Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding.
Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding.
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DOI：
10.1016/s0021-9258(19)74235-5
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发表时间：
1993-12
期刊：
影响因子：
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通讯作者：
R. Gopalakrishna;Zhen Hai Chen;U. Gundimeda
中科院分区：
文献类型：
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作者：
R. Gopalakrishna;Zhen Hai Chen;U. Gundimeda
由于蛋白巯基的S-亚硝基化是一氧化氮（NO）诱导的细胞调节机制之一，并且由于蛋白激酶C（PKC）具有影响其激酶活性的关键巯基残基，我们确定了NO是否可以调节这种酶。用纯化的PKC和NO生成剂S-亚硝基半胱氨酸进行了初步研究。该药物以Ca（2+）和氧依赖性方式降低PKC的磷酸转移酶活性，IC 50为75 μ M。佛波酯结合仅在较高浓度（> 100 μ M）的S-亚硝基半胱氨酸下受到部分影响。这种失活的PKC被阻断的NO清除剂氧合血红蛋白或逆转的二硫苏糖醇。这是可能的，NO最初诱导邻位硫醇的S-亚硝基化，然后氧化形成分子内二硫化物。其他NO生成剂，如S-亚硝基-N-乙酰青霉胺和硝普钠，以及真正的NO气体，诱导类似类型的PKC修饰。在完整的B16黑色素瘤细胞与S-亚硝基半胱氨酸治疗的PKC活性迅速下降，在细胞质和膜观察。与纯化的PKC的实验不同，在用S-亚硝基半胱氨酸处理的完整细胞中，佛波酯结合也降低到与PKC活性相等的速率。通过在试管中用二硫苏糖醇处理匀浆或通过从完整细胞中去除NO生成源，这些修饰很容易逆转。为了确定完整细胞内产生的有限量的NO是否可以诱导这种类型的PKC修饰，用脂多糖和Ca 2+离子载体A23187处理巨噬细胞系IC-21以诱导NO的产生。随着这些细胞中NO生成的增加（3-12小时），观察到PKC活性和佛波酯结合的平行和不可逆的降低。一氧化氮合酶的特异性抑制剂，NG-单甲基-L-精氨酸，抑制NO的产生和PKC失活。在使用纯化的酶或完整细胞的实验中，cAMP依赖性蛋白激酶活性没有降低。可以想象，由于二硫键的形成，有限时间的NO产生诱导PKC可逆失活，而NO的慢性产生可诱导PKC不可逆失活。PKC的可逆或不可逆失活可能分别部分影响NO介导的细胞保护或细胞毒性作用。
Since S-nitrosylation of protein thiols is one of the cellular regulatory mechanisms induced by nitric oxide (NO), and since protein kinase C (PKC) has critical thiol residues which influence its kinase activity, we have determined whether NO could regulate this enzyme. Initial studies were carried out with purified PKC and the NO-generating agent S-nitrosocysteine. This agent decreased phosphotransferase activity of PKC in a Ca(2+)- and oxygen-dependent manner with an IC50 of 75 microM. Phorbol ester binding was affected partially only at higher concentrations (> 100 microM) of S-nitrosocysteine. This inactivation of PKC was blocked by the NO scavenger oxyhemoglobin or reversed by dithiothreitol. It is likely that NO initially induced an S-nitrosylation of vicinal thiols, which were then oxidized to form an intramolecular disulfide. Other NO-generating agents such as S-nitroso-N-acetylpenicillamine and sodium nitroprusside, as well as authentic NO gas, induced similar types of PKC modifications. In intact B16 melanoma cells treated with S-nitrosocysteine a rapid decrease in PKC activity in both cytosol and membrane was observed. Unlike in experiments with purified PKC, in intact cells treated with S-nitrosocysteine the phorbol ester binding also decreased to a rate equal to that of PKC activity. These modifications were readily reversed by treating the homogenates with dithiothreitol in test tubes or by removing the NO-generating source from intact cells. To determine whether the limited amounts of NO generated within the intact cells could induce this type of PKC modification, the macrophage cell line IC-21 was treated with lipopolysacharide and Ca2+ ionophore A23187 to induce the NO production. With an increase in generation of NO (3-12-h period) in these cells, a parallel and irreversible decrease in PKC activity and phorbol ester binding was observed. A specific inhibitor for NO synthase, NG-monomethyl-L-arginine, inhibited both the production of NO and PKC inactivation. In experiments using purified enzyme or intact cells there was no decrease in cAMP-dependent protein kinase activity. Conceivably, NO production for limited time induces a reversible inactivation of PKC due to the formation of a disulfide bridge(s), whereas the chronic production of NO could induce irreversible inactivation of PKC. The reversible or irreversible inactivations of PKC may in part influence NO-mediated cytoprotective or cytotoxic actions, respectively.