Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H 2 O 2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H 2 O 2 -induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with STATs (signal transducers and activators of transcription) are a class of transcription factors bearing SH2 domains that become activated upon tyrosine phosphorylation. STATs are often activated by members of the JAK family of protein-tyrosine kinases (PTKs) in response to cytokine stimulation. This activation mechanism involves the SH2 domain-dependent recruitment of the STATs to tyrosine-phosphorylated cytokine receptors. The STATs then become phosphorylated by receptorassociated JAKs, which induces their dimerization via reciprocal SH2-phosphotyrosine interaction. STAT dimers then enter the nucleus and bind to specific DNA elements, thereby activating the transcription of a number of genes. The JAK-STAT pathway has been the subject of many recent comprehensive reviews (17-21). STAT3, a well characterized 92-kDa protein, has been shown to become activated by both epidermal growth factor and interleukin-6 in human A-431 cells (22). Because the ROI generated in response to various external stimuli can play a role both as regulators of transcription factors, including nuclear factors B (2, 23) and AT (24), and as inhibitors of protein-tyrosine phosphatases (PTPases) (25-27), we have investigated whether H 2 O 2 and other oxidizing agents could modulate STAT3 function in human lymphocytes. Enhanced phosphotyrosine accumulation could then result from the combined effects of increased phosphorylation and decreased dephosphorylation. Moreover, the DNA binding activity of STATs is known to depend primarily on tyrosine phosphorylation (19, 28 -31), although serine phosphorylation is also important in modulating the binding affinity of STAT3 (32-34). Here we show for the first time that STAT3 is phosphorylated on tyrosine residue(s), translocated to the nucleus, and elicited to bind to specific DNA elements upon lymphocyte treatment with H 2 O 2 . An additive effect between H 2 O 2 and vanadate was also evidenced suggesting that inhibition of tyrosine phosphatase(s)

Neutrophils are mobilized to the vascular wall during vessel inflammation. Published data are conflicting on phagocytic nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activation during the hypertensive state, and the capacity of angiotensin II (Ang II) to modulate the intracellular redox status has not been analyzed in neutrophils. We here describe that Ang II highly stimulates endogenous and extracellular O 2 ؊ production in these cells, consistent with the translocation to the cell membrane of the cytosolic components of NADPH oxidase, p47 phox , and p67 phox . The Ang II-dependent O 2 ؊ production was suppressed by specific inhibitors of AT1 receptors, of the p38MAPK and ERK1/2 pathways, and of flavin oxidases. Furthermore, Ang II induced a robust phosphorylation of p38MAPK, ERK1/2, and JNK1/2 (particularly JNK2), which was hindered by inhibitors of NADPH oxidase, tyrosine kinases, and ROS scavengers. Ang II increased cytosolic Ca 2؉ levels-released mainly from calcium stores-enhanced the syn- IntroductionAngiotensin II (Ang II), the main peptide hormone of the reninangiotensin system, induces leukocyte recruitment to the vessel wall, which constitutes a hallmark of the early stages of atherosclerosis and several hypertensive diseases. 1 In addition, it plays a regulatory role on blood pressure and circulation volume and on the proliferation of vascular smooth muscle cells. 2 Ang II acts through high-affinity cell surface receptors (AT1), which are linked to pathways classically associated with G-protein-coupled and tyrosine-kinase-mediated responses. 3 Although most studies on Ang II have been carried out in smooth muscle and endothelial cells, experimental evidence has been obtained on its effects on circulating cells. AT1 receptors for Ang II have been found recently in circulating neutrophils 4 and human peripheral monocytes, 5 and Ang II-induced cell activation in the latter has been reported. 6 In this context, the migration of leukocytes from blood to sites of inflammation and their adhesion to endothelial cells are primary events taking place during the acute inflammatory response and the pathogenesis of vascular diseases. 7 Because chronic inflammation of vessel walls is a hallmark of atherosclerosis, 8 and reactive oxygen species (ROS) such as superoxide anion (O 2 Ϫ ) and H 2 O 2 constitute the main intermediary molecules responsible for inflammation, 9 a link between atherosclerosis and ROS production has been postulated. 10 Nicotinamideadenine dinucleotide phosphate (NADPH) oxidase of phagocytes catalyzes the reduction of oxygen to O 2 Ϫ . In resting cells this enzyme is inactive, and its components are distributed between the cytosol and the membrane of secretory vesicles. When phagocytic cells are activated, the enzyme's cytosolic components associate to membrane-bound components and assemble into catalytically active NADPH oxidase. 11 It has been reported that Ang II-induced hypertrophy of vascular smooth muscle cells is mediated by both O 2 Ϫ and H 2 O 2 . 12 In addition, ROS ha...

A ligand-insensitive form of the human epidermal growth factor receptor (EGFR) was enriched by Ca2+-dependent calmodulin-affinity chromatography purification. The basic amphiphilic segment Arg645-Arg-Arg-His-Ile-Val-Arg-Lys-Arg-Thr654-Leu-Arg-Arg-Le u-Leu-Gln 660, located within the cytoplasmic juxtamembrane domain of this receptor, was purified as a fusion protein with glutathione S-transferase and shown to bind calmodulin in a Ca2+-dependent manner. An apparent dissociation constant of 0.4 microM calmodulin (Kd'(CaM)) and an apparent affinity constant of 0.5 microM free Ca2+ (Ka'(Ca)) were measured for this binding process. Binding of calmodulin at the juxtamembrane site prevented the phosphorylation of residue Thr-654 by protein kinase C, and an apparent inhibition constant of 0.5-1 microM calmodulin (Ki'(CaM)) was determined. Conversely, phosphorylation of this site by protein kinase C prevented its subsequent interaction with calmodulin. We therefore propose that cross talk between signaling pathways mediated by calmodulin and protein kinase C occurs at the juxtamembrane domain of the EGFR. This calmodulin-binding sequence is highly conserved among protein tyrosine kinases of the vertebrate EGFR family.