Ligand-independent GLUT4 translocation induced by guanosine 5'-O-(3-thiotriphosphate) involves tyrosine phosphorylation

To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPgammaS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPgammaS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPgammaS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPgammaS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPgammaS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPgammaS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPgammaS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).     

TGF-beta inhibits IL-2-induced tyrosine phosphorylation and activation of Jak-1 and Stat 5 in T lymphocytes

The major yolk protein precursor in mosquito oocytes, vitellogenin (Vg), is internalized by a 205-kDa membrane-bound receptor (VgR). Recently, VgR has been isolated permitting the production of polyclonal anti-VgR antibodies. To elucidate the pathway of VgR internalization and recycling in mosquito oocytes during Vg uptake, we carried out an immunogold electron-microscopic study, labeling both Vg and VgR in ultrathin frozen sections of ovarian tissue. VgR immunolabeling demonstrated that the oocyte plasma membrane was subdivided into microdomains, with VgR being located between and at the lower portions of the oocyte microvilli. During the early stages of internalization, Vg and VgR were observed together in coated pits, coated vesicles, and early endosomes. Fusion of early endosomes created transitional yolk bodies (TYB) in which Vg and VgR became segregated. VgR label was present in the numerous tubular compartments that protruded from the TYBs. These tubular organelles extended to and fused with the plasma membrane, suggesting that they represented the vehicle for VgR recycling. Vg label was not observed in the tubular compartments. Instead, Vg accumulated in the core of the TYB, a region free of VgR label. Mature yolk bodies (MYB) were heavily labeled for Vg, but completely lacked any VgR label, indicating that MYB are storage compartments that do not participate in receptor recycling. Thus, our immunocytochemical data clearly visualize the steps in Vg/VgR internalization, dissociation, sorting, and recycling of the receptor to the plasma membrane.     

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)     

Apolipoprotein E (ApoE) peptide regulates tau phosphorylation via two different signaling pathways

OBJECTIVE: The aim of this study was to compare levels of emotional and behavioural problems and competencies among immigrant and non-immigrant adolescents, and to determine factors that may contribute to any differences reported. METHOD: Subjects were selected randomly from students aged 12-16 years attending a high school with a high proportion of immigrants in Perth, Western Australia. Parents completed the Child Behaviour Checklist (CBCL), and students completed the Youth Self-Report (YSR) and a Personal History Questionnaire. RESULTS: On univariate analyses, non-immigrant adolescents had significantly higher CBCL and YSR scores than immigrant adolescents. Multivariate analyses suggested that CBCL scores were predicted by a number of variables other than immigration, including family intactness, socioeconomic status (SES) and gender. Higher YSR scores were predicted by non-intact families, school setting and non-immigrant status, and higher competencies scores were predicted by higher SES and parents not being immigrants. CONCLUSIONS: In assessing the effects of immigration on adolescent mental health, it is important to control for factors associated with adolescent behavioural and emotional problems and to use multiple informants. Overall, immigrant adolescents report fewer total and externalizing problems and fewer competencies than native-born adolescents. This finding may reflect strict immigration policies or cultural differences in definitions of psychopathology and the social expectations for adolescents' behaviour.     

The synapse-associated protein rapsyn regulates tyrosine phosphorylation of proteins colocalized at nicotinic acetylcholine receptor clusters

Protein tyrosine phosphorylation has been suggested to play an important role in the clustering of the nicotinic acetylcholine receptor (AChR) at the developing neuromuscular junction. Recent studies have shown that the 43-kDa synapse-associated protein rapsyn induces clustering of the AChR in heterologous expression systems. In this study we examined whether tyrosine phosphorylation is involved in this rapsyn-induced AChR clustering. Rapsyn-induced AChR clusters in fibroblasts contain phosphotyrosine, as detected using immunofluorescent labeling with anti-phosphotyrosine antibodies. No anti-phosphotyrosine staining of rapsyn clusters is seen in the absence of AChR expression, indicating that the AChR is required for the appearance of phosphotyrosine at clusters. In addition, coexpression of rapsyn with the AChR induces the tyrosine phosphorylation of the beta amd delta subunits of the AChR. Surprisingly, mutation of the tyrosine phosphorylation sites in the AChR did not inhibit rapsyn-induced clustering of the AChR and clusters of the mutant AChRs still contained high levels of phosphotyrosine. Experiments with single AChR subunits demonstrate that the alpha subunit of the AChR appears to be necessary and sufficient for codistribution of phosphotyrosine with rapsyn-induced clusters of AChR subunits. Finally, transfection of cells with rapsyn activates cellular protein tyrosine kinase activity, resulting in the tyrosine phosphorylation of several membrane-associated proteins. These results suggest that rapsyn may therefore regulate clustering at least in part by regulating the tyrosine phosphorylation of cellular proteins.     

