Gopalakrishna R, Gundimeda U, Anderson W B, Colburn N H, Slaga T J
School of Medicine, University of Southern California, Los Angeles, California, 90033, USA.
Arch Biochem Biophys. 1999 Mar 15;363(2):246-58. doi: 10.1006/abbi.1999.1100.
Since tumor promoter benzoyl peroxide (BPO) mimics phorbol esters in some aspects, its effects on protein kinase C (PKC) were previously studied. However, in those studies due to the presence of thiol agents in the PKC preparations, the sensitive reaction of BPO with redox-active cysteine residues in PKC was not observed. In this study, by excluding thiol agents present in the purified PKC preparation, low concentrations of BPO modified PKC, resulting in the loss of both kinase activity and phorbol ester binding (IC50 = 0. 2 to 0.5 microM). This modification, which was not dependent on transition metals, was totally blocked by a variety of thiol agents including GSH, which directly reacted with BPO. Substoichiometric amounts of BPO (0.4 mol/mol of PKC) oxidized two sulfhydryls in PKC and inactivated the enzyme which was readily reversed by dithiothreitol. The regulatory domain having zinc thiolate structures supporting the membrane-inserting region provided the specificity for PKC reaction with BPO, which partitioned into the membrane. Unlike H2O2, BPO did not induce the generation of the Ca2+/lipid-independent activated form of PKC. Other redox-sensitive enzymes such as protein kinase A, phosphorylase kinase, and protein phosphatase 2A required nearly 25- to 100-fold higher concentrations of BPO for inactivation. BPO also inactivated PKC in a variety of cell types. In the JB6 (30 P-) nonpromotable cell line and other normal cell lines, where BPO was more cytotoxic, it readily inactivated PKC due to a slow reversibility of this inactivation by the cell. However, in the JB6 (41 P+) promotable cell line, C3H10T1/2 and B16 melanoma cells, where BPO was less cytotoxic, it did not readily inactivate PKC due to a rapid reversibility of this inactivation by an endogenous mechanism. Nevertheless, BPO inactivated PKC at an equal rate in the homogenates prepared from all these cell types. Inclusion of NADPH reversed this inactivation in the homogenates to a different extent, presumably due to a difference in distribution of a protein disulfide reductase, which reverses this oxidative modification. BPO-induced modification of PKC occurred independent of the cellular status of GSH. However, externally added GSH and cell-impermeable thiol agents prevented the BPO-induced modification of PKC. Since BPO readily partitions into membranes, its reaction with redox-cycling thiols of membrane proteins such as PKC may trigger epigenetic events to prevent cytotoxicity, but favor tumor promotion.
由于肿瘤促进剂过氧化苯甲酰（BPO）在某些方面模拟佛波酯，此前对其对蛋白激酶C（PKC）的影响进行了研究。然而，在那些研究中，由于PKC制剂中存在硫醇试剂，未观察到BPO与PKC中具有氧化还原活性的半胱氨酸残基的敏感反应。在本研究中，通过去除纯化的PKC制剂中存在的硫醇试剂，低浓度的BPO修饰了PKC，导致激酶活性和佛波酯结合丧失（IC50 = 0.2至0.5 microM）。这种修饰不依赖于过渡金属，被包括谷胱甘肽（GSH）在内的多种硫醇试剂完全阻断，GSH可直接与BPO反应。亚化学计量的BPO（0.4摩尔/摩尔PKC）氧化了PKC中的两个巯基并使酶失活，二硫苏糖醇可轻易逆转这种失活。具有支持膜插入区域的硫醇锌结构的调节结构域为PKC与BPO的反应提供了特异性，BPO可分配到膜中。与过氧化氢不同，BPO不会诱导产生不依赖Ca2+/脂质的PKC活化形式。其他对氧化还原敏感的酶，如蛋白激酶A、磷酸化酶激酶和蛋白磷酸酶2A，失活所需的BPO浓度要高近25至100倍。BPO还能使多种细胞类型中的PKC失活。在JB6（30 P-）非促癌细胞系和其他正常细胞系中，BPO的细胞毒性更强，由于细胞对这种失活的逆转缓慢，它很容易使PKC失活。然而，在JB6（41 P+）促癌细胞系、C3H10T1/2和B16黑色素瘤细胞中，BPO的细胞毒性较小，由于内源性机制可快速逆转这种失活，它不容易使PKC失活。尽管如此，BPO在由所有这些细胞类型制备的匀浆中以相同的速率使PKC失活。加入NADPH可不同程度地逆转匀浆中的这种失活，可能是由于蛋白二硫键还原酶分布的差异，该酶可逆转这种氧化修饰。BPO诱导的PKC修饰与细胞内谷胱甘肽的状态无关。然而，外部添加的谷胱甘肽和细胞不可渗透的硫醇试剂可防止BPO诱导的PKC修饰。由于BPO很容易分配到膜中，它与膜蛋白（如PKC）的氧化还原循环硫醇的反应可能会引发表观遗传事件以防止细胞毒性，但有利于肿瘤促进。
