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PubMed2005

Control of dendritic arborization by the phosphoinositide-3'-kinase-Akt-mammalian target of rapamycin pathway.

The molecular mechanisms that determine the size and complexity of the neuronal dendritic tree are unclear. Here, we show that the phosphoinositide-3' kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) signaling pathway promotes the growth and branching of dendrites in cultured hippocampal neurons. Constitutively active mutants of Ras, PI3K, and Akt, or RNA interference (RNAi) knockdown of lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome Ten), induced growth and elaboration of dendrites that was blocked by mTOR inhibitor rapamycin and/or by overexpression of eIF-4E binding protein 1 (4E-BP1), which inhibits translation of 5' capped mRNAs. The effect of PI3K on dendrites was lost in more mature neurons (>14 d in vitro). Dendritic complexity was reduced by inhibition of PI3K and by RNAi knockdown of mTOR or p70 ribosomal S6 kinase (p70S6K, an effector of mTOR). A rapamycin-resistant mutant of mTOR "rescued" the morphogenetic effects of PI3K in the presence of rapamycin. By regulating global and/or local protein translation, and as a convergence point for multiple signaling pathways, mTOR could play a central role in the control of dendrite growth and branching during development and in response to activity.

Animals↗
PubMed2004

Insulin regulates cleavage of procaspase-9 via binding of X chromosome-linked inhibitor of apoptosis protein in HT-29 cells.

Insulin significantly reduced tumor necrosis factor (TNF)-alpha-induced cleavage of procaspase-8, -9, and -3 and poly(ADP-ribose) polymerase when observed for up to 24 hours in a dose-dependent manner. Signaling pathways responsible for the inhibitory effects of insulin were investigated by using protein kinase inhibitors. Both phosphatidylinositol 3'-kinase (PI3K) and mitogen-activated protein kinase kinase pathways mediate the ability of insulin to decrease the TNF-alpha-induced cleavage of procaspase-8. In contrast, only the PI3K inhibitor reversed the effect of insulin on the TNF-alpha-induced cleavage of procaspase-9. Moreover, insulin decreased the apoptotic level induced by TNF-alpha, whereas the PI3K inhibitor enhanced it. The protein level of Apaf-1, an activator of procaspase-9, remained constant with the application of agents affecting the cleavage of procaspase-9. In examining another regulator of cleaved caspase-9, X chromosome-linked inhibitor of apoptosis protein (XIAP), we observed that TNF-alpha treatment induced fragmentation of XIAP, which was also enhanced by the PI3K inhibitor. In addition, XIAP was coimmunoprecipitated with procaspase-9. The treatment with TNF-alpha reduced the level of XIAP precipitated with procaspase-9, whereas insulin reversed this effect. Moreover, PI3K and Akt inhibitors, but not mammalian target of rapamycin inhibitor, inhibited the effect of insulin on the coprecipitation of procaspase-9 and XIAP. Our data suggest that insulin decreases the TNF-alpha-induced cleavage of procaspase-9 and subsequent apoptosis by regulating XIAP via the PI3K/Akt pathway.

Apoptosis↗
PubMed2003

Signalling pathways involved in the short-term potentiation of dopamine release by BDNF.

Brain-derived neurotrophic factor (BDNF) has been shown to modulate synaptic plasticity in the corpus striatum in vitro by activation of the tyrosine kinase linked receptor, TrkB. However, the signalling pathways that mediate this modulation of plasticity are poorly understood. Three proteins mediating signalling pathways are activated by the binding of BDNF to TrkB: phosphoinositol-3 kinase (PI3K); Ras-MEK and phospholipase C-gamma (PLCgamma). The present study investigates which of these pathways are necessary for BDNF-mediated potentiation of synaptic output of dopamine from slices and synaptosomes of rat corpus striatum. The results indicate that activation of the PI3K and Ras-MEK pathways, but not PLCgamma, are involved. Inhibitors of transcription and translation had no effect on the potentiation of depolarisation-stimulated (15 mM KCl) dopamine release mediated by BDNF.