Prolactin stimulates phosphorylation of the human T-cell antigen receptor complex and ZAP-70 tyrosine kinase: a potential mechanism for its immunomodulation

Prolactin (PRL) is an immunomodulatory hormone which promotes T-cell activation and proliferation. However, the intracellular mechanisms of this action in normal lymphocytes are unknown. Because the PRL receptor (PRLR) activates several signals also activated by the T-cell antigen receptor (TCR)/CD3 complex, we evaluated whether signaling "cross-talk" occurs between these distinct receptors. Using human thymocytes, human peripheral blood lymphocytes and the rat Nb2 lymphoma T-cell, we found that PRL induced rapid phosphorylation of multiple, TCR/CD3 complex proteins, an event required for lymphocyte activation. Two of these phosphorylated proteins were identified to be CD3 epsilon and ZAP-70 tyrosine kinase, molecules essential for TCR function. Further, PRL induced tyrosyl phosphorylation of ZAP-70 in each population of T-lymphocytes tested, demonstrating for the first time that ZAP-70 is a target of PRL action. Taken together, our results suggest that the PRLR directly affects T-lymphocyte activation by means of signaling cross-talk with the TCR/CD3 complex.     

Stimulation of Stat5 by granulocyte colony-stimulating factor (G-CSF) is modulated by two distinct cytoplasmic regions of the G-CSF receptor

In a manner similar to many other cytokines, treatment of cells with granulocyte CSF (G-CSF) has been shown to induce the tyrosine phosphorylation of the STAT proteins. Activation of Stat1 and Stat5 by G-CSF requires the membrane-proximal cytoplasmic domain of the receptor, including box1 and box2, while G-CSF-stimulated tyrosine phosphorylation of Stat3 also requires a region distal to box 2. In this study, we show that although the membrane-proximal 55 amino acids of the G-CSF receptor are sufficient for activation of Stat5, the maximal rate of Stat5 activation requires an additional 30 amino acids of the cytoplasmic domain. In contrast, the distal carboxyl-terminal region of the receptor appears to down-regulate Stat5 activation in that deletion of this carboxyl terminus results in increased amplitude and prolonged duration of Stat5 activation by G-CSF. Significantly, expression of a truncated dominant-negative Stat5 protein in hemopoietic cells not only inhibits G-CSF-dependent cell proliferation, but also suppresses cell survival upon G-CSF withdrawal. We further show that a potential protein tyrosine phosphatase may play a critical role in the down-regulation of G-CSF-stimulated Stat5 activation. These results demonstrate that two distinct cytoplasmic regions of the G-CSF receptor are involved in the regulation of the intensity and duration of Stat5 activation, and that Stat5 may be an important player in G-CSF-mediated cell proliferation and survival.     

Activation of separate calcium and A-kinase-dependent pathways by ACTH

Although cAMP has long been regarded as the primary intracellular messenger for ACTH-stimulated cortisol secretion, a requirement for Ca2+ is well established. However, a specific mechanism which couples ACTH receptor activation to increased intracellular calcium concentration in the adrenal cortical cell has not been elucidated. Here, we present evidence for a specific model in which ACTH at picomolar concentrations induces cAMP which acts through kinase-dependent and independent pathways to stimulate cortisol secretion. Along one of these pathways, cAMP acts directly to depolarize cells by inhibition of a specific non-inactivating K+ channel (I(AC)). This model provides a specific mechanism whereby cAMP-mediated inhibition of I(AC) is tightly coupled to depolarization-dependent Ca2+ entry and cortisol secretion. Ca2+ and cAMP are dual second messengers in the ACTH signalling pathway that are linked through I(AC) K+ channels.     

