Nerve growth factor induces a succession of increases in isoprenylated methylated small GTP-binding proteins of PC-12 phyochromocytoma cells

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 carboxyl [methyl-³H]ester formation and levels of [α-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 [α-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 [α-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 [α-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). Taken together the results show that NGF induces a succession of changes in prenylated methylated small GTP binding-proteins which are synchronized with the processes by which PC-12 cells acquire a neuron-like phenotype. Prenylation methylation and GTP-binding proteins are therefore required in these processes.