Schalkwijk C G, van der Heijden M A, Bunt G, Maas R, Tertoolen L G, van Bergen en Henegouwen P M, Verkleij A J, van den Bosch H, Boonstra J
Centre for Biomembranes and Lipid Enzymology, Department of Molecular Cell Biology, Utrecht University, The Netherlands.
Biochem J. 1996 Jan 1;313 ( Pt 1)(Pt 1):91-6. doi: 10.1042/bj3130091.
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.
85 kDa的胞质型磷脂酶A2（cPLA2）优先催化从磷脂的sn-2位水解花生四烯酸。cPLA2可被细胞外刺激如凝血酶、血小板衍生生长因子和表皮生长因子（EGF）激活：cPLA2的完全激活需要细胞内Ca2+浓度升高以及丝裂原活化蛋白（MAP）激酶对Ser-505的磷酸化。由于EGF可引起细胞内[Ca2+]（[Ca2+]i）升高和MAP激酶激活，我们研究了这些途径在EGF诱导的cPLA2激活中的作用。对两种表达不同数量EGF受体的细胞系（HERc13和HER14）的特性分析表明，二者均对EGF产生反应而激活MAP激酶，但只有HER14的[Ca2+]i升高。在本研究中，我们将这两种细胞系作为工具来阐明每条途径在cPLA2激活中的作用。我们发现，在体外，如在胞质组分中所测，EGF刺激两种细胞系中的cPLA2活性，但在体内，如在用[3H]花生四烯酸预标记的细胞中通过3H释放所测，EGF仅刺激HER14中的cPLA2活性。通过添加离子载体A23187，可在HERc13细胞中恢复后一种激活作用。有趣的是，只有当EGF刺激先于A23187添加时才观察到这种效应。然而，在相同条件下MAP激酶的磷酸化情况是相同的。我们得出结论，EGF对cPLA2的最大激活需要二者，且顺序如下：MAP激酶激活，随后[Ca2+]i浓度升高。