Animals↗
PubMed2005

Involvement of PI3K and MAPK signaling in bcl-2-induced vascular endothelial growth factor expression in melanoma cells.

We have previously demonstrated that bcl-2 overexpression in tumor cells exposed to hypoxia increases the expression of vascular endothelial growth factor (VEGF) gene through the hypoxia-inducible factor-1 (HIF-1). In this article, we demonstrate that exposure of bcl-2 overexpressing melanoma cells to hypoxia induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2 proteins. On the contrary, no modulation of these pathways by bcl-2 was observed under normoxic conditions. When HIF-1alpha expression was reduced by RNA interference, AKT and ERK1/2 phosphorylation were still induced by bcl-2. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways reduced the induction of VEGF and HIF-1 in response to bcl-2 overexpression in hypoxia. No differences were observed between control and bcl-2-overexpressing cells in normoxia, in terms of VEGF protein secretion and in response to PI3K and MAPK inhibitors. We also demonstrated that RNA interference-mediated down-regulation of bcl-2 expression resulted in a decrease in the ERK1/2 phosphorylation and VEGF secretion only in bcl-2-overexpressing cell exposed to hypoxia but not in control cells. In conclusion, our results indicate, for the first time, that bcl-2 synergizes with hypoxia to promote expression of angiogenesis factors in melanoma cells through both PI3K- and MAPK-dependent pathways.

Apoptosis↗
PubMed1999

HIV-1 Nef plays an essential role in two independent processes in CD4 down-regulation: dissociation of the CD4-p56(lck) complex and targeting of CD4 to lysosomes.

Human immunodeficiency virus type 1 (HIV-1) Nef down-regulates CD4 by triggering rapid endocytosis of cell surface CD4. To better understand how Nef induces CD4 down-regulation, we generated a series of Nef mutants with small in-frame deletions in the coding region. Three classes of mutants were obtained. The first class produces neither CD4 down-regulation nor dissociation of the CD4-p56(lck) complex. The second class induces CD4 down-regulation in cells lacking p56(lck) expression, but not in cells with p56(lck);these mutants fail to dissociate CD4 from p56lck. These results show that Nef-mediated CD4 dissociation from p56(lck) is important for CD4 down-regulation. The third class of mutants is able to dissociate the CD4-p56(lck) complex but fails to down-regulate surface CD4; internalized CD4 molecules are recycled back to the cell surface. This result suggests that Nef diverts the CD4 recycling pathway to a degradative pathway. We also demonstrate that Nef associates with phosphatidylinositol-3-kinase (PI3K) activity, which is known to be involved in several aspects of membrane trafficking. However, Nef mutants that cause internalized CD4 to be recycled do not associate with PI3K activity; thus Nef-associated PI3K activity might be involved in the latter process of targeting CD4 to a degradative pathway. We conclude that HIV-1 Nef plays a critical role in multiple processes in CD4 down-regulation: (i) disrupting the CD4-p56(lck) complex on the cell surface to allow CD4 internalization and (ii) diverting the internalized CD4 to a lysosomal pathway for its degradation, likely through a PI3K activity.

CD4 Antigens↗
PubMed1998

Analysis of signal transduction pathways regulating cytokine-mediated Fc receptor activation on human eosinophils.

Igs can be potent stimulants of eosinophil activation since interaction with IgA or IgG-coated particles can lead to eosinophil degranulation. We have investigated the comparative roles of mitogen-activated protein (MAP) kinases (MAPKs; ERK1/2 and p38) and phosphatidylinositol-3 kinase (PI3K) in the priming and regulation of Fc receptor functioning on human eosinophils utilizing a MAPK kinase (MEK) inhibitor (PD98059), a p38 inhibitor SB203580, and the widely used PI3K inhibitors wortmannin and LY294002. We demonstrate that priming of human eosinophils with Th2-derived cytokines, IL-4 and IL-5, differentially activate phosphotyrosine-associated PI3K and ERK and p38 MAP kinases. This activation can be inhibited by pre-incubation with wortmannin or LY294002, PD98059, and SB203580, respectively. Analysis of the effects of the inhibitors on rosette formation between human eosinophils and IgA- or IgG-coated beads revealed that activation of MEK was not required for IgA binding after priming with IL-4 or IL-5. However, inhibition of MEK did inhibit IL-5-primed binding of IgG-beads. The rosette formation of primed eosinophils with IgA-beads could be completely inhibited by wortmannin and LY294002 treatment, demonstrating a critical role for PI3K. Interestingly, inhibition of the p38 pathway also resulted in a complete blockade of IgA rosette formation. This work demonstrates regulatory control by inside-out signaling of Fc receptors by various cytokines on human eosinophils. Thus in vivo the local production of Th2-derived cytokines will regulate the effector functions of Fc receptors.

