CA-2+-INDEPENDENT AND PHOSPHOLIPID-INDEPENDENT ACTIVATION OF PROTEIN KINASE-C BY SELECTIVE OXIDATIVE MODIFICATION OF THE REGULATORY DOMAIN
CA-2+-INDEPENDENT AND PHOSPHOLIPID-INDEPENDENT ACTIVATION OF PROTEIN KINASE-C BY SELECTIVE OXIDATIVE MODIFICATION OF THE REGULATORY DOMAIN
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DOI：
10.1073/pnas.86.17.6758
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发表时间：
1989-09-01
影响因子：
11.1
通讯作者：
ANDERSON, WB
中科院分区：
文献类型：
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作者：
GOPALAKRISHNA, R;ANDERSON, WB
纯化的蛋白激酶C对H2 O2氧化失活的敏感性被发现通过单独的高浓度（5 nM）或低浓度（约10 nM）的Ca 2+而增加。50 μ M）浓度沿着磷脂酰丝氨酸和二酰基甘油，以及甚至在不存在Ca 2+的情况下通过促肿瘤的佛波酯。这表明蛋白激酶C的膜结合和/或催化活性形式相对更容易氧化失活。虽然蛋白激酶C的调节和催化结构域都容易氧化失活，但在温和的氧化条件下，通过用ATP/Mg 2+保护催化位点，获得了调节结构域的选择性修饰。在这些条件下，有一个损失的佛波酯结合的Ca 2 +/磷脂刺激的激酶活性。然而，这种修饰形式的酶表现出Ca 2 + /磷脂非依赖性激酶活性的增加。这表明调节结构域的选择性氧化修饰可能会消除激活对Ca 2+和脂质的需求。完整的C6神经胶质瘤或B16黑色素瘤细胞与H2 O2的治疗导致在时间和温度依赖性的降低Ca 2 +/磷脂依赖性蛋白激酶C的活性随着伴随的瞬时增加的氧化修饰的蛋白激酶C的亚型表现出活性的情况下，Ca 2+和磷脂。由于蛋白激酶C最初可以通过温和的氧化修饰活化，随后通过进一步氧化失活，因此蛋白激酶C响应于H2 O2的这种双重活化-失活表明了影响细胞事件的有效开/关信号机制。
The susceptibility of purified protein kinase C to oxidative inactivation by H2O2 was found to be increased by Ca2+ either alone at a high (5 nM) concentration or at a low (.apprxeq. 50 .mu.M) concentration along with phosphatidylserine and diacylglycerol and by tumor-promoting phorbol esters even in the absence of Ca2+. This suggested that the membrane-bound and/or catalytically active form of protein kinase C is relatively more susceptible to oxidative inactivation. Although both the regulatory and catalytic domains of protein kinase C were susceptible to oxidative inactivation, a selective modification of the regulatory domain was obtained under mild oxidative conditions by protecting the catalytic site with ATP/Mg2+. Under these conditions there was a loss of both phorbol ester binding the Ca2+/phospholipid-stimulated kinase activity. However, this modified form of enzyme exhibited an increase in Ca2+ / phospholipid-independent kinase activity. This suggests that selective oxidative modification of the regulatory domain may negate the requirement for Ca2+ and lipids for activation. Treatment of intact C6 glioma or B16 melanoma cells with H2O2 resulted in a time- and temperature-dependent decrease in Ca2+/phospholipid-dependent protein kinase C activity along with a concomitant transient increase in an oxidatively modified isoform of protein kinase C exhibited activity in the absence of Ca2+ and phospholipids. Since protein kinase C can initially be activated by mild oxidative modification and subsequently inactivated by further oxidation, this dual activation-inactivation of protein kinase C in response to H2O2 suggests an effective on/off signal mechanism to influence cellular events.