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PubMed2004

Bile acids regulate gluconeogenic gene expression via small heterodimer partner-mediated repression of hepatocyte nuclear factor 4 and Foxo1.

Bile acid homeostasis is tightly controlled by the feedback mechanism in which an atypical orphan nuclear receptor (NR) small heterodimer partner (SHP) inactivates several NRs such as liver receptor homologue-1 and hepatocyte nuclear factor 4. Although NRs have been implicated in the transcriptional regulation of gluconeogenic genes, the effect of bile acids on gluconeogenic gene expression remained unknown. Here, we report that bile acids inhibit the expression of gluconeogenic genes, including glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase, and fructose 1,6-bis phosphatase in an SHP-dependent fashion. Cholic acid diet decreased the mRNA levels of these gluconeogenic enzymes, whereas those of SHP were increased. Reporter assays demonstrated that the promoter activity of phosphoenolpyruvate carboxykinase and fructose 1,6-bis phosphatase via hepatocyte nuclear factor 4, or that of G6Pase via the forkhead transcription factor Foxo1, was down-regulated by treatment with chenodeoxicholic acid and with transfected SHP. Remarkably, Foxo1 interacted with SHP in vivo and in vitro, which led to the repression of Foxo1-mediated G6Pase transcription by competition with a coactivator cAMP response element-binding protein-binding protein. These findings reveal a novel mechanism by which bile acids regulate gluconeogenic gene expression via an SHP-dependent regulatory pathway.

Animals↗
PubMed2006

Structural basis and functional consequence of Helicobacter pylori CagA multimerization in cells.

Helicobacter pylori cagA-positive strains are associated with gastric adenocarcinoma. The cagA gene product CagA is delivered into gastric epithelial cells where it localizes to the plasma membrane and undergoes tyrosine phosphorylation at the EPIYA-repeat region, which contains the EPIYA-A segment, EPIYA-B segment, and Western CagA-specific EPIYA-C or East Asian CagA-specific EPIYA-D segment. In host cells, CagA specifically binds to and deregulates SHP-2 phosphatase via the tyrosine-phosphorylated EPIYA-C or EPIYA-D segment, thereby inducing an elongated cell shape known as the hummingbird phenotype. In this study, we found that CagA multimerizes in cells in a manner independent of its tyrosine phosphorylation. Using a series of CagA mutants, we identified a conserved amino acid sequence motif (FPLXRXXXVXDLSKVG), which mediates CagA multimerization, within the EPIYA-C segment as well as in a sequence that located immediately downstream of the EPIYA-C or EPIYA-D segment. We also found that a phosphorylation-resistant CagA, which multimerizes but cannot bind SHP-2, inhibits the wild-type CagA-SHP-2 complex formation and abolishes induction of the hummingbird phenotype. Thus, SHP-2 binds to a preformed and tyrosinephosphorylated CagA multimer via its two Src homology 2 domains. These results, in turn, indicate that CagA multimerization is a prerequisite for CagA-SHP-2 interaction and subsequent deregulation of SHP-2. The present work raises the possibility that inhibition of CagA multimerization abolishes pathophysiological activities of CagA that promote gastric carcinogenesis.

Amino Acid Motifs↗
PubMed2006

A discoidin domain receptor 1/SHP-2 signaling complex inhibits alpha2beta1-integrin-mediated signal transducers and activators of transcription 1/3 activation and cell migration.

Regulation of cell migration is an important step for the development of branching tubule morphogenesis in collagen gel. Here, we showed that discoidin domain receptor (DDR) 1a/b inhibited collagen-induced tyrosine phosphorylation of signal transducers and activators of transcription (Stat) 1/3 and cell migration triggered by alpha2beta1-integrin. Overexpression of DDR1a/b increased the interaction of DDR1 with SHP-2 and up-regulated the tyrosine phosphatase activity of SHP-2. Expression of catalytically inactive SHP-2 in DDR1-transfected cells restored the tyrosine phosphorylation of Stat3 and cell migration. We demonstrated that the Src homology-2 (SH2)-SH2 and phosphotyrosyl phosphatase (PTP) domains of SHP-2 were responsible for interaction with DDR1 and that both tyrosine phosphorylation sites 703 and 796 of DDR1 were essential for it to bind with SHP-2. Mutation of tyrosine 703 or 796 of DDR1 abolished the ability of DDR1 to inhibit the tyrosine phosphorylation of Stat1 and Stat3 and restored collagen-induced cell migration and hepatocyte growth factor-induced branching tubulogenesis in collagen gel. Together, these results demonstrate that SHP-2 is required for the DDR1-induced suppression of Stat1 and Stat3 tyrosine phosphorylation, cell migration, and branching tubulogenesis.

