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PubMed2000

Factor VIIa and thrombin induce the expression of Cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor VIIa x tissue factor-induced signal transduction.

Extracellular interactions of plasma clotting factor VIIa (FVIIa) with tissue factor (TF) on cell surfaces trigger the intracellular signaling events. At present, it is unclear how these signals influence phenotype. To elucidate this, we have used cDNA microarray technology to examine changes in transcriptional program in human fibroblasts in response to exposure to FVIIa. cDNA microarrays revealed that FVIIa binding to TF up-regulated the expression of Cyr61 and CTGF (connective tissue growth factor), the genes that encode extracellular matrix signaling proteins Cyr61 and CTGF, respectively. Northern blot analysis confirmed that FVIIa binding to TF markedly increased the expression of Cyr61 and CTGF in a time- and dose-dependent manner. FVIIa catalytic activity is required for the gene induction. In addition to FVIIa, thrombin also induced the expression of Cyr61 and CTGF. Hirudin abolished the thrombin-induced expression of these mRNAs but not the FVIIa-induced expression. FVIIa-induced expression of Cyr61 appears not to involve the currently known protease-activated receptors (PARs), whereas thrombin-induced expression involves the activation of PAR1 and possibly an additional PAR. Various intracellular signaling pathway inhibitors exhibited different inhibitory pattern on FVIIa and thrombin-induced up-regulation of Cyr61. Cyr61 and CTGF could act as downstream mediators of FVIIa x TF in affecting various biological processes.

Cell Line↗

PubMed2000

Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling.

The mechanism of p53-mediated apoptosis after cellular stress remains poorly understood. Evidence suggests that p53 induces cell death by a multitude of molecular pathways involving activation of target genes and transcriptionally independent direct signaling. Mitochondria play a key role in apoptosis. We show here that a fraction of p53 protein localizes to mitochondria at the onset of p53-dependent apoptosis but not during p53-independent apoptosis or p53-mediated cell cycle arrest. The accumulation of p53 to mitochondria is rapid (within 1 h after p53 activation) and precedes changes in mitochondrial membrane potential, cytochrome c release, and procaspase-3 activation. Immunoelectron microscopy and immuno-fluorescence-activated cell sorter analysis of isolated mitochondria show that the majority of mitochondrial p53 localizes to the membranous compartment, whereas a fraction is found in a complex with the mitochondrial import motor mt hsp70. After induction of ectopic p53 without additional DNA damage in p53-deficient cells, p53 again partially localizes to mitochondria, preceding the onset of apoptosis. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL abrogates stress signal-mediated mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback signaling loop between p53 and mitochondrial apoptotic regulators. Importantly, bypassing the nucleus by targeting p53 to mitochondria using import leader fusions is sufficient to induce apoptosis in p53-deficient cells. We propose a model where p53 can contribute to apoptosis by direct signaling at the mitochondria, thereby amplifying the transcription-dependent apoptosis of p53.

Apoptosis↗

PubMed2001

Estradiol-induced nongenomic calcium signaling regulates genotropic signaling in macrophages.

Estradiol (E(2)) exerts not only genotropic but also nongenomic actions through nuclear estrogen receptors (ER). Here, we provide a novel paradigm for nongenomic E(2) signaling independent of nuclear ER. E(2) induces a rapid rise in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) through membrane estrogen receptors in murine RAW 264.7 macrophages. This E(2)-induced Ca(2+) signaling is not prevented by different ER blockers and cannot directly activate stably transfected c-fos promoter or the mitogen-activated protein kinases p38, ERK1/2, and SAPK/JNK, or NO production. However, the E(2)-induced rise in [Ca(2+)](i) specifically down-regulates the serum-stimulated activation of c-fos promoter and ERK1/2, and conversely, it specifically up-regulates lipopolysaccharide-stimulated activation of c-fos promoter, p38, and NO production. The E(2)-changed activation of c-fos promoter can be prevented by an intracellular Ca(2+) chelator. Our data indicate that E(2)-induced nongenomic Ca(2+) signaling through membrane ER is able to specifically modulate genotropic signaling pathways with impact on macrophage activation.

