Biomedical subjects

R A Roth

Publications and source records attributed to R A Roth.

At least 55 records · Page 3Linked to original sources

PubMed1998

Evidence of insulin-stimulated phosphorylation and activation of the mammalian target of rapamycin mediated by a protein kinase B signaling pathway.

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

3T3 Cells↗

PubMed1998

Construction and characterization of a conditionally active version of the serine/threonine kinase Akt.

Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.

3T3 Cells↗

PubMed1998

Development of an assay for bioactive insulin.

A method to detect the biological activity of serum insulin has been developed. This method, called a bioactive insulin assay, determines the ability of serum insulin to stimulate the autophosphorylation of insulin receptors in an intact cell system. For this, intact Chinese hamster ovary cells which overexpress the human insulin receptor are treated with serum and then lysed. Autophosphorylation of the insulin receptors is then measured by a two-site immunofluorometric assay using monoclonal anti-insulin receptor antibodies and europium-labeled anti-phosphotyrosine antibodies. The detection limit of this assay is 1 microU/ml of insulin. Dilution and recovery test inter- and intraassay coefficient variations are permissible. The amount of insulin determined by this assay correlated well with the amount of insulin detected by a traditional immunological assay for insulin (r = 0.94, P < 0.001). In the case of a mutant insulin, the insulin from a Wakayama subject, the biologically active insulin was found to constitute 9% of the immunologically reactive insulin. Since this assay specifically measures the amount of biologically active insulin present in serum, it should be particularly useful in monitoring active insulin in patients with various mutant insulins.

Animals↗

PubMed1998

Protein kinase C modulates the insulin-stimulated increase in Akt1 and Akt3 activity in 3T3-L1 adipocytes.

In the present studies, we have compared the properties of two members of the Akt family of ser/thr kinases, Akt1 and Akt3. First, we demonstrate that both 3T3-L1 fibroblasts and adipocytes express Akt3 mRNA by RT-PCR and sequencing of the resultant PCR product. Second, we show that insulin stimulates the enzymatic activity of Akt1 and Akt3 15- and 7-fold, respectively. We then investigated the ability of protein kinase C to regulate Akt1 and 3. Neither enzyme was activated by stimulation of protein kinase C, however, the insulin-stimulated increases in activity of both isozymes were found to be comparably inhibited by prior protein kinase C activation. Since this inhibition could have resulted from an interaction of the pleckstrin homology domain of the Akt with protein kinase C, we also examined the ability of a mutant Akt1 lacking this domain to be regulated by this enzyme. The insulin-stimulated increase in enzymatic activity of this mutant Akt was regulated by PKC activation like the wild type enzyme. These results indicate that Akt1 and 3 are similarly stimulated by insulin and this stimulation is inhibited by prior activation of protein kinase C through a mechanism that is independent of the presence of the pleckstrin homology domain.

3T3 Cells↗

PubMed1998

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate.

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.

3T3 Cells↗

PubMed1998

Impairment of human neutrophil oxidative burst by polychlorinated biphenyls: inhibition of superoxide dismutase activity.

We report evidence of a novel mechanism by which polychlorinated biphenyls might act as potent inducers of inflammation. Aroclor 1242 (A1242), a polychlorinated biphenyl mixture, and 2,2',4,4'-tetrachlorobiphenyl (PCB47), a constituent of A1242 that produces the same patterns of effects, impaired the oxidative burst of human neutrophils by inhibiting the antioxidant enzyme superoxide dismutase, which converts O2- to H2O2. Pre-incubation of neutrophils with A1242 or PCB47 before stimulation with phorbol 12-myristate 13-acetate heightened the respiratory burst, producing a significant increase in intracellular O2- production along with a significant decrease in H2O2 production compared with unexposed agonist-stimulated neutrophils. This was also evident in a physiologically relevant situation in which neutrophils pre-incubated with A1242 were subsequently stimulated with a combination of N-formyl-L-methionyl-L-leucyl-L-phenylalanine and tumor necrosis factor-alpha. Incubation of bovine copper-zinc superoxide dismutase (EC 1.15.1.1) with A1242 or PCB47 in a cell-free system reversed the enzyme-mediated inhibition of 6-hydroxydopamine autoxidation, indicating that polychlorinated biphenyls inhibited superoxide dismutase activity. Low superoxide dismutase activity in neutrophils leads to imbalances between production of free radicals and antioxidant defense mechanisms, which can in turn induce tissue damage and hasten the onset of neutrophil apoptosis.