3T3 Cells↗
PubMed1993

Duodenal findings on ultrasound in children with Schonlein-Henoch purpura and gastrointestinal symptoms.

Abdominal ultrasonic examination was performed in 14 patients with Schonlein-Henoch purpura (SHP). Thickening of the duodenal wall was observed in nine (82%) of the 11 with such gastrointestinal (GI) symptoms as severe abdominal pain or bleeding. The thickened duodenal wall showed a high echogenicity. Enlargement of the duodenal lumen was seen in seven (64%) patients with GI symptoms. These findings had been observed in four patients before SHP was diagnosed on the basis of the peculiar skin lesions. In three cases of SHP without GI symptoms, those changes were absent. Four cases of ulcerative colitis, three of bacterial enterocolitis (two Yersinia and one Klebsiella), and five without SHP and any GI problems did not exhibit such duodenal abnormalities. On subsequent endoscopic study, mucosal edema and multiple hemorrhagic erosions were seen, especially at the second portion of the duodenum in two cases of SHP. Biopsy specimens from the duodenum of those cases showed leukocytoclastic vasculitis, suggested by the ultrasound (US) findings. It is important to consider the duodenal changes carefully when US is performed in patients with severe GI symptoms of unknown origin. The characteristic duodenal findings described suggest the differential diagnosis of SHP, which usually requires no surgical intervention.

Adolescent↗
PubMed1994

Heterosexual transmission of hepatitis C virus and the possible role of coexistent human immunodeficiency virus infection in the index case. A multicentre study of 423 pairings.

OBJECTIVES: To define the role that sexual transmission plays in the spread of hepatitis C virus (HCV) infection, and to examine the influence of coexistent human immunodeficiency virus (HIV) infection on this mode of transmission. DESIGN: A multicentre, seroprevalence study of anti-HCV performed in the stable heterosexual partners (SHP) of index cases reactive for anti-HCV. SETTING: Department of Internal Medicine and Section of Gastroenterology of three University Hospitals, Spain. SUBJECTS: A total of 423 stable heterosexual partners of index cases reactive for anti-HCV. This included a group of 142 intravenous drug users (IVDU), 120 of whom were coinfected with HIV. Additionally, 2886 first-time voluntary blood donors selected at random were included to compare the prevalence of anti-HCV. MAIN OUTCOME MEASURES: Serum samples were screened for anti-HCV by a commercially available, second-generation enzyme-linked immunoassay. Tests repeatedly reactive for anti-HCV were analysed by a four-antigen, recombinant immunoblot assay. Anti-HIV was tested by enzyme immunoassay and Western blot was used for confirmation of positive cases. RESULTS: The prevalence of anti-HCV, was 7.1% in SHP and 1.2% in random donors (P < 0.001). This prevalence was higher in SHP of index cases coinfected with HIV in comparison with that shown in the SHP of index cases only reactive for the anti-HCV (9.1 vs. 6.3%; P = 0.2), particularly when a younger and more homogeneous group such as the SHP of IVDU index cases was considered alone (9.2 vs. 0%; P = 0.1). However, the SHP of IVDU index cases coinfected with HIV were almost three times more likely to be infected with HIV than HCV (24.2 vs. 9.2%). CONCLUSIONS: These data suggest that HCV infection may be sexually transmitted but with low efficiency, and this could be increased in the presence of coexistent HIV infection in the index case.

Adolescent↗
PubMed2006

Rifampicin induction of CYP3A4 requires pregnane X receptor cross talk with hepatocyte nuclear factor 4alpha and coactivators, and suppression of small heterodimer partner gene expression.

