ABSTRACT
SAM68 (Src-associated in mitosis 68 kDa) is a member of the signal transduction of activator RNA novel gene family coding for proteins postulated to be involved in signal transduction and activation of RNA. It has been implicated through its phosphorylation status in the control of the transition from the G(1) to the S phases during mitosis. However, the implication and role of SAM68 in nonproliferative cells are unknown. The present study was initiated to examine the role of SAM68 in the phagocytic responses of the terminally differentiated human neutrophils. The results obtained show that SAM68 is present in human neutrophils and that it is tyrosine phosphorylated in response to stimulation by monosodium urate crystals or by ligation of CD32. Stimulation of neutrophils by these agonists decreases the association of SAM68 with Sepharose-conjugated poly-U beads. Additionally, the amount of immunoprecipitable SAM68 was modulated differentially after stimulation by monosodium urate crystals or by CD32 engagement indicating that the posttranslational modifications and/or protein associations of SAM68 induced by these two agonists differed. The results of this study provide evidence for an involvement of SAM68 in signal transduction by phagocytic agonists in human neutrophils and indicate that SAM68 may play a role in linking the early events of signal transduction to the posttranscriptional modulation of RNA.
Subject(s)
Neutrophils/immunology , Neutrophils/metabolism , RNA-Binding Proteins/physiology , Receptors, IgG/immunology , Receptors, IgG/metabolism , Uric Acid/pharmacology , Adaptor Proteins, Signal Transducing , Adult , Crystallization , DNA-Binding Proteins , Humans , Isoflurophate/pharmacology , Ligands , Microspheres , Neutrophils/drug effects , Neutrophils/enzymology , Phagocytosis/drug effects , Phosphorylation/drug effects , Poly U/metabolism , Precipitin Tests , Protease Inhibitors/pharmacology , Protein Binding/drug effects , Protein Binding/immunology , RNA-Binding Proteins/isolation & purification , RNA-Binding Proteins/metabolism , Signal Transduction/drug effects , Signal Transduction/immunology , Subcellular Fractions/drug effects , Subcellular Fractions/enzymology , Subcellular Fractions/metabolism , Tyrosine/metabolism , Up-Regulation/immunologyABSTRACT
The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10-6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubation (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered. Tyrosine phosphorylation of Cbl in response to CD32 cross-linking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphorylation on serine/threonine residues. However, tyrosine phosphorylation of Syk and of the receptor itself were not affected. Furthermore, the mobilization of intracellular calcium stimulated by CD32 cross-linking was totally abrogated by calyculin. Finally, the stimulation of superoxide production observed in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events regulate the signaling pathways activated by CD32 cross-linking in neutrophils and identify a novel mechanism of modulation of the functional responsiveness of human neutrophils to CD32 cross-linking.
Subject(s)
Enzyme Inhibitors/pharmacology , Neutrophils/enzymology , Neutrophils/immunology , Phosphoprotein Phosphatases/antagonists & inhibitors , Receptors, IgG/immunology , Receptors, IgG/metabolism , Serine/metabolism , Threonine/metabolism , Tyrosine/metabolism , Adult , Calcium/antagonists & inhibitors , Calcium/metabolism , Calcium Signaling/drug effects , Calcium Signaling/immunology , Electrophoresis, Polyacrylamide Gel , Humans , Marine Toxins , Neutrophil Activation/drug effects , Neutrophils/drug effects , Neutrophils/metabolism , Oncogene Protein v-cbl , Oxazoles/pharmacology , Phosphorylation/drug effects , Receptors, IgG/physiology , Retroviridae Proteins, Oncogenic/isolation & purification , Retroviridae Proteins, Oncogenic/metabolism , Superoxides/metabolismABSTRACT
Reactive oxygen species (ROS), particularly hydroxyl radical (HO*), increase neutrophil adherence to hypoxanthine-xanthine oxidase (HX-XO)-treated human umbilical vein endothelial cells (HUVEC) in culture. This adherence is inhibited by the tyrosine kinase inhibitors genistein (30 microM) and herbimycin A (0.9 microM), suggesting the involvement of tyrosine kinase. Phosphorylation of several HUVEC proteins in the range of 120-130 and 70 kDa was found to depend on the XO concentration and stimulation time. This phosphorylation was inhibited by the antioxidants dimethylthiourea (DMTU, 0.75 to 7.5 mM) and pentoxifylline (Ptx, 0.1 mM), and by the iron chelators desferrioxamine (DF, 1 mM) and hydroxybenzyl ethylene diamine (HBED, 0.5 mM), suggesting the involvement of HO*. Three tyrosine-phosphorylated proteins, focal adhesion kinase (p125FAK), paxillin (PAX) and p130cas were isolated and characterized by immunoprecipitation and western blotting. Antioxidants and iron chelators reduced their phosphorylation. HUVEC treated with ROS for 15 min showed actin stress fiber formation. Cytochalasin D (5 microM) inhibited tyrosine phosphorylation and PMN-HUVEC adherence, showing the importance of cytoskeleton integrity in these two functions. In conclusion, HO*, which is involved in increased PMN-HUVEC adhesion, also increases tyrosine phosphorylation on three major cytoskeleton proteins which seem to play a role in this adhesion.
Subject(s)
Cell Adhesion Molecules/metabolism , Cytochalasin D/pharmacology , Cytoskeletal Proteins/metabolism , Endothelium, Vascular/metabolism , Phosphoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Proteins , Reactive Oxygen Species/metabolism , Cell Adhesion , Cells, Cultured , Crk-Associated Substrate Protein , Cytoskeleton/metabolism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Focal Adhesion Kinase 1 , Focal Adhesion Protein-Tyrosine Kinases , Humans , Hydroxyl Radical/metabolism , Hypoxanthine/metabolism , Immunohistochemistry , Iron Chelating Agents/pharmacology , Microscopy, Fluorescence , Neutrophils/metabolism , Paxillin , Phosphorylation , Phosphotyrosine/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Retinoblastoma-Like Protein p130 , Xanthine Oxidase/metabolismABSTRACT
We investigated reactive oxygen species (ROS) involvement in polymorphonuclear neutrophilic leukocyte (neutrophil) apoptosis triggering. Neutrophils were incubated with xanthine oxidase (XO), which produces superoxide anion (O2.-) and hydrogen peroxide (H2O2) or glucose oxidase (GO), which produces only H2O2. Both XO and GO accelerated apoptosis when compared to spontaneously aged neutrophils. Catalase inhibited both spontaneous apoptosis and XO- or GO-accelerated apoptosis, but superoxide dismutase did not. Hydrogen peroxide can enter the cell, thus generating intracellular oxidation, which was observed by flow cytometry. Furthermore, the intracellular reduced glutathione content fell in the presence of XO or GO; however, apoptosis was not accelerated in the presence of buthionine sulfoximine (BSO), suggesting that the fall in glutathione in the presence of XO or GO is a consequence of oxidative stress but not a trigger of apoptosis. Hydrogen peroxide can react with iron to form hydroxyl radicals (HO.); we observed that two iron chelators, deferoxamine and hydroxybenzyl ethylenediamine (HBED), both inhibited spontaneous and accelerated apoptosis, suggesting that HO. may mediate neutrophil apoptosis.