Androstadienes↗
PubMed2005

LY294002 and rapamycin co-operate to inhibit T-cell proliferation.

1. T-cell proliferation is critical for mounting an effective adaptive immune response. It is regulated by signals through the T-cell receptor, through co-stimulation and through cytokines such as interleukin-2 (IL-2). Phosphatidylinositol 3-kinase (PI3K) lies downstream of each of these pathways and has been directly implicated in the regulation of lymphocyte proliferation. 2. In this study, we have shown that PI3K regulates cyclin D2 and cyclin D3, the first cell cycle proteins induced in T-cell proliferation, transcriptionally and post-transcriptionally. In T-lymphoblasts, LY294002, a PI3K inhibitor, prevents the induction of both D-type cyclin mRNA and protein, while rapamycin inhibits the induction of protein. Rapamycin inhibits mammalian target of rapamycin (mTOR), which lies downstream of PI3K. 3. Furthermore, our data show that the combination of LY294002 and rapamycin results in a co-operative inhibition of T-cell proliferation. This co-operation occurs in Kit225 cells stimulated with IL-2, and also in resting peripheral blood lymphocytes stimulated with antibodies to the T-cell receptor in the presence and absence of antibodies to CD28. 4. These data indicate that PI3K regulates T-cell proliferation in response to diverse stimuli, and suggest that combinations of inhibitors, perhaps isoform-selective, may be useful as alternative immunosuppressive therapies.

Cell Line, Tumor↗
PubMed2004

Involvement of protein kinase A in cannabinoid receptor-mediated protection from oxidative neuronal injury.

CB1 cannabinoid receptors (CB1Rs) are involved in protecting the brain from ischemia and related disorders. However, the underlying protective mechanisms are incompletely understood. We investigated the effect of CB1R activation on oxidative injury, which has been implicated in neuronal death after cerebral ischemia and neurodegenerative disorders, in mouse cortical neuron cultures. The CB1R agonist Win 55212-2 [R-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] reduced neuronal death, measured by lactate dehydrogenase release, in cultures treated with 50 microM FeCl2, and its protective effect was attenuated by the CB1R antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-cichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride]. The endocannabinoid anandamide reproduced the effect of Win 55212-2, as did the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Neuronal injury was more severe after in vitro or in vivo administration of FeCl2 to CB1R-knockout compared with wild-type mice. Win 55212-2 reduced the formation of reactive oxidative species in cortical neuron cultures treated with FeCl2, consistent with an antioxidant action. Pertussis toxin reduced CB1R-mediated protection, which points to a protective mechanism that involves signaling through G(i/o) proteins. Since CB1R-activated G protein signaling inhibits protein kinase A but activates phosphatidylinositol 3-kinase (PI3K), we tested the involvement of these pathways in CB1R-mediated neuroprotection. Dibutyryl-cyclic adenosine monophosphate (dbcAMP) blocked protection by Win 55212-2, whereas the PI3K inhibitor wortmannin did not, and the effect of dbcAMP was inhibited by the protein kinase A inhibitor H89 [N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide] (> or =10 nM). CB1R-induced, SR141716A-, pertussis toxin-, and dbcAMP-sensitive protection was also observed for two other oxidative insults, exposure to H2O2 or buthionine sulfoximine. Therefore, receptor-stimulated inhibition of protein kinase A seems to be required for the neuroprotective effect of CB1R activation against oxidative neuronal injury.