Animals↗

PubMed2004

Suppressors of cytokine signaling 4 and 5 regulate epidermal growth factor receptor signaling.

Suppressors of cytokine signaling (SOCS) are Src homology-2-containing proteins originally identified as negative regulators of cytokine signaling. Accumulating evidence indicates a role for SOCS proteins in the regulation of additional signaling pathways including receptor tyrosine kinases. Notably, SOCS36E, the Drosophila ortholog of mammalian SOCS5, was recently implicated as a negative regulator of the Drosophila ortholog of EGFR. In this study, we aimed at characterizing the role of SOCS5 in the negative regulation of EGFR. Here we show that the expression of SOCS5 and its closest homolog SOCS4 is elevated in cells following treatment with EGF, similar to several negative feedback regulators of EGFR whose expression is up-regulated upon receptor activation. The expression of SOCS5 led to a marked reduction in EGFR expression levels by promoting EGFR degradation. The reduction in EGFR levels and EGF-induced signaling in SOCS5-expressing cells requires both the Src homology-2 and SOCS box domains of SOCS5. Interestingly, EGFR is degraded by SOCS5 prior to EGF treatment in a ligand- and c-Cbl-independent manner. SOCS5 can associate with EGFR and can also bind the ElonginBC protein complex via its SOCS box, which may recruit an E3 ubiquitin ligase to promote EGFR degradation. Thus, we have characterized a novel function for SOCS5 in regulating EGFR and discuss its potential role in controlling EGFR homeostasis.

Animals↗

PubMed2005

Sustained extracellular signal-regulated kinase1/2 mitogen-activated protein kinase signalling is related to increased p21 expression in cholesteatoma epithelium.

CONCLUSION: These results show for the first time that the RAS/RAF/ERK1/2 MAPK signalling pathway is active and involved in p21-mediated cell cycle arrest in human cholesteatoma epithelium. OBJECTIVE: In a previous report we have demonstrated that the epithelium in human cholesteatoma is characterized by high p53-dependent p21 expression. The RAS/RAF/extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) signalling pathway can induce p21 expression and subsequent cell cycle arrest via p53-dependent or -independent mechanisms. We designed the present study to investigate whether the RAS/RAF/ERK1/2 MAPK signalling pathway is involved in p53-dependent and p21-mediated cell cycle arrest in human cholesteatoma. MATERIAL AND METHODS: A total of 18 cholesteatoma samples and 18 paired control retro-auricular skin samples were immunohistochemically stained for p53, p21, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2. Positive cells were counted by means of digital image analysis. Double-label fluorescence immunohistochemistry was performed to demonstrate co-expression of p21 and pERK1/2. RESULTS: Protein expression of p53, p21 and pERK1/2 differed significantly between cholesteatoma epithelium and retro-auricular skin (p <0.01). In cholesteatoma, co-expression of p21 and pERK1/2 was prominent, whereas in retro-auricular skin there was hardly any co-expression. Positive correlations were found between p53 and p21 (p =0.003) and between p21 and pERK1/2 (p =0.013).

Antibodies, Monoclonal↗

PubMed2006

Hepatic growth hormone resistance during sepsis is associated with increased suppressors of cytokine signaling expression and impaired growth hormone signaling.

