Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi-endosomal fraction
Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi-endosomal fraction
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DOI：
10.1046/j.1432-1327.1999.00123.x
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发表时间：
1999-02-01
期刊：
影响因子：
--
通讯作者：
Benelli, C
中科院分区：
文献类型：
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作者：
Geoffroy, V;Fouque, F;Benelli, C
在体内和无细胞系统中检查了 Ca2+/磷脂依赖性蛋白激酶（蛋白激酶 C、PKC）刺激肝脏高尔基体内体 (GE) 部分中 cAMP 磷酸二酯酶 (PDE) 活性的能力。向大鼠注射 4β-佛波醇 12-肉豆蔻酸酯 13-乙酸酯（一种已知的 PKC 激活剂）可导致 GE 级分中 PKC 活性快速显着增加（10 分钟+ 325%），同时蛋白质免疫印迹中观察到的 PKC α 同工型丰度也增加。同时，4β-佛波醇12-肉豆蔻酸酯13-乙酸酯处理引起GE组分中cAMP PDE活性的时间依赖性增加（30分钟时96%）。将蛋白激酶 A (PKA) 催化亚基添加到对照和 4 β-佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理的大鼠的 GE 级分中，导致 PDE 活性相应增加 (130-150%)，表明 PKA 可能不参与 4 β-佛波醇 12-肉豆蔻酸酯 13-乙酸酯的体内效应。相反，添加纯化的 PKC 使对照大鼠的 GE 级分中的 PDE 活性增加（两倍），但仅轻微影响 4 β-佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理的大鼠的 GE 级分中的活性。 GE 级分中大约 50% 的 Triton-X-100 溶解的 cAMP PDE 活性用抗 PDE3 抗体进行免疫沉淀。在 DEAE-Sephacel 色谱上，PDE 的三个峰依次被洗脱出来：一个早期峰，受 cGMP 刺激，受 erythro-9（2-羟基-3-壬基）腺嘌呤（EHNA）抑制； 2 型 PDE 的选择性抑制剂；和两个延迟的活性峰，它们被 cGMP 和 cilostamide（3 型 PDE 抑制剂）有效抑制。通过 HPLC 对峰 I 进行进一步表征，分辨出一个被 5 μM cGMP 激活（三倍）并被 25 μM EHNA 抑制（87%）的主峰，以及一个对 EHNA 和西洛酰胺不敏感的次要峰。 4 beta-佛波醇 12-肉豆蔻酸酯 13-乙酸酯处理导致与 DEAE-Sephacel 峰 I 相关的活性选择性增加（2.5 倍），而不改变 K-m 值。这些结果表明 PKC 选择性激活 GE 组分中的 PDE2（cGMP 刺激的亚型）。
The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+ 325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 mu M cGMP and inhibited (87%) by 25 mu M EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K-m value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.