H]arachidonic acid. This latter activation can be restored in HERc13 cells by the addition of the ionophore A23187. Interestingly, this effect is only observed when EGF stimulation precedes A23187 addition. The phosphorylation of MAP kinase, however, was identical under identical conditions. We conclude that a maximal cPLA2 activation by EGF requires both, and in this order: MAP kinase activation followed by a rise in [Ca2+](i) concentration. Chemicals/CAS: Arachidonic Acid, 506-32-1; Ca(2+)-Calmodulin Dependent Protein Kinase, EC 2.7.1.123; Calcimycin, 52665-69-7; Calcium, 7440-70-2; Epidermal Growth Factor, 62229-50-9; Ionophores; Phospholipases A, EC 3.1.1.-
Subject
3T3 Cells
Animals
Arachidonic Acid
Ca(2+)-Calmodulin Dependent Protein Kinase
Calcimycin
Calcium
Cytosol
Enzyme Activation
Epidermal Growth Factor
Intracellular Fluid
Ionophores
Mice
Phospholipases A
Phosphorylation
Time Factors
Animalia
Tritium
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TNO identifier
233147
ISSN
0264-6021
Source
Biochemical Journal, 313 (313), 91-96
Document type
article