22 results on '"Wilde JI"'
Search Results
2. The thiazolidinedione pioglitazone increases cholesterol biosynthetic gene expression in primary cortical neurons by a PPARgamma-independent mechanism.
- Author
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Cocks G, Wilde JI, Graham SJ, Bousgouni V, Virley D, Lovestone S, and Richardson J
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- 2010
- Full Text
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3. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology.
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Alexander EK, Kennedy GC, Baloch ZW, Cibas ES, Chudova D, Diggans J, Friedman L, Kloos RT, LiVolsi VA, Mandel SJ, Raab SS, Rosai J, Steward DL, Walsh PS, Wilde JI, Zeiger MA, Lanman RB, and Haugen BR
- Published
- 2012
4. Assembly of nuclear dimers of PI3K regulatory subunits is regulated by the Cdc42-activated tyrosine kinase ACK.
- Author
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Clayton NS, Fox M, Vicenté-Garcia JJ, Schroeder CM, Littlewood TD, Wilde JI, Krishnan K, Brown MJB, Crafter C, Mott HR, and Owen D
- Subjects
- Cell Nucleus enzymology, HEK293 Cells, Humans, Phosphorylation, Protein Multimerization, Signal Transduction, Phosphatidylinositol 3-Kinases metabolism, Protein-Tyrosine Kinases metabolism
- Abstract
Activated Cdc42-associated kinase (ACK) is an oncogenic nonreceptor tyrosine kinase associated with poor prognosis in several human cancers. ACK promotes proliferation, in part by contributing to the activation of Akt, the major effector of class 1A phosphoinositide 3-kinases (PI3Ks), which transduce signals via membrane phosphoinositol lipids. We now show that ACK also interacts with other key components of class 1A PI3K signaling, the PI3K regulatory subunits. We demonstrate ACK binds to all five PI3K regulatory subunit isoforms and directly phosphorylates p85α, p85β, p50α, and p55α on Tyr607 (or analogous residues). We found that phosphorylation of p85β promotes cell proliferation in HEK293T cells. We demonstrate that ACK interacts with p85α exclusively in nuclear-enriched cell fractions, where p85α phosphorylated at Tyr607 (pTyr607) also resides, and identify an interaction between pTyr607 and the N-terminal SH2 domain that supports dimerization of the regulatory subunits. We infer from this that ACK targets p110-independent p85 and further postulate that these regulatory subunit dimers undertake novel nuclear functions underpinning ACK activity. We conclude that these dimers represent a previously undescribed mode of regulation for the class1A PI3K regulatory subunits and potentially reveal additional avenues for therapeutic intervention., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article,, (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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5. Does addition of BRAF V600E mutation testing modify sensitivity or specificity of the Afirma Gene Expression Classifier in cytologically indeterminate thyroid nodules?
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Kloos RT, Reynolds JD, Walsh PS, Wilde JI, Tom EY, Pagan M, Barbacioru C, Chudova DI, Wong M, Friedman L, LiVolsi VA, Rosai J, Lanman RB, and Kennedy GC
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- Amino Acid Substitution genetics, Amino Acid Substitution physiology, Biopsy, Fine-Needle, Cytological Techniques, DNA Mutational Analysis, Diagnosis, Differential, Gene Expression Profiling classification, Glutamic Acid genetics, HT29 Cells, Humans, Proto-Oncogene Proteins B-raf physiology, Reproducibility of Results, Sensitivity and Specificity, Thyroid Nodule genetics, Thyroid Nodule pathology, Valine genetics, Genetic Testing methods, Mutation, Missense physiology, Proto-Oncogene Proteins B-raf genetics, Thyroid Nodule diagnosis
- Abstract
