Activation of protein phosphorylation by oxidants in vascular endothelial cells: identification of tyrosine phosphorylation of caveolin

The colonial protochordate Botryllus schlosseri possesses a historecognition system which has long invited comparison to the vertebrate MHC. Upon contact, colonies either fuse or reject one another in a manner resembling graft acceptance or rejection in vertebrates. This response is controlled by a single highly polymorphic genetic region, the FuHC locus. Colonial protochordates such as B. schlosseri are among the closest relatives of the vertebrate lineage, and therefore may possess a recognizable MHC homologue. Since linkage between heat shock protein 70 (HSP70) genes and MHC appears to be conserved within the vertebrate lineage, we have analyzed HSP70 genes from B. schlosseri as a first step toward isolating the historecognition locus. Two HSP70 genes (HSP70.1 and HSP70.2) have been cloned and sequenced, and exhibit 93.6% sequence identity within the predicted coding regions. The B. schlosseri genes share a number of characteristics with vertebrate MHC-linked HSP70 genes: Northern blotting and sequence analysis suggest that the protochordate genes are cytoplasmically-expressed heat-inducible members of the HSP70 gene family (FAGAN and WEISSMAN 1996). However, unlike vertebrate MHC-linked HSP70 genes, HSP70.1 and HSP70.2 are not closely linked (FAGAN and WEISSMAN 1997). Furthermore, neither is closely linked to the locus determining historecognition (FAGAN and WEISSMAN 1997). These results do not support the hypothesis that the B. schlosseri FuHC locus is an MHC homolog. A discussion of the implications of these results for evolution of the vertebrate MHC is included.     

Signal transduction by the IL-1 type I receptor: evidence for the involvement of a receptor-coupled protein kinase

A novel serine/threonine specific protein kinase was found to be associated with the type I IL-1 receptor in the murine T cell lines D10N and EL-4. This kinase was identified in immunoprecipitates from IL-1 stimulated T-cells by its ability to phosphorylate exogenous substrates in the presence of radiolabeled ATP. An endogenous protein, most likely a member of the IL-1 R1 complex, was also phosphorylated. The activation of the kinase is specific for IL-1, neither TNF nor phorbol esters were able to activate the IL-1 RI associated kinase activity. The IL-1 receptor antagonist had no intrinsic activity and inhibited the activation of the kinase. The activation of the kinase was rapid and detectable after 30 seconds of IL-1 stimulation. A minimal model of the IL-RI signal transduction complex is discussed, presenting this novel serine/threonine kinase as a constituent of the complex.     

Thrombin receptor activation stimulates astrocyte proliferation and reversal of stellation by distinct pathways: involvement of tyrosine phosphorylation

Treatment of cultured type-1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half-maximal concentrations of thrombin required for each response are 500 and 2 pM, respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein-coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half-maximal concentrations of 30 microM for an increase in cell number and DNA synthesis and 100 nM for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10-fold greater than those required for half-maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55-65, 106, 110-115, and 120-130 kDa. The response was rapid (< 1 min) and transient with a peak response after approximately 2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin- or SFLLRNP-mediated reversal of stellation at concentrations of up to 1 microM. In contrast, 1 microM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 microM herbimycin A corresponded to inhibition of receptor-induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor-activated tyrosine kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Varicella-zoster virus Fc receptor gE glycoprotein: serine/threonine and tyrosine phosphorylation of monomeric and dimeric forms