Adult↗

PubMed1998

Alpha-naphthylisothiocyanate causes neutrophils to release factors that are cytotoxic to hepatocytes.

Administration of alpha-naphthylisothiocyanate (ANIT) to rats causes acute liver injury characterized in part by hepatocellular damage and marked neutrophil infiltration, effects that resemble drug-induced cholangiolitic hepatitis in people. ANIT-induced liver injury is neutrophil dependent. Moreover, ANIT can activate neutrophils in vitro. Since neutrophil-derived proteases can mediate hepatocellular killing, we hypothesized that ANIT stimulates neutrophils to release proteolytic enzymes that are cytotoxic to hepatic parenchymal cells. To test this hypothesis, neutrophils were isolated from Sprague-Dawley rats and incubated with ANIT for 6-24 h. ANIT (6-50 microM) was not toxic to neutrophils as indicated by the lack of lactate dehydrogenase release into the incubation medium. The conditioned medium from ANIT-treated neutrophils (ANCM) was collected, centrifuged, added to isolated hepatocytes, and incubated for 8, 16, or 24 h. Conditioned medium collected from neutrophils exposed to 25 or 50 microM ANIT for 16-24 h caused hepatocellular damage as indicated by the release of alanine aminotransferase into the culture medium. The concentration of ANIT in ANCM was nondetectable (0.5 microM). Analysis of ANCM indicated the presence of both cathepsin G and elastase activities. Inhibitors of these enzymes afforded protection against ANCM-induced hepatocellular injury. These results indicate that ANIT causes neutrophils to release toxic proteases which cause hepatocellular damage in vitro.

1-Naphthylisothiocyanate↗

PubMed1998

The response of pulmonary vascular endothelial cells to monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro and in vivo.

Monocrotaline pyrrole (MCTP) causes pulmonary vascular endothelial cell (EC) injury followed by progressive pulmonary vascular leak in vivo and the inhibition of EC proliferation in vitro. It was hypothesized that MCTP inhibits cell proliferation in vitro by interfering with cell cycle progression in a cycle phase-specific manner. Furthermore, it was proposed that early alterations in MCTP-induced lung injury leading to hypertension were associated with a similar inhibition of EC proliferation. Subconfluent cultures of bovine pulmonary artery endothelial cells (BECs) were synchronized with aphidicolin (APH), a reversible G1-S phase inhibitor. Upon removal of APH, BECs were exposed to MCTP (5 micrograms/ml) or its vehicle for a 4-h interval corresponding to either the G1-S, S-G2, or G2 through mitosis (M) phases of the cell cycle. Fluorescence-activated cell sorting (FACS) was used to identify MCTP-induced changes in cell cycle progression in BECs, and the transit of S phase cells through the cycle was characterized through the incorporation of bromodeoxyuridine (BrdU). Synchronized BECs exposed to MCTP between mid-S-G2 or G2 through M were briefly delayed in G2-M at 12 h but underwent cell division by 24 h. By contrast, BECs treated with MCTP immediately after release from APH block became arrested in G2-M at 24 h and showed evidence of continued DNA synthesis and hypertetraploidy, but they did not divide. In vivo, MCTP (3.5 mg/kg i.v.) administration caused an increase in arterial EC BrdU incorporation between Days 3 and 7, but no increase in EC density. During this same interval, pulmonary vascular permeability increased and persisted. In summary, MCTP inhibits cell proliferation in a cell cycle phase-dependent manner in vitro. The results suggest that a similar mechanism could occur in vivo and may be associated with delayed EC repair, a process that could contribute to persistent pulmonary vascular leak.

Animals↗

PubMed1998

Cytotoxicity of naphthylisothiocyanates in rat hepatocyte-neutrophil cocultures.

