Snoek G T, Rosenberg I, de Laat S W, Gitler C
Biochim Biophys Acta. 1986 Aug 21;860(2):336-44. doi: 10.1016/0005-2736(86)90530-4.
The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.
通过5-[¹²⁵I]碘萘-1-叠氮化物对酶的光标记程度，确定了脂质组成在纯化的蛋白激酶C与大单层囊泡相互作用中的作用。当蛋白激酶C暴露于磷脂酰胆碱（PC）脂质体时，其仅被轻微标记。向PC脂质体中添加佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）或二酰基甘油，在低钙浓度下显著增强了蛋白激酶C的标记。在含有2%磷脂酰丝氨酸（PS）的脂质体中，观察到蛋白激酶C的光标记进一步增强。在低钙浓度下，添加PMA或二酰基甘油时，该酶与这些脂质体的结合增加。脂质体中PS水平超过2%并没有增强蛋白激酶C的结合。然而，当以碱性组蛋白为底物测量蛋白激酶C的酶活性时，在PC与PS比例为1的脂质体中获得了最大磷酸化。蛋白激酶C从溶液转移到脂质体表面的过程可以通过其用5-碘萘-1-叠氮化物标记来监测，这一事实促使我们确定其他细胞质蛋白是否也具有这一特性。HeLa细胞的细胞质蛋白与PC脂质体的相互作用产生的标记很微量，无论是否添加钙。当HeLa细胞细胞质蛋白与含有PS的脂质体相互作用时，在不同的蛋白中观察到选择性的5-碘萘-1-叠氮化物光标记。添加钙以及PMA或二酰基甘油改变了其中一些但不是所有这些蛋白的标记。这些结果表明，所开发的方法可能有助于鉴定在佛波酯或诱导二酰基甘油生成的生长因子刺激细胞过程中转移到膜上的蛋白。这些结果还表明质膜（或任何细胞内膜）的磷脂组成在调节特定磷脂依赖性蛋白，特别是蛋白激酶C的激活过程中发挥作用。
