Biomedical subjects

A Arcaro

Publications and source records attributed to A Arcaro.

11 recordsLinked to original sources

PubMed2006

Potent inhibition of small-cell lung cancer cell growth by simvastatin reveals selective functions of Ras isoforms in growth factor signalling.

The impact of the 3-hydroxy-3methylglutaryl CoA reductase inhibitor simvastatin on human small-cell lung cancer (SCLC) cell growth and survival was investigated. Simvastatin profoundly impaired basal and growth factor-stimulated SCLC cell growth in vitro and induced apoptosis. SCLC cells treated with simvastatin were sensitized to the effects of the chemotherapeutic agent etoposide. Moreover, SCLC tumour growth in vivo was inhibited by simvastatin. These responses correlated with the inhibition of stem cell factor (SCF)-stimulated activation of extracellular signal-regulated kinase (Erk), protein kinase B (PKB) and ribosomal S6 kinase by simvastatin. Constitutive activation of the Erk pathway was sufficient to rescue SCLC cell from the effects of simvastatin. The drug did not directly affect activation of c-Kit or its localization to lipid rafts, but in addition to its ability to block Ras membrane localization, it selectively downregulated H-Ras protein levels at the post-translational level. Downregulation of either H- or K-Ras by RNA interference (RNAi) did not impair Erk activation by growth factors, whereas an RNAi specific for N-Ras inhibited activation of Erk, PKB and SCLC cell growth. Together our data demonstrate that inhibiting Ras signalling with simvastatin potently disrupts growth and survival in human SCLC cells.

Animals↗

PubMed2001

CD8beta endows CD8 with efficient coreceptor function by coupling T cell receptor/CD3 to raft-associated CD8/p56(lck) complexes.

The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.

Amino Acid Sequence↗

PubMed2001

Novel cross talk between MEK and S6K2 in FGF-2 induced proliferation of SCLC cells.

Here, we show that fibroblast growth factor-2 (FGF-2) induces proliferation of H-510 and H-69 small cell lung cancer (SCLC) cells. However, the optimal response to FGF-2 was obtained at 10-fold lower concentrations in H-510 cells. This correlated with the selective activation of the mitogen-activated protein kinase kinase (MEK) pathway in H-510, but not H-69 cells. Moreover, inhibition of MEK with PD098059 blocked FGF-2-induced proliferation in H-510 cells only. Similarly, ribosomal protein S6 kinase 2 (S6K2), a recently identified homologue of S6K1 was activated by FGF-2 in H-510, but not H-69 cells. This activation was independent of phosphatidylinositol-3 kinase, but was sensitive to inhibition of the MEK pathway. These data suggest that S6K2 is a novel downstream target of MEK. The potency of FGF-2 in H-510 cells might reflect this additional MEK/S6K2 signalling. In contrast to S6K2, S6K1 was activated in both SCLC cell lines. Inhibition of the mammalian target of rapamycin with 10 ng/ml rapamycin blocked S6K1 activation and proliferation of both lines. However, even at 100 ng/ml, rapamycin only partially inhibited S6K2. Strikingly, this correlated with inhibition of MEK signalling. Our data indicate that S6K1, and possibly S6K2, are involved in FGF-2-induced SCLC cell growth, a notion supported by the overexpression and higher baseline activity of both isoforms in SCLC lines, as compared to normal human type-II pneumocytes.

Amino Acid Sequence↗

PubMed2000

Essential role of CD8 palmitoylation in CD8 coreceptor function.

To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.

4-Aminobenzoic Acid↗

PubMed2000

Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors.

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2alpha and PI3K-C2beta are two recently identified members of the large PI3K family. Both enzymes are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidylinositol 4-monophosphate as lipid substrates. Little is understood about how the catalytic activity of either enzyme is regulated in vivo. In this study, we demonstrate that PI3K-C2alpha and PI3K-C2beta represent two downstream targets of the activated epidermal growth factor (EGF) receptor in human carcinoma-derived A431 cells. Stimulation of quiescent cultures with EGF resulted in the rapid recruitment of both enzymes to a phosphotyrosine signaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3K-C2alpha and PI3K-C2beta immunoreactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca(2+) as an essential divalent cation in lipid kinase assays and since the catalytic activity of PI3K-C2alpha is refractory to the inhibitor wortmannin, these properties were used to confirm the recruitment of each PI3K isozyme to the activated EGF receptor complex. To examine this interaction in greater detail, PI3K-C2beta was chosen for further investigation. EGF and platelet-derived growth factor also stimulated the association of PI3K-C2beta with their respective receptors in other cells, including epithelial cells and fibroblasts. The use of EGF receptor mutants and phosphopeptides derived from the EGF receptor and Erb-B2 demonstrated that the interaction with recombinant PI3K-C2beta occurs through E(p)YL/I phosphotyrosine motifs. The N-terminal region of PI3K-C2beta was found to selectively interact with the EGF receptor in vitro, suggesting that it mediates the association of this PI3K with the receptor. However, the mechanism of this interaction remains unclear. We conclude that class II PI3K enzymes may contribute to the generation of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biological activity.

Binding Sites↗

PubMed1999

Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85alpha Src homology 2 domains with phosphoinositides and inositol polyphosphates.

