Search

Treatment of cultured hippocampal neurons with high concentrations of short-chain acyl ceramide derivatives, such as N-hexanoyl-D-sphingosine (C(6)-Cer), results in apoptotic cell death. We now show that death-associated protein (DAP) kinase plays an important role in mediating this effect. Upon incubation with C(6)-Cer, DAP kinase levels are elevated as early as 1 h after treatment, reaching levels 2-3-fold higher than untreated cells after 4 h. Neurons cultured from DAP kinase-deficient mice were significantly less sensitive to apoptosis induced by C(6)-Cer or by ceramide generated by high concentrations of nerve growth factor. A peptide corresponding to the 17 amino acids at the C terminus of DAP kinase protected wild type neurons from C(6)-Cer-induced death and from death induced by the addition of exogenous bacterial neutral sphingomyelinase, whereas a scrambled peptide had no protective effect, implying that the DAP kinase C-terminal tail inhibits the function of DAP kinase. Together, these data demonstrate that DAP kinase plays a central role in ceramide-induced cell death in neurons, but the pathway in which DAP kinase is involved is not the only one via which ceramide can induce apoptosis.

Mammalian cells contain a large number of iso-acceptor transfer RNAs, some of which are present in very small amounts as compared to the major species, and until now the function of these minor tRNA species has not been clarified. We have found that extracts from mouse cells treated by interferon require specifically the addition of some minor species of tRNA to allow proper translation of exogenous messenger RNA. By a series of chromatographic steps, we have purified minor Leu-tRNA species required for Mengo virus RNA translation from the other Leu-tRNAs, and identified their cognate codons. Globin mRNA and Mengo RNA require different minor Leu-tRNA species. This represents the first system in which translation of different mRNA is shown to be completely dependent on the addition of minor iso-acceptor tRNA which cannot be replaced by major species, even if these recognize the same codons.