1-Naphthylisothiocyanate (ANIT) produces cholangiolitic hepatitis in rats. This injury is characterized by periportal bile duct and hepatic parenchymal cell necrosis with inflammatory cell involvement. In contrast, 2-naphthylisothiocyanate (BNIT) does not induce cholangiolitic hepatitis. The mechanism(s) involved in ANIT-induced hepatic injury remain to be elucidated. To investigate this difference in toxicity further, we examined the cytotoxicity of ANIT and BNIT in primary rat hepatocyte cultures. Since neutrophils (PMNs) are required for the development of ANIT-induced cholangiolitic hepatitis in vivo, we also examined the potential for PMNs to modulate ANIT and BNIT cytotoxicity in rat hepatocyte-PMN cocultures. Both ANIT and BNIT injured rat hepatocytes within the range of concentrations examined (0-100 microM); however, BNIT was more potent. The presence of PMNs did not significantly influence the hepatocellular injury produced by either naphthylisothiocyanate (NIT). In an attempt to clarify the disparity between these results in vitro and observations reported in vivo, we examined, in hepatocyte PMN cocultures, the cytotoxic potential of bile collected from NIT-treated rats. Bile from BNIT-treated rats was markedly more cytotoxic to hepatocytes than was bile from ANIT-treated rats. As was observed in earlier experiments, the inclusion of PMNs had no effect on the hepatocellular toxicity of bile from NIT-treated rats. These findings prompted evaluation of the effect of NITs on rat PMNs. ANIT (1 and 10 microM) had no effect on phorbal myristate acetate (PMA)-induced superoxide production by PMNs, whereas BNIT (1 and 10 microM) markedly inhibited PMA-induced superoxide production. In contrast, ANIT and BNIT were equally effective at inhibiting f-met-leu-phe (fMLP)-induced PMN degranulation (beta-glucuronidase release). Altogether, the relative NIT toxicity observed in hepatocyte primary cultures is contrary to reports of hepatotoxic potential of these NITs in vivo. The PMN-dependence of ANIT hepatotoxicity in vivo was not reproduced in hepatocyte-PMN cocultures exposed to ANIT, suggesting that the PMN dependence in vivo involves factors not present in hepatocyte PMN cocultures. The greater PMN inhibitory effect of BNIT may, in part, underlie its inability to elicit the PMN-dependent liver injury in vivo that characterizes ANIT-induced cholangiolitic hepatitis.

Animals↗

PubMed1998

Divergent effects of exercise on metabolic and mitogenic signaling pathways in human skeletal muscle.

The molecular signaling mechanisms by which muscle contractions lead to changes in glucose metabolism and gene expression remain largely undefined. We assessed whether exercise activates MAP kinase proteins (ERK1/2, SEK1, and p38 MAP kinase) as well as Akt and PYK2 in skeletal muscle from healthy volunteers obtained during and after one-leg cycle ergometry at approximately 70% VO2max. Exercise led to a marked increase in ERK1/2 phosphorylation, which rapidly decreased to resting levels upon recovery. Exercise increased phosphorylation of SEK1 and p38 MAP kinase to a lesser extent than ERK1/2. In contrast to ERK1/2, p38 MAP kinase phosphorylation was increased in nonexercised muscle upon cessation of exercise. Phosphorylation of the transcription factor CREB was increased in nonexercised muscle upon cessation of exercise. Exercise did not activate Akt or increase tyrosine phosphorylation of PYK2. Thus, exercise has divergent effects on parallel MAP kinase pathways, of which only p38 demonstrated a systemic response. However, our data do not support a role of Akt or PYK2 in exercise/contraction-induced signaling in human skeletal. Activation of the different MAP kinase pathways by physical exercise appears to be important in the regulation of transcriptional events in skeletal muscle.

Blood Glucose↗

PubMed1998

Comparison of the signaling abilities of the cytoplasmic domains of the insulin receptor and the insulin receptor-related receptor in 3T3-L1 adipocytes.

In the present work a chimeric receptor containing the intracellular domain of the insulin receptor-related receptor (IRR) and the extracellular domain of the colony stimulating factor-1 (CSF-1) receptor was expressed in 3T3-L1 adipocytes and compared with the parallel chimeric receptor containing the cytoplasmic domain of the insulin receptor (IR). Both chimeric receptors exhibited CSF-stimulated tyrosine kinase activity when assayed in vitro after in vivo activation comparable to that of the endogenous IR present in these cells. No cross-activation of the expressed chimeric and endogenous receptors was observed. The cytoplasmic domain of the IRR was found to 1) mediate activation of the Ser/Thr kinase Akt/PKB, 2) stimulate glucose uptake, 3) inhibit lipolysis, and 4) stimulate glycogen synthase, all with a potency comparable to those of the expressed CSF-1R/IR chimera and the endogenous insulin receptors. These results indicate that despite the extensive differences in sequence between the cytoplasmic domains of the IRR and IR, the elements required for insulin-specific responses have been conserved in this distinct member of the insulin receptor family.