Bile acids and drugs activate pregnane X receptor (PXR) to induce CYP3A4, which is the predominant cytochrome P450 enzyme expressed in the liver and intestine and plays a critical role in detoxifying bile acids and drugs, and protecting against cholestasis. The aim of this study is to investigate the molecular mechanism of PXR cross talk with other nuclear receptors and coactivators in regulating human CYP3A4 gene transcription. Rifampicin dose dependently induced the CYP3A4 but inhibited small heterodimer partner (SHP) mRNA expression levels in primary human hepatocytes. Rifampicin strongly stimulated PXR and hepatocyte nuclear factor 4alpha (HNF4alpha) interaction, and CYP3A4 reporter activity, which was further stimulated by peroxisome proliferators-activated receptorgamma co-activator 1alpha (PGC-1alpha) and steroid receptor coactivator-1 (SRC-1) but inhibited by SHP. Mutation of the putative HNF4alpha binding site in the distal xenobiotic responsive element module did not affect CYP3A4 basal promoter activity and synergistic stimulation by PXR and HNF4alpha. Chromatin immunoprecipitation assays revealed that rifampicin-activated PXR recruited HNF4alpha and SRC-1 to the CYP3A4 chromatin. On the other hand, SHP reduced PXR recruitment of HNF4alpha and SRC-1 to the CYP3A4 chromatin. The human SHP promoter was stimulated by HNF4alpha and PGC-1alpha. Upon activation by rifampicin, PXR inhibited SHP promoter activity. Results suggest that PXR strongly induces CYP3A4 gene transcription by interacting with HNF4alpha, SRC-1, and PGC-1alpha. PXR concomitantly inhibits SHP gene transcription and maximizes the PXR induction of the CYP3A4 gene in human livers. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs.

Antibiotics, Antitubercular↗
PubMed2002

Identification and characterization of a novel heme-associated cell surface protein made by Streptococcus pyogenes.

Analysis of the genome sequence of a serotype M1 group A Streptococcus (GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in Escherichia coli and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The shp gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein.

Animals↗
PubMed2002

Induction of short heterodimer partner 1 precedes downregulation of Ntcp in bile duct-ligated mice.

Cholestasis is associated with retention of bile acids and reduced expression of the Na(+)/taurocholate cotransporter (Ntcp), the major hepatocellular bile acid uptake system. This study aimed to determine whether downregulation of Ntcp in obstructive cholestasis 1) is a consequence of bile acid retention and 2) is mediated by induction of the transcriptional repressor short heterodimer partner 1 (SHP-1). To study the time course for the changes in serum bile acid levels as well as SHP-1 and Ntcp steady-state mRNA levels, mice were subjected to common bile duct ligation (CBDL) for 3, 6, 12, 24, 72, and 168 h and compared with sham-operated controls. Serum bile acid levels were determined by radioimmunoassay. SHP-1 and Ntcp steady-state mRNA expression were assessed by Northern blotting. In addition, Ntcp protein expression was studied by Western blotting and immunofluorescence microscopy. Increased SHP-1 mRNA expression paralleled elevations of serum bile acid levels and was followed by downregulation of Ntcp mRNA and protein expression in CBDL mice. Maximal SHP-1 mRNA expression reached a plateau phase after 6-h CBDL (12-fold; P < 0.001) and preceded the nadir of Ntcp mRNA levels (12%, P < 0.001) by 6 h. In conclusion, bile acid-induced expression of SHP-1 may, at least in part, mediate downregulation of Ntcp in CBDL mice. These findings support the concept that downregulation of Ntcp in cholestasis limits intracytoplasmatic accumulation of potentially toxic bile acids.

Animals↗
PubMed2006

Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes.

