Murray-Whelan R, Reid J D, Piuz I, Hezareh M, Schlegel W
Foundation Pour Recherches Medicales, Department of Medicine, University of Geneva, Switzerland.
Eur J Biochem. 1995 May 15;230(1):164-9. doi: 10.1111/j.1432-1033.1995.tb20547.x.
The mechanisms of activation of cytoplasmic phospholipase A2 (cPLA2) are complex and incompletely defined. In Chinese hamster ovary (CHO) cells, receptor stimulation of cPLA2 is due to the interaction of pathways involving the alpha subunits of at least two guanine-nucleotide-binding (G) proteins, G alpha i2 and G alpha q. Activation of cPLA2 is inhibited by pertussis toxin and G alpha i2 mutants. In addition, activation of phospholipase C via G alpha q results in increased intracellular calcium ([Ca2+]i) and activation of protein kinase C, both of which interact with and activate cPLA2. The present study was undertaken to analyze the mechanism of interaction of G alpha i2 with the phospholipase-C-stimulated pathway in the activation of cPLA2. We addressed this question using a dominant negative G alpha i2 mutant, [G203T]G alpha i2, in which Gly203 is mutated to Thr. [G203T]G alpha i2 inhibits ATP receptor activation of cPLA2. The effect of [G203T]G alpha i2 was specific to G alpha i2-activated pathways, as shown by its lack of effect on other purinergic receptor stimulated pathways: ATP stimulation of [Ca2+]i or mitogen-activated protein kinase phosphorylation is unaltered by [G203T]G alpha i2. We addressed the possibility that the activation of cPLA2 by Ca2+ and/or protein kinase C is dependent on G alpha i2. Activation of cPLA2 by the Ca2+ ionophore, ionomycin, was inhibited by 61 +/- 9% (n = 5) in [G203T]G alpha i2-expressing cells; however the ionomycin-induced [Ca2+]i rise was unaffected by [G203T]G alpha i2. Thus, [G203T]G alpha i2. specifically inhibits Ca2+ activation of cPLA2. In contrast, activation of cPLA2 via protein kinase C by phorbol 12-myristate 13-acetate was unaffected by [G203T]G alpha i2. Our results demonstrate that Ca2+ but not phorbol ester activation of cPLA2 in CHO cells is G alpha i2-dependent. The possibility is discussed that G alpha i2 is downstream of Ca2+ but upstream of protein kinase C activation of cPLA2.
细胞质磷脂酶A2（cPLA2）的激活机制复杂且尚未完全明确。在中国仓鼠卵巢（CHO）细胞中，cPLA2的受体刺激是由于至少两种鸟嘌呤核苷酸结合（G）蛋白的α亚基Gαi2和Gαq所涉及的信号通路相互作用。百日咳毒素和Gαi2突变体可抑制cPLA2的激活。此外，通过Gαq激活磷脂酶C会导致细胞内钙浓度（[Ca2+]i）升高以及蛋白激酶C激活，这两者都会与cPLA2相互作用并激活它。本研究旨在分析Gαi2在cPLA2激活过程中与磷脂酶C刺激信号通路的相互作用机制。我们使用一种显性负性Gαi2突变体[G203T]Gαi2来解决这个问题，其中甘氨酸203突变为苏氨酸。[G203T]Gαi2可抑制cPLA2的ATP受体激活。[G203T]Gαi2对Gαi2激活的信号通路具有特异性作用，因为它对其他嘌呤能受体刺激的信号通路没有影响：[G203T]Gαi2不会改变ATP对[Ca2+]i的刺激或丝裂原活化蛋白激酶的磷酸化。我们探讨了Ca2+和/或蛋白激酶C对cPLA2的激活是否依赖于Gαi2的可能性。在表达[G203T]Gαi2的细胞中，钙离子载体离子霉素对cPLA2的激活被抑制了61±9%（n = 5）；然而，离子霉素诱导的[Ca2+]i升高不受[G203T]Gαi2的影响。因此，[G203T]Gαi2特异性抑制Ca2+对cPLA2的激活。相比之下，佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯通过蛋白激酶C对cPLA2的激活不受[G203T]Gαi2的影响。我们的结果表明，CHO细胞中Ca2+而非佛波酯对cPLA2的激活是Gαi2依赖性的。文中讨论了Gαi2可能位于Ca2+下游但在蛋白激酶C激活cPLA2的上游的可能性。
