Oxidative stress links diverse neuropathological conditions that include stroke, Parkinson's disease, and Alzheimer's disease and has been modeled in vitro with various paradigms that lead to neuronal cell death following the increased accumulation of reactive oxygen species. For example, immortalized neurons and immature primary cortical neurons undergo cell death in response to depletion of the antioxidant glutathione, which can be elicited by administration of glutamate at high

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... . We have demonstrated previously that this glutamate-induced oxidative toxicity requires activation of the mitogen-activated protein kinase member ERK1/2, but the mechanisms by which this activation takes place in oxidatively stressed neurons are still not fully known. In this study, we demonstrate that during oxidative stress, ERK-directed phosphatases of both the serine/threonine-and tyrosine-directed classes are selectively and reversibly inhibited via a mechanism that is dependent upon the oxidation of cysteine thiols. Furthermore, the impact of ERK-directed phosphatases on ERK1/2 activation and oxidative toxicity in neurons was tested in a neuronal cell line and in primary cortical cultures. Overexpression of the highly ERK-specific phosphatase MKP3 and its catalytic mutant, MKP3 C293S, were neuroprotective in transiently transfected HT22 cells and primary neurons. The neuroprotective effect of the MKP3 C293S mutant, which enhances ERK1/2 phosphorylation but blocks its nuclear translocation, demonstrates the necessity for active ERK1/2 nuclear localization for oxidative toxicity in neurons. Together, these data implicate the inhibition of endogenous ERK-directed phosphatases as a mechanism that leads to aberrant ERK1/2 activation and nuclear accumulation during oxidative toxicity in neurons. Oxidative stress is a common feature of a diverse range of neuropathological conditions, including stroke, Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis (1, 2). Glutamate-induced oxidative toxicity provides an excellent model for studying the effects of oxidative stress in immortalized neurons and in primary neuronal cultures (3-5). In this model, inhibition of a glutamate/cystine antiporter, known as system x c Ϫ , leads to decreased accumulation of intracellular free cysteine, a necessary precursor of glutathione, and to eventual glutathione depletion. As a result, reactive oxygen species (ROS) 1 accumulate and activate cellular signaling events that contribute to neuronal cell death. We have shown previously that oxidative toxicity causes a delayed, sustained activation of extracellular signal-regulated kinase (ERK) 1/2 that is necessary for neuronal cell death (6 -8), but the mechanisms by which oxidative stress drives ERK1/2 activation have not been fully elucidated. The regulation of ERK1/2 phosphorylation and activation reflects a subtle balance between ERK-directed kinase and phosphatase activity. A diverse range of phosphatases directed against phospho-serine/threonine, phospho-tyrosine, or both have been identified as negative regulators of ERK1/2. Protein phosphatase 2A (PP2A) (9, 10) and strial-enriched phosphatase (11), among numerous others, can function as ERK-directed phosphatases in neurons. Dynamic changes in ERK-directed phosphatase activity can result from several mechanisms, including ERK-dependent up-regulation of phosphatase expression (12), phosphorylation-dependent increases in phosphatase stability (13), and protein-protein interaction-dependent activation of phosphatase activity (14). Collectively, these events have been shown to function in a negative feedback loop that terminates ERK signaling (12). Recently, the role of oxidative stress in the regulation of phosphatase activity has received much attention (15) (16) (17) . Several phosphatases have been shown to be redox-sensitive and can be either reversibly or irreversibly inhibited, depending upon the degree and mechanism of oxidation (15, 18). Oxidative phosphatase inhibition can impact various cellular signaling pathways and accounts for a mechanism now referred to as oxidative signaling. The extent to which oxidative inhibition of phosphatases plays a role in driving signaling events during neurotoxicity remains relatively unexplored. We sought to characterize the effect of glutamate-induced oxidative toxicity on ERK-directed phosphatases in primary neuronal cultures. In this study, we show that endogenous ERK-directed phosphatase activity is specifically and reversibly inhibited during oxidative stress, consistent with a mechanism involving the oxidation of cysteine thiols. This inhibition of ERK-directed phosphatases correlates with an increase in phosphorylated ERK levels. We show that this aggregate phosphatase activity is likely composed of PP2A and a vanadate-sensitive component.