Growth hormone (GH) stimulates STAT5 phosphorylation by JAK2, which activates IGF-I and serine protease inhibitor 2.1 (Spi 2.1) transcription, whereas STAT5 dephosphorylation by protein tyrosine phosphatases (PTPs) terminates this signal. We hypothesized that the inhibitory effects of TNF on GH signaling and gene transcription were responsible for hepatic GH resistance. CWSV-1 hepatocytes were treated with TNF, pervanadate (a PTP inhibitor), or both, before GH stimulation. Total and tyrosine-phosphorylated JAK2, STAT5, ERK1/2, SHP-1 and SHP-2, IGF-I, and Spi 2.1 mRNA levels were measured. GH stimulated STAT5 and ERK1/2 phosphorylation, IGF-I, and Spi 2.1 mRNA expression. TNF attenuated JAK2/STAT5 and ERK1/2 phosphorylation and IGF-I and Spi 2.1 mRNA expression following GH stimulation. SHP-1 and SHP-2 protein levels were unaltered by TNF or GH, and the GH-induced increase in SHP-1 PTP activity was not further increased by TNF. In TNF-treated cells, pervanadate restored STAT5 and ERK1/2 phosphorylation to control levels following GH stimulation but did not restore IGF-I or Spi 2.1 mRNA induction. Cells transfected with a Spi 2.1 promoter-luciferase vector demonstrate a 50-fold induction in luciferase activity following GH stimulation or cotransfection with a constitutively active STAT5 vector. TNF prevented the induction of Spi 2.1 promoter activity by GH and the STAT5 construct. We conclude that TNF does not inhibit GH activity by inducing SHP-1 or -2 expression and that correction of GH signaling defects in TNF-treated cells by pervanadate does not restore GH-induced gene expression. The inhibitory effects of TNF on GH-mediated gene transcription appear independent of STAT5 activity and previously identified abnormalities in JAK2/STAT5 signaling.

Animals↗
PubMed1996

Increased excretion of tumor necrosis factor alpha and interleukin 1 beta in urine from patients with IgA nephropathy and Schönlein-Henoch purpura.

Urinary proteins (5 mg/ml) collected from a group of 16 patients including 13 with IgA nephropathy and 3 with Schönlein-Henoch purpura (SHP) and from a control group consisting of 6 patients with diabetic nephropathy, 5 patients with hypertensive nephrosclerosis, and 5 healthy hospital staff members were studied for the contents of interleukins (IL) 1 beta, 2, 4, 6, and 12 and tumor necrosis factor alpha (TNF-alpha). Eleven patient with IgA nephropathy or SHP (11/16) but only 1 of the controls (1/16) had TNF-alpha activity in urinary proteins (p < 0.01). The IL-1 beta activity exhibited a similar tendency but to a lesser extent (10 of 16 patients with IgA nephropathy or SHP vs. 2 of 16 with other conditions, p < 0.05). Conversely, the detection rates of IL-2, IL-4, and IL-6 in both groups were not significantly different. IL-12 was not found in any of the samples from both groups. Sera and nonpurified urine samples from the same individuals were also measured for cytokines. IL-1 beta, IL-2, IL-4, and IL-12 were absent in all these samples, but TNF-alpha was found in four of the serum samples from patients with IgA nephropathy. Urinary proteins (2 mg/ml) were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereby peptides of 52, 49, 45, 34, 30, and 11 kD could be demonstrated in the patients with IgA nephropathy or SHP. Urinary proteins (200 micrograms/ml from patients with IgA nephropathy or SHP exerted a mitogen-like effect on the normal human mononuclear cells, as demonstrated by 3H-thymidine incorporation. In addition, these urinary proteins (400 micrograms/ml) enhanced the proliferative activity of the cultured rat glomerular mesangial cells. The exaggerated proliferation of rat glomerular mesangial cells exerted by urine proteins from 2 patients with active disease was markedly suppressed after treatment with glucocorticoids/cyclophosphamide. These results suggest that patients with IgA nephropathy or SHP can excrete excessive amounts of TNF-alpha and IL-1 beta in the urine. The inconsistent presence of these two cytokines in urine and serum may indicate that they can be produced locally and that they are implicated in the development of mesangial inflammation and glomerular damage.

Adolescent↗
PubMed2005

Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcgammaRIIA and alphaIIbbeta3.

The low-affinity IgG receptor, FcgammaRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcgammaRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017-78 caused the tyrosine phosphorylation of FcgammaRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCgamma2. These early events were dependent on Src family kinases but independent of either TxA(2) or the engagement of the alpha(IIb)beta(3) integrin. During the lag phase prior to platelet aggregation, FcgammaRIIA, Syk, LAT, and PLCgamma2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017-78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcgammaRIIA, Syk, LAT, and PLCgamma2 were rephosphorylated in an RGDS-sensitive, and therefore alpha(IIb)beta(3)-dependent, manner. Additionally, TxA(2) release, 5-hydroxytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited alpha(IIb)beta(3) -mediated re-phosphorylation. Therefore, S. sanguis -bound IgG cross links FcgammaRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of alpha(IIb)beta(3) leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA(2) release and consequent platelet aggregation.

