Evidence that a GTP binding protein regulates phosphorylation of the CD3 antigen in human T lymphocytes

The role of guanine nucleotide binding regulatory proteins (G proteins) in the regulation of phosphorylation of the gamma subunit of the CD3 antigen has been examined. CD3 gamma chain phosphorylation in isolated T cell microsomes was stimulated by the G protein activator guanosine 5'-0 thiotriphosphate (GTP gamma S), but cyclic adenosine monophosphate and guanosine 5'-diphosphate were ineffective at inducing gamma chain phosphorylation. The effect of GTP gamma S was rapid and transient; a half maximal effect was observed with 50 microM of the nucleotide. gamma polypeptide phosphorylated in vitro in GTP gamma S stimulated microsomes incorporated phosphate on Serines 123 and 126. These data are consistent with the involvement of a G protein in the signalling mechanisms that regulate the phosphorylation of the CD3 gamma chain.     

Capsaicin induces cofilin dephosphorylation in human intestinal cells: the triggering role of cofilin in tight-junction signaling

Previously, we demonstrated that capsaicin induces tight-junction (TJ) opening in human intestinal Caco-2 cells. In order to clarify the mechanism underlying the TJ opening action of capsaicin, we performed a proteomics study on capsaicin-treated Caco-2 cells. Phosphorylated cofilin was decreased significantly by capsaicin treatment. In addition, capsaicin induced Ca2+ influx in Caco-2 cells and there was a clear correlation between Ca2+) influx and cofilin dephosphorylation (activation). The Ca2+-chelating reagent EGTA blocked the cofilin dephosphorylation induced by both capsaicin and ionomycin, suggesting that the dephosphorylation was mediated by Ca2+ influx. Finally, transepithelial electrical resistance measurements showed that TJ opening accompanied cofilin dephosphorylation. Our data suggest that TJ opening is mediated by cofilin dephosphorylation, which is caused by capsaicin stimuli, including Ca2+ influx. This is the first report of capsaicin action via the dephosphorylation of cofilin in human intestinal cells.     

Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes

The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.     

Novel PtdIns(3)P-binding protein Etf1 functions as an effector of the Vps34 PtdIns 3-kinase in autophagy

Autophagy is the process whereby cytoplasmic cargo (e.g., protein and organelles) are sequestered within a double membrane-enclosed transport vesicle and degraded after vesicle fusion with the vacuole/lysosome. Current evidence suggests that the Vps34 phosphatidylinositol 3-kinase is essential for macroautophagy, a starvation-induced autophagy pathway (Kihara et al., 2001). Here, we characterize a requirement for Vps34 in constitutive autophagy by the cytoplasm-to-vacuole targeting (Cvt) pathway. First, we show that transient disruption of phosphatidylinositol (PtdIns) 3-phosphate (PtdIns[3]P) synthesis through inactivation of temperature-sensitive Vps34 or its upstream activator, Vps15, blocks the Cvt and macroautophagy pathways. Yet, PtdIns(3)P-binding FYVE domain-containing proteins, which mediate carboxypeptidase Y (CPY) transport to the vacuole by the CPY pathway, do not account for the requirement of Vps34 in autophagy. Using a genetic selection designed to isolate PtdIns(3)P-binding effectors of Vps34, we identify Etf1, an uncharacterized type II transmembrane protein. Although Etf1 does not contain a known 3-phosphoinositide-binding domain (i.e., FYVE or Phox), we find that Etf1 interacts with PtdIns(3)P and that this interaction requires a basic amino acid motif (KKPAKK) within the cytosolic region of the protein. Moreover, deletion of ETF1 or mutation of the KKPAKK motif results in strong sorting defects in the Cvt pathway but not in macroautophagy or in CPY sorting. We propose that Vps34 regulates the CPY, Cvt, and macroautophagy pathways through distinct sets of PtdIns(3)P-binding effectors and that Vps34 promotes protein trafficking in the Cvt pathway through activation/localization of the effector protein Etf1.     

The ORF4a protein of human coronavirus 229E functions as a viroporin that regulates viral production

In addition to a set of canonical genes, coronaviruses encode additional accessory proteins. A locus located between the spike and envelope genes is conserved in all coronaviruses and contains a complete or truncated open reading frame (ORF). Previously, we demonstrated that this locus, which contains the gene for accessory protein 3a from severe acute respiratory syndrome coronavirus (SARS-CoV), encodes a protein that forms ion channels and regulates virus release. In the current study, we explored whether the ORF4a protein of HCoV-229E has similar functions. Our findings revealed that the ORF4a proteins were expressed in infected cells and localized at the endoplasmic reticulum/Golgi intermediate compartment (ERGIC). The ORF4a proteins formed homo-oligomers through disulfide bridges and possessed ion channel activity in both Xenopus oocytes and yeast. Based on the measurement of conductance to different monovalent cations, the ORF4a was suggested to form a non-selective channel for monovalent cations, although Li⁺ partially reduced the inward current. Furthermore, viral production decreased when the ORF4a protein expression was suppressed by siRNA in infected cells. Collectively, this evidence indicates that the HCoV-229E ORF4a protein is functionally analogous to the SARS-CoV 3a protein, which also acts as a viroporin that regulates virus production. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Activation of Wnt signaling arrests effector differentiation in human peripheral and cord blood-derived T lymphocytes

