Haklai R, Lerner S, Kloog Y
Department of Biochemistry, George S. Wise Faculty of Life Sciecnes, Tel Aviv University, Israel.
Neuropeptides. 1993 Jan;24(1):11-25. doi: 10.1016/0143-4179(93)90036-a.
Pheochromocytoma (PC-12) cells exposed to nerve growth factor (NGF) acquire a sympathetic neuron-like phenotype. This NGF-response is blocked by methylation inhibitors and can be mimicked by the farnesylated methylated small GTP-binding protein p21ras. The implicated involvement of prenylation, methylation and a small GTP-binding protein in the NGF-response has been studied by directly measuring 3H-mevalonic acid (MVA)-metabolites incorporated into proteins, protein carboxy [methyl-3H]ester formation and levels of [alpha-32P]GTP-binding proteins in NGF-induced PC-12 cells. We demonstrate that NGF induces a 2-3-fold increase in 21-24 kDa methylated membrane proteins that incorporate 3H-MVA-metabolites, and bind GTP. Levels of [alpha-32P]GTP-binding in these proteins were increased by 2-3-fold. Methylation and membrane association of the small GTP-binding proteins were blocked by lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which also enhanced their labeling by 3H-MVA-metabolites. Cycloheximide reduced the levels of [methyl-3H] labeled 21-24 kDa proteins and of the overlapping [alpha-32P]GTP binding-proteins. About 70% of the [methyl-3H]-groups found in these proteins were recovered from two dimensional gel blots in nine distinct spots of [alpha-32P]GTP-binding proteins. Taken together these results strongly suggest that in PC-12 cells, NGF induces an increase in the synthesis of prenylated methylated small GTP-binding proteins. The efficacy of lovastatin blockage of protein methylation and enhancement of 3H-MVA-metabolites incorporation into GTP-binding proteins was lower in NGF-induced cells than in controls. This suggests that NGF also induces an increase in HMG-CoA reductase activity. At the early phase of the NGF response in PC-12 cells (15 min-1 h), the levels of two small GTP-binding proteins (molecular mass of 21-22 kDa and 23-24 kDa) were increased. Thus, at least two proteins, of which one but not the other may be p21ras, appear to be involved in the early response. After a lag period of 24 h with NGF, a second more robust phase of increase in methylated small GTP-binding proteins was apparent. This relatively late response, which was almost completed within 24 h, may reflect involvement of small GTP-binding proteins in neurite-outgrowth and in the functional activity of the differentiated cells. Many small GTP-binding proteins were increased during the second phase, precluding electrophoretic separation of all of them. 3 proteins, however, were well separated (one 23-24 kDa protein and two 21-22 kDa proteins).(ABSTRACT TRUNCATED AT 400 WORDS)
暴露于神经生长因子（NGF）的嗜铬细胞瘤（PC - 12）细胞会获得类似交感神经元的表型。这种NGF反应被甲基化抑制剂阻断，并且可被法尼基化甲基化的小GTP结合蛋白p21ras模拟。通过直接测量掺入蛋白质中的3H - 甲羟戊酸（MVA）代谢物、蛋白质羧基[甲基 - 3H]酯的形成以及NGF诱导的PC - 12细胞中[α - 32P]GTP结合蛋白的水平，研究了异戊二烯化、甲基化和小GTP结合蛋白在NGF反应中的潜在参与情况。我们证明，NGF诱导21 - 24 kDa甲基化膜蛋白增加2 - 3倍，这些蛋白掺入3H - MVA代谢物并结合GTP。这些蛋白中[α - 32P]GTP结合水平增加了2 - 3倍。3 - 羟基 - 3 - 甲基戊二酰辅酶A（HMG - CoA）还原酶抑制剂洛伐他汀阻断了小GTP结合蛋白的甲基化和膜结合，同时也增强了它们被3H - MVA代谢物的标记。放线菌酮降低了[甲基 - 3H]标记的21 - 24 kDa蛋白以及重叠的[α - 32P]GTP结合蛋白的水平。在这些蛋白中发现的约70%的[甲基 - 3H]基团，从二维凝胶印迹中[α - 32P]GTP结合蛋白的九个不同斑点中回收。综合这些结果强烈表明，在PC - 12细胞中，NGF诱导法尼基化甲基化的小GTP结合蛋白合成增加。洛伐他汀对蛋白甲基化的阻断以及增强3H - MVA代谢物掺入GTP结合蛋白的效果在NGF诱导的细胞中比在对照细胞中更低。这表明NGF还诱导HMG - CoA还原酶活性增加。在PC - 12细胞中NGF反应的早期阶段（15分钟 - 1小时），两种小GTP结合蛋白（分子量为21 - 22 kDa和23 - 24 kDa）的水平增加。因此，至少有两种蛋白似乎参与了早期反应，其中一种可能是p21ras，另一种不是。在NGF作用24小时的延迟期后，甲基化小GTP结合蛋白出现第二个更强有力的增加阶段。这个相对较晚的反应在24小时内几乎完成，可能反映了小GTP结合蛋白参与神经突生长和分化细胞的功能活动。在第二阶段许多小GTP结合蛋白增加，无法通过电泳将它们全部分离。然而，有3种蛋白得到了很好的分离（一种23 - 24 kDa蛋白和两种21 - 22 kDa蛋白）。（摘要截断于400字）
