leupeptins and pervanadate

To assess the role of nuclear factor kappaB (NFKB) in cellular radiosensitivity, three different IkappaB-alpha (also known as NFKBIA) expression plasmids, i.e., S-IkappaB (mutations at (32, 36)Ser), Y-IkappaB (a mutation at (42)Tyr), and SY-IkappaB, were constructed and introduced into human brain tumor M054 cells. The clones were named as M054-S8, M054-Y2 and M054-SY4, respectively. Compared to the parental cell line, M054-S8 and M054-Y2 cells were more sensitive to X rays while M054-SY4 cells exhibited the greatest sensitivity. After treatment with N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, the X-ray sensitivity of M054-S8 and M054-SY4 cells did not change, while that of M054-Y2 cells and the parental cells was enhanced. An increase in X-ray sensitivity accompanied by a decrease in translocation of NFKB to the nucleus in parental cells was observed after treatment with pervanadate, an inhibitor of tyrosine phosphatase, as well as in M054-S8 and M054-SY4 cells. Repair of potentially lethal damage (PLD) was observed in the parental cells but not in the clones. Four hours after irradiation (8 Gy), the expression of TP53 and phospho-p53 ((15)Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IkappaB-alpha phosphorylation at serine or tyrosine acts independently in sensitizing cells to X rays. NFKB may play a role in determining radiosensitivity and PLD repair in malignant glioma cells; TP53 may also be involved.

Topics: Cell Survival; Cloning, Molecular; DNA Repair; Gene Expression Regulation, Neoplastic; Glioma; Humans; Leupeptins; Mutagenesis, Site-Directed; NF-kappa B; Phosphorylation; Radiation Tolerance; Transfection; Tumor Cells, Cultured; Vanadates

Tumor necrosis factor activates nuclear transcription factor kappaB (NF-kappaB) by inducing serine phosphorylation of the inhibitory subunit of NF-kappaB (IkappaBalpha), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-kappaB and induces tyrosine phosphorylation of IkappaBalpha (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049-31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787-798). Whether PV also induces IkappaBalpha degradation and whether degradation is required for NF-kappaB activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IkappaBalpha degradation and NF-kappaB activation. PV activated NF-kappaB, as determined by DNA binding, NF-kappaB-dependent reporter gene expression, and phosphorylation and degradation of IkappaBalpha. Maximum degradation of IkappaBalpha occurred at 180 min, followed by NF-kappaB-dependent IkappaBalpha resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IkappaBalpha degradation and NF-kappaB activation, suggesting that the IkappaBalpha degradation is required for NF-kappaB activation. PV did not induce serine phosphorylation of IkappaBalpha but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IkappaBalpha. Like TNF, however, PV induced phosphorylation and degradation of IkappaBalpha, and subsequent NF-kappaB activation, which could be blocked by N-tosyl-L-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-kappaB activation and IkappaBalpha degradation. Overall, our studies demonstrate that PV activates NF-kappaB, which, unlike TNF, requires tyrosine phosphorylation of IkappaBalpha and its degradation.

Topics: Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; HeLa Cells; Humans; I-kappa B Proteins; Leupeptins; Multienzyme Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Proteasome Endopeptidase Complex; Protein Tyrosine Phosphatases; Transcription, Genetic; Tumor Necrosis Factor-alpha; Tyrosine; U937 Cells; Vanadates

STAT5b (signal transducer and activator of transcription 5b) is a key mediator of the effects of plasma GH pulses on male-specific liver gene expression. STAT5b is activated in liver cells in vivo by physiological pulses of GH and then is rapidly deactivated. Investigation of the cellular events involved in this activation/deactivation cycle using the rat liver cell line CWSV-1 established that a brief exposure to GH and the associated activation of JAK2 (Janus kinase 2) tyrosine kinase activity are both necessary and sufficient to initiate all of the downstream steps associated with STAT5b activation by tyrosine phosphorylation and the subsequent deactivation of both JAK2 kinase and STAT5b. JAK2 signaling to STAT5b at the conclusion of a GH pulse could be sustained by the protein synthesis inhibitor cycloheximide or by the proteasome inhibitor MG132, indicating that termination of this JAK2-catalyzed STAT activation loop requires synthesis of a labile or GH-inducible protein factor and is facilitated by the proteasome pathway. This factor may be a phosphotyrosine phosphatase, since the phosphatase inhibitor pervanadate both sustained GH pulse-induced JAK2 signaling to STAT5b and blocked the rapid deactivation of phosphorylated STAT5b (t(1/2) = 8.8 +/- 0.9 min) seen in its absence. Finally, the serine kinase inhibitor H7 blocked down-regulation of JAK2 signaling to STAT5b in a manner that enabled cells to respond to a subsequent GH pulse without the need for the approximately 3-h interpulse interval normally required for full recovery of GH pulse responsiveness. Termination of GH pulse-induced STAT5b signaling is thus a complex process that involves multiple biochemical events. These are proposed to include the down-regulation of JAK2 signaling to STAT5b via a cycloheximide- and H7-sensitive step, proteasome-dependent degradation of a key component or regulatory factor, and dephosphorylation leading to deactivation of the receptor-kinase signaling complex and its STAT5b substrate via the action of a phosphotyrosine phosphatase.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cell Line; Cycloheximide; Cysteine Endopeptidases; DNA-Binding Proteins; Enzyme Activation; Enzyme Inhibitors; Growth Hormone; Janus Kinase 2; Leupeptins; Liver; Milk Proteins; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Receptors, Somatotropin; Signal Transduction; STAT5 Transcription Factor; Trans-Activators; Vanadates