The canonical Wnt/β-catenin signaling pathway plays an important role in thymocyte development and T cell migration, but little is known about its role in naive-to-effector differentiation in human peripheral T cells. We show that activation of Wnt/β-catenin signaling arrests human peripheral blood and cord blood T lymphocytes in the naive stage and blocks their transition into functional T effector cells. Wnt signaling was induced in polyclonally activated human T cells by treatment either with the glycogen synthase kinase 3β inhibitor TWS119 or the physiological Wnt agonist Wnt-3a, and these T cells preserved a naive CD45RA(+)CD62L(+) phenotype compared with control-activated T cells that progressed to a CD45RO(+)CD62L(-) effector phenotype, and this occurred in a TWS119 dose-dependent manner. TWS119-induced Wnt signaling reduced T cell expansion, as a result of a block in cell division, and impaired acquisition of T cell effector function, measured by degranulation and IFN-γ production in response to T cell activation. The block in T cell division may be attributed to the reduced IL-2Rα expression in TWS119-treated T cells that lowers their capacity to use autocrine IL-2 for expansion. Collectively, our data suggest that Wnt/β-catenin signaling is a negative regulator of naive-to-effector T cell differentiation in human T lymphocytes. The arrest in T cell differentiation induced by Wnt signaling might have relevant clinical applications such as to preserve the naive T cell compartment in Ag-specific T cells generated ex vivo for adoptive T cell immunotherapy.     

Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation

Synthesis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a signaling phospholipid, is primarily carried out by phosphatidylinositol 4-phosphate 5-kinase [PI(4)P5K], which has been reported to be regulated by RhoA and Rac1. Unexpectedly, we find that the GTPgammaS-dependent activator of PI(4)P5Kalpha is the small G protein ADP-ribosylation factor (ARF) and that the activation strictly requires phosphatidic acid, the product of phospholipase D (PLD). In vivo, ARF6, but not ARF1 or ARF5, spatially coincides with PI(4)P5Kalpha. This colocalization occurs in ruffling membranes formed upon AIF4 and EGF stimulation and is blocked by dominant-negative ARF6. PLD2 similarly translocates to the ruffles, as does the PH domain of phospholipase Cdelta1, indicating locally elevated PI(4,5)P2. Thus, PI(4)P5Kalpha is a downstream effector of ARF6 and when ARF6 is activated by agonist stimulation, it triggers recruitment of a diverse but interactive set of signaling molecules into sites of active cytoskeletal and membrane rearrangement.     

Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells

The antioxidant-responsive element (ARE) plays an important role in the induction of phase II detoxifying enzymes including NADPH:quinone oxidoreductase (NQO1). We report herein that activation of the human NQO1-ARE (hNQO1-ARE) by tert-butylhydroquinone (tBHQ) is mediated by phosphatidylinositol 3-kinase (PI3-kinase), not extracellular signal-regulated kinase (Erk1/2), in IMR-32 human neuroblastoma cells. Treatment with tBHQ significantly increased NQO1 protein without activation of Erk1/2. In addition, PD 98059 (a selective mitogen-activated kinase/Erk kinase inhibitor) did not inhibit hNQO1-ARE-luciferase expression or NQO1 protein induction by tBHQ. Pretreatment with LY 294002 (a selective PI3-kinase inhibitor), however, inhibited both hNQO1-ARE-luciferase expression and endogenous NQO1 protein induction. In support of a role for PI3-kinase in ARE activation we show that: 1) transfection of IMR-32 cells with constitutively active PI3-kinase selectively activated the ARE in a dose-dependent manner that was completely inhibited by treatment with LY 294002; 2) pretreatment of cells with the PI3-kinase inhibitors, LY 294002 and wortmannin, significantly decreased NF-E2-related factor 2 (Nrf2) nuclear translocation induced by tBHQ; and 3) ARE activation by constitutively active PI3-kinase was blocked completely by dominant negative Nrf2. Taken together, these data clearly show that ARE activation by tBHQ depends on PI3-kinase, which lies upstream of Nrf2.     

Phosphatidylinositol 3-kinase regulates differentiation of H9c2 cardiomyoblasts mainly through the protein kinase B/Akt-independent pathway.