Recently, it has been shown that large doses of all-trans-retinol (vitamin A) can potentiate the hepatotoxicity of several organic chemicals in the rat. Whether retinol pretreatment can alter the acute hepatotoxicity of an inorganic chemical, such as cadmium, is unknown. Therefore, the objective of this study was to determine how retinol might affect the acute toxicity of cadmium chloride (CdCl2) and to elucidate possible mechanisms. Cadmium exposure can induce acute, lethal hepatocellular necrosis in rodents, as well as lesions in the lung, kidney, testis, and gastrointestinal tract. In the present studies, male Sprague-Dawley rats were pretreated with retinol (75 mg/kg/day, po) for 7 consecutive days. One day after the last dose of retinol, animals were given a single injection of CdCl2 (2.5 to 4.0 mg/kg, iv). Cadmium chloride administration to unpretreated control rats caused extensive hepatic, renal, pulmonary, and testicular toxicity at 6, 24, and 48 hr postdosing as evaluated by plasma enzymes and/or histopathology. In retinol-pretreated rats, a significant attenuation of CdCl2-induced tissue injury was observed. Since the inducible cadmium-binding protein metallothionein (MT) is often an essential aspect of cadmium tolerance, its content in tissue was assessed using the cadmium-hemoglobin assay. Interestingly, retinol pretreatment significantly increased MT in the liver by sevenfold, but had no effect on lung, kidney, testicular, or pancreatic MT content. Although this increase in hepatic MT was much less than that induced by CdCl2, it was additive to the induction of CdCl2. Furthermore, the tissue distribution of cadmium was significantly altered by retinol pretreatment. The liver accumulated more cadmium, while less cadmium was found in the lung, kidney, and testis in retinol-pretreated rats than in controls. In monolayers of primary isolated hepatocytes, CdCl2-induced toxicity was significantly reduced in cells isolated from retinol-pretreated rats compared to those isolated from control rats. The dose response was shifted to the right and the in vitro cadmium LC50 was increased by in vivo retinol exposure from 1.1 +/- 0.1 to 2.4 +/- 0.04 microM. From these data it is concluded that the induction of hepatic MT is an essential aspect of retinol-induced tolerance to CdCl2 hepatotoxicity, as well as toxicity in other tissues.     

Human olfactory neuroepithelial cells: tyrosine phosphorylation and process extension are increased by the combination of IL-1beta, IL-6, NGF, and bFGF

Olfactory neuroepithelial cells (ONC) grown from biopsies of human donors are a novel cell culture system that may facilitate studies into normal and disease-related human neurobiology. We further characterized the expression of cell surface markers and intermediate filaments, and responses to neurotrophic factors by ONC. ONC are positive for cell surface markers N-CAM, PSA-N-CAM, neutral endopeptidase, N-aminopeptidase, NGF low-affinity receptor homologue (CD40), and transferrin receptor by flow cytometry for the intermediate filament proteins peripherin, vimentin, and NF-H by immunocytochemistry. Responses to neurotrophic factors measured were process outgrowth, cytoskeletal protein expression, and protein phosphorylation. Process outgrowth was increased by interleukin-beta 164-171 (IL-1beta) or by the combination of IL-1beta, interleukin-6 (IL-6), nerve growth factor (NGF), and basic fibroblast growth factor (bFGF). This combination of IL-1beta, IL-6, NGF, and bFGF (16NF) increased expression of two cytoskeletal proteins, NF-H protein and microtubule-associated protein tau. Application of the individual neurotrophic factors IL-1beta, IL-6, NGF, and bFGF increased protein phosphorylation, while 16NF produced an immediate increase in tyrosine phosphorylation of several proteins (MW of 40-80, 120, 150, and 190 kDa). The 16NF combination appears to act through a tyrosine-kinase-mediated pathway to induce process extension and increase NF-H expression. The ONC culture has the potential to be further explored to examine the relationship among process outgrowth, protein phosphorylation, and synergy between neurotrophin and cytokine receptor systems.     

Coordinated regulation of the tyrosine phosphorylation of Cbl by Fyn and Syk tyrosine kinases

Cross-linking of the T cell antigen receptor (TCR)-CD3 complex induces rapid tyrosine phosphorylation and activation of Src (Lck and Fyn) and Syk (Syk and Zap-70) family protein tyrosine kinases (PTKs) which, in turn, phosphorylate multiple intracellular substrates. Cbl is a prominent PTK substrate suggesting a pivotal role for it in early signal transduction events. However, the regulation of Cbl function and tyrosine phosphorylation in T cells by upstream PTKs remains poorly understood. In the present study, we used genetic and biochemical approaches to demonstrate that Cbl directly interacts with Syk and Fyn via its N-terminal and C-terminal regions, respectively. Tyr-316 of Syk was required for the interaction with Cbl as well as for the maximal tyrosine phosphorylation of Cbl. However, both wild-type Syk and Y316F-mutated Syk phosphorylated equally well the C-terminal fragment of Cbl in vivo, suggesting the existence of an alternative, N terminus-independent mechanism for the Syk-induced tyrosine phosphorylation of Cbl. This mechanism appears to involve Fyn, since, in addition to its association with the C-terminal region of Cbl, Fyn also associated with Syk and enhanced the Syk-induced tyrosine phosphorylation of Cbl. These findings implicate Fyn as an adaptor protein that facilitates the interaction between Syk and Cbl, and suggest that Src and Syk family PTKs coordinately regulate the tyrosine phosphorylation of Cbl.     

