The neurotrophin receptor, p75NTR, protects PC12 pheochromocytoma cells from oxidant stress.1
p75ICD, a cytoplasmic fragment liberated upon ligand binding to the extracellular domain of p75NTR, can substitute for the holoreceptor in this regard. Previous studies have demonstrated that p75ICD phosphorylation plays a role in its activity and in determining which of several p75NTR-triggered signaling pathways is activated.3
We have used site-directed mutagenesis to generate single and double mutants of p75ICD at Y337 and Y366. The mutants involve substitution of F for Y and therefore cannot be phosphorylated at the mutated position. These studies demonstrate that p75NTR-negative PC12 cells transfected with wildtype or Y366F p75ICD are more resistant to oxidative stress than cells that are transfected with Y337F or Y337F/Y366F double mutant p75ICD. As both wildtype and Y366F p75ICD mutant can be phosphorylated on Y337 and both Y337F and Y337F/Y366F cannot, our results suggest that phosphorylation of Y337 of p75ICD plays an essential role in the p75ICD-mediated protection of PC12 cells from cell death due to oxidant stress.
Interestingly, as phosphorylation of Y337 is essential for ubiquitination of p75NTR prior to its degradation,4
protein levels of the p75ICD Y366F mutant might be expected to be lower than that of the other mutants. On the other hand, in the Y337F mutant, none of Y337 is phosphorylated and thus the p75ICD protein level might be predicted to be highest compared to the other three mutants. However, on average, there were no significant differences in p75ICD content among the cells expressing the four different p75ICD sequences ().
Phosphorylation of p75NTR has been hypothesized to involve transfer of a phosphate from MAPK p38 to p75NTR; there is evidence that p38 can interact directly with the fifth and sixth alpha helices of p75NTR in the C-terminal death domain.8
Interestingly, Y366 is located between helix 2 and 3 of p75ICD in the open pocket for p38. However, our studies demonstrate no correlation between protective activity of phosphorylation site mutants of p75ICD and cellular content of phosphorylated p38 (). Of interest in this regard, members of the MAPK family, including p38 and ERK1,2, have been shown, like p75NTR, to be pro- or anti-apoptotic in neuronal systems and to be critical in oxidant-induced apoptosis in hepatocytes in culture.9
Furthermore, p38 activation is critical for 6-OHDA-induced apoptosis in the CNS-derived tyrosine hydroxylase expressing B65 cell line.11
This complex relationship between activation of any of the MAPKs and the life or death of the cell may account for our observation, shown in and , that mutants with similar susceptibility to 6-OHDA-induced death have different levels at baseline of activation of the MAPKs p38 and ERK 1,2 relative to wildtype p75ICD cells.
The time course of ERK activation is of particular interest in light of the observation of Luo and DeFranco12
that activation of ERK in response to oxidative stress is biphasic and consists of an early, brief, compensatory, protective phase followed by a late, prolonged, cell-injurious phase. Mock- and Y337F-transfected cells demonstrate both of these phases of ERK activation and are most susceptible to 6-OHDA-induced death. Cells transfected with wildtype p75NTR have high levels of activated ERK at baseline and demonstrate a paradoxical decrease in activated ERK at the usual time of the injurious phase of ERK activation. This is perhaps responsible for the protection of these cells from 6-OHDA-induced death. Y366F- and double mutant-transfected cells exhibit ERK activation time courses that do not fit a conventional pattern. Y366F-transfected cells demonstrate early increased ERK activation that increases in magnitude with time, without return to baseline. Given the resistance of these cells to 6-OHDA-induced cell death, it is tempting to speculate that the return to baseline between ERK activation phases is necessary for the transition of p-ERK from protective to injurious. Double mutant-transfected cells demonstrate high baseline levels of ERK activation and early-onset progressively decreasing ERK activation.
Phosphorylation of p75NTR has been shown to enhance NF-κB activation.8
Our studies demonstrate that activation of NF-κB is greatest in the Y366F mutant (), implying that phosphorylation of Y366 may inhibit and phosphorylation of Y337 may enhance activation of NF-κB.
Several markers of apoptosis were examined in the present study. Exposure to 6-OHDA results in nuclear margination and fragmentation ( and B) and cleavage of caspase-3 () and PARP () in all transfectants. It is interesting that the fractional caspase-3 activation and fractional PARP cleavage for each transfectant do not correlate absolutely and quantitatively with one another or with resistance to oxidant stress. There are two possible, non-mutually exclusive explanations for this. First, it likely that cell death in this model, as in others,15
occurs by a combination of apoptosis and necrosis and the relative incidence of these two mechanisms of cell death differ among the transfectants. Second, while useful for the binary decision of whether cells in a given model die by apoptosis or necrosis, Western blotting does not allow for precise kinetic or quantitative comparisons of PARP cleavage from cell line to cell line or tissue to tissue.16
The significance of the differing roles in the anti-oxidant effects of p75ICD of phosphorylation of p75ICD at Y337 and Y366, respectively, for Alzheimer's disease is not clear. Oxidative injury to cholinergic central neurons plays a role in this disorder. In familial Alzheimer's disease, mutation of presenilin, a γ-secretase for which p75NTR is a substrate, prevents release of p75ICD. However, sporadic Alzheimer's disease, the more common of the two, does not involve mutation of presenilin. Our studies suggest that altered phosphorylation of p75ICD could also result in the enhanced likelihood of apoptotic death of neurons subjected to oxidative stress.