Animals↗
PubMed2006

Developmental acquisition of T3-sensitive Na-K-ATPase stimulation by rat alveolar epithelial cells.

Late in gestation, the developing air space epithelium switches from chloride and fluid secretion to sodium and fluid absorption. Absorption requires Na-K-ATPase acting in combination with apical sodium entry mechanisms. Hypothyroidism inhibits perinatal fluid resorption, and thyroid hormone [triiodothyronine (T3)] stimulates adult alveolar epithelial cell (AEC) Na-K-ATPase. This study explored the developmental regulation of Na-K-ATPase by T3 in fetal rat distal lung epithelial (FDLE) cells. T3 increased Na-K-ATPase activity in primary FDLE cells from gestational day 19 [both primary FDLE cells at embryonic day 19 (E19) and the cell line FD19 derived from FDLE cells at E19]. However, T3 did not increase the Na-K-ATPase activity in less mature FDLE cells, including primary E17 and E18 FDLE cells and the cell line FD18 (derived from FDLE cells at E18). Subsequent experiments assessed the T3 signal pathway to define whether it was similar in the late FDLE and adult AEC and to determine the site of the switch in responsiveness to T3. As in adult AEC, in the FD19 cell line, the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin blocked the T3-induced increase in Na-K-ATPase activity and plasma membrane quantity. T3 caused a parallel increase in phosphorylation of Akt at Ser473 in FDLE cells from E19, but not from E17 or E18. In the FD18 cell line, transient expression of a constitutively active mutant of the PI3K catalytic p110 subunit significantly augmented the Na-K-ATPase activity and the cell surface expression of Na-K-ATPase alpha(1) protein. In conclusion, FDLE cells from E17 and E18 lacked T3-sensitive Na-K-ATPase activity but acquired this response at E19. The developmental stimulation of Na-K-ATPase by T3 in rat FDLE cells requires activation of PI3K, and the acquisition of T3 responsiveness may be at PI3K or upstream in the signaling pathway.

Animals↗
PubMed2006

Activities of the protein kinases STK, PI3K, MEK, and ERK are required for the development of the head organizer in Hydra magnipapillata.

The development of the hydra's head and its hypostome has been studied at the molecular level. Many genes have been cloned from hydra as potential candidates that control the development of its head. Much work was performed on the mechanisms controlling expression of these genes in the position-dependent manner. Moreover, there have been data to support the involvement of three main signaling pathways that involve PKC, SRC, and PI3K kinases in the regulation of the head formation and in the expression of several head-specific genes. In this report, we present data supporting the participation of these three signaling pathways on the development of the hypostome. We used grafting experiments and inhibitors of the specific kinases to show the participation of these enzymes in hypostome formation. From our results, we postulate that these signal transduction pathways regulate the very early stages of the head development, most likely at the point when the cells start to differentiate to form the head organizer.

Animals↗
PubMed2002

Phospholipase C axis is the preferential pathway leading to PKC activation following PTH or PTHrP stimulation in human term placenta.

Parathyroid-related peptide (PTHrP) is abundant in human syncytiotrophoblast where it was suggested to play an important role in maternal-fetal calcium homeostasis. On the other hand, parathyroid hormone (PTH), another hypercalcemic factor, would be implicated in the maintenance of the mother's calcium balance. In many cells, these hormones are associated to G-coupled receptors and activate protein kinase (PKC). Thus, the first aim of this study was to determine the cellular pathway (phospholipase; PLC and phosphatidyl-inositol-3 kinase; PI3K) leading to the activation of PKC following a PTH or PTHrP stimulation in brush border (BBM) and basal plasma membranes (BPM) of human term placenta. Both peptides were shown to be potent modulators of the PKC activity in these membranes with optimal concentrations of 10(-8)M and 10(-9)M for hPTH and hPTHrP, respectively. Furthermore, the use of bisindolylmaleimide (BIM), a non-selective PKC inhibitor, serves to demonstrate the specificity of the PKC-dependent MARCKS-psd phosphorylation. While LY-294002, a PI3K inhibitor failed to counteract the hPTH- and hPTHrP-induced PKC stimulation in BBM and BPM, U-73122, a PLC inhibitor, totally abolished the PKC stimulation by hPTH and hPTHrP. Taken together, these data suggest that the activation of PKC by hPTH or hPTHrP, in BBM and BPM, is preferentially associated to the PLC pathway rather than the PI3K's.