OBJECTIVE: During sepsis, a two- to four-fold increase in circulating growth hormone (GH) is seen with 40-50% reductions in plasma insulin-like growth factor (IGF)-I. The suppressors of cytokine signaling (SOCS), inhibitors of cytokine, and growth factor signaling via the janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway have been implicated in the development of hepatic GH resistance. In this study we examine the effects of sepsis on GH-induced IGF-I expression and potential mechanisms for GH resistance. DESIGN: Prospective experimental study. SETTING: University research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were randomized to laparotomy alone (control) or implantation of fecal agar pellets inoculated with Escherichia coli and Bacteroides fragilis (sepsis). GH was injected intravenously to assess hepatic IGF-I synthesis and GH signaling. MEASUREMENTS AND MAIN RESULTS: Plasma IGF-I was measured in both groups at baseline (4 hrs postoperatively) and then again at 12 hrs and 24 hrs after GH administration. Basal IGF-I levels were similar in both groups, but controls had a 35% increase in IGF-I at 12 hrs, whereas septic rats demonstrated reductions in circulating IGF-I at 12 and 24 hrs after GH. Hepatic expression of SOCS-1, -2, -3, and cytokine-inducible SH2-containing protein (CIS) were determined at 1, 4, 8, and 24 hrs in septic and control rats by Northern blot. SOCS-1, SOCS-3, and CIS messenger RNA in liver were increased from 4 to 8 hrs after the induction of sepsis (p < .05 for SOCS-1 and -3). Total GH receptor (GHR), JAK2, and STAT5 signaling proteins and the time course of STAT5 activation were also measured in liver after recombinant human GH administration by immunoblot and electrophoretic mobility shift analysis. Levels of total GHR, JAK2, and STAT5 were unaltered in liver from septic rats. However, phosphorylated STAT5 and STAT5 DNA binding were significantly reduced 30 mins after GH administration in liver from septic rats. CONCLUSIONS: Sepsis diminished STAT5 phosphorylation and activity in liver as well as plasma IGF-I following GH administration. Hepatic messenger RNA expression of SOCS-1, SOCS-3, and CIS was transiently increased during abdominal sepsis and temporally associated with the development of hepatic GH resistance.

Analysis of Variance↗

PubMed2006

4-Hydroxy-2-nonenal adduction of extracellular signal-regulated kinase (Erk) and the inhibition of hepatocyte Erk-Est-like protein-1-activating protein-1 signal transduction.

4-Hydroxy-2-nonenal (4-HNE) is a major lipid peroxidation (LPO) product formed during oxidative stress. 4-HNE is highly reactive toward cellular nucleophiles and is implicated in the evolution of numerous pathologies associated with oxidative stress and LPO. Recent evidence suggests that chronic prooxidant exposure results in the loss of extracellular signal-regulated kinase (Erk)-1/2 phosphorylation in vivo, a signaling pathway associated with cellular proliferation, survival, and homeostasis. Immunodetection and molecular analysis were used in this study to evaluate the hypothesis that 4-HNE modification of Erk-1/2 inhibits constitutive Erk-Est-like protein (Elk)-1-activating protein (AP)-1 signaling. Primary rat hepatocytes treated with subcytotoxic, pathologically relevant concentrations of 4-HNE demonstrated a concentration-dependent loss of constitutive Erk-1/2 phosphorylation, activity, and nuclear localization. These findings were consistent with iron-induced intracellular LPO, which also resulted in a concentration-dependent decrease in hepatocyte Erk-1/2 phosphorylation and activity. 4-HNE and iron-induced inhibition of Erk-1/2 was inversely correlated with the accumulation of 4-HNE-Erk-1/2 monomer adducts. 4-HNE treatment of hepatocytes decreased nuclear total and phosphorylated Erk-1/2, Elk-1, and AP-1 phosphorylation as well as cFos and cJun activities. The cytosolic modification of unphosphorylated Erk-1/2 was evaluated in vitro using molar ratios of inactive Erk-2 to 4-HNE consistent with increasing oxidative stress in vivo. Liquid chromatography combined with tandem mass spectrometry confirmed monomer adduct formation and identified the major adduct species at the histidine 178 residue within the kinase phosphorylation lip. These novel results show that the formation of 4-HNE-Erk-1/2 monomer-adducts results in the inhibition of Erk-Elk-AP-1 signaling in hepatocytes and implicates the His 178 residue with the mechanism of inhibition.

Aldehydes↗

PubMed2006

Gene 33/RALT is induced by hypoxia in cardiomyocytes, where it promotes cell death by suppressing phosphatidylinositol 3-kinase and extracellular signal-regulated kinase survival signaling.

