Kolesnick R N, Clegg S
Department of Medicine, Memorial Sloan-Kettering Cancer Center, Cornell University Medical College, New York 10021.
J Biol Chem. 1988 May 15;263(14):6534-7.
It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C. Further, the failure of phorbol esters to activate this system might account for some differences between these agents.
有人提出，鞘氨醇碱可能作为蛋白激酶C的生理抑制剂。由于在GH3垂体细胞中，1,2 - 二酰基甘油而非佛波酯通过鞘磷脂酶增强鞘磷脂降解（科尔斯尼克，R.N.（1987年）《生物化学杂志》262, 16759 - 16762），因此评估了佛波酯、1,2 - 二酰基甘油和鞘磷脂酶对蛋白激酶C激活的影响。在基础条件下，蛋白激酶C的无活性胞质形式占主导。1,2 - 二酰基甘油刺激蛋白激酶C短暂重新分布到细胞膜。1,2 - 二辛酰甘油（200微克/毫升）将胞质蛋白激酶C活性从72皮摩尔·分钟-1·10(6)细胞-1降至对照的67%，即48皮摩尔·分钟-1·10(6)细胞-1，并在刺激4分钟后将膜结合活性从6皮摩尔·分钟-1·10(6)细胞-1提高到对照的430%，即25皮摩尔·分钟-1·10(6)细胞-1。此后，蛋白激酶C活性回到胞质。相比之下，佛波酯12 - O - 十四酰佛波醇-13 - 乙酸酯（TPA）刺激其重新分布到细胞膜，且不会回到胞质。外源性鞘磷脂酶将膜结合的蛋白激酶C活性降至对照的30%，但未改变胞质活性。在佛波酯诱导的重新分布完成后添加鞘磷脂酶，可使活性恢复到胞质中。在这些研究中，TPA（10(-8) M）将胞质活性降至对照的62%，并将膜结合的蛋白激酶C活性提高到对照的650%。鞘磷脂酶将胞质活性恢复到对照的84%，并将膜结合活性降至对照的297%。同样，游离鞘氨醇碱、鞘氨醇、二氢鞘氨醇和植物鞘氨醇可逆转佛波酯诱导的蛋白激酶C重新分布。由于1,2 - 二酰基甘油激活鞘磷脂酶，且鞘磷脂酶的作用可逆转蛋白激酶C激活，这些研究表明，涉及鞘磷脂酶的途径可能构成蛋白激酶C的生理负效应系统。此外，佛波酯未能激活该系统可能解释了这些试剂之间的一些差异。
