Calcium-independent binding to interfacial phorbol esters causes protein kinase C to associate with membranes in the absence of acidic lipids.
Calcium-independent binding to interfacial phorbol esters causes protein kinase C to associate with membranes in the absence of acidic lipids.
复制标题
与界面佛波酯的钙独立结合导致蛋白激酶 C 在没有酸性脂质的情况下与膜结合。
DOI：
10.1021/bi952031q
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Newton,AC
中科院分区：
文献类型：
--
作者：
Mosior,M;Newton,AC
通过检测这类激活剂与蛋白激酶C βII的直接结合，阐述了佛波醇酯与常规蛋白激酶C的相互作用机制。结合测量表明，佛波酯的主要作用是增加几个数量级的蛋白激酶C的膜的亲和力。相对增加线性依赖于佛波酯在膜中的摩尔分数，与1摩尔%佛波12-肉豆蔻酸酯13-乙酸酯（PMA）增加蛋白激酶C的膜协会约4个数量级的发现所示的效力。为了比较，二酰基甘油（DG），也激活蛋白激酶C，通过增加酶的膜亲和力，是2个数量级低于PMA在改变蛋白激酶C的膜亲和力。与佛波醇酯的显着高亲和力的相互作用，使我们能够测量蛋白激酶C的PMA在中性膜中的直接结合，从而评估钙离子的影响，在没有钙离子对酶的相互作用与酸性脂质的佛波醇酯的相互作用。改变Ca 2+浓度超过5个数量级对固定在磷脂酰胆碱膜中的PMA与蛋白激酶C的直接相互作用没有影响。因此，钙离子结合位点的膜协会和佛波酯结合位点不相互作用变构。最后，用一种不受Scatchard图限制的方法测定了蛋白激酶C与佛波醇酯的解离常数：相对于膜脂，解离常数为1.5 × 10 ~（-5）mol %。总之，我们的数据表明：（1）蛋白激酶C与佛波酯的直接结合，在不存在与酸性脂质的相互作用的情况下，对蛋白激酶C与膜的缔合中涉及的自由能变化提供了主要贡献;（2）这种相互作用不受Ca 2+的调节。
The mechanism of interaction of phorbol esters with conventional protein kinase Cs was addressed by examining the direct binding of this class of activators to protein kinase C βII. Binding measurements reveal that the major role of phorbol esters is to increase the affinity of protein kinase C for membranes by several orders of magnitude. The relative increase depends linearly on the mole fraction of phorbol esters in membranes, with the potency illustrated by the finding that 1 mol % phorbol 12-myristate 13-acetate (PMA) increases protein kinase C's membrane association by approximately 4 orders of magnitude. For comparison, diacylglycerol (DG), which also activates protein kinase C by increasing the enzyme's membrane affinity, is 2 orders of magnitude less effective than PMA in altering protein kinase C's membrane affinity. The remarkably high-affinity interaction with phorbol esters allowed us to measure the direct binding of protein kinase C to PMA in neutral membranes and, thus, to evaluate the effect of Ca2+on the phorbol ester interaction in the absence of Ca2+effects on the enzyme's interaction with acidic lipids. Changing the Ca2+concentration over 5 orders of magnitude had no effect on the direct interaction of protein kinase C with PMA immobilized in phosphatidylcholine membranes. Thus, the Ca2+-binding site for membrane association and the phorbol ester-binding site do not interact allosterically. Lastly, a method that does not have the limitations of the Scatchard plot for analysis of amphitropic proteins was used to determine the dissociation constant of protein kinase C from phorbol esters: expressed relative to membrane lipids, the dissociation constant is 1.5 × 10-5mol %. In summary, our data reveal that (1) the direct binding of protein kinase C to phorbol esters, in the absence of interactions with acidic lipids, provides a major contribution to the free energy change involved in the association of protein kinase C with membranes and (2) this interaction is not regulated by Ca2+.