Ischemia in the heart deprives cardiomyocytes of oxygen, triggering cell death (myocardial infarction). Ischemia and its cell culture model, hypoxia, elicit a stress response program that contributes to cardiomyocyte death; however, the molecular components required to promote this process remain nebulous. Gene 33 is a 50-kDa cytosolic adapter protein that suppresses signaling from receptor Tyr kinases of the epidermal growth factor receptor/ErbB family. Here we show that adenoviral expression of Gene 33 swiftly stimulates cardiomyocyte death coincident with reduced Akt and extracellular signal-regulated kinase (ERK) signaling. Subjecting cardiomyocytes to hypoxia and then reoxygenation induces gene 33 mRNA and Gene 33 protein. RNA interference experiments indicate that endogenous Gene 33 reduces Akt and ERK signaling and is required for maximal hypoxia-induced cardiomyocyte death. Gene 33 levels are also strikingly increased in myocardial ischemic injury and infarction. Our results identify a new role for Gene 33 as a component in the molecular pathophysiology of ischemic injury.

Adenoviridae↗

PubMed1998

Tumor promoter arsenite activates extracellular signal-regulated kinase through a signaling pathway mediated by epidermal growth factor receptor and Shc.

Although arsenite is an established carcinogen, the mechanisms underlying its tumor-promoting properties are poorly understood. Previously, we reported that arsenite treatment leads to the activation of the extracellular signal-regulated kinase (ERK) in rat PC12 cells through a Ras-dependent pathway. To identify potential mediators of the upstream signaling cascade, we examined the tyrosine phosphorylation profile in cells exposed to arsenite. Arsenite treatment rapidly stimulated tyrosine phosphorylation of several proteins in a Ras-independent manner, with a pattern similar to that seen in response to epidermal growth factor (EGF) treatment. Among these phosphorylated proteins were three isoforms of the proto-oncoprotein Shc as well as the EGF receptor (EGFR). Tyrosine phosphorylation of Shc allowed for enhanced interactions between Shc and Grb2 as identified by coimmunoprecipitation experiments. The arsenite-induced tyrosine phosphorylation of Shc, enhancement of Shc and Grb2 interactions, and activation of ERK were all drastically reduced by treatment of cells with either the general growth factor receptor poison suramin or the EGFR-selective inhibitor tyrphostin AG1478. Down-regulation of EGFR expression through pretreatment of cells with EGF also attenuated ERK activation and Shc tyrosine phosphorylation in response to arsenite treatment. These results demonstrate that the EGFR and Shc are critical mediators in the activation of the Ras/ERK signaling cascade by arsenite and suggest that arsenite acts as a tumor promoter largely by usurping this growth factor signaling pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Selenium regulates cyclooxygenase-2 and extracellular signal-regulated kinase signaling pathways by activating AMP-activated protein kinase in colon cancer cells.

Epidemiologic and experimental evidences indicate that selenium, an essential trace element, can reduce the risk of a variety of cancers. Protection against certain types of cancers, particularly colorectal cancers, is closely associated with pathways involving cyclooxygenase-2 (COX-2). We found that AMP-activated protein kinase (AMPK), which functions as a cellular energy sensor, mediates critical anticancer effects of selenium via a COX-2/prostaglandin E(2) signaling pathway. Selenium activated AMPK in tumor xenografts as well as in colon cancer cell lines, and this activation seemed to be essential to the decrease in COX-2 expressions. Transduction with dominant-negative AMPK into colon cancer cells or application of cox-2(-/-)-negative cells supported the evidence that AMPK is an upstream signal of COX-2 and inhibits cell proliferation. In HT-29 colon cancer cells, carcinogenic agent 12-O-tetradecanoylphorbol-13-acetate (TPA) activated extracellular signal-regulated kinase (ERK) that led to COX-2 expression and selenium blocked the TPA-induced ERK and COX-2 activation via AMPK. We also showed the role of a reactive oxygen species as an AMPK activation signal in selenium-treated cells. We propose that AMPK is a novel and critical regulatory component in selenium-induced cancer cell death, further implying AMPK as a prime target of tumorigenesis.

