Ras proteins, nitrosylation and homocysteine metabolism.

Elevated levels of homocysteine and S-adenosylmethionine are causal risk factors for several neurological disorders. Homocysteine is a sulfur-containing, nonproteinogenic, neurotoxic amino acid biosynthesized during methyl cycles after demethylation of S-adenosylmethionine (SAM) to adenosylhomocysteine (SAH) and subsequent hydrolysis of SAH into homocysteine and adenosine. PC12 pheochromocytoma cells expressing a dominant inhibitory mutant of Ha-ras (M-M17-26, PC12 pheochromocytoma cells expressing a mutant v-ras gene (MVR) and PC12 cells transfected with normal c-rasH (M-CR3B) has been used to investigate the role of nitrosylation and farnesylation of Ras on the production of homocysteine and the activities of the redox-sensitive transcription factors NF-kB and c-Fos. We found that under serum and nerve growth factor withdrawal conditions undifferentiated apoptotic M-CR3B cells accumulated more homocysteine, than M-M17-26 cells and the production of homocysteine decreased under the action of manumycin (inhibitor of farnesyltransferase) and increased in the presence of L-NAME (inhibitor of nitric oxide synthase). Furthermore, we have shown that manumycin increased the activity of c-Fos in the M-CR3B cells and decreased the activity of NF-kB, while L-NAME reduced the activities of both transcription factors, and accelerated apoptosis of M-CR3B cells. In contrast to the M-CR3B cells, in M-M17-26 cells manumycin did not change the activity of c-Fos or the activity of NF-kB. Moreover, we have shown that L-NAME significantly changes the SAM/SAH ratio in both MCR and MVR cells. Moreover, these alterations have reciprocal character; in the MCR cells, the SAM/SAH ratio was raised, whereas in the MVR cells this ratio was reduced. We conclude that trophic factor withdrawal stimulates Ras, which apparently through the Rac/NADPH oxidase system induces permanent oxidative stress, modulates the activities of NF-kB and c-Fos, induces production of homocysteine and accelerates apoptosis. Nitrosylation of Ras is necessary for maintaining the survival of PC12 cells, while farnesylation of Ras stimulates apoptosis under withdrawal conditions. Besides, our results suggest that in conditions of a low level of nitric oxide PC12 cells with mutated oncogenic Ras produce more ROS than cells with wild type Ras and switch homocysteine metabolism toward to transsulfuration.