Proinflammatory cytokines regulate antigen-independent T-cell activation by two separate calcium-signaling pathways in multiple sclerosis patients

OBJECTIVE: The study examined the prevalence of low birth weight among biracial infants of black and white parents, by region of the United States. METHODS: Using the national linked live birth-death infant file for 1991, low birth weight (<2,500 g) was examined among 50,980 biracial singleton infants according to parental race (black mother-white father vs. white mother-black father). RESULTS: Nationally, the rate of low birth weight was 31% higher in the black mother-white father group (8.4%) than in the white mother-black father group (6.4%). The difference was smaller in the Northeast, reflecting a high rate (9.8%) for biracial infants of Puerto-Rican white mothers. The difference in the West was larger (75%), due to both a high rate in the black mother-white father group (9.1%) and a low rate for the white mother-black father group (5.2%), and persisted after controlling for parental education and a variety of maternal risk factors. CONCLUSIONS: Further studies are needed to identify the maternal factors involved in the regional differences in the prevalence of low birth weight among biracial infants.     

Anchorage mediated by integrin alpha6beta4 to laminin 5 (epiligrin) regulates tyrosine phosphorylation of a membrane-associated 80-kD protein

Detachment of basal keratinocytes from basement membrane signals a differentiation cascade. Two integrin receptors alpha6beta4 and alpha3beta1 mediate adhesion to laminin 5 (epiligrin), a major extracellular matrix protein in the basement membrane of epidermis. By establishing a low temperature adhesion system at 4 degrees C, we were able to examine the exclusive role of alpha6beta4 in adhesion of human foreskin keratinocyte (HFK) and the colon carcinoma cell LS123. We identified a novel 80-kD membrane-associated protein (p80) that is tyrosine phosphorylated in response to dissociation of alpha6beta4 from laminin 5. The specificity of p80 phosphorylation for laminin 5 and alpha6beta4 was illustrated by the lack of regulation of p80 phosphorylation on collagen, fibronectin, or poly-L-lysine surfaces. We showed that blocking of alpha3beta1 function using inhibitory mAbs, low temperature, or cytochalasin D diminished tyrosine phosphorylation of focal adhesion kinase but not p80 phosphorylation. Therefore, under our assay conditions, p80 phosphorylation is regulated by alpha6beta4, while motility via alpha3beta1 causes phosphorylation of focal adhesion kinase. Consistent with a linkage between p80 dephosphorylation and alpha6beta4 anchorage to laminin 5, we found that phosphatase inhibitor sodium vanadate, which blocked the p80 dephosphorylation, prevented the alpha6beta4-dependent cell anchorage to laminin 5 at 4degreesC. In contrast, adhesion at 37 degrees C via alpha3beta1 was unaffected. Furthermore, by in vitro kinase assay, we identified a kinase activity for p80 phosphorylation in suspended HFKs but not in attached cells. The kinase activity, alpha6beta4, and its associated adhesion structure stable anchoring contacts were all cofractionated in the Triton-insoluble cell fraction that lacks alpha3beta1. Thus, regulation of p80 phosphorylation, through the activities of p80 kinase and phosphatase, correlates with alpha6beta4-SAC anchorage to laminin 5 at 4 degrees C in epithelial cells of the skin and intestine. Transmembrane signaling through p80 is an early tyrosine phosphorylation event responsive to and possibly required for anchorage to laminin 5 by HFK and LS123 epithelial cells.     

Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.     

CCKA receptor activation stimulates p130(Cas) tyrosine phosphorylation, translocation, and association with Crk in rat pancreatic acinar cells

p130(Cas) (Crk-associated substrate), because of its structure as an adapter protein, can interact when tyrosine-phosphorylated with a large number of cellular proteins and therefore be an important modulator of downstream signals. A number of growth factors, lipids, and a few G protein-coupled receptors can stimulate p130(Cas) tyrosine phosphorylation. Recent studies show that tyrosine phosohorylation of intracellular proteins by the hormone/neurotransmitter cholecystokinin (CCK) in rat pancreatic acinar cells may be an important signaling cascade. In this study, we show in rat dispersed pancreatic acini CCK-8 rapidly stimulates tyrosine phosphorylation of p130(Cas), reaching a maximum (6.6 +/- 1. 4)-fold increase with a half-maximal effect at 0.3 nM. Activation of protein kinase C by TPA or increases in [Ca2+]i by the calcium ionophore A23187 stimulated p130(Cas) phosphorylation. Blockade of CCK increases in [Ca2+]i or PKC activity did not alter CCK-8-stimulated p130(Cas) phosphorylation; however, simultaneous blockage of both cascades caused a 50% inhibition. Partial inactivation by C. botulinum toxin of the small GTP-binding protein Rho caused a 41 +/- 12% decrease in the CCK-stimulated p130(Cas) phosphorylation. Disruption of the actin cytoskeleton with cytochalasin D, but not the microtubule network with colchicine, completely inhibited CCK-8-stimulated p130(Cas) phosphorylation. Total p130(Cas) under basal conditions was largely localized (70 +/- 2%) in the membrane fraction, and stimulation with CCK-8 induced total p130(Cas) translocation from the cytosolic fraction. CCK stimulation also caused a (5 +/- 1)-fold increase in p130(Cas) tyrosine phosphorylated in the plasma membrane. Treatment with tyrphostin B44 inhibited CCK-8-stimulated p130(Cas) phosphorylation, but it had no effect on p130(Cas) translocation. CCK-8 caused rapid formation of a p130(Cas)-Crk complex. In conclusion, our results demonstrate CCKA receptor activation causes rapid tyrosine phosphorylation of p130(Cas) through PLC-dependent and -independent mechanisms that require the participation of the small GTP-binding protein Rho and the integrity of the actin cytoskeleton, but not the microtubule network. Moreover, CCKA receptor activation causes translocation of p130(Cas) to the membrane and an increase in membrane tyrosine-phosphorylated p130(Cas). The translocation to the membrane does not require antecedent tyrosine phosphorylation. CCKA activation promotes the rapid formation of a p130(Cas)-Crk complex. These results suggest that p130(Cas) is likely an important modulator of downstream signals activated by CCK-8, possibly involved in regulating numerous cellular effects, such as effects on cell growth or cell shape.     

Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.     

Lipopolysaccharide induction of tissue factor in THP-1 cells involves Jun protein phosphorylation and nuclear factor kappaB nuclear translocation

Many Caenorhabditis elegans genes exist in operons in which polycistronic precursors are processed by cleavage at the 3' ends of upstream genes and trans splicing 100 to 400 nucleotides away, at the 5' ends of downstream genes, to generate monocistronic messages. Of the two spliced leaders, SL1 is trans spliced to the 5' ends of upstream genes, whereas SL2 is reserved for downstream genes in operons. However, there are isolated examples of what appears to be a different sort of operon, in which trans splicing is exclusively to SL1 and there is no intercistronic region; the polyadenylation signal is only a few base pairs upstream of the trans-splice site. We have analyzed the processing of an operon of this type by inserting the central part of mes-6/cks-1 into an SL2-type operon. In this novel context, cks-1 is trans spliced only to SL1, and mes-6 3'-end formation occurs normally, demonstrating that this unique mode of processing is indeed intrinsic to this kind of operon, which we herein designate "SL1-type." An exceptionally long polypyrimidine tract found in the 3' untranslated regions of the three known SL1-type operons is shown to be required for the accumulation of both upstream and downstream mRNAs. Mutations of the trans-splice and poly(A) signals indicate that the two processes are independent and in competition, presumably due to their close proximity, raising the possibility that production of upstream and downstream mRNAs is mutually exclusive.     

Protection against myocardial ischemic/reperfusion injury by inhibitors of two separate pathways of Na+ entry

Critics of Kohlberg's moral theory today focus on the content of his theory and more specifically on its justice-orientated moral concept. This has led to the well-known 'justice-care debate'. The purpose of this article is to critically examine the validity of Kohlberg's moral theory for research in nursing ethics from a caring perspective (referring to the content) as well as from a cognitive-structural perspective (referring to the basic assumptions of the model). The analysis points to the usefulness and value of the cognitive-structural model to empirically study nurses' ethical behaviour; the content of Kohlberg's model, however, needs to be adapted by adding a caring perspective as well as some personal and situational variables. An adjusted version of Kohlberg's model is proposed and discussed.