Adaptor Proteins, Signal Transducing↗
PubMed1988

Influences of pressure surrounding the heart and intracardiac pressure on the diastolic coronary pressure-flow relation in excised canine heart.

We investigated the change in the instantaneous diastolic left coronary pressure-flow relation (DPFR) when the pressure surrounding the heart (SHP), right heart pressure (RHP), and left heart pressure (LHP) were systematically varied. Eight excised and maximally vasodilated canine hearts placed in an air-tight chamber were used. To obtain a capacitance-free DPFR, coronary perfusion pressure was slowly decreased (about 2 mm Hg/sec) during a prolonged diastole. The zero-flow pressure (Pf = 0) and the slope of the DPFR were analyzed. The mean values of the slope did not change significantly throughout the interventions. The mean value of Pf = 0 in the control state (SHP = RHP = LHP = 0 mm Hg) was 6.0 +/- 2.0 mm Hg (mean +/- SD, n = 8), significantly higher than the venous outflow pressure, RHP (p less than 0.001), and the other two pressures (p less than 0.001). When SHP was raised to 15 and 30 mm Hg, while the other pressures remained at 0 mm Hg, the mean values of Pf = 0 increased to 20.9 +/- 2.4 and 35.6 +/- 3.1 mm Hg (p less than 0.001 and p less than 0.0005, respectively, vs. control). The mean values of Pf = 0 when only RHP was elevated to 15 and 30 mm Hg were 16.0 +/- 1.5 and 29.3 +/- 1.5 mm Hg (p less than 0.001 and p less than 0.0005 vs. control). On elevation of LHP to 15 and 30 mm Hg, the mean values of Pf = 0 were 12.0 +/- 2.8 and 17.3 +/- 3.6 mm Hg (p less than 0.01 and p less than 0.01 vs. control). When both SHP and LHP were almost evenly elevated to about 15 and 30 mm Hg, the mean values of Pf = 0 were raised to 22.0 +/- 2.9 and 35.3 +/- 3.2 mm Hg, respectively. These mean values were not significantly different from those when only SHP was elevated to the comparable levels. The observation that Pf = 0 exceeded RHP in the control state and that RHP, which was elevated above the preceding Pf = 0, was identical with the present Pf = 0 supports the vascular waterfall mechanism when RHP is low. Furthermore, the evidence that the degree of DPFR shift was almost linearly dependent on the SHP level rather than on the LHP level indicates that the pressure on the epicardial side is one of the factors that determines the pressure at the top of the vascular waterfall.

Animals↗
PubMed2005

Functional analysis of leukemia-associated PTPN11 mutations in primary hematopoietic cells.

PTPN11 encodes the protein tyrosine phosphatase SHP-2, which relays signals from growth factor receptors to Ras and other effectors. Germline PTPN11 mutations underlie about 50% of Noonan syndrome (NS), a developmental disorder that is associated with an elevated risk of juvenile myelomonocytic leukemia (JMML). Somatic PTPN11 mutations were recently identified in about 35% of patients with JMML; these mutations introduce amino acid substitutions that are largely distinct from those found in NS. We assessed the functional consequences of leukemia-associated PTPN11 mutations in murine hematopoietic cells. Expressing an E76K SHP-2 protein induced a hypersensitive pattern of granulocyte-macrophage colony-forming unit (CFU-GM) colony growth in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3) that was dependent on SHP-2 catalytic activity. E76K SHP-2 expression also enhanced the growth of immature progenitor cells with high replating potential, perturbed erythroid growth, and impaired normal differentiation in liquid cultures. In addition, leukemia-associated SHP-2 mutations conferred a stronger phenotype than a germline mutation found in patients with NS. Mutant SHP-2 proteins induce aberrant growth in multiple hematopoietic compartments, which supports a primary role of hyperactive Ras in the pathogenesis of JMML.

Amino Acid Substitution↗
PubMed2002

The small heterodimer partner interacts with the liver X receptor alpha and represses its transcriptional activity.