Adult↗
PubMed2004

The small GTP-binding protein, Rhes, regulates signal transduction from G protein-coupled receptors.

The Ras homolog enriched in striatum, Rhes, is the product of a thyroid hormone-regulated gene during brain development. Rhes and the dexamethasone-induced Dexras1 define a novel distinct subfamily of proteins within the Ras family, characterized by an extended variable domain in the carboxyl terminal region. We have carried this study because there is a complete lack of knowledge on Rhes signaling. We show that in PC12 cells, Rhes is targeted to the plasma membrane by farnesylation. We demonstrate that about 30% of the native Rhes protein is bound to GTP and this proportion is unaltered by typical Ras family nucleotide exchange factors. However, Rhes is not transforming in murine fibroblasts. We have also examined the role of Rhes in cell signaling. Rhes does not stimulate the ERK pathway. By contrast, it binds to and activates PI3K. On the other hand, we demonstrate that Rhes impairs the activation of the cAMP/PKA pathway by thyroid-stimulating hormone, and by an activated beta2 adrenergic receptor by a mechanism that suggests uncoupling of the receptor to its cognate heterotrimeric complex. Overall, our results provide the initial insights into the role in signal transduction of this novel Ras family member.

Animals↗
PubMed2001

The multisubstrate adapter Gab1 regulates hepatocyte growth factor (scatter factor)-c-Met signaling for cell survival and DNA repair.

Hepatocyte growth factor (scatter factor) (HGF/SF) is a pleiotrophic mediator of epithelial cell motility, morphogenesis, angiogenesis, and tumorigenesis. HGF/SF protects cells against DNA damage by a pathway from its receptor c-Met to phosphatidylinositol 3-kinase (PI3K) to c-Akt, resulting in enhanced DNA repair and decreased apoptosis. We now show that protection against the DNA-damaging agent adriamycin (ADR; topoisomerase IIalpha inhibitor) requires the Grb2-binding site of c-Met, and overexpression of the Grb2-associated binder Gab1 (a multisubstrate adapter required for epithelial morphogenesis) inhibits the ability of HGF/SF to protect MDCK epithelial cells against ADR. In contrast to Gab1 and its homolog Gab2, overexpression of c-Cb1, another multisubstrate adapter that associates with c-Met, did not affect protection. Gab1 blocked the ability of HGF/SF to cause the sustained activation of c-Akt and c-Akt signaling (FKHR phosphorylation). The Gab1 inhibition of sustained c-Akt activation and of cell protection did not require the Gab1 pleckstrin homology or SHP2 phosphatase-binding domain but did require the PI3K-binding domain. HGF/SF protection of parental MDCK cells was blocked by wortmannin, expression of PTEN, and dominant negative mutants of p85 (regulatory subunit of PI3K), Akt, and Pak1; the protection of cells overexpressing Gab1 was restored by wild-type or activated mutants of p85, Akt, and Pak1. These findings suggest that the adapter Gab1 may redirect c-Met signaling through PI3K away from a c-Akt/Pak1 cell survival pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Amino acids require glucose to enhance, through phosphoinositide-dependent protein kinase 1, the insulin-activated protein kinase B cascade in insulin-resistant rat adipocytes.