3T3 Cells↗

PubMed1998

Insulin-stimulated Akt kinase activity is reduced in skeletal muscle from NIDDM subjects.

The serine/threonine kinase Akt (PKB/Rac) has been implicated as playing a role in the insulin-signaling pathway to glucose transport. Little is known regarding the regulation of Akt kinase activity in insulin-sensitive tissues, such as skeletal muscle, or whether this regulation is altered in insulin-resistant states such as NIDDM. We examined the effect of insulin on Akt kinase activity in skeletal muscle from six NIDDM patients and six healthy subjects. Whole-body insulin sensitivity, assessed by the euglycemic-hyperinsulinemic clamp, was significantly lower in NIDDM subjects (P < 0.001), and this was accompanied by impaired in vitro insulin-stimulated glucose transport in skeletal muscle. In both groups, insulin induced a significant increase in Akt kinase activity, but the response to maximal insulin (60 nmol/l) was markedly reduced in skeletal muscle from NIDDM subjects (66% of control levels, P < 0.01). Impaired Akt kinase activity was not accompanied by decreased protein expression of Akt. Instead, a trend toward increased Akt expression was noted in skeletal muscle from NIDDM subjects (P < 0.1). These parallel defects in insulin-stimulated Akt kinase activity and glucose transport in diabetic skeletal muscle suggest that reduced Akt kinase activity may play a role in the development of insulin resistance in NIDDM.

Biological Transport↗

PubMed1998

Binding of SH2 containing proteins to the insulin receptor: a new way for modulating insulin signalling.

Prior studies have established a role in insulin action for the tyrosine phosphorylation of substrates and their subsequent complexing with SH2 containing proteins. More recently, SH2 proteins have been identified which can tightly bind to the tyrosine phosphorylated insulin receptor. The major protein identified so far (called Grb-IR or Grb10) of this type appears to be present in at least 3 isoforms, varying in the presence of a pleckstrin homology domain and in the sequence of its amino terminus. The binding of this protein to the insulin receptor appears to inhibit signalling by the receptor. The present review will discuss the current knowledge of the structure and function of this protein.

Amino Acid Sequence↗

PubMed1997

Modulation of insulin receptor substrate-1 tyrosine phosphorylation and function by mitogen-activated protein kinase.

Increased serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been observed in several systems to correlate with a decreased ability of the insulin receptor to tyrosine-phosphorylate this endogenous substrate and to inhibit its subsequent association with phosphatidylinositol 3-kinase. In the present studies we have examined the potential role of the mitogen-activated protein (MAP) kinase in the increased serine phosphorylation of IRS-1 observed in human embryonic kidney cells treated with an activator of protein kinase C, phorbol 12-myristate 13-acetate. First, recombinantly produced kinase was shown to phosphorylate intact IRS-1 in a way that decreased the ability of isolated insulin receptor to phosphorylate the tyrosines recognized by the SH2 domains of the phosphatidylinositol 3-kinase. Second, an inhibitor of MAP kinase activation, PD98059, blocked the phorbol 12-myristate 13-acetate-induced inhibition of the insulin-stimulated increase in IRS-1 associated phosphatidylinositol 3-kinase. Third, activation of MAP kinase in intact cells via a regulatable upstream kinase, a RAF:estrogen receptor construct, could also inhibit the insulin-stimulated increase in IRS-1-associated phosphatidylinositol 3-kinase. Fourth, an in gel kinase assay showed that MAP kinase was the primary renaturable kinase in cell extracts capable of phosphorylating an IRS-1 fusion protein. Finally, IRS-1 was found to associate in coprecipitation studies with endogenous MAP kinase. These studies implicate MAP kinase as one of the kinases capable of phosphorylating and regulating IRS-1 tyrosine phosphorylation.