The small heterodimer partner SHP (NR0B2) is an unusual nuclear receptor that lacks the typical DNA binding domain common to most nuclear receptors. SHP has been reported to act as a corepressor for several nuclear receptors, but its exact mechanism of action is still elusive. Here we show that SHP can interact with the liver X receptors LXRalpha (NR1H3) and LXRbeta (NR1H2), as demonstrated by glutathione-S-transferase pull-down assays, mammalian two-hybrid, and coimmunoprecipitation experiments. In transfection assays, SHP inhibits the expression of an artificial reporter driven by an LXR-response element and represses the transcriptional activation by LXR of the human ATP-binding cassette transporter 1 (ABCA1) promoter. Treatment of Caco-2 cells with bile acids, which activate farnesoid X receptor and subsequently induce SHP, leads to the repression of the human ABCG1 gene, an established LXR target gene. These results demonstrate that SHP is able to interact with LXR and to modulate its transcriptional activity.

Caco-2 Cells↗
PubMed2004

Interaction between insulin-like growth factor-I receptor and alphaVbeta3 integrin linked signaling pathways: cellular responses to changes in multiple signaling inputs.

Integrins are heterodimeric transmembrane proteins that mediate cell attachment to extracellular matrix, migration, division, and inhibition of apoptosis. Because growth factors are also important for these processes, there has been interest in cooperative signaling between growth factor receptors and integrins. IGF-I is an important growth factor for vascular cells. One integrin, alphaVbeta3, that is expressed in smooth muscle cells modulates IGF-I actions. Ligand occupancy of alphaVbeta3 is required for IGF-I to stimulate cell migration and division. Src homology 2 containing tyrosine phosphatase (SHP-2) is a tyrosine phosphatase whose recruitment to signaling molecules is stimulated by growth factors including IGF-I. If alphaVbeta3 ligand occupancy is inhibited, there is no recruitment of SHP-2 to alphaVbeta3 and its transfer to downstream signaling molecules is blocked. Ligand occupancy of alphaVbeta3 stimulates tyrosine phosphorylation of the beta3-subunit, resulting in recruitment of SHP-2. This transfer is mediated by an insulin receptor substrate-1-related protein termed DOK-1. Subsequently, SHP-2 is transferred to another transmembrane protein, SHPS-1. This transfer requires IGF-I receptor-mediated tyrosine phosphorylation of SHPS-1, which contains two YXXL motifs that mediate SHP-2 binding. The transfer of SHP-2 to SHPS-1 is also required for recruitment of Shc to SHPS-1. Ligand occupancy of alphaVbeta3 results in sustained Shc phosphorylation and enhanced Shc recruitment. Shc activation results in induction of MAPK. Inhibition of the Shc/SHPS-1 complex formation results in failure to achieve sustained MAPK activation and an attenuated mitogenic response. Thus, within the vessel wall, a mechanism exists whereby ligand occupancy of the alphaVbeta3 integrin is required for assembly of a multicomponent membrane signaling complex that is necessary for cells to respond optimally to IGF-I.

Animals↗
PubMed2006

An intracellular multi-effector complex mediates somatostatin receptor 1 activation of phospho-tyrosine phosphatase eta.

The receptor-like phosphotyrosine phosphatase eta (PTPeta) is an important intracellular effector of the cytostatic action of SST. Here we characterize, in Chinese hamster ovary-k1 cells, the intracellular pathway that from somatostatin receptor 1 (SSTR1), leads to the activation of PTPeta and that involves, in a multimeric complex and sequential activation, the tyrosine kinases Janus kinase (JAK) 2 and Src, and the cytosolic phosphotyrosine phosphatase SHP-2. We show that inhibitors of JAK2 and Src and dominant-negative mutants of SHP-2 and Src abolished the SSTR1-mediated PTPeta activation, suggesting that all these effectors participate in the activation of PTPeta. In basal conditions, JAK2 forms a multimeric complex with SHP-2, Src and PTPeta. In response to SST, JAK2 is activated in a G protein-dependent manner, dissociates from and phosphorylates SHP-2, increasing its activity. Subsequently, SHP-2 dissociates from Src, dephosphorylates the Src inhibitory tyrosine-529, and causes an autocatalytical increase of the phosphorylation of Src tyrosine 418, located inside its kinase activation loop. Active Src, in turn, controls the activity of PTPeta, via a direct interaction and phosphorylation of the phosphatase. These data for the first time depict an intracellular pathway involving a precise sequence of interactions and cross-activation among tyrosine phosphatases and kinases acting upstream of PTPeta. In particular the sequential activation of JAK2, SHP-2, and Src conveys the molecular signaling from SSTR1 to the activation of this phosphatase that is responsible for the final biological effects of SST.