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.

3T3 Cells↗

PubMed1999

Insulin activates the alpha isoform of class II phosphoinositide 3-kinase.

The novel class II phosphoinositide (PI) 3-kinases are characterized by the presence of a C-terminal C2 domain, but little is known about their regulation. We find insulin causes a rapid 2-3-fold increase in the activity of PI 3-kinase C2alpha (PI3K-C2alpha) in CHO-IR cells, 3T3-L1 adipocytes, and fully differentiated L5L6 myotubes. No insulin-induced activation of PI3K-C2alpha was observed in cell types known to have low responsiveness to insulin including HEK 293 cells, 3T3-L1 preadipocytes, and undifferentiated L5L6 myoblasts. The mechanism of activation of PI3K-C2alpha by insulin differs from that of class Ia PI 3-kinases in that insulin stimulation did not cause PI3K-C2alpha to associate with IRS-1 or insulin receptor. PI3K-C2alpha existed as a doublet, and insulin stimulation caused a redistribution from the lower molecular weight band to the higher molecular weight band, suggesting phosphorylation-induced bandshift. Consistent with this, in vitro phosphatase treatment reduced the intensity of the upper band back to that seen in unstimulated cells. This suggests that insulin-induced phosphorylation could play a role in regulation of the activity of PI3K-C2alpha. The finding that insulin activates PI3K-C2alpha in cell types known to possess a wide range of responses to insulin suggests that PI3K-C2alpha is a novel component of insulin-stimulated signaling cascades.

Animals↗

PubMed1998

Human phosphoinositide 3-kinase C2beta, the role of calcium and the C2 domain in enzyme activity.

The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2beta) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2beta utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4, 5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2beta, like the Class I PI 3-kinases, but unlike PI 3-kinase C2alpha, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2beta is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions.

Animals↗

PubMed1998

The small GTP-binding protein Rac promotes the dissociation of gelsolin from actin filaments in neutrophils.

Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma-thio]triphosphate (GTPgammaS) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgammaS-loaded ialpha2 and the beta1gamma2 subunits of heterotrimeric G-proteins (Gialpha2 and Gbeta1gamma2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgammaS-loaded Gialpha2 or Gbeta1gamma2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.

Actins↗

PubMed1994

Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates actin rearrangements in fibroblasts.

Various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to correlate with the synthesis of PtdIns(3,4,5)P3; however, the significance of this rise in second messenger levels is not clear. We show here that wortmannin inhibits platelet-derived growth factor (PDGF)-mediated production of PtdIns(3,4,5)P3 in human foreskin fibroblasts with an IC50 of about 5 nM. A similar inhibition was observed in in vitro assays (IC50 approximately 1 nM) with phosphatidylinositol 3-kinase immunoprecipitated by antibodies directed against the 85 kDa subunit (p85). On the other hand, wortmannin did not affect PDGF-mediated phosphorylation of p85 as detected by immunoprecipitation with anti-phosphotyrosine antibodies, and did not dissociate the complex of p85 and the catalytic subunit (p110) of phosphatidylinositol 3-kinase. These results are consistent with a direct, specific inhibition of the enzyme by wortmannin at concentrations relevant for its previously reported effects on cellular responses. When stimulated with PDGF, human foreskin fibroblasts form circular structures of filamentous actin. Preincubation of these cells with wortmannin inhibits PDGF-mediated actin rearrangements, suggesting a need for PtdIns(3,4,5)P3 formation as a signal for this cell response.

3T3 Cells↗

PubMed1993

Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses.

Phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) is rapidly produced upon exposure of neutrophils to the chemoattractant N-formylmethionyl-leucylphenylalanine (fMLP), and has been proposed to act as a second messenger mediating actin polymerization and respiratory-burst activity. Here we present evidence that wortmannin, a known inhibitor of respiratory-burst activity, acts on PtdIns 3-kinase, the enzyme producing PtdInsP3 from PtdIns(4,5)P2. Pretreatment of 32P-labelled human neutrophils with 100 nM wortmannin totally abolished fMLP-mediated PtdInsP3 production, raised PtdInsP2 levels, and did not affect cellular PtdInsP and PtdIns contents. The inhibitory effect on PtdInsP3 formation in intact cells was dose-dependent, with an IC50 of approximately 5 nM. Similar results were obtained with PtdIns 3-kinase immunoprecipitated by antibodies against the p85 regulatory subunit: wortmannin totally inhibited PtdIns3P production in immunoprecipitates at concentrations of 10-100 nM (IC50 approximately 1 nM). These results illustrate the direct and specific inhibition of PtdIns 3-kinase by wortmannin. Since agonist-mediated respiratory-burst activation is most sensitive to wortmannin (IC50 = 12 nM), this suggests that agonist-mediated PtdInsP3 formation is indispensable for this cell response. Neutrophils pretreated with wortmannin develop oscillatory changes in F-actin content, but actin polymerization in response to fMLP is not inhibited. This, and the absence of PtdInsP3 under these conditions, are in agreement with a modulatory role for PtdInsP3 in cytoskeletal rearrangements, but imply that PtdInsP3 production is not a primary event triggering elongation of actin filaments in neutrophils.

Androstadienes↗