scholarly journals Characterization and identification of an epidermal-growth-factor-activated phospholipase A2

1992 ◽  
Vol 287 (1) ◽  
pp. 37-43 ◽  
Author(s):  
M Spaargaren ◽  
S Wissink ◽  
L H K Defize ◽  
S W de Laat ◽  
J Boonstra
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase A2 ◽  
Molecular Mass ◽  
Egf Receptor ◽  
Gel Filtration ◽  
Partial Purification ◽  
Pla2 Activity ◽  
Epidermal Growth ◽  
Cytosolic Pla2

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.

scholarly journals The tyrosine kinase activity of the epidermal-growth-factor receptor is necessary for phospholipase A2 activation

1990 ◽  
Vol 267 (2) ◽  
pp. 461-465 ◽  
Author(s):  
H J Goldberg ◽  
M M Viegas ◽  
B L Margolis ◽  
J Schlessinger ◽  
K L Skorecki
Keyword(s):  
Arachidonic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Phospholipase A2 ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Pla2 Activity ◽  
Tyrosine Kinase Activity ◽  
Epidermal Growth

We have previously reported that epidermal growth factor (EGF) activates phospholipase A2 (PLA2) independently of phospholipase C (PLC) in renal mesangial cells. In this study we use NIH 3T3 cell lines transfected with the normal EGF receptor (HER14 cells) or with EGF receptor defective in tyrosine kinase activity (K721A cells), to determine whether the intrinsic tyrosine kinase activity of the EGF receptor is required for the PLC-independent activation of PLA2. Intact cells were preincubated with EGF or other ligands, and then PLA2 activity was assayed in cell-free extracts with 1-stearoyl-2-[14C]arachidonyl phosphatidylcholine as the substrate. In HER14 cells, EGF increased PLA2 activity by 226 +/- 30%, and the tumour promoter phorbol myristate acetate (PMA) increased activity by 223 +/- 30%. The effect of EGF was not mediated through protein kinase C (PKC), whose activation by EGF requires tyrosine kinase activity, since raising intracellular Ca2+ alone with the Ca2+ ionophore A23187 did not mimic its effect, and the effect of EGF persisted in PKC-down-regulated cells. In K721A cells EGF was ineffective, whereas PMA was still active. Furthermore, in intact HER14 cells prelabelled with [14C]arachidonate, EGF-stimulated release of [14C]arachidonic acid was synergistic with A23187, but was unaccompanied by a rise in [14C]diacylglycerol. EGF had no effect on [14C]arachidonic acid release in intact K721A cells. We conclude that the tyrosine kinase activity of the EGF receptor is necessary for the PLC-independent stimulation of PLA2 by EGF.

scholarly journals Modulation of phospholipase A2 activity by epidermal growth factor (EGF) in CHO cells transfected with human EGF receptor. Role of receptor cytoplasmic subdomain

1991 ◽  
Vol 274 (3) ◽  
pp. 715-721 ◽  
Author(s):  
S Clark ◽  
M Dunlop
Keyword(s):  
Arachidonic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase A2 ◽  
Egf Receptor ◽  
Control Cell ◽  
Cell Types ◽  
Positive Control ◽  
Prostaglandin Production ◽  
Epidermal Growth

Activation of phospholipase A2 (PLA2) in response to external stimuli may play a pivotal role in signal-transduction pathways via the generation of important cellular intermediates, including prostaglandins. Epidermal growth factor (EGF) has been shown to modulate prostaglandin production, possibly via direct activation of PLA2 or indirectly via interaction with a PLA2-modifying protein such as lipocortin I. We have investigated these pathways with two CHO cell-lines, one (CHOwt) transfected with the full-length human EGF receptor and the second (CHO 11) with a deletion mutant, delta 990, that has lost the autophosphorylation sites and part of the internalization domain. CHOwt cells responded to EGF with a rapid rise in lysophosphatidylcholine and arachidonic acid release concomitant with an increase in prostaglandin production. However, in the non-internalizing CHO 11 cells no such activation of PLA2 was observed. This was not due to an intrinsic lack of PLA2 in these cells, as PLA2 activation was shown on melittin addition, nor was this difference due to a defect in intracellular pathways, as arachidonic acid was released from both cell types by Ca2+ and protein kinase C modulators. However, only in CHOwt cells were these responses potentiated by concomitant addition of EGF. Thus the cytoplasmic subdomain of the EGF receptor, containing the major sites of autophosphorylation and the internalization domain, seems to be involved in the activation of PLA2 by EGF. In addition, we have shown that phosphorylation of lipocortin I is unlikely to play a role in PLA2 activation. In CHOwt cells and a positive control cell line, A431, activation of PLA2 was complete by 10 min, at which time there was no evidence of lipocortin I phosphorylation.

scholarly journals Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells

1993 ◽  
Vol 295 (3) ◽  
pp. 763-766 ◽  
Author(s):  
A P Maxwell ◽  
H J Goldberg ◽  
A H N Tay ◽  
Z G Li ◽  
G S Arbus ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Phospholipase A2 ◽  
Mesangial Cells ◽  
Mrna Levels ◽  
Glomerular Mesangial Cells ◽  
Pla2 Activity ◽  
Mrna Accumulation ◽  
Epidermal Growth