AMP-Activated Protein Kinases↗

PubMed2005

Flt3 tandem duplication mutations cooperate with Wnt signaling in leukemic signal transduction.

Activating Flt3 mutations occur in about 30% of patients with acute myeloid leukemia (AML), often as in-frame internal tandem duplication (ITD) at the juxtamembrane domain of the receptor. These mutations transform hematopoietic cell lines and primary mouse bone marrow. Here, we analyzed the interaction between oncogenic Flt3-ITD mutations and the Wingless-type (Wnt) signaling pathway in the myeloid progenitor cell line 32D. Microarray analyses revealed higher mRNA expression of Frizzled-4, a receptor for Wnt ligands in 32D/Flt3-ITD cells. Findings were verified by quantitative realtime reverse transcription-polymerase chain reaction (RT-PCR) and on the protein level. Compared with 32D/Flt3-WT (wild-type) cells, 32D/Flt3-ITD cells also showed greatly enhanced beta-catenin protein levels, irrespective of their exposure to Wnt3a, a ligand inducing the canonical Wnt signal transduction pathway. In addition, 5 of 7 AML samples with Flt3-ITD mutations expressed high beta-catenin protein levels, whereas patients with wild-type Flt3 did not. Also, Flt3-ITD induced enhanced T-cell factor (TCF)-dependent transcriptional activity and the induction of the Wnt target gene c-myc. In the presence of Flt3-WT or Flt3-ITD signaling, Wnt3a slightly increased 32D cell proliferation. However, transfection experiments with dominant-negative (dn) TCF4 revealed a strong dependence of Flt3-ITD-mediated clonogenic growth on TCF activity. Taken together, our results indicate that Flt3-ITD and Wnt-dependent signaling pathways synergize in myeloid transformation.

Animals↗

PubMed2005

Melanocortinergic modulation of cholecystokinin-induced suppression of feeding through extracellular signal-regulated kinase signaling in rat solitary nucleus.

Signals from the gut and hypothalamus converge in the caudal brainstem to control ingestive behavior. We have previously shown that phosphorylation of ERK1/2 in the solitary nucleus (NTS) is necessary for food intake suppression by exogenous cholecystokinin (CCK). Here we test whether this intracellular signaling cascade is also involved in the integration of melanocortin-receptor (MCR) mediated inputs to the caudal brainstem. Using fourth ventricular-cannulated rats and Western blotting of NTS tissue, we show that the MC4R agonist melanotan II (MTII) rapidly and dose-dependently increases phosphorylation of both ERK1/2 and cAMP response element-binding protein (CREB). Sequential administration of fourth ventricular MTII and peripheral CCK at doses that alone produced submaximal stimulation of pERK1/2 produced an additive increase. Prior fourth ventricular administration of the MC4R antagonist SHU9119 completely abolished the CCK-induced increases in pERK and pCREB and, in freely feeding rats, SHU9119 significantly increased meal size and satiety ratio. Prior administration of the MAPK kinase inhibitor U0126 abolished the capacity of MTII to suppress 2-h food intake and significantly decreased MTII-induced ERK phosphorylation in the NTS. Furthermore, pretreatment with the cAMP inhibitor, cAMP receptor protein-Rp isomer, significantly attenuated stimulation of pERK induced by either CCK or MTII. The results demonstrate that activation of the ERK pathway is necessary for peripheral CCK and central MTII to suppress food intake. The cAMP-->ERK-->CREB cascade may thus constitute a molecular integrator for converging satiety signals from the gut and adiposity signals from the hypothalamus in the control of meal size and food intake.

Animals↗

PubMed2005

Ontogeny of rapid estrogen-mediated extracellular signal-regulated kinase signaling in the rat cerebellar cortex: potent nongenomic agonist and endocrine disrupting activity of the xenoestrogen bisphenol A.

