diamide and pervanadate

Band 3 (AE1), the most prominent polypeptide of the human erythrocyte membrane, becomes heavily tyrosine phosphorylated following treatment of intact cells with protein tyrosine phosphatase inhibitors such as diamide, pervanadate, vanadate, or N-ethylmaleimide (NEM). The mechanism underlying this tyrosine phosphorylation is thought to involve the sequential action of two protein tyrosine kinases, Syk (p72syk) and Lyn (p53/56lyn). While Lyn catalysed phosphorylation appears to be strictly dependent on prior phosphorylation of Tyr8 and 21 of band 3 by Syk, little is known about the mechanism of induction of Syk phosphorylation. Data presented here show that both the fraction of Syk that associates with the membrane and the extent of phosphorylation of band 3 differ in response to the above inhibitors. While diamide and NEM stimulate syk translocation to the membrane during their induction of band 3 tyrosine phosphorylation, pervanadate and vanadate induce no change in kinase distribution. Moreover, diamide and NEM-induced Syk recruitment to the membrane are phosphotyrosine independent and involve their preferential association with Triton X-100-insoluble membrane skeletons. Together these data reveal a complex process controlling the association and catalytic activity of protein tyrosine kinases syk and lyn with the human erythrocyte membrane.

Topics: Anion Exchange Protein 1, Erythrocyte; Cytoskeleton; Diamide; Enzyme Precursors; Erythrocytes; Ethylmaleimide; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Luminescent Measurements; Oxidation-Reduction; Phosphorylation; Protein-Tyrosine Kinases; src-Family Kinases; Syk Kinase; Vanadates

Recent studies from our laboratory have indicated that protein tyrosine phosphatase (PTPase) inhibitors can down-modulate the tumor necrosis factor (TNF)-mediated activation of the nuclear transcription factor NF-kappa B in ML-1a, a monocytic cell line (Singh and Aggarwal, J. Biol. Chem. 1995: 270: 10631). Since TNF is one of the major inducers of various adhesion molecules in human endothelial cells and their expression is known to require the activation of NF-kappa B, we examined the effect of PTPase inhibitors on the TNF-mediated induction of intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and endothelial leukocyte adhesion molecule (ELAM)-1. Like ML-1a, human dermal microvessel endothelial cells (MVEC) treated with TNF rapidly activated (within 30 min) NF-kappa B; this effect was completely abolished by co-treatment with phenylarsine oxide (PAO), a specific inhibitor of PTPase. The induction of ICAM-1, VCAM-1, and ELAM-1 by TNF in MVEC occurred within 6 h and was also completely down-regulated by PAO in a dose-dependent manner. PAO was found to be effective even when added 3 h after TNF, suggesting a rapid mode of action of this inhibitor. Besides PAO, other inhibitors of PTPase, including pervanadate and diamide, also blocked TNF-dependent NF-kappa B activation and induction of all the three adhesion proteins. Consistent with these results, the attachment of monocytes to MVEC was also blocked by the PTPase inhibitors. Thus, overall, our results demonstrate that a PTPase is involved either directly or indirectly in the pathway leading to the induction of endothelial cell adhesion molecules by TNF. Because of their role in cell adhesion, PTPase may provide a novel target of drug development for treatment of inflammation, atherogenesis, and tumor metastasis.

Topics: Arsenicals; Cell Adhesion; Cells; Cells, Cultured; Diamide; DNA-Binding Proteins; E-Selectin; Endothelium, Vascular; Enzyme Inhibitors; Humans; Intercellular Adhesion Molecule-1; Monocytes; NF-kappa B; Protein Tyrosine Phosphatases; Signal Transduction; Tumor Necrosis Factor-alpha; Vanadates; Vascular Cell Adhesion Molecule-1

Most of the inflammatory and proviral effects of tumor necrosis factor (TNF) are mediated through the activation of the nuclear transcription factor NF-kappa B. How TNF activates NF-kappa B, however, is not well understood. We examined the role of protein phosphatases in the TNF-dependent activation of NF-kappa B. Treatment of human myeloid ML-1a cells with TNF rapidly activated (within 30 min) NF-kappa B; this effect was abolished by treating cells with inhibitors of protein-tyrosine phosphatase (PTPase), including phenylarsine oxide (PAO), pervanadate, and diamide. The inhibition was dependent on the dose and occurred whether added before or at the same time as TNF. PAO also inhibited the activation even when added 15 min after the TNF treatment of cells. In contrast to inhibitors of PTPase, okadaic acid and calyculin A, which block serine-threonine phosphatase, had no effect. The effect of PTPase inhibitors was not due to the modulation of TNF receptors. Since both dithiothreitol and dimercaptopropanol reversed the inhibitory effect of PAO, critical sulfhydryl groups in the PTPase must be involved in NF-kappa B activation by TNF. PTPase inhibitors also blocked NF-kappa B activation induced by phorbol ester, ceramide, and interleukin-1 but not that activated by okadaic acid. The degradation of I kappa B protein, a critical step in NF-kappa B activation, was also abolished by the PTPase inhibitors as revealed by immunoblotting. Thus, overall, we demonstrate that PTPase is involved either directly or indirectly in the pathway leading to the activation of NF-kappa B.

Topics: Adenosine Triphosphate; Arsenicals; Base Sequence; Ceramides; Diamide; DNA-Binding Proteins; Dose-Response Relationship, Drug; Ethers, Cyclic; Humans; I-kappa B Proteins; In Vitro Techniques; Interleukin-1; Marine Toxins; Molecular Sequence Data; NF-kappa B; NF-KappaB Inhibitor alpha; Okadaic Acid; Oligodeoxyribonucleotides; Oxazoles; Protein Tyrosine Phosphatases; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Vanadates