Phosphatidylinositol 3-kinase (PI3-kinase) is known to be a crucial regulator of muscle differentiation. However, its downstream pathway for this function is quite obscure. In this experiment we demonstrated the regulatory mechanism of the differentiation of H9c2 cardiomyoblasts, focusing on PI3-kinase, protein kinase B/Akt (PKB/Akt) and p42/44 mitogen-activated protein kinase (p42/44 MAPK). When H9c2 cells stably transfected with a constitutively active p110 (H9c2-p110*), a constitutively active PKB/Akt (H9c2-Akt), and an empty vector (H9c2-con) were induced to differentiate, H9c2-p110* cells differentiated fastest, followed by H9c2-Akt cells. H9c2-con cells differentiated at the slowest rate. Consistent with this result, LY294002 completely blocked differentiation of all these transfected cell lines, whereas PD098059 had no effect on their differentiation. When H9c2-p110* cells were transiently transfected with a dominant negative form of PKB/Akt, differentiation was not affected. Taken together, we concluded that PI3-kinase, but not p42/44 MAPK, regulates differentiation of H9c2 cardiomyoblasts mainly through the PKB/Akt-independent pathway.     

Pertussis toxin-sensitive and insensitive intracellular signalling pathways in undifferentiated 3T3-L1 cells stimulated by insulin converge with phosphatidylinositol 3-kinase upstream of the Ras mitogen-activated protein kinase cascade.

We have previously reported that pertussis toxin (PTX)-sensitive GTP binding protein (G-protein) and phosphatidylinositol 3-kinase (PI 3-K) are involved in adipocyte differentiation of 3T3-L1 cells induced by insulin/dexamethasone/methylisobutyl xanthine. The aim of this study was to examine the effect of PTX on the tyrosine kinase cascade stimulated by insulin acting through insulin-like growth factor-I (IGF-I) receptors in undifferentiated 3T3-L1 cells. A high level of mitogen-activated protein kinase (MAPK) activation was sustained for up to 4 h after insulin treatment, and mobility shifted and tyrosine phosphorylated MAPK was also detected. MAPK kinase activity measured by the incorporation of 32P into kinase-negative recombinant MAPK was enhanced by insulin treatment. We previously discovered that insulin activates Ras and that this is mediated by wortmannin-sensitive PI 3-K. Tyrosine-phosphorylation of IRS-1 and Shc also occurred in response to insulin. Subsequently, we investigated the effects of PTX on the activation of these proteins by insulin. Interestingly, treating 3T3-L1 cells with PTX attenuates the activation by insulin of both the Ras-MAPK cascade and PI 3-K. In contrast, neither tyrosine-phosphorylation of IRS-1 and Shc nor the interaction between IRS-1 and PI 3-K is sensitive to PTX. However, activation of the Ras-MAPK cascade and tyrosine-phosphorylation of Shc by epidermal growth factor are insensitive to PTX. These results indicate that there is another pathway which regulates PI 3-K and Ras-MAPK, independent of the pathway mediated by IGF-I receptor kinase. These findings suggest that in 3T3-L1 fibroblasts, PTX-sensitive G-proteins cross-talk with the Ras-MAPK pathway via PI 3-K by insulin acting via IGF-I receptors.     

Characterization of human T lymphocytes that express the C3b receptor

The presence of the C3b receptor (C3bR) on human peripheral blood T lymphocytes was recognized by the capacity of rabbit F(ab')2 anti-C3bR and tetramethylrhodamine isothiocyanate (TRITC)-conjugated goat F(ab')2 anti-rabbit F(ab')2 to stain 14.5 +/- 3.7% (mean +/- SEM; n = 5) of lymphocytes forming rosettes with sheep erythrocytes (E). The F(ab')2 anti-C3bR also blocked the capacity of peripheral blood lymphocytes stained with OKT11 to form rosettes with bovine E bearing C3b and immunoprecipitated a single membrane protein having a m.w. of approximately 250,000 from detergent lysates of 125I-labeled, purified T cells. Measurement by fluorescent flow cytometry of the quantitative expression of the C3bR indicated that T cells had slightly more antigenic sites/cell than did E and approximately 10-fold fewer sites than were present on B cells. The surface constituents of the peripheral blood T cells expressing the C3bR were assessed in an assay that employed simultaneously three markers: rosette formation with sheep E, TRITC staining with anti-C3bR and fluorescein isothiocyanate (FITC)-staining with a panel of monoclonal antibodies or with aggregated IgG. Among lymphocytes forming rosettes with sheep E and expressing the C3bR, 99.6 +/- 0.4%, 65.0 +/- 5.8%, 17.2 +/- 6.2%, and 15.3 +/- 5.0% of the cells expressed antigens detected by OKT3, OKT4, OKT8, and OKM1 monoclonal antibodies, respectively. Ninety-seven per cent of the C3bR-bearing T cells were also capable of specifically binding aggregated IgG, indicating the presence of Fc receptors for IgG (Fc gamma R) on these cells. The T cells expressing the C3bR had large nuclei, thin rims of basophilic cytoplasm and no azurophilic granules. Thus, the C3bR is present on some T cells, all of which have a typical lymphocyte morphology, the T3 antigen and the Fc gamma R.     

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.     

Phosphatidylinositol 3-kinase interacts with the adaptor protein Dab1 in response to Reelin signaling and is required for normal cortical lamination

Reelin is a large secreted signaling protein that binds to two members of the low density lipoprotein receptor family, the apolipoprotein E receptor 2 and the very low density lipoprotein receptor, and regulates neuronal positioning during brain development. Reelin signaling requires activation of Src family kinases as well as tyrosine phosphorylation of the intracellular adaptor protein Disabled-1 (Dab1). This results in activation of phosphatidylinositol 3-kinase (PI3K), the serine/threonine kinase Akt, and the inhibition of glycogen synthase kinase 3beta, a protein that is implicated in the regulation of axonal transport. Here we demonstrate that PI3K activation by Reelin requires Src family kinase activity and depends on the Reelin-triggered interaction of Dab1 with the PI3K regulatory subunit p85alpha. Because the Dab1 phosphotyrosine binding domain can interact simultaneously with membrane lipids and with the intracellular domains of apolipoprotein E receptor 2 and very low density lipoprotein receptor, Dab1 is preferentially recruited to the neuronal plasma membrane, where it is phosphorylated. Efficient Dab1 phosphorylation and activation of the Reelin signaling cascade is impaired by cholesterol depletion of the plasma membrane. Using a neuronal migration assay, we also show that PI3K signaling is required for the formation of a normal cortical plate, a step that is dependent upon Reelin signaling.     

Phosphatidylinositol 3 kinase–Akt signaling serves as a circadian output in the retina

The daily rhythm of L-type voltage-gated calcium channels (L-VGCCs) is part of the cellular mechanism underlying the circadian regulation of retina physiology and function. However, it is not completely understood how the circadian clock regulates L-VGCC current amplitudes without affecting channel gating properties. The phosphatidylinositol 3 kinase–protein kinase B (PI3K–Akt) signaling pathway has been implicated in many vital cellular functions especially in trophic factor-induced ion channel trafficking and membrane insertion. Here, we report that PI3K–Akt signaling participates in the circadian phase-dependent modulation of L-VGCCs. We found that there was a circadian regulation of Akt phosphorylation on Thr308 that peaked at night. Inhibition of PI3K or Akt significantly decreased L-VGCC current amplitudes and the expression of membrane-bound L-VGCCα1D subunit only at night but not during the subjective day. Photoreceptors transfected with a dominant negative Ras had significantly less expression of phosphorylated Akt and L-VGCCα1D subunit compared with non-transfected photoreceptors. Interestingly, both PI3K–Akt and extracellular signal-related kinase were downstream of Ras, and they appeared to be parallel and equally important pathways to regulate L-VGCC rhythms. Inhibition of either pathway abolished the L-VGCC rhythm indicating that there were multiple mechanisms involved in the circadian regulation of L-VGCC rhythms in retina photoreceptors.     

Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor

Mouse fibroblast cells overexpressing phosphatidylinositol transfer protein alpha [PI-TPalpha; sense PI-TPalpha (SPIalpha) cells] show a significantly increased rate of proliferation and an extreme resistance toward ultraviolet- or tumor necrosis factor-alpha-induced apoptosis. The conditioned medium (CM) from SPIalpha cells or the neutral lipid extract from CM stimulated the proliferation of quiescent wild-type NIH3T3 cells. CM was also highly effective in increasing resistance toward induced apoptosis in both wild-type cells and the highly apoptosis-sensitive SPIbeta cells (i.e., wild-type cells overexpressing PI-TPbeta). CM from SPIalpha cells grown in the presence of NS398, a specific cyclooxygenase-2 (COX-2) inhibitor, expressed a diminished mitogenic and antiapoptotic activity. This strongly suggests that at least one of the bioactive factor(s) is an eicosanoid. In accordance, SPIalpha cells express enhanced levels of COX-1 and COX-2. The antiapoptotic activity of CM from SPIalpha cells tested on SPIbeta cells was inhibited by approximately 50% by pertussis toxin and suramin as well as by SR141716A, a specific antagonist of the cannabinoid 1 receptor. These inhibitors had virtually no effect on the COX-2-independent antiapoptotic activity of CM from SPIalpha cells. The latter results imply that PI-TPalpha mediates the production of a COX-2-dependent eicosanoid that activates a G-protein-coupled receptor, most probably a cannabinoid 1-like receptor.