In addition to regulating estrogen receptor-dependent gene expression, 17beta-estradiol (E(2)) can directly influence intracellular signaling. In primary cultured cerebellar neurons, E(2) was previously shown to regulate growth and oncotic cell death via rapid stimulation of ERK1/2 signaling. Here we show that ERK1/2 signaling in the cerebellum of neonatal and mature rats was rapidly responsive to E(2) and during development to the environmental estrogen bisphenol A (BPA). In vivo dose-response analysis for each estrogenic compound was performed by brief (6-min) intracerebellar injection, followed by rapid fixation and phosphorylation-state-specific immunohistochemistry to quantitatively characterize changes in activated ERK1/2 (pERK) immunopositive cell numbers. Beginning on postnatal d 8, E(2) significantly influenced the number of pERK-positive cells in a cell-specific manner that was dependent on concentration and age but not sex. In cerebellar granule cells on postnatal d 10, E(2) or BPA increased pERK-positive cell numbers at low doses (10(-12) to 10(-10) M) and at higher (10(-7) to 10(-6) M) concentrations. Intermediate concentrations of either estrogenic compound did not modify basal ERK signaling. Rapid E(2)-induced increases in pERK immunoreactivity were specific to the ERK1/2 pathway as demonstrated by coinjection of the mitogen-activated, ERK-activating kinase (MEK)1/2 inhibitor U0126. Coadministration of BPA (10(-12) to 10(-10) M) with 10(-10) M E(2) dose-dependently inhibited rapid E(2)-induced ERK1/2 activation in developing cerebellar neurons. The ability of BPA to act as a highly potent E(2) mimetic and to also disrupt the rapid actions of E(2) at very low concentrations during cerebellar development highlights the potential low-dose impact of xenoestrogens on the developing brain.

Aging↗

PubMed2006

Phosphate stimulates matrix Gla protein expression in chondrocytes through the extracellular signal regulated kinase signaling pathway.

Whereas increasing evidence suggests that inorganic phosphate (Pi) may act as a signaling molecule in mineralization-competent cells, its mechanisms of action remain largely unknown. The aims of the present work were to determine whether Pi regulates expression of matrix Gla protein (MGP), a mineralization inhibitor, in growth plate chondrocytes and to identify the involved signaling pathways. Chondrogenic ATDC5 cells and primary growth plate chondrocytes were used. Messenger RNA and protein analyses were performed by quantitative PCR and Western blotting, respectively. The activation and role of MAPKs were, respectively, determined by Western blotting and the use of specific inhibitors. Immunohistological detection of ERK1/2 was performed in rib organ cultures from newborn mice. The results indicate that Pi markedly stimulates expression of MGP in ATDC5 cells and primary growth plate chondrocytes. Investigation of the involved intracellular signaling pathways reveals that Pi activates ERK1/2 in a cell-specific manner, because the stimulation was observed in ATDC5 and primary chondrocytes, MC3T3-E1 osteoblasts, and ST2 stromal cells, but not in L929 fibroblasts or C2C12 myogenic cells. Accordingly, immunohistological detection of ERK1/2 phosphorylation in rib growth plates revealed a marked signal in chondrocytes. Finally, a specific ERK1/2 inhibitor, UO126, blocks Pi-stimulated MGP expression in ATDC5 cells, indicating that ERK1/2 mediates, mainly, the effects of Pi. These data demonstrate, for the first time, that Pi regulates MGP expression in growth plate chondrocytes, thereby suggesting a key role for Pi and ERK1/2 in the regulation of bone formation.

Animals↗

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↗

PubMed2005

Suppressors of cytokine signaling regulate angiotensin II-activated Janus kinase-signal transducers and activators of transcription pathway in renal cells.