The role(s) played by protein tyrosine kinases (PTKs) in the regulation of insulin secretion from pancreatic beta cells is not clear. We have examined the effects of glucose, the major physiological insulin secretagogue, on the tyrosine phosphorylation state of islet proteins, and assessed beta cell insulin secretory responses in the presence of PTK inhibitors. Under basal conditions islets contained many proteins phosphorylated on tyrosine residues, and glucose (20 mM; 5-15 min) was without demonstrable effect on the pattern of tyrosine phosphorylation, in either the absence or presence of the protein tyrosine phosphatase (PTP) inhibitor, sodium pervanadate (PV). PV alone (100 microM) increased tyrosine phosphorylation of several islet proteins. The PTK inhibitors genistein (GS) and tyrphostin A47 (TA47) inhibited islet tyrosine kinase activities and glucose-, 4alpha ketoisocaproic acid (KIC)- and sulphonylurea-stimulated insulin release, without affecting glucose metabolism. GS and TA47 also inhibited protein serine/threonine kinase activities to a limited extent, but had no effect on Ca2+, cyclic AMP- or phorbol myristate acetate (PMA)-induced insulin secretion from electrically permeabilised islets. These results suggest that PTK inhibitors exert their inhibitory effects on insulin secretion proximal to Ca2+ entry and it is proposed that they act at the site of the voltage-dependent Ca2+ channel which regulates Ca2+ influx into beta cells following nutrient- and sulphonylurea-induced depolarisation.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcium; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Genistein; Glucose; Insulin; Insulin Secretion; Ion Transport; Islets of Langerhans; Isoflavones; Leupeptins; Phenylmethylsulfonyl Fluoride; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tyrphostins; Vanadates

The suppression of male-specific, GH pulse-induced, liver transcription in adult female rats has been linked to the down-regulation of STAT5b activation by the female plasma pattern of near-continuous GH exposure. The mechanism underlying this down-regulation was studied in the rat liver cell line CWSV-1, where continuous GH suppressed the level of activated (tyrosine- phosphorylated) STAT5b to approximately 10-20% of the maximal GH pulse-induced STAT5b signal within 3 h. In contrast to the robust JAK2 kinase-dependent STAT5b activation loop that is established by a GH pulse, JAK2 kinase signaling to individual STAT5b molecules was found to be short lived in cells treated with GH continuously. Moreover, maintenance of the low-level STAT5b signal required ongoing protein synthesis and persisted for at least 7 days provided that GH was present in the culture continuously. Increased STAT5b DNA-binding activity was observed in cells treated with the proteasome inhibitor MG132, suggesting that at least one component of the GH receptor (GHR)-JAK2-STAT5b signaling pathway becomes labile in response to continuous GH treatment. The phosphotyrosine phosphatase inhibitor pervanadate fully reversed the down-regulation of STAT5b DNA-binding activity in continuous GH-treated cells by a mechanism that involves both increased STAT5b activation and decreased STAT5b dephosphorylation. Moreover, the requirement for ongoing GH stimulation and active protein synthesis to maintain STAT5b activity in continuous GH-treated cells were both eliminated by pervanadate treatment, suggesting that phosphotyrosine dephosphorylation may be an obligatory first step in the internalization/degradation pathway for the GHR-JAK2 complex. Finally, the sustaining effect of the serine kinase inhibitor H7 on GH pulse-induced JAK2 signaling to STAT5b was not observed in continuous GH-treated cells. These findings suggest a model where continuous GH exposure of liver cells down-regulates the STAT5b pathway by a mechanism that involves enhanced dephosphorylation of both STAT5b and GHR-JAK2, with the latter step leading to increased internalization/degradation of the re-ceptor-kinase complex.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cells, Cultured; Cycloheximide; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Down-Regulation; Electrophoresis; Female; Genistein; Growth Hormone; Growth Inhibitors; Janus Kinase 2; Leupeptins; Liver; Male; Milk Proteins; Protein Synthesis Inhibitors; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Rats; Signal Transduction; STAT5 Transcription Factor; Staurosporine; Trans-Activators; Vanadates