Over-expression of protein tyrosine phosphatase 1 (PTP1) alters IL-3-dependent growth and tyrosine phosphorylation

Interleukin-3 (IL-3) is a hematopoietic growth factor receptor which stimulates the proliferation of multilineage progenitor cells. It is known that IL-3 stimulates tyrosine phosphorylation while transducing a mitogenic signal. The signal transduction pathways activated by the IL-3 receptor, however, are not fully understood. In this study a protein tyrosine phosphatase has been over-expressed in the IL-3 dependent, murine myeloid progenitor cell line, 32D cl3 in order to test whether altering the levels of tyrosine phosphorylation would change IL-3 stimulated proliferation. These cells were transfected with a metal-inducible expression vector containing a rat cDNA encoding PTP1. A low basal level of rat PTP1 message and protein was detected in cells transfected with the PTP1 vector, and zinc treatment resulted in a three- to fourfold increase in the amount of PTP1 message, protein and catalytic activity. Over-expression of PTP1 resulted in a two- to threefold decrease in IL-3 stimulated proliferation. Cells over-expressing PTP1 also exhibited decreased levels of tyrosine phosphorylation; phosphorylation of the IL-3 receptor beta subunit and the Shc protein were both dramatically decreased. Thus, PTP1 over-expression negatively modulated IL-3 signal transduction. To identify potential substrates of PTP1, 32D cl3 cells were transfected with a catalytically inactive PTP1 mutant, PTP1(C/S). Three tyrosine-phosphorylated proteins of MW 140, 79 and 69 k coprecipitated with PTP1(C/S). We believe that the 140 kDa protein represents the beta subunit of the IL-3 receptor. In addition, a GST-fusion protein containing active PTP1 dephosphorylated the beta-subunit in an in vitro assay. By immunofluorescent microscopy over-expressed PTP1(C/S) co-localized largely with calnexin, an endoplasmic reticulum-associated protein. Immunofluorescent microscopy also indicated that PTP1(C/S) and the beta subunit co-localized at discrete sites at the plasma membrane and around a cytoplasmic organelle where most of the beta subunit was located. These observations suggest PTP1 over-expression may down-regulate the growth response to IL-3 through dephosphorylation of the IL-3 receptor, perhaps in an intracellular compartment, thereby inhibiting propagation of the IL-3 mitogenic signal.     

Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase

Previously, we have found that human liver annexin V (hA-V; in earlier reports referred as Endonexin II) is a specific hepatitis B surface antigen (HBsAg) binding protein. In this study, we demonstrate that transfection of rat hepatoma FTO 2B cells, a cell line that is not infectable by hepatitis B virus (HBV) and does not express hA-V, with a construct containing the hA-V gene, resulted in hA-V expressing cells susceptible to HBV infection. After in vitro infection, transfected FTO cells (assigned as FTO 9.1 cells) expressing hA-V in cultures were shown to contain HBV-precore/core, X mRNAs, and covalently closed circular (ccc) DNA as detected by polymerase chain reaction (PCR). The presence of HBV ccc and replicative intermediate DNA was also demonstrated by Southern blot hybridization assay. HBV DNA secreted in the culture medium was also evident as determined by quantitative branched DNA (bDNA) assay. HBsAg and hepatitis B core antigen (HBcAg) could also be detected by an immunocytochemical method in 10% to 15% of the cells at day 3 and day 5 after infection. Infectivity of in vitro-propagated HBV was demonstrated by infection of the naive FTO 9.1 cells with the culture supernatant from HBV-carrier cultures. In contrast to primary cultures of human hepatocytes and FTO 9.1 cells, primary rat and mouse hepatocytes, as well as rat hepatoma cell lines that do not express hA-V, are not susceptible to HBV infection. These findings suggest that hA-V plays a key role in the initial step of HBV infection and that the species-specific susceptibility to HBV infection and replication in hepatocytes is associated with the expression of hA-V.     

