Kv2.1, the primary delayed rectifying potassium channel in neurons, is extensively regulated by phosphorylation. Previous reports have described Kv2.1 phosphorylation events affecting channel gating and the impact of this process on cellular excitability. Kv2.1, however, also provides the critical exit route for potassium ions during neuronal apoptosis via p38 MAPK-dependent membrane insertion, resulting in a pronounced enhancement of K ؉ currents. Here, electrophysiological and viability studies using Kv2.1 channel mutants identify a p38 phosphorylation site at Ser-800 (S800) that is required for Kv2.1 membrane insertion, K ؉ current surge, and cell death. In addition, a phospho-specific antibody for S800 detects a p38-dependent increase in Kv2.1 phosphorylation in apoptotic neurons and reveals phosphorylation of S800 in immunopurified channels incubated with active p38. Consequently, phosphorylation of Kv2.1 residue S800 by p38 leads to trafficking and membrane insertion during apoptosis, and remarkably, the absence of S800 phosphorylation is sufficient to prevent completion of the cell death program.apoptosis ͉ ion channel ͉ MAPK

OBJECTIVETo establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in β-cells.RESEARCH DESIGN AND METHODSGlucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR.RESULTSPax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content.CONCLUSIONSPax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression.

Changes in the levels of O-linked N-acetylglucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer's and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5-and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30-107 %) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by ~30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.

The commercially available Jak2 inhibitor, alpha-cyano-3,4-dihydroxy-N-benzylcinnamide (AG490), has been used extensively to study Jak2 kinase function. While alpha-cyano-3,4-dihydroxy-N-benzylcinnamide is a potent Jak2 inhibitor, it can inhibit a number of other kinase signaling pathways as well. To circumvent this problem, we sought to identify novel small molecule inhibitors of Jak2 tyrosine kinase activity. For this, we constructed a homology model of the Jak2 kinase domain and identified solvent accessible pockets on the surface of the structure. Using the DOCK program, we tested 6451 compounds of known chemical structure in silico for their ability to interact with a pocket positioned adjacent to the activation loop. We attained the top seven scoring compounds from the National Cancer Institute and tested their ability to inhibit Jak2 autophosphorylation in vitro. Using Western blot analysis, we found that one of the compounds, 1,2,3,4,5,6-hexabromocyclohexane, was able to potently, and directly, inhibit Jak2 autophosphorylation. Characterization of this compound revealed that it inhibits Jak2 tyrosine autophosphorylation in both a time- and concentration-dependent manner. It greatly reduced growth hormone-mediated Jak2 autophosphorylation but did not block autophosphorylation of the epidermal growth factor receptor. Furthermore, doses as high as 100 muM were not toxic to cells as measured by their ability to exclude propidium iodide. As such, we believe that this compound could serve as a lead compound for a new generation of Jak2 inhibitors and, perhaps, be useful in elucidating the mechanisms of Jak2 kinase function.