Characterization and identification of an epidermal-growth-factor-activated phospholipase A2.
The production of arachidonic acid (AA), which is involved in mitogenic signalling by epidermal growth factor (EGF), is most directly accomplished by the action of phospholipase A2 (PLA2). We demonstrate that EGF treatment of intact NEF cells rapidly activates a cytosolic PLA2, as measured in cell-free extracts by the release of radiolabelled AA from exogenously added 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine. Activation of PLA2 by EGF resulted in an enhanced Vmax. and no change in Km. The PLA2 activity was eluted in a single peak at 0.4 M-NaCl from a Mono Q anion-exchange column, and migrated with an approximate molecular mass of 70 kDa on a Superose 12 gel-filtration column. The EGF-activated PLA2 activity co-migrated with the basal PLA2 activity upon gel filtration, and persisted after partial purification, which indicates that the activation is due to a stable modification of the enzyme. The EGF-stimulated PLA2 is Ca(2+)-dependent, with maximal activity at micromolar concentrations of Ca2+, has a pH optimum at 9, associates with the particulate cell fraction in a Ca(2+)-dependent fashion, and is selective for arachidonoyl at the sn-2 position. These data demonstrate the EGF-induced activation of a PLA2, which is similar to a recently cloned high-molecular-mass AA-selective cytosolic PLA2, thus providing a link between EGF-receptor tyrosine kinase activation and AA metabolism.
Spaargaren
M
M
Department of Molecular Cell Biology, University of Utrecht, The Netherlands.
Wissink
S
S
Defize
L H
LH
de Laat
S W
SW
Boonstra
J
J
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002451:Cell Compartmentation; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D066246:ErbB Receptors / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D015398:Signal Transduction; D013379:Substrate Specificity
D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium