In DO11.10 T-cell hybridoma, ERK1/2-RSK pathway was shown to phosphorylate Nur77 at residue 354. An alanine substitution at this site impairs Nur77 nuclear export and apoptosis 26. To see if this might be true in DP cells, we used 16610D9 cells, a CD4+CD8+ thymoma cell line that possesses many characteristics of primary DP thymocytes 48. As shown in Fig. 6A, 16610D9 cells express very little endogenous Nur77 (lanes 1, 4, 7 and 10).

Infection of these cells with Nur77 retrovirus led to expression of Nur77 in the nuclear compartment (lanes 2 and 5). However, very little Nur77 was found in the mitochondria/cytoplasmic fractions unless PMA/ionomycin were added (see lane 11 versus lane 8). Interestingly, expression of Nur77(354A) mutant (mutation verified by sequencing) led to constitutive translocation of Nur77 Fulvestrant to the mitochondria/cytoplasmic fraction (lane 9). These data show that phosphorylation of Nur77 at residue 354 might have a different effect in DP cells from DO11.10 cells and that regulation of Nur77 nuclear transport and its association with Bcl-2 is more complicated than initially thought. Nur77 has been reported as the target of numerous kinases including protein

kinase A, PKC, Akt, JNK, ERK5 and p90 ribosomal S6 kinase 23, 24, 49–51. Phosphorylation of Nur77 by these proteins was demonstrated in various in vitro and in vivo experiments. However, the functional consequence of Nur77 phosphorylation remains controversial. Here, we report that the PKC proteins regulate Nur77 phosphorylation and nuclear/cytoplasmic translocation NVP-LDE225 order in thymocytes during apoptosis that mimics negative selection. Chemical inhibition of PKC proteins prevented Nur77 and family member Nor-1 from targeting the mitochondria and their targeting of Bcl-2. In contrast, inhibition of AKT, JNK, ERK1/2 and p38 did not affect the subcellular localization of Nur77 family proteins in thymocytes. These results are different from mitochondria

translocation of Nur77 induced by the retinoid analog CD437, which requires activation of the JNK and inhibition of the AKT pathways 23. Inhibition of ERK1/2 was also C-X-C chemokine receptor type 7 (CXCR-7) recently reported to block Nur77 mitochondria translocation in DO11.10 T-cell hybridoma cells 26. The discrepancy with our results is most likely due to the differences in the cells used. Consistent with this, we found that alanine mutation at Nur77 residue 354, which impairs mitochondrial translocation in DO11.10 cells, causes constitutive translocation of Nur77 in 16610D9 CD4+CD8+ cells. Thus, the involvement of kinase pathway(s) in Nur77 mitochondria translocation is cell type and stimulus specific. Though calcium signals alone were adequate in causing Nur77 to be localized to the mitochondria, these levels may be inadequate for binding Bcl-2, as no Bcl-2/Nur77 interaction could be detected in ionomycin treated thymocytes. In addition, ionomycin could not induce Nor-1 to any appreciable levels.