Signal transducers and activators of transcription (STATs) reside in a latent state in the cytoplasm of the cell, but accumulate in the nucleus in response to cytokines or growth factors. Localization in the nucleus occurs following STAT tyrosine phosphorylation and dimerization. In this report we demonstrate a direct interaction of importin-a5 with tyrosine-phosphorylated STAT1 dimers, and provide evidence that a nuclear localization signal (NLS) exists in an inactive state within a STAT1 monomer. A mutation in STAT1 leucine 407 (L407A) is characterized, which generates a protein that is accurately tyrosine phosphorylated in response to interferon, dimerizes and binds DNA, but does not localize to the nucleus. The import defect of STAT1(L407A) appears to be a consequence of the inability of this protein to be recognized by its import shuttling receptor. In addition, we demonstrate that STAT1 binding to speci®c target DNA effectively blocks importin-a5 binding. This result may play a role in localizing STAT1 to its destination in the nucleus, and in releasing importin-a5 from STAT1 for recycling back to the cytoplasm.

STAT2 is a transcription factor critical to the signal transduction pathway of type I interferons (e.g. IFN␣). STAT2 resides primarily in the cytoplasm and is tyrosine-phosphorylated after IFN␣ binds to cell surface receptors. In response to tyrosine phosphorylation STAT2 rapidly localizes to the nucleus and acquires the ability to bind specific DNA targets in association with two other proteins, STAT1 and IFN regulatory factor-9 (IRF-9). To elucidate the mechanisms that regulate cellular localization of STAT2, we investigated STAT2 nuclear trafficking both prior to tyrosine phosphorylation and after phosphorylation. Prior to phosphorylation, STAT2 is primarily resident in the cytoplasm, however, we found that it dynamically shuttles between nuclear and cytoplasmic compartments. The nuclear translocation of latent unphosphorylated STAT2 was found to be dependent on its constitutive association with IRF-9, and the export of STAT2 from the nucleus was contingent upon the function of an intrinsic nuclear export signal within the carboxyl terminus of STAT2. STAT2 export could be inhibited with leptomycin B, indicating a nuclear export signal within STAT2 is recognized by the CRM1 exportin carrier. In contrast, following tyrosine phosphorylation, STAT2 dimerizes with phosphorylated STAT1 and accumulates in the nucleus. In the absence of STAT1, STAT2 does not accumulate in the nucleus. In addition, subsequent to nuclear import of phosphorylated STAT2, it redistributes to the cytoplasm within an hour coordinate with its dephosphorylation in the nucleus. The regulation of STAT2 nuclear trafficking is distinct from the previously characterized STAT1 factor.The signal transducers and activators of transcription (STATs) 1 remain the only characterized DNA binding factors that are regulated directly by tyrosine phosphorylation (1-4). Tyrosine phosphorylation confers new properties to the STAT factors by inducing dimerization via reciprocal phosphotyrosine and Src homology 2 (SH2) domain interactions. This conformational change can contribute to nuclear translocation and is essential for binding to specific DNA targets. The STAT1 and STAT2 factors are tyrosine-phosphorylated in the cytoplasm by Janus kinases (JAKs) activated in response to type I interferon (IFN) (5, 38). The phosphorylated STATs subsequently dimerize and localize to the nucleus. The STAT2 factor is unique among the STATs in that it is associated constitutively with a distinct transcription factor, interferon regulatory factor-9 (IRF-9) (7-9). After phosphorylation, a trimeric complex forms of STAT1⅐STAT2⅐IRF-9, known as IFN-stimulated gene factor 3 (ISGF3), that binds to a specific IFN-stimulated response element in the promoters of responsive genes (reviewed in Refs. 10 -12). A successful innate immune response to viral infection requires the induced expression of genes by ISGF3. Targeted gene disruptions of STAT1, STAT2, or IRF-9 clearly demonstrate that animals lacking one of these factors succumb to infection (13-15).STATs serve as a link between the ...

BackgroundReactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target.MethodsNADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H2O2-induced carboxy-H2-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.ResultsNigral dopaminergic neurons in situ expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.ConclusionsA two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.

Oxidative stress is a key contributor to the pathogenesis of Parkinson's disease. NADPH oxidase is a membrane enzyme responsible for the oxidative burst in activated phagocytes. NADPH oxidase also exists in many types of non‐phagocytic cells including vascular cells and neurons, with a wide distribution throughout the brain. Although the microglial NADPH oxidase is known to participate in dopaminergic neurotoxicity, whether NADPH oxidase also exists in the dopamine neurons in the substantia nigra pars compacta (SNpc) and whether it contributes to the ROS generation has not been explored. We have examined the role of NADPH oxidase in MPP+ induced oxyradical production in immortalized dopaminergic cell line (N27). MPP+ dose dependently led to upregulation of ROS in N27 cells, which was attenuated by 40% with treatment with NADPH oxidase inhibitors. N27 cells were observed to express mRNA for all the necessary components of the NADPH oxidase, Nox2, p22, p40, p47 and p67, as well as Nox1 and Nox4. SiRNA‐mediated knockdown of p22, a critical stabilizing subunit of the NADPH oxidase, reduced the amount of ROS generated. Immunocytochemical examination of mouse dopaminergic neurons in sections of the SNpc revealed that these neurons express Nox2, p47 and p67. While our results suggest that NADPH oxidase is a contributor to ROS generation in dopaminergic neurons, the functional role of this process remains to be defined. Supported by NIH AA016654 and The Neuroscience Program at the UCD.