Receptor-mediated endocytosis of IL-8: a fluorescent microscopic evidence and implication of the process in ligand-induced biological response in human neutrophils

Interleukin 8 (IL-8), a neurophil-activating and chemotactic cytokine, is known to play a key role in the pathogenesis of a large number of neutrophil-driven inflammatory diseases. Although the cytokine is rapidly internalized at 37 degrees C with its receptors, there was no direct evidence for the ligand-induced endocytosis of the receptor or that of the interaction of receptor ligand complex at 37 degrees C. As a result, our understanding about the regulation of Il-8 induced biological response is very limited. In the present study, using FITC-IL-8 conjugate as a probe, we have demonstrated the time- and temperature-dependent endocytosis of IL-8 under fluorescent microscope. We have also shown that the bright fluorescent light on the surface of neutrophils gradually disappears and it becomes almost dark after 120 min of incubation. Monodansyl cadaverine (MDC, 900 microM), however, was found to retain the fluorescent light of FITC coupled with Il-8 on the cells. MDC and ouabain (2.5 mM) can inhibit the ligand induced endocytosis by 76% and 96%, respectively, compared to control. With respect to control, IL-8 induced biological responses e.g. IL-8 directed migration, intracellular Ca2+ release and superoxide release are significantly reduced by 77%, 94% and 76%, respectively, in presence of MDC. The study presents a direct visual evidence of the time and temperature-dependent receptor-mediated endocytosis of IL-8 which is inhibited by MDC and ouabain. This information is useful for understanding the ligand receptor interaction at 37 degrees C and may be useful for developing anti-inflammatory agents against IL-8.     

Activation of the leukocyte NADPH oxidase by phorbol ester requires the phosphorylation of p47PHOX on serine 303 or 304

The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.     

Regulation of cell morphology in B lymphocytes by IL-4: evidence for induced cytoskeletal changes

Lymphocyte activation is often accompanied by changes in cell morphology, for example, in cell adhesion or motility. IL-4 is a cytokine exerting many effects on B lymphocytes. In this study, we show that stimulation with LPS in combination with IL-4, but not LPS or IL-4 alone, results in a pronounced dendritic morphology of B cells. Using a culture system in which Abs directed to B cell surface markers are immobilized on the tissue culture plastic, we find that cell spreading can be mediated by a variety of Abs, including anti-CD44, -CD23, -LFA-1, -VLA-4, -ICAM-1, and -Ig. B cells stimulated with anti-Ig Abs plus IL-4, or anti-CD40 Abs in the presence or absence of IL-4, are also induced to spread, while IL-2, IL-5, or IL-10 in combination with LPS or alone fail to induce this. Spreading correlates with induction of tight cell aggregation. It is sensitive to cytochalasin B, indicating a requirement for intact actin cytoskeleton. CD44 is selectively detected in the detergent-insoluble fraction of cell lysates prepared from LPS plus IL-4-stimulated B cell cultures after Ab cross-linking of CD44, suggesting a membrane protein-cytoskeleton interaction. Interestingly, electron microscopy studies reveal induction of microvilli-like structures on LPS plus IL-4-stimulated blasts, suggesting that IL-4 can influence cell morphology on an ultra-structural level. In summary, our data show that stimulation with LPS plus IL-4 or ligation of CD40 is capable of inducing dramatic morphologic changes in murine B cells, which correlates with in vitro induction of strong cell adhesion.     

Abnormal myelocytic cell development in interleukin-2 (IL-2)-deficient mice: evidence for the involvement of IL-2 in myelopoiesis

Mice lacking interleukin-2 (IL-2) developed a severe hematopoietic disorder characterized by the abnormal development of myeloid cells and neutropenia. Analysis of the bone marrow of IL-2-deficient (IL-2(-/-)) mice showed that the number of mature polymorphonuclear cells was decreased by 65% to 75%, and granulocyte/macrophage precursor cells were reduced by 50%. Bone marrow cells from IL-2(-/-) mice were unable to sustain myelopoiesis in lethally irradiated mice and in long-term bone marrow cultures (LTBMC). The addition of exogenous IL-2 to LTBMC of IL-2(-/-) cells partially restored hematopoietic progenitor activity. In the bone marrow of wild-type mice, immature (Mac-1(lo)) myeloid cells, including myeloblasts and promyelocytes, constitutively expressed the beta-chain of the IL-2R, and the number of Mac-1(lo)IL-2Rbeta+ cells was increased by twofold to threefold in IL-2(-/-) mice. During culture in the presence of IL-2 and the absence of stromal cells, Mac-1(lo)IL-2Rbeta+ immature myeloid cells proliferated and gave rise to mature granulocytes and macrophages. Collectively, these observations indicate that defective myelopoiesis in IL-2(-/-) mice is at least in part a consequence of their direct dependency on IL-2, and by regulating the growth of immature myeloid cells, IL-2 plays an important role in the homeostatic regulation of myelocytic cell generation.     