AIMS/HYPOTHESIS: Amino acids are well known to activate the mammalian target of the rapamycin (mTOR) pathway in synergy with insulin to regulate cell functions. Despite recent important advances, the mTOR signalling pathway is poorly understood. Our previous results revealed a new pathway in which amino acids permit insulin-induced activation of the protein kinase B (PKB)/mTOR pathway in freshly isolated adipocytes when phosphatidylinositol 3-kinase (PI3K) is inhibited. The aim of this study was to further investigate this pathway at the molecular level. METHODS: We studied the effect of amino acids on PKB phosphorylation in different cellular models or in freshly isolated adipocytes incubated in different buffers, after a time course of insulin and amino acids and in the presence of pharmacological inhibitors. To investigate the potential role of amino acids in insulin action, the effect on glucose transport in obese rat adipocytes following a high-fat diet was assessed. RESULTS: Insulin-induced PKB phosphorylation is restored by amino acids in the presence of wortmannin in adipose tissue explants and freshly isolated adipocytes, but not in cultured adipocytes or hepatocytes. Moreover, amino acids require the presence of glucose to phosphorylate PKB and to partially rescue glucose transport in a PI3K-independent manner. The results also suggest that the amino acids act through the phosphoinositide-dependent protein kinase 1. In addition, amino acids were seen to improve insulin-stimulated glucose transport in adipocytes from high-fat-fed rats. CONCLUSIONS/INTERPRETATION: This study suggests that amino acids could enhance adipocyte insulin signalling in pathophysiological situations such as insulin resistance associated with obesity.

Adipocytes↗
PubMed2001

Regulation of intracellular trafficking of the EGF receptor by Rab5 in the absence of phosphatidylinositol 3-kinase activity.

Rab5 and phosphatidylinositol 3-kinase (PI3K) have been proposed to co-regulate receptor endocytosis by controlling early endosome fusion. However, in this report we demonstrate that inhibition of epidermal growth factor (EGF)-stimulated PI3K activity by expression of the kinase-deficient PI3K p110 subunit (p110delta kin) does not block the lysosomal targeting and degradation of the EGF receptor (EGFR). Moreover, inhibition of total PI3K activity by wortmannin or LY294002 significantly enlarges EGFR-containing endosomes and dissociates the early-endosomal autoantigen EEA1 from membrane fractions. However, this does not block the lysosomal targeting and degradation of EGFR. In contrast, transfection of cells with mutant Rab5 S34N or microinjection of anti-Rabaptin5 antibodies inhibits EGFR endocytosis. Our results, therefore, demonstrate that PI3K is not universally required for the regulation of receptor intracellular trafficking. The present work suggests that the intracellular trafficking of EGFR is controlled by a novel endosome fusion pathway that is regulated by Rab5 in the absence of PI3K, rather than by the previously defined endosome fusion pathway that is co-regulated by Rab5 and PI3K.

Androstadienes↗
PubMed2004

Effects of PI3K and p42/p44 MAPK on overexpression of vascular endothelial growth factor in hepatocellular carcinoma.

AIM: To study the relationship between hypoxia or epidermal growth factor (EGF) and the overexpression of vascular endothelial growth factor (VEGF) in hepatocellular carcinoma (HCC) and the signal transduction pathway of the transcription of VEGF in hepatoma cells. METHODS: Cobalt chloride and recombinant human EGF were used to stimulate the hepatoma cell lines HepG(2). VEGF mRNA was detected by using of semi-quantitative polymerase chain reaction (RT-PCR). Specific inhibitors of phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen activated protein kinase (MAPK) were used to observe the effects of the two kinases on the regulation of the transcription of VEGF in hepatoma cells. RESULTS: The expression of VEGF mRNA in HepG(2) cells cultured in serum-free medium was 0.117. However, 100 mumol/L cobalt chloride for 24 h increased the expression of VEGF mRNA and VEGF mRNA increased gradually with the increase of the concentration and duration of cobalt chloride. Also, 25 ng/mL recombinant human EGF stimulated the expression of VEGF in HepG(2) cells and the expression increased with the increase of EGF concentration. 5 mumol/L LY294002 inhibited the expression of VEGF stimulated by cobalt chloride or recombinant human EGF and the inhibition decreased step by step with increase of the concentration of LY294002. But even 20 mumol/L LY294002 could not completely block the expression of VEGF. In contrast, PD98059 had no inhibitory effects on the transcription of VEGF stimulated by cobalt chloride or recombinant human EGF. CONCLUSION: The overexpression of VEGF in HCC could be promoted by hypoxia and EGF expression in HCC. The signal transduction pathway of VEGF transcription in HepG(2) cells may be through PI3K pathway, but not through p42/p44 MAPK pathway.

Carcinoma, Hepatocellular↗
PubMed2006

SRC-family tyrosine kinases in wound- and ligand-induced epidermal growth factor receptor activation in human corneal epithelial cells.

PURPOSE: The authors have previously demonstrated that wounding of human corneal epithelial cells (HCECs) transactivates epidermal growth factor (EGF) receptor (EGFR) and its downstream signaling pathways and that this EGFR signaling is required for epithelial wound healing. In this study, the authors sought to identify the underlying mechanisms for EGFR transactivation in response to wounding in HCECs. METHODS: SV40-immortalized HCEC (THCE) monolayer was wounded and allowed to heal in the presence or absence of a selective inhibitor of the Src family kinases PP2 and EGFR ligand heparin-binding EGF-like growth factor (HB-EGF). Wound closure was monitored by photographing of the injury immediately or 24 hours after wounding. Activation of EGFR in THCE cells and in primary HCECs was analyzed by immunoprecipitation of EGFR, followed by Western blotting with phosphotyrosine antibody. Phosphorylation of extracellular signal-regulated kinase (ERK), AKT (a major substrate of phosphatidylinositol 3'-kinase [PI3K]), Src at tyrosine Y416, and EGFR at Y845 was analyzed by Western blotting with antibodies specific to phosphorylated proteins. Effects of PP2 on THCE cell migration were determined by Boyden chamber migration assay. RESULTS: Among several inhibitors tested, PP2 blocked wound-induced EGFR phosphorylation in THCE cells. PP2 at 12.5 microM effectively inhibited EGFR transactivation in response to wounding and to the phosphorylation of ERK and AKT in THCE cells and primary HCECs. Consistent with the inhibition of EGFR transactivation, PP2 also attenuated epithelial migration and wound closure with or without exogenously added HB-EGF. PP2 at a concentration as high as 50 microM exhibited no effects on HB-EGF induced ERK phosphorylation. On the other hand, AKT phosphorylation was much more sensitive to PP2 than ERK or EGFR phosphorylation because 3.13 microM PP2 effectively inhibited wound- or HB-EGF-induced AKT phosphorylation. CONCLUSIONS: These results suggest that Src kinase mediates wound-induced EGFR transactivation and participates in a pathway to activate the PI3K-AKT pathway downstream of EGFR in HCECs.

Blotting, Western↗
PubMed2006

Protease-activated receptor-2 simultaneously directs beta-arrestin-1-dependent inhibition and Galphaq-dependent activation of phosphatidylinositol 3-kinase.

Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor (GPCR) activated upon proteolytic cleavage of its N-terminus by a number of serine proteases. We have previously reported that formation of a beta-arrestin-dependent signaling scaffold is required for PAR-2-stimulated activation of extracellular signal regulated kinases 1 and 2 and chemotaxis. beta-Arrestin-dependent pathways downstream of some GPCRs have been shown to function independently and sometimes in opposition to classic signaling through heterotrimeric G-proteins; however, this possibility has not been addressed with respect to PAR-2. Here we demonstrate that PAR-2 can increase PI3K activity through a Galphaq/Ca(2+)-dependent pathway involving PYK2 and a Src-family kinase, while inhibiting PI3K activity through a beta-arrestin-dependent mechanism, and that beta-arrestin-1 can directly associate with and inhibit the catalytic activity of p110alpha. Using size exclusion chromatography and co-immunoprecipitation, we demonstrate that the PI3K is recruited into a scaffolding complex containing PAR-2 and beta-arrestins. Inhibition of PI3K activity blocks PAR-2-stimulated chemotaxis, and beta-arrestin-1 colocalizes with p85 within the pseudopodia, suggesting that beta-arrestin-1 association with PI3K may spatially restrict its enzymatic activity and that this localized inhibition may be crucial for PAR-2-stimulated chemotaxis.

Animals↗