Vaccinia H1-related phosphatase (VHR) is classified as a dual specificity phosphatase. Unlike typical dual specificity phosphatases, VHR lacks the MAPK-binding domain and shows poor activity against MAPKs. We found that EGF receptor (EGFR) was a direct substrate of VHR and that overexpression of VHR down-regulated EGFR phosphorylation, particularly at Tyr-992 residue. Expression of VHR inhibited the activation of phospholipase C␥ and protein kinase C, both downstream effectors of Tyr-992 phosphorylation of EGFR. Decreasing VHR expression by RNA interference caused higher EGFR phosphorylation at Tyr-992. In addition to EGFR, VHR also directly dephosphorylated ErbB2. Consistent with these results, suppression of VHR augmented the foci formation ability of H1299 non-small cell lung cancer (NSCLC) cells, whereas overexpression of VHR suppressed cell growth in both two-and three-dimensional cultures. Expression of VHR also suppressed tumor formation in a mouse xenograft model. Furthermore, VHR expression was significantly lower in NSCLC tissues in comparison to that in normal lung tissues. Collectively, this study shows that down-regulation of VHR expression enhances the signaling of ErbB receptors and may be involved in NSCLC pathogenesis.Among protein modifications, tyrosine phosphorylation is extensively used only in multicellular, eukaryotic organisms. Protein-tyrosine phosphorylation plays an important role in signaling transduction pathways that are involved in embryogenesis, development, and homeostasis. Disorders in proteintyrosine phosphorylation are found in many human diseases from cancer to immune disorders. Although protein phosphorylation is a balanced action of protein kinases and phosphatases, the experimental data of protein phosphatases is proportionally much less than that of protein kinases. Dual specificity phosphatases (DUSPs) 3 are structurally related to protein-tyrosine phosphatases (PTP) and are initially implicated in the down-regulation of MAPKs (1). Distinct from PTPs, which have a deep catalytic cleft; DUSPs have shallow catalytic sites, which permit the less stringent phospho-amino acid specificity of DUSPs (2-4). Several DUSPs including MAPK phosphatases (MKP)-1 to -7, M3/6 (also called VH5), and VHR have been shown to inactivate one or several MAPKs (5-12). The expression of certain DUSPs is increased by mitogenic signaling (5, 13-15). Both ERK and JNK pathways induce the expression of MKPs (15-17). The induction of MKP expression by MAPK signaling may, in turn, lead to the down-regulation of MAPK activities. Recently, many newly identified DUSPs were found to have little or no phosphatase activity against MAPKs, indicating that MAPK inactivation is not the sole function of DUSPs (18 -21). These novel DUSPs are smaller in size compared with MKPs and lack the MAPK-binding domain. These groups of DUSPs have been classified as atypical DUSPs (1). Others' and our recent studies reveal that atypical DUSPs may play a critical role in the regulation of signaling triggered by protein tyrosine...
Oxidative stress in the brain has been increasingly associated with the development of numerous human neurological diseases. Microglia, activated upon neuronal injury or inflammatory stimulation, are known to release superoxide anion (*O(2) (-)), hydrogen peroxide (H(2)O(2)), and nitric oxide (NO), thereby further contributing to oxidative neurotoxicity. The reaction of NO and *O(2) (-), forming the toxic peroxynitrite (ONOO(-)), has been proposed to play a pathogenic role in neuronal injury. However, the interactions between H(2)O(2) and NO during oxidative stress, which may promote or diminish cell death, is less clear. In this study, we explored oxidative neurotoxicity induced by H(2)O(2) plus NO in primary cultures of rat cerebral cortex neurons. As the mechanisms may involve reactions between H(2)O(2) and NO, we monitored the production of ONOO(-)and reactive oxygen species (ROS) throughout the experiments. Results indicated that the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) and H(2)O(2) by themselves elicited neuronal death in a concentration- and time-dependent manner. Sublytic concentrations of H(2)O(2) plus SNAP were sufficient to induce neuronal apoptosis as determined by DNA laddering and fluorescent staining of apoptotic nuclei. Transient ONOO(-)increase was accompanied by rapid H(2)O(2) decay and NO production, whereas ROS slowly decreased following treatment. Furthermore, p38 mitogen-activated protein kinase (MAPK) activation and the cleavage of caspase-3 were observed. Conversely, inhibition of p38 MAPK and caspase-3 significantly reduced apoptotic death induced by H(2)O(2) plus SNAP. These data suggest that H(2)O(2) and NO act synergistically to induce neuronal death through apoptosis in which activation of p38 MAPK and caspase-3 is involved.
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