Phosphorylation

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work ...

Our recent study has shown that beetroot juice protects against N-nitrosodimethylamine (NDEA)-induced liver injury and increases the activity of phase II enzymes, suggesting the activation of the nuclear factor erythroid-2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. The aim of the present study was to further explore the mechanism of the activity of beetroot by evaluating the cytoprotective effects of its major component. The influence of betanin (BET) on the activation of Nrf2 and the expression of GSTA, GSTP, GSTM, GSTT, NQO1 and HO-1 was assessed in two hepatic cell lines: non-tumour THLE-2 and hepatoma-derived HepG2 cell lines. The level of the tumour suppressor p53 in both cell lines and the methylation of GSTP in HepG2 cells were also evaluated. Treatment of both cell lines with 2, 10 and 20 μm of BET resulted in the translocation of Nrf2 from the cytosol to the nucleus. The mRNA and nuclear protein levels of Nrf2 and the binding of Nrf2 to ARE sequence...

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDN...

NKX3.1, a member of the NK class of homeodomain proteins, is expressed primarily in the adult prostate and has growth suppression and differentiating effects in prostate epithelial cells. A C-->T polymorphism at nucleotide 154 (NKX3.1 C154T) is present in approximately 11% of healthy men with equal distribution among whites and blacks. In a cohort of 1253 prostate cancer patients and age-matched controls, the presence of the polymorphism was associated with a 1.8-fold risk of having stage C or D prostate cancer or Gleason score > or =7 (confidence interval, 1.01-3.22). The NKX3.1 C154T polymorphism codes for a variant protein that contains an arginine-to-cysteine substitution at amino acid 52 (R52C) adjacent to a protein kinase C phosphorylation site at serine 48. Substitution of cysteine for arginine 52 or of alanine for serine 48 (S48A) reduced phosphorylation at serine 48 in vitro and in vivo. Phosphorylation of wild-type NKX3.1, but not of NKX3.1 R52C or NKX3.1 S48A, dimin...

Coiled bodies are nuclear organelles that contain components of at least three RNA-processing pathways: pre-mRNA splicing, histone mRNA 3′- maturation, and pre-rRNA processing. Their function remains unknown. However, it has been speculated that coiled bodies may be sites of splicing factor assembly and/or recycling, play a role in histone mRNA 3′-processing, or act as nuclear transport or sorting structures. To study the dynamics of coiled bodies in living cells, we have stably expressed a U2B"–green fluorescent protein fusion in tobacco BY-2 cells and in Arabidopsis plants. Time-lapse confocal microscopy has shown that coiled bodies are mobile organelles in plant cells. We have observed movements of coiled bodies in the nucleolus, in the nucleoplasm, and from the periphery of the nucleus into the nucleolus, which suggests a transport function for coiled bodies. Furthermore, we have observed coalescence of coiled bodies, which suggests a mechanism for the decrease in coiled bo...

Membrane-permeable calmodulin inhibitors, such as the napthalenesulfonamide derivatives W-7/W-13, trifluoperazine, and calmidazolium, are used widely to investigate the role of calcium/calmodulin (Ca 2+/CaM) in living cells. If two chemically different inhibitors ...

Vanadate, an inhibitor of tyrosine phosphatases, has insulin-mimetic properties. It has been shown that acute vanadate administration enhances glucose uptake independently of phosphatidylinositol (PI) 3-kinase and p38 MAPK. However, therapeutic vanadate use requires chronic administration, and this could potentially involve a different signaling pathway(s). Thus, we examined the mechanisms by which chronic vanadate exposure (16 h) stimulates glucose uptake in primary cultures of adult cardiomyocytes. The effect of vanadate on the activation of insulin-signaling molecules was evaluated 60 min after its withdrawal and in the absence of insulin. We therefore evaluated the persistent effect of vanadate on the insulin-signaling cascade. Our results demonstrate that preincubation with low vanadate concentrations (25-75 microM) induces a dose-dependent increase in glucose uptake. The augmentation of this process was not due to alterations in GLUT1 or GLUT4 protein levels, transcription, or...

Phosphorylation of the transcriptional coactivator YAP1 is a key event in defining Hippo signaling outputs. Previous studies demonstrated that phosphorylation of YAP1 at serine 127 (S127) sequesters YAP1 in the cytoplasm and consequently inhibits YAP1 transcriptional activity. Mammalian tissue-culture experiments suggest that downstream of MST1/2 signaling, LATS1/2 function as YAP1-S127 kinases. However, studies of Mst1/2 knockout mouse models revealed that the identity of the physiological YAP1-S127 kinase(s) in certain tissues, such as the intestine, remains unknown. We show that mammalian NDR1/2 kinases phosphorylate YAP1 on S127 and thereby negatively regulate YAP1 activity in tissue-cultured cells. By studying NDR1/2-deficient mice, we demonstrate the in vivo relevance of NDR1/2-mediated regulation of YAP1. Specifically, upon loss of NDR1/2 in the intestinal epithelium, endogenous S127 phosphorylation is decreased whereas total YAP1 levels are increased. Significantly, ablation...