We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications.

scholarly journals Distinct structural specificities for functional coupling of the epidermal growth factor receptor to calcium-signalling versus phospholipase A2 responses

1991 ◽  
Vol 275 (3) ◽  
pp. 563-567 ◽  
Author(s):  
N Hack ◽  
B L Margolis ◽  
A Ullrich ◽  
J Schlessinger ◽  
K L Skorecki
Keyword(s):  
Arachidonic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Tyrosine Kinase ◽  
Phospholipase A2 ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Functional Coupling ◽  
Tyrosine Kinase Activity ◽  
Epidermal Growth

Activation of phospholipase C (PLC), leading to a rise in cytosolic Ca2+, and of phospholipase A2 (PLA2) leading to a release of arachidonic acid, are among the early transmembrane signalling events that have been demonstrated in response to occupancy of the epidermal growth factor (EGF) receptor. The tyrosine kinase activity of the receptor has been shown to be necessary for both of these responses. This requirement for the tyrosine kinase activity could conceivably implicate a role for receptor autophosphorylation in the activation of PLA2. We now demonstrate that coupling of the EGF receptor to PLA2 was not impaired in a deletion mutant (CD126) devoid of the 126 amino acids from the C-terminus which include four major autophosphorylation sites. Functional coupling of the EGF receptor to PLA2 was demonstrated using three different experimental designs: (1) release of [14C]arachidonic acid from prelabelled intact cells. (2) release of [3H]arachidonic acid from prelabelled cells permeabilized with glass beads, and (3) direct measurement of PLA2 enzymic activity in cell-free extracts using an ‘in vitro’ assay employing exogenous phospholipid substrate. Functional coupling of the EGF receptor to PLA2 occurred despite the absence of a demonstrable Ca(2+)-signalling response and the detection of diminished but persistent PLC-gamma phosphorylation on tyrosine residues in the CD126 deletion mutants. These results point to a clear distinction in the biochemical mechanism and role for receptor autophosphorylation in functional coupling of the EGF receptor to PLA2 activation versus Ca2+ signalling.

scholarly journals Interleukin-1 activates a novel protein kinase that phosphorylates the epidermal-growth-factor receptor peptide T669

1994 ◽  
Vol 302 (3) ◽  
pp. 897-905 ◽  
Author(s):  
M Kracht ◽  
M Shiroo ◽  
C J Marshall ◽  
J J Hsuan ◽  
J Saklatvala
Keyword(s):  
Growth Factor ◽  
Protein Kinase ◽  
Epidermal Growth Factor ◽  
Map Kinase ◽  
Egf Receptor ◽  
Interleukin 1 ◽  
Gel Filtration ◽  
Growth Factor Receptor ◽  
Protein Band ◽  
Epidermal Growth

We have isolated from KB cells stimulated with interleukin-1 (IL-1) a protein kinase that phosphorylates a peptide (T669) based on the sequence around T669 of the epidermal growth factor (EGF) receptor. The enzyme, which had an apparent molecular mass of 45 kDa on gel-filtration chromatography, was purified 170,000-fold from cytosolic extracts by sequential chromatography on Mono Q, Mono S, phenyl-Sepharose, Superose 12, ATP-Sepharose and Mono Q. The enzyme activity co-chromatographed at the last step with a 45 kDa protein band that stained for phosphotyrosine. This peak fraction also contained some actin and a 60 kDa protein that stained weakly for phosphotyrosine. The T669 peptide is a substrate for mitogen-activated protein (MAP) kinase. Amounts of IL-1-induced T669 kinase and activated recombinant p42 MAP kinase having equal activity on T669 peptide were compared on commonly used MAP kinase substrates. T669 kinase was two or three orders of magnitude less active on myelin basic protein or microtubule-associated protein-2 than was MAP kinase. The IL-1-induced T669 kinase did not react with antiserum to p42/p44 MAP kinase. It was inactivated by treatment with protein phosphatase 2A or protein phosphotyrosine phosphatase 1B, so it may be regulated by dual phosphorylation in similar fashion to MAP kinase. The dephosphorylated enzyme was not re-activated by MAP kinase kinase. This novel enzyme could lie on a kinase cascade induced by IL-1. It may be responsible for phosphorylating T669 of the EGF receptor.

947: Chemopreventive Effects of COX-2 Inhibitor and Epidermal Growth Factor (EGF)-Receptor Kinase Inhibitor on Rat Urinary Bladder Tumorigenesis

2004 ◽  
Vol 171 (4S) ◽  
pp. 251-251
Author(s):  
Kazunori Hattori ◽  
Katsuyuki Iida ◽  
Akira Johraku ◽  
Sadamu Tsukamoto ◽  
Taeko Asano ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Urinary Bladder ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Receptor Kinase ◽  
Rat Urinary Bladder ◽  
Epidermal Growth ◽  
Cox 2
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