Suppressors of cytokine signaling (SOCS) family is constituted by cytokine-inducible proteins that modulate receptor signal transduction via tyrosine kinases, mainly the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. Differential SOCS expression was noted in renal cells that were incubated with inflammatory stimuli, but the role of SOCS in the pathogenesis of renal diseases is not yet well defined. Because angiotensin II (Ang II) plays a key role in renal disease, SOCS proteins were studied as a novel mechanism involved in the negative regulation of Ang II-mediated processes. Systemic Ang II infusion for 3 d increased the renal mRNA expression of SOCS-3 and SOCS-1. SOCS protein synthesis was found in glomerular mesangial area and tubules. In cultured mesangial cells and tubular epithelial cells, Ang II induced a rapid and transient SOCS-3 and SOCS-1 expression in parallel with JAK2 and STAT1 activation. In both cell types, overexpression of SOCS proteins prevented the STAT activation in response to Ang II. SOCS expression observed in Ang II-infused rats and in Ang II-stimulated cells was significantly inhibited by treatment with AT(1) but not AT(2) receptor antagonist and was attenuated in mesangial cells from AT(1a)-deficient mice, demonstrating the implication of AT(1) in those responses. In SOCS-3 knockdown studies, antisense oligonucleotides inhibited the expression of SOCS-3 and increased the Ang II-induced STAT activation and c-Fos/c-Jun expression, then resulting in a more severe renal damage. These results suggest that SOCS proteins may act as negative regulators of Ang II signaling in renal cells and implicate SOCS as important modulators of renal damage.

Angiotensin II↗

PubMed2006

Ligation of CD28 stimulates the formation of a multimeric signaling complex involving grb-2-associated binder 2 (gab2), SRC homology phosphatase-2, and phosphatidylinositol 3-kinase: evidence that negative regulation of CD28 signaling requires the gab2 pleckstrin homology domain.

Grb-2-associated binder (Gab)2 is a scaffolding adaptor protein that has been reported to promote growth factor and cytokine receptor signal transduction, but inhibit TCR-mediated signaling events. In this study, we show that ligation of CD28 by its natural ligand B7-1/CD80, induces tyrosine phosphorylation of Gab2 and its coassociation with Src homology phosphatase (SHP)-2 and class IA PI3K in Jurkat cells. Overexpression of wild-type Gab2 revealed a negative role in regulation of CD3/CD28 induction of the transcription factors NF-kappaB and AP-1. To characterize this inhibitory function further, we used Gab2 mutants unable to bind either PI3K or SHP-2 and a PH domain deletion mutant. Although PI3K has previously been implicated as necessary for Gab2-mediated inhibition of TCR signaling, Gab2 mutants defective in their ability to bind PI3K or SHP-2 retained their inhibitory function, whereas deletion of the PH domain ablated the inhibitory effect of Gab2. Together, these data demonstrate that CD28 stimulation of T cells is sufficient to induce an inhibitory multimeric signaling complex involving Gab2, SHP-2, and PI3K. Furthermore, the inhibitory capacity of Gab2 is strictly dependent upon the integrity of its PH domain, suggesting phosphoinositide-mediated membrane recruitment is important to Gab2 function in T cells.

Adaptor Proteins, Signal Transducing↗

PubMed1991

Signal peptide analogs derived from two chloroplast precursors interact with the signal recognition system of the chloroplast envelope.

We have used synthetic peptides representing segments of the signal sequences of preferredoxin (pFd) and the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pS) to study interactions with the signal sequence recognition system at the chloroplast surface. Peptides representing the COOH-terminal 30 amino acids of the pFd and pS signal peptides were able to completely and reversibly inhibit the import of their homologous precursors into isolated chloroplasts at a 2.5 microM concentration. Import was blocked at the level of precursor binding to the chloroplast. This inhibition of precursor binding and import was not due to disruption of chloroplast integrity as incubation of isolated chloroplasts with the peptides did not cause measurable perturbation of the envelope membranes. The peptides also were able to block the import of the heterologous precursor protein, suggesting that pS and pFd share a common signal sequence recognition system. Visualization of the bound peptides at the chloroplast surface by indirect immunofluorescence microscopy using antipeptide antibodies gave a marked punctate staining pattern. This pattern is consistent with the localization of chloroplast import receptor(s) at contact zones between the inner and outer envelope membranes.

Amino Acid Sequence↗