Evidence for IL-12-activated Ca2+ and tyrosine signaling pathways in human neutrophils

The cytokine IL-12 is proposed to play a bridging role between innate and adaptive immunity. Here we demonstrate that IL-12 binds specifically to human neutrophils. This binding leads to a transient increase in 1) intracellular free calcium due to its release from membrane-enclosed stores and its influx from extracellular medium, 2) actin polymerization, and 3) tyrosine phosphorylation. IL-12 treatment also leads to a concentration-dependent increase in reactive oxygen metabolite production. The effect of IL-12 is blocked by neutralizing Abs to IL-12. Inhibition of either calcium transient or tyrosine phosphorylation causes inhibition of reactive oxygen metabolite production. However, inhibition of actin polymerization enhances IL-12-induced oxidase activation. Our data suggest 1) a direct role for IL-12 in the activation of human neutrophils, and 2) a calcium-dependent signaling pathway for IL-12.     

Cooperative interaction between alpha- and beta-chains of hepatocyte growth factor on c-Met receptor confers ligand-induced receptor tyrosine phosphorylation and multiple biological responses

Hepatocyte growth factor (HGF) is a heterodimeric molecule composed of the alpha-chain containing the N-terminal hairpin domain, four kringle domains, and the serine protease-like beta-chain. We prepared HGF/NK4 and HGF/beta from the entire HGF after single-cut digestion with elastase. HGF/NK4 contains the N-terminal hairpin and four kringle domains, while HGF/beta is composed of the C-terminal 16 amino acids of the alpha-chain and the entire beta-chain, linked by a disulfide bridge. HGF/NK4 competitively inhibited the binding of 125I-HGF to the receptor, and affinity cross-linking analysis indicated that HGF/NK4 alone can bind to the c-Met receptor. In contrast, HGF/beta alone did not competitively inhibit the binding of 125I-HGF to the receptor and did not bind to the c-Met/HGF receptor. Scatchard analysis and affinity cross-linking experiments indicated that HGF/beta specifically binds to c-Met in the presence of HGF/NK4 but not HGF/NK2. Neither HGF/NK4 nor HGF/beta alone induced mitogenic, motogenic (cell scattering), and morphogenic (induction of branching tubulogenesis) responses; however, HGF/beta did induce these biological responses in the presence of HGF/NK4. Consistent with these results, although neither HGF/NK4 alone nor HGF/beta alone induced tyrosine phosphorylation of the c-Met/HGF receptor, HGF/beta induced tyrosine phosphorylation of the receptor when c-Met/HGF receptor was occupied by HGF/NK4. These results indicate that HGF/beta binds to the c-Met/HGF receptor that is occupied by HGF/NK4 and induces receptor tyrosine phosphorylation and the subsequent biological activities of HGF. We propose that there exists a unique cooperative interaction between alpha- and beta-chains, this interaction leading to beta-chain-dependent receptor tyrosine phosphorylation and subsequent biological responses.     

Integrin signalling and tyrosine phosphorylation: just the FAKs?

PURPOSE: Physical rehabilitation is one of the major forms of treatment of chronic low back pain. The ability of some patients to cooperate is limited by pain. Since 1992 continuous epidural analgesia has been combined with a physical rehabilitation programme for patients with chronic low back pain who have been unable to make progress with conventional physical rehabilitation due to severity of pain. METHOD: This study reports a series of 46 consecutive patients with chronic back pain admitted over a 6 month period to a 5-day inpatient rehabilitation programme. A lumbar epidural catheter was inserted and bupivacaine 0.125% was infused at a rate that produced analgesia without sensory or motor deficit over a period of 5 days. An intensive mobilizing physiotherapy programme was instituted. Physical and psychological parameters were measured on day 1, after 1 week, after 1 month and after 1 year. RESULTS: Time to complete a 50 m walk, time from sitting to standing, and spinal flexion were improved at 1 week and 1 month, but only time to complete the walk remained improved at 1 year. In Goldberg's General Health Questionnaire 28 scores were improved for social dysfunction, somatic symptoms, anxiety and insomnia, and depression, at 1 week and 1 month but only social dysfunction remained improved at 1 year. Using a Visual Analogue Scale pain ratings were unaltered after 1 year. CONCLUSION: Continuous 5 day epidural analgesia combined with intensive physiotherapy may offer a means of initial rehabilitation of chronic low back pain. The initial benefit was most marked at 1 week, with benefit still evident after 1 month. However, the benefit decreased with time. This technique may be of value as part of a more comprehensive programme of physical and psychological rehabilitation.