Bidirectional control of phospholipase A2 activity by Ca2+/calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and casein kinase II.
Bidirectional control of phospholipase A2 activity by Ca2+/calmodulin-dependent protein kinase II, cAMP-dependent protein kinase, and casein kinase II.
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Ca2/钙调蛋白依赖性蛋白激酶 II、cAMP 依赖性蛋白激酶和酪蛋白激酶 II 对磷脂酶 A2 活性的双向控制。
DOI：
10.1073/pnas.88.15.6770
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发表时间：
1991
影响因子：
11.1
通讯作者：
Greengard,P
中科院分区：
文献类型：
--
作者：
Piomelli,D;Greengard,P
在从大鼠脑分离的突触终末（突触体）的制备中，磷脂酶A2（PLA 2）的活性，在信号转导中起中心作用的磷脂水解酶，通过与60 mM K+或与Ca（2+）选择性离子载体离子霉素孵育以Ca（2+）依赖性方式被抑制。碱性磷酸酶处理的抑制作用表明，这种抑制作用是由于突触体蛋白底物的磷酸化。当裂解的突触体孵育与Ca 2 +/钙调素（CaM），纯化的Ca 2 +/CaM依赖性蛋白激酶II（Ca 2 +/CaM依赖性PK II）和ATP，PLA 2活性裂解物中几乎被废除在10分钟内。这种效果是伴随着一个显着降低的Vmax的酶和很少或没有变化的Km。此外，Ca 2 +/CaM与ATP，但没有外源性的Ca 2 +/CaM依赖的PK II部分抑制PLA 2的活性，这种效果被阻止处理的裂解物与Ca 2 +/CaM依赖的PK II的选择性肽抑制剂。相反，与4 β-佛波醇12-肉豆蔻酸酯13-乙酸酯或裂解的突触体与纯化的蛋白激酶C的完整的突触体的孵育有很少或没有影响PLA 2的活性。结果强烈提示，在完整神经末梢中观察到的PLA 2活性的Ca（2+）依赖性抑制是通过激活多功能Ca（2+）/CaM依赖性PK II产生的。一种膜渗透性腺苷酸环化酶激活剂，毛喉素，增强PLA 2活性在完整的突触体，和cAMP依赖性蛋白激酶增强PLA 2活性在溶解的突触体。此外，另一种广谱蛋白激酶存在于突触末梢，酪蛋白激酶II，也增强了PLA 2活性溶解突触体。两种蛋白激酶的作用均与Km降低相关，而Vmax无变化。结果表明，突触终末的PLA 2活性受不同信号转导通路的双向控制。此外，相互拮抗作用的Ca 2 +/CaM依赖的PK II和PLA 2途径提供了一个可能的分子机制，双向调节神经递质的释放。
In preparations of synaptic terminals (synaptosomes) isolated from rat brain, the activity of phospholipase A2 (PLA2), a phospholipid hydrolase that serves a central function in signal transduction, was inhibited in a Ca(2+)-dependent manner by incubation with 60 mM K+ or with the Ca(2+)-selective ionophore ionomycin. Reversal by alkaline phosphatase treatment suggested that this inhibitory effect resulted from phosphorylation of a synaptosomal protein substrate. When lysed synaptosomes were incubated with Ca2+/calmodulin (CaM), purified Ca2+/CAM-dependent protein kinase II (Ca2+/CaM-dependent PK II) and ATP, PLA2 activity in lysates was nearly abolished within 10 min. This effect was accompanied by a marked decrease in the Vmax of the enzyme and little or no change in the Km. Furthermore, Ca2+/CaM with ATP but without exogenous Ca2+/CaM-dependent PK II partially inhibited PLA2 activity, and this effect was prevented by treating the lysates with a selective peptide inhibitor of Ca2+/CaM-dependent PK II. In contrast, incubation of intact synaptosomes with 4 beta-phorbol 12-myristate 13-acetate or of lysed synaptosomes with purified protein kinase C had little or no effect on PLA2 activity. The results strongly suggest that the Ca(2+)-dependent inhibition of PLA2 activity observed in intact nerve endings was produced by activation of the multifunctional Ca2+/CaM-dependent PK II. A membrane-permeable adenylyl cyclase activator, forskolin, enhanced PLA2 activity in intact synaptosomes, and cAMP-dependent protein kinase potentiated PLA2 activity in lysed synaptosomes. Furthermore, another broad-spectrum protein kinase present in synaptic terminals, casein kinase II, also potentiated PLA2 activity in lysed synaptosomes. The effects of both protein kinases were associated with a decrease in Km and no change in Vmax. The results suggest that PLA2 activity in synaptic terminals is subject to bidirectional control by distinct signal transduction pathways. Moreover, mutually antagonistic effects of the Ca2+/CaM-dependent PK II and PLA2 pathways provide a possible molecular mechanism for bidirectional modulation of neurotransmitter release.