Animals↗
PubMed1999

Differentiation-dependent prolactin responsiveness and stat (signal transducers and activators of transcription) signaling in rat ovarian cells.

PRL activates an important cytokine signaling cascade that is obligatory for maintaining luteal cell function in the rat ovary. To determine when specific components of this cascade are expressed and can be activated by PRL, we analyzed the expression of receptor subtypes (short, PRL-R(s), and long, PRL-R(L)), the presence and kinetics of Stat (signal transducer and activator of transcription) activation using the PRL-response element (PRL-RE) of the alpha2M (alpha2-macroglobulin) gene, and the content and hormonal regulation of three specific modulators of cytokine signaling; the tyrosine phosphatases (SHP-1 and SHP-2), and the protein inhibitor of activated Stat3 (PIAS-3). These components were analyzed in differentiating granulosa/ luteal cells of hypophysectomized (H) rats and in corpora lutea of pregnant rats. Levels of PRL-R mRNAs increased as granulosa cells differentiated and reached maximal levels in luteal cells of pregnant rats where levels of PRL-R(s) approached those of PRL-R(L). The relative concentrations shifted from a 27-fold excess of PRL-R(L) in preovulatory granulosa cells to a 3.7-fold difference in luteal cells during midgestation. Despite the increased PRL-R(L) expression in differentiated granulosa cells, PRL did not stimulate detectable activation of Stats. Rather PRL activation of Stat5, principally Stat5b, occurred in association with luteinization. In contrast, granulosa cells of untreated immature and H rats contained a high level of DNA binding activity, which was shown to be comprised entirely of activated, phosphorylated Stat3. Treatment with estrogen and FSH reduced the amount of phosphorylated Stat3 and abolished its ability to bind DNA, an effect temporally related to increased PIAS-3. Expression of SHP-1 (but not SHP-2) was also hormonally regulated; SHP-1 mRNA and protein were high in granulosa cells of H rats, decreased by estrogen and FSH, and subsequently increased dramatically with luteinization. Of particular note, SHP-1 was localized in cytoplasm of granulosa cells in atretic follicles but was distinctly nuclear in luteal cells, indicative of different functional roles. Collectively, these results indicate that Stat3 and Stat5 are activated by distinct cytokine-signaling pathways modulated through differentiation-dependent transcriptional regulation of signaling pathway components and mediate distinct functional processes in the rat ovary: early follicle growth and atresia vs. luteinization.

Animals↗
PubMed1999

Analysis of functional domains of angiotensin II type 2 receptor involved in apoptosis.

We previously demonstrated that the intracellular third loop (i3 loop) of angiotensin II type 2 receptor (AT2) plays a key role in mediating the biological functions of this receptor. To determine which residues are important for AT2 signaling, mutated receptors with serial deletions within the i3 loop were stably expressed in PC12 cells. Deletion of residues 240-244 within the intermediate portion of the i3 loop resulted in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases (ERK), and SHP-1 activation. In contrast to well characterized heptahelical receptors, the AT2 functions were not affected by deletions of the amino- or carboxyl-terminal portions of the i3 loop. Alanine substitutions further demonstrated that lysine 240, asparagine 242, and serine 243 are key residues for AT2-induced apoptosis, ERK inhibition, and SHP-1 activation. To examine whether a functional link exists between activation of SHP-1 and apoptosis, we used a catalytically inactive SHP-1 mutant and demonstrated that preventing SHP-1 activation strongly attenuates AT2-induced ERK inhibition and apoptosis. Our data demonstrate that the intermediate portion of the i3 loop is important for AT2 function and that SHP-1 is a proximal effector of the AT2 receptor that is implicated in the inhibition of ERKs and in the apoptotic effect of this receptor.

Alanine↗