Kazanietz M G, Barchi J J, Omichinski J G, Blumberg P M
Molecular Mechanisms of Tumor Promotion Section, NCI, National Institutes of Health, Bethesda, Maryland 20892-4255, USA.
J Biol Chem. 1995 Jun 16;270(24):14679-84. doi: 10.1074/jbc.270.24.14679.
Binding of phorbol esters to protein kinase C (PKC) has been regarded as dependent on phospholipids, with phosphatidylserine being the most effective for reconstituting binding. By using a purified single cysteine-rich region from PKC delta expressed in Escherichia coli we were able to demonstrate that specific binding of [3H]phorbol 12,13-dibutyrate to the receptor still takes place in the absence of the phospholipid cofactor. However, [3H]phorbol 12,13-dibutyrate bound to the cysteine-rich region with 80-fold lower affinity in the absence than in the presence of 100 micrograms/ml phosphatidylserine. Similar results were observed with the intact recombinant PKC delta isolated from insect cells. When different phorbol derivatives were examined, distinct structure-activity relations for the cysteine-rich region were found in the presence and absence of phospholipid. Our results have potential implications for PKC translocation, for inhibitor design, and for PKC structural determination.
佛波酯与蛋白激酶C（PKC）的结合被认为依赖于磷脂，其中磷脂酰丝氨酸对重组结合最为有效。通过使用在大肠杆菌中表达的PKCδ纯化的富含单个半胱氨酸的区域，我们能够证明在没有磷脂辅因子的情况下，[3H]佛波醇12,13 - 二丁酸酯与受体的特异性结合仍然会发生。然而，在不存在100微克/毫升磷脂酰丝氨酸的情况下，[3H]佛波醇12,13 - 二丁酸酯与富含半胱氨酸区域的结合亲和力比存在时低80倍。从昆虫细胞中分离出的完整重组PKCδ也观察到了类似结果。当检测不同的佛波醇衍生物时，在有和没有磷脂的情况下，发现富含半胱氨酸区域有不同的构效关系。我们的结果对PKC易位、抑制剂设计和PKC结构测定具有潜在意义。