Animals↗

PubMed1997

Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612.

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.

Amino Acid Sequence↗

PubMed1997

Neither platelet activating factor nor leukotrienes are critical mediators of liver injury after lipopolysaccharide administration.

The intravenous administration of lipopolysaccharide (LPS) to rats results in liver lesions characterized by the infiltration of both platelets and neutrophils into the liver and by midzonal hepatocellular necrosis. The liver injury is dependent on neutrophils, platelets and the coagulation system, as removal or inhibition of any of these components inhibits the development of hepatocellular necrosis. Platelet activating factor (PAF) and the cysteinyl leukotrienes (LTs) are potent inflammatory lipids that are critical for the development of some LPS-mediated alterations. To test the hypothesis that PAF, alone or in combination with LTs, contributes to the development of liver injury during LPS exposure, we conducted studies with the PAF receptor antagonist, WEB 2086, and the 5-lipoxygenase inhibitor, Zileuton. Female, Sprague-Dawley rats were pretreated with WEB 2086 (10 mg/kg, i.p.) alone or with Zileuton (40 mg/kg, p.o.) 1 h before the administration of LPS (4 mg/kg, i.v.) or its saline vehicle. Treatment with WEB 2086, alone or in combination with Zileuton, did not inhibit LPS-mediated hepatic neutrophil infiltration or liver injury, as assessed by histologic evaluation and increases in plasma alanine aminotransferase activity. Pretreatment with these agents also had no effect on the activation of the coagulation system or on the thrombocytopenia induced by LPS. These results suggest that PAF, alone or in combination with 5-lipoxygenase products, is not a critical mediator of LPS-induced hepatocellular injury in this model.

Administration, Oral↗

PubMed1997

Juxtamembrane tyrosine residues couple the Eph family receptor EphB2/Nuk to specific SH2 domain proteins in neuronal cells.

Eph-related receptor tyrosine kinases have been implicated in the control of axonal navigation and fasciculation. To investigate the biochemical mechanisms underlying such functions, we have expressed the EphB2 receptor (formerly Nuk/Cek5/Sek3) in neuronal NG108-15 cells, and have observed the tyrosine phosphorylation of multiple cellular proteins upon activation of EphB2 by its ligand, ephrin-B1 (formerly Elk-L/Lerk2). The activated EphB2 receptor induced the tyrosine phosphorylation of a 62-64 kDa protein (p62[dok]), which in turn formed a complex with the Ras GTPase-activating protein (RasGAP) and SH2/SH3 domain adaptor protein Nck. RasGAP also bound through its SH2 domains to tyrosine-phosphorylated EphB2 in vitro, and complexed with activated EphB2 in vivo. We have localized an in vitro RasGAP-binding site to conserved tyrosine residues Y604 and Y610 in the juxtamembrane region of EphB2, and demonstrated that substitution of these amino acids abolishes ephrin-B1-induced signalling events in EphB2-expressing NG108-15 cells. These tyrosine residues are followed by proline at the + 3 position, consistent with the binding specificity of RasGAP SH2 domains determined using a degenerate phosphopeptide library. These results identify an EphB2-activated signalling cascade involving proteins that potentially play a role in axonal guidance and control of cytoskeletal architecture.

Adaptor Proteins, Signal Transducing↗

PubMed1997

C5a-dependent up-regulation in vivo of lung vascular P-selectin.

Acute lung injury in rats following systemic activation of complement by i.v. infusion of cobra venom factor (CVF) is known to be P-selectin dependent. In the current studies, infusion of CVF caused the appearance in plasma of C5a (as revealed by ELISA analysis) together with neutrophil chemotactic activity, which was totally blocked by addition in vitro of anti-rat C5a. Using a detector 125I-labeled antibody to rat P-selectin, we have demonstrated quantitative up-regulation in vivo of lung vascular P-selectin in a time-dependent manner after infusion of CVF. This up-regulation was almost completely blocked by prior complement depletion or by the infusion of anti-rat C5a. Platelet depletion did not affect CVF-induced up-regulation of lung vascular P-selectin, indicating that platelets were not the source of P-selectin. These results demonstrate that complement and, specifically, C5a are necessary for up-regulation of lung vascular P-selectin after systemic activation of complement.

Animals↗