Objective: The purpose of this study was to determine the frequency of BRAF mutation in cytologically indeterminate thyroid nodules and to investigate whether adding the BRAF test improves diagnostic accuracy of the Afirma Gene Expression Classifier (GEC)., Design: BRAF V600E mutational status was determined for DNA extracted from cytologically benign (n = 40), indeterminate (n = 208), and malignant (n = 48) fine-needle aspiration specimens previously categorized by GEC as molecularly Benign or Suspicious. Analytical performance of the BRAF assay was assessed to establish reproducibility and limits of detection. Molecular testing results were correlated with blinded expert histopathological diagnoses., Results: The BRAF assay detected mutations reproducibly to 2.5% mutant allele frequency. The prevalence of BRAF mutations in cytologically benign specimens was 2 of 40 (5.0%, 95% confidence interval [CI], 0-16) and in cytologically malignant specimens was 36 of 48 (75.0%, 95% CI, 60-86). In the cytologically indeterminate category, 10.1% of specimens were BRAF+: 2 of 95 were subcategorized as atypia of undetermined significance or follicular lesion of undetermined significance (2.1%, 95% CI, 0-7); 1 of 70 as follicular neoplasm or suspicious for follicular neoplasm (1.4%, 95% CI, 0-9); and 18 of 43 as suspicious for malignancy (41.9%, 95% CI, 27-58). All BRAF+ specimens were classified as Suspicious by the GEC., Conclusions: BRAF mutations are uncommon in nodules with atypia of undetermined significance or follicular lesion of undetermined significance or follicular neoplasm or suspicious for follicular neoplasm cytology. Most cytologically indeterminate nodules that proved to be malignant were also BRAF-, and all nodules that were false-negative by GEC were also BRAF-. Similarly, all BRAF+ specimens were also GEC Suspicious. Neither GEC test sensitivity nor specificity was improved by addition of BRAF mutation testing.
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- 2013
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6. Analytical performance verification of a molecular diagnostic for cytology-indeterminate thyroid nodules.
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Walsh PS, Wilde JI, Tom EY, Reynolds JD, Chen DC, Chudova DI, Pagan M, Pankratz DG, Wong M, Veitch J, Friedman L, Monroe R, Steward DL, Lupo MA, Lanman RB, and Kennedy GC
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- Biopsy, Fine-Needle, Case-Control Studies, Diagnosis, Differential, Efficiency, Humans, Models, Biological, Molecular Diagnostic Techniques standards, Predictive Value of Tests, Reproducibility of Results, Sensitivity and Specificity, Specimen Handling methods, Thyroid Nodule blood, Thyroid Nodule genetics, Diagnostic Techniques, Endocrine, Molecular Diagnostic Techniques methods, Thyroid Nodule diagnosis, Thyroid Nodule pathology
- Abstract
Objective: Our objective was to verify the analytical performance of the Afirma gene expression classifier (GEC) in the classification of cytologically indeterminate thyroid nodule fine-needle aspirates (FNAs)., Design: Analytical performance studies were designed to characterize the stability of RNA in FNAs during collection and shipment, analytical sensitivity as applied to input RNA concentration and malignant/benign FNA mixtures, analytical specificity (i.e. potentially interfering substances) as tested on blood and genomic DNA, and assay performance studies including intra-nodule, intraassay, inter-assay, and inter-laboratory reproducibility., Results: RNA content within FNAs preserved in FNAProtect is stable for up to 6 d at room temperature with no changes in RNA yield (P = 0.58) or quality (P = 0.56). FNA storage and shipping temperatures were found to have no significant effect on GEC scores (P = 0.55) or calls (100% concordance). Analytical sensitivity studies demonstrated tolerance to variation in RNA input (5-25 ng) and to the dilution of malignant FNA material down to 20%. Analytical specificity studies using malignant samples mixed with blood (up to 83%) and genomic DNA (up to 30%) demonstrated negligible assay interference with respect to false-negative calls, although benign FNA samples mixed with relatively high proportions of blood demonstrated a potential for false-positive calls. The test is reproducible from extraction through GEC result, including variation across operators, runs, reagent lots, and laboratories (sd of 0.158 for scores on a >6 unit scale)., Conclusions: Analytical sensitivity, analytical specificity, robustness, and quality control of the GEC were successfully verified, indicating its suitability for clinical use.
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- 2012
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7. Molecular classification of thyroid nodules using high-dimensionality genomic data.
- Author
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Chudova D, Wilde JI, Wang ET, Wang H, Rabbee N, Egidio CM, Reynolds J, Tom E, Pagan M, Rigl CT, Friedman L, Wang CC, Lanman RB, Zeiger M, Kebebew E, Rosai J, Fellegara G, LiVolsi VA, and Kennedy GC
- Subjects
- Algorithms, Artificial Intelligence, Biopsy, Fine-Needle, Gene Expression Regulation, Genetic Variation, Humans, Oligonucleotide Array Sequence Analysis, RNA, Messenger genetics, ROC Curve, Reproducibility of Results, Thyroid Nodule classification, Thyroid Nodule pathology, Transcription, Genetic, Genomics methods, Thyroid Nodule genetics, Thyroid Nodule surgery
- Abstract
Objective: We set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA)., Design: We used mRNA expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology., Results: Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30-fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, we demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples., Conclusions: The FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs.
- Published
- 2010
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8. Targeting gut T cell Ca2+ release-activated Ca2+ channels inhibits T cell cytokine production and T-box transcription factor T-bet in inflammatory bowel disease.
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Di Sabatino A, Rovedatti L, Kaur R, Spencer JP, Brown JT, Morisset VD, Biancheri P, Leakey NA, Wilde JI, Scott L, Corazza GR, Lee K, Sengupta N, Knowles CH, Gunthorpe MJ, McLean PG, MacDonald TT, and Kruidenier L
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- Adult, Aged, Animals, Calcium Channel Blockers pharmacology, Cell Line, Tumor, Humans, Inflammatory Bowel Diseases metabolism, Intestinal Mucosa immunology, Intestinal Mucosa metabolism, Intestinal Mucosa pathology, Jurkat Cells, Middle Aged, Organ Culture Techniques, Patch-Clamp Techniques, Rats, T-Box Domain Proteins physiology, T-Lymphocyte Subsets pathology, Young Adult, Calcium Channels metabolism, Cytokines antagonists & inhibitors, Cytokines biosynthesis, Inflammatory Bowel Diseases immunology, Inflammatory Bowel Diseases pathology, T-Box Domain Proteins antagonists & inhibitors, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism
- Abstract
Prolonged Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels is crucial in activating the Ca(2+)-sensitive transcription factor NFAT, which is responsible for directing T cell proliferation and cytokine gene expression. To establish whether targeting CRAC might counteract intestinal inflammation, we evaluated the in vitro effect of a selective CRAC inhibitor on T cell cytokine production and T-bet expression by lamina propria mononuclear cells (LPMC) and biopsy specimens from inflammatory bowel disease (IBD) patients. The inhibitory activity of the CRAC blocker was investigated through patch-clamp experiments on rat basophilic leukemia cells and fluorometric imaging plate reader intracellular Ca(2+) assays using thapsigargin-stimulated Jurkat T cells and its detailed selectivity profile defined using a range of in vitro radioligand binding and functional assays. Anti-CD3/CD28-stimulated LPMC and biopsy specimens from 51 patients with IBD were cultured with a range of CRAC inhibitor concentrations (0.01-10 microM). IFN-gamma, IL-2, IL-8, and IL-17 were analyzed by ELISA. T-bet was determined by immunoblotting. We found that the CRAC blocker concentration-dependently inhibited CRAC current in rat basophilic leukemia cells and thapsigargin-induced Ca(2+) influx in Jurkat T cells. A concentration-dependent reduction in T-bet expression and production of IFN-gamma, IL-2, IL-17, but not IL-8, was observed in IBD LPMC and biopsy specimens treated with the CRAC inhibitor. In conclusion, we provide evidence that the suppression of CRAC channel function may dampen the increased T cell response in the inflamed gut, thus suggesting a promising role for CRAC inhibitor drugs in the therapeutic management of patients with IBD.
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- 2009
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9. Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis.
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Janas E, Priest R, Wilde JI, White JH, and Malhotra R
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- Antibodies, Monoclonal, Murine-Derived, Antigen-Antibody Reactions, Apoptosis, Biological Transport, Cell Line, Cell Membrane metabolism, Cholesterol metabolism, Humans, Octoxynol, Rituximab, Signal Transduction, Antibodies, Monoclonal pharmacology, Antigens, CD20 immunology, Antineoplastic Agents pharmacology, B-Lymphocytes immunology, Calcium metabolism, Lipids immunology
- Abstract
Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.
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- 2005
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10. Glycoproteins VI and Ib-IX-V stimulate tyrosine phosphorylation of tyrosine kinase Syk and phospholipase Cgamma2 at distinct sites.
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Suzuki-Inoue K, Wilde JI, Andrews RK, Auger JM, Siraganian RP, Sekiya F, Rhee SG, and Watson SP
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- Agammaglobulinaemia Tyrosine Kinase, Animals, COS Cells, Chlorocebus aethiops, Intracellular Signaling Peptides and Proteins, Mice, Phospholipase C gamma, Phosphorylation, Platelet Aggregation, Ristocetin pharmacology, Syk Kinase, Type C Phospholipases physiology, Tyrosine metabolism, von Willebrand Factor pharmacology, Enzyme Precursors chemistry, Enzyme Precursors metabolism, Platelet Glycoprotein GPIb-IX Complex metabolism, Platelet Membrane Glycoproteins metabolism, Protein-Tyrosine Kinases chemistry, Protein-Tyrosine Kinases metabolism, Type C Phospholipases chemistry, Type C Phospholipases metabolism
- Abstract
Glycoproteins GPVI and GPIb-IX-V stimulate robust tyrosine phosphorylation of Syk and PLCg2 (phospholipase Cg2) in washed platelets, but only the former stimulates pronounced activation of phospholipase. Using phospho-specific antibodies, we demonstrate that GPVI, but not GPIb-IX-V, stimulates significant tyrosine phosphorylation of Syk at the autophosphorylation site pY525/526, a marker of Syk activity. In addition, GPVI stimulates tyrosine phosphorylation of PLCg2 at Tyr753 and Tyr759, whereas GPIb-IX-V only induces significant phosphorylation at Tyr753. Both receptors stimulate tyrosine phosphorylation of Btk at the regulatory Tyr223 and Tyr551. Syk and Btk phosphorylate peptides from PLCg2 containing Tyr753 and Tyr759 respectively, suggesting that they may stimulate phosphorylation at these sites in phospholipase. Studies using PLCg2-deficient platelets demonstrated that phospholipase is not required for the activation of integrin aIIbb3 by GPIb-IX-V. Our results demonstrate fundamental differences between GPVI and GPIb-IX-V in the regulation of tyrosine phosphorylation of Syk and PLCg2 consistent with the functional impairment of phospholipase in signalling by GPIb-IX-V.
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- 2004
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11. c-Cbl negatively regulates platelet activation by glycoprotein VI.
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Auger JM, Best D, Snell DC, Wilde JI, and Watson SP
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- Adaptor Proteins, Vesicular Transport physiology, Animals, Blood Platelets, Down-Regulation, Humans, Mice, Mice, Inbred Strains, Phosphorylation, Platelet Aggregation drug effects, Platelet Membrane Glycoproteins agonists, Proto-Oncogene Proteins deficiency, Proto-Oncogene Proteins c-cbl, Signal Transduction, Thrombin pharmacology, Ubiquitin-Protein Ligases deficiency, Platelet Activation drug effects, Platelet Membrane Glycoproteins pharmacology, Proto-Oncogene Proteins physiology, Ubiquitin-Protein Ligases physiology
- Abstract
Background: The adapter protein c-Cbl has emerged as having a potential role in negative regulation of immune receptor signaling. The major platelet-signaling receptor for collagen, glycoprotein VI (GpVI), is associated with the Fc receptor (FcR) gamma-chain, and signals through a similar pathway to immune receptors. c-Cbl is tyrosine-phosphorylated in response to stimulation of GpVI, whereas phosphorylation of c-Cbl in thrombin-activated platelets is dependent on fibrinogen binding to the integrin GpIIb/IIIa., Objective: To investigate the role of c-Cbl in platelet signaling., Methods: Murine platelets lacking functional c-Cbl or Src family kinases were analyzed., Results: Phosphorylation of c-Cbl through GpVI is reduced in murine platelets deficient in the Src-family kinases Fyn and Lyn, demonstrating that they lie upstream of c-Cbl phosphorylation. Phosphorylation of several proteins of the GpVI-signaling pathway, including the FcR gamma-chain, Syk and phospholipase Cgamma2 (PLCgamma2), is increased in the absence of c-Cbl. In line with this, aggregation is potentiated in response to the GpVI-specific collagen-related peptide (CRP) after a slight delay. A delay in potentiation is also seen in response to stimulation by thrombin., Conclusions: These observations demonstrate that c-Cbl negatively regulates platelet responses to GpVI agonists and to thrombin, with the latter effect possibly being mediated downstream of GpIIb/IIIa. c-Cbl may play a physiological role in helping to prevent unwanted platelet activation in vivo.
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- 2003
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12. Vav1, but not Vav2, contributes to platelet aggregation by CRP and thrombin, but neither is required for regulation of phospholipase C.
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Pearce AC, Wilde JI, Doody GM, Best D, Inoue O, Vigorito E, Tybulewicz VL, Turner M, and Watson SP
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- Animals, Blood Platelets drug effects, Blood Platelets metabolism, Carrier Proteins pharmacology, Enzyme Activation drug effects, Humans, Mice, Mice, Knockout, Oncogene Proteins genetics, Phospholipase C gamma, Phosphorylation, Platelet Adhesiveness drug effects, Platelet Membrane Glycoproteins physiology, Proto-Oncogene Proteins genetics, Proto-Oncogene Proteins c-vav, Signal Transduction drug effects, Thrombin pharmacology, Type C Phospholipases drug effects, Cell Cycle Proteins, Oncogene Proteins physiology, Peptides, Platelet Aggregation drug effects, Proto-Oncogene Proteins physiology, Type C Phospholipases metabolism
- Abstract
We have investigated the role of the Rho and Rac family small guanine triphosphate (GTP) exchange factors (RhoGEFs), Vav1 and Vav2, in the activation of platelets by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled collagen receptor GPVI and by the G protein-coupled receptor agonist thrombin. The glycoprotein VI (GPVI)-specific agonist collagen-related peptide (CRP) and thrombin stimulated tyrosine phosphorylation of Vav1 but not Vav2 in human platelets. Surprisingly, however, CRP did not activate the low-molecular-weight G protein Rac and stimulated only a small increase in activity of p21-associated kinase 2 (PAK2), despite the fact that both proteins are regulated downstream of Vav1 in other cells. Further, activation of Rac and PAK2 by thrombin was maintained in platelets from mice deficient in Vav1. Activation of phospholipase C (PLC) by GPVI and thrombin was unaltered in Vav1-, Vav2-, and Vav1/Vav2-deficient platelets. A weak inhibition of late-stage aggregation to CRP and thrombin was observed in platelets deficient in Vav1 but not Vav2, whereas spreading on fibrinogen was not changed. The present results demonstrate that neither Vav1 nor Vav2 lie upstream of PLC or Rac in platelets, highlighting an important difference in their role in signaling by ITAM-coupled receptors in other cell types. The present study has provided evidence for a possible role of Vav1 but not Vav2 in the later stages of platelet aggregation.
- Published
- 2002
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13. Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cgamma2 regulation in platelets.
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Wonerow P, Obergfell A, Wilde JI, Bobe R, Asazuma N, Brdicka T, Leo A, Schraven B, Horejsí V, Shattil SJ, and Watson SP
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- Binding Sites, Blood Platelets drug effects, Blood Platelets physiology, Cell Adhesion drug effects, Cell Adhesion physiology, Cell Membrane chemistry, Fibrinogen pharmacology, Glycolipids blood, Humans, Membrane Lipids blood, Phospholipase C gamma, Phosphorylation, Phosphotyrosine metabolism, Signal Transduction, Blood Platelets enzymology, Cell Membrane enzymology, Isoenzymes blood, Platelet Glycoprotein GPIIb-IIIa Complex pharmacology, Platelet Membrane Glycoproteins pharmacology, Type C Phospholipases blood
- Abstract
The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates alphaIIbbeta3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCgamma2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and alphaIIbbeta3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCgamma2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.
- Published
- 2002
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14. Regulation of phospholipase C gamma isoforms in haematopoietic cells: why one, not the other?
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Wilde JI and Watson SP
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- Animals, B-Lymphocytes immunology, Blood Platelets physiology, Isoenzymes chemistry, Macromolecular Substances, Mice, Models, Biological, Phospholipase C gamma, Protein Isoforms chemistry, Protein Isoforms metabolism, Protein Structure, Tertiary, Protein-Tyrosine Kinases physiology, Receptor Protein-Tyrosine Kinases physiology, Signal Transduction, T-Lymphocytes immunology, Type C Phospholipases chemistry, Blood Cells physiology, Isoenzymes metabolism, Type C Phospholipases metabolism
- Abstract
Phospholipase C gamma (PLCgamma) isoforms are critical for the generation of calcium signals in haematopoietic systems in response to the stimulation of immune receptors. PLCgamma is unique amongst phospholipases in that it is tightly regulated by the action of a number of tyrosine kinases. It is itself directly phosphorylated on a number of tyrosines and contains several domains through which it can interact with other signalling proteins and lipid products such as phosphatidylinositol 3,4,5-trisphosphate. Through this network of interactions, PLCgamma is activated and recruited to its substrate, phosphatidylinositol 4,5-bisphosphate, at the membrane. Both isoforms of PLCgamma, PLCgamma1 and PLCgamma2, are present in haematopoietic cells. The signalling cascade involved in the regulation of these two isoforms varies between cells, though the systems are similar for both PLCgamma1 and PLCgamma2. We will compare these cascades for both PLCgamma1 and PLCgamma2 and discuss possible reasons as to why one form of PLCgamma and not the other is required for signalling in specific haematopoietic cells, including T lymphocytes, B lymphocytes, platelets, and mast cells.
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- 2001
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15. Phosphatidylinositol 3-kinase-dependent translocation of phospholipase Cgamma2 in mouse megakaryocytes is independent of Bruton tyrosine kinase translocation.
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Bobe R, Wilde JI, Maschberger P, Venkateswarlu K, Cullen PJ, Siess W, and Watson SP
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- Agammaglobulinaemia Tyrosine Kinase, Agammaglobulinemia enzymology, Androstadienes pharmacology, Animals, Blood Platelets drug effects, Blood Platelets enzymology, Calcium metabolism, Cell Membrane drug effects, Cell Membrane metabolism, Cells, Cultured, Enzyme Inhibitors pharmacology, Enzyme Precursors metabolism, Humans, Intracellular Signaling Peptides and Proteins, Isoenzymes chemistry, Megakaryocytes drug effects, Mice, Mice, Mutant Strains, Models, Biological, Phosphatidylinositol Phosphates metabolism, Phosphoinositide-3 Kinase Inhibitors, Phospholipase C gamma, Protein Transport, Proteins pharmacology, Syk Kinase, Thrombin pharmacology, Type C Phospholipases chemistry, Wortmannin, X Chromosome, src Homology Domains, Carrier Proteins, Isoenzymes metabolism, Megakaryocytes enzymology, Phosphatidylinositol 3-Kinases physiology, Protein-Tyrosine Kinases metabolism, Type C Phospholipases metabolism
- Abstract
Activation of the collagen receptor glycoprotein VI (GPVI) by a collagen-related peptide (CRP) induces stimulation of platelets and megakaryocytes through the phosphatidylinositol (PI) 3-kinase-dependent pathway leading to activation of Bruton tyrosine kinase (Btk) and phospholipase Cgamma2 (PLCgamma2). Here, we present evidence that both proteins undergo PI 3-kinase-dependent translocation to the plasma membrane on CRP stimulation that is markedly inhibited by wortmannin and LY294002. Translocation of PLCgamma2 but not Btk is also seen in megakaryocytes from X-linked immunodeficiency mice, which have a mutation that reduces the affinity of the pleckstrin homology (PH) domain of Btk for PI 3,4,5-trisphosphate (PI 3,4,5-P3). Activation of PC12 cells by epidermal growth factor (EGF) results in increased PI 3-kinase activity and high PI 3,4,5-P3 levels that trigger translocation of the green fluorescent protein (GFP)-labeled PH of Btk, but not the GFP-labeled PH and tandem Src homology 2 (SH2) domains of PLCgamma2. In contrast to the results with CRP, the G protein-coupled receptor agonist thrombin stimulates PI 3-kinase-independent translocation of Btk but not PLCgamma2. In conclusion, these results demonstrate that in mouse megakaryocytes, CRP leads to PI 3-kinase-dependent translocation of PLCgamma2 and Btk that are independent of one another, whereas thrombin only induces translocation of Btk through a pathway that is independent of PI 3-kinase activity.
- Published
- 2001
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16. Serine 727 phosphorylation and activation of cytosolic phospholipase A2 by MNK1-related protein kinases.
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Hefner Y, Borsch-Haubold AG, Murakami M, Wilde JI, Pasquet S, Schieltz D, Ghomashchi F, Yates JR 3rd, Armstrong CG, Paterson A, Cohen P, Fukunaga R, Hunter T, Kudo I, Watson SP, and Gelb MH
- Subjects
- Animals, Base Sequence, CHO Cells, Cricetinae, DNA Primers, Enzyme Activation, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins, Molecular Sequence Data, Mutagenesis, Phospholipases A2, Phosphorylation, Protein Serine-Threonine Kinases genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Phospholipases A metabolism, Protein Serine-Threonine Kinases metabolism, Serine metabolism
- Abstract
We have previously reported that in thrombin-stimulated human platelets, cytosolic phospholipase A(2) (cPLA2) is phosphorylated on Ser-505 by p38 protein kinase and on Ser-727 by an unknown kinase. Pharmacological inhibition of p38 leads to inhibition of cPLA2 phosphorylation at both Ser-505 and Ser-727 suggesting that the kinase responsible for phosphorylation on Ser-727 is activated in a p38-dependent pathway. By using Chinese hamster ovary, HeLa, and HEK293 cells stably transfected with wild type and phosphorylation site mutant forms of cPLA2, we show that phosphorylation of cPLA2 at both Ser-505 and Ser-727 and elevation of Ca(2+) leads to its activation in agonist-stimulated cells. The p38-activated protein kinases MNK1, MSK1, and PRAK1 phosphorylate cPLA2 in vitro uniquely on Ser-727 as shown by mass spectrometry. Furthermore, MNK1 and PRAK1, but not MSK1, is present in platelets and undergo modest activation in response to thrombin. Expression of a dominant negative form of MNK1 in HEK293 cells leads to significant inhibition of cPLA2-mediated arachidonate release. The results suggest that MNK1 or a closely related kinase is responsible for in vivo phosphorylation of cPLA2 on Ser-727.
- Published
- 2000
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17. Interaction of linker for activation of T cells with multiple adapter proteins in platelets activated by the glycoprotein VI-selective ligand, convulxin.
- Author
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Asazuma N, Wilde JI, Berlanga O, Leduc M, Leo A, Schweighoffer E, Tybulewicz V, Bon C, Liu SK, McGlade CJ, Schraven B, and Watson SP
- Subjects
- Blood Platelets metabolism, Collagen pharmacology, Cyclic AMP Receptor Protein pharmacology, GRB2 Adaptor Protein, Humans, Intracellular Signaling Peptides and Proteins, Phosphorylation, Proteins physiology, Receptors, Collagen, Receptors, IgG physiology, Type C Phospholipases physiology, Adaptor Proteins, Signal Transducing, Blood Platelets drug effects, Carrier Proteins physiology, Crotalid Venoms pharmacology, Integrins metabolism, Lectins, C-Type, Membrane Proteins, Phosphoproteins physiology
- Abstract
The snake venom toxin convulxin activates platelets through the collagen receptor glycoprotein VI (GPVI)/Fc receptor gamma-chain (FcR gamma-chain) complex leading to tyrosine phosphorylation and activation of the tyrosine Syk and phospholipase Cgamma2 (PLCgamma2). In the present study, we demonstrate that convulxin is a considerably more powerful agonist than collagen or the GPVI-selective collagen-related peptide (CRP). Confirmation that the response to convulxin is mediated solely via Syk was provided by studies on Syk-deficient platelets. The increase in phosphorylation of the FcR gamma-chain is associated with marked increases in tyrosine phosphorylation of downstream proteins including Syk, linker for activation of T cells (LAT), SLP-76, and PLCgamma2. The transmembrane adapter LAT coprecipitates with SLP-76 and PLCgamma2, as well as with a number of other adapter proteins, some of which have not been previously described in platelets, including Cbl, Grb2, Gads, and SKAP-HOM. Gads is constitutively associated with SLP-76 and is probably the protein bridging its association with LAT. There was no detectable association between Grb2 and SLP-76 in control or stimulated cells, suggesting that the interaction of LAT with Grb2 is present in a separate complex to that of LAT-Gads-SLP-76. These results show that the trimeric convulxin stimulates a much greater phosphorylation of the FcR gamma-chain and subsequent downstream responses relative to CRP and collagen, presumably because of its ability to cause a greater degree of cross-linking of GPVI. The adapter LAT appears to play a critical role in recruiting a number of other adapter proteins to the surface membrane in response to activation of GPVI, presumably at sites of glycolipid-enriched microdomains, enabling an organized signaling cascade that leads to platelet activation.
- Published
- 2000
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18. ADP-induced platelet shape change: an investigation of the signalling pathways involved and their dependence on the method of platelet preparation.
- Author
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Wilde JI, Retzer M, Siess W, and Watson SP
- Subjects
- Amides pharmacology, Animals, Apyrase pharmacology, Blood Platelets drug effects, Blood Platelets physiology, Calcium antagonists & inhibitors, Calcium pharmacology, Calcium Signaling drug effects, Calcium Signaling physiology, Cell Size drug effects, Chelating Agents pharmacology, Egtazic Acid pharmacology, Enzyme Inhibitors pharmacology, Humans, Intracellular Signaling Peptides and Proteins, Kinetics, Mice, Platelet Activation drug effects, Platelet Activation physiology, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases pharmacology, Pyridines pharmacology, Receptors, Purinergic P2 metabolism, Receptors, Purinergic P2Y1, Receptors, Purinergic P2Y12, Signal Transduction drug effects, Signal Transduction physiology, rho-Associated Kinases, Adenosine Diphosphate pharmacology, Blood Platelets cytology, Egtazic Acid analogs & derivatives, Membrane Proteins
- Abstract
Shape change is an important early event in platelet activation. In this study we show that the Ca2+ chelator BAPTA and the Rho-kinase inhibitor Y-27632 inhibit ADP-induced myosin light chain (MLC) phosphorylation and platelet shape change through distinct pathways and with distinct kinetics. Ca2+ is largely responsible for the initial onset of shape change, whilst Rho-kinase plays a major role in the maintenance of the response. The relative contribution of these two pathways to each stage of the response was dependent on the method of platelet preparation, but in all cases shape change was shown to be downstream of the P2Y1 receptor. Similar observations were made in murine platelets. The shape change response was modulated via changes in cAMP levels, possibly via the P2TAC receptor, but not by tyrosine phosphorylation. We conclude that ADP-induced shape change occurs via the P2Y1 receptor, which can be differentially coupled to Rho-kinase and Ca2+-linked pathways dependent on the method of platelet preparation.
- Published
- 2000
- Full Text
- View/download PDF
19. Up-regulation of p21- and RhoA-activated protein kinases in human pregnant myometrium.
- Author
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Moore F, Da Silva C, Wilde JI, Smarason A, Watson SP, and López Bernal A
- Subjects
- ADP Ribose Transferases pharmacology, Clostridium botulinum metabolism, Female, Humans, Myometrium drug effects, Phosphorylation, Pregnancy, Botulinum Toxins, Myometrium enzymology, Oncogene Protein p21(ras) metabolism, Protein Kinases metabolism, Up-Regulation, rhoA GTP-Binding Protein metabolism
- Abstract
The role of small ras homologous GTP-binding proteins in the regulation of smooth muscle contractility has become increasingly apparent but there is still little information about the presence of these proteins in human uterine smooth muscle. Messenger RNAs for p21-activated protein kinase isoforms (PAK1, PAK2, and PAK3) were detectable in both nonpregnant and pregnant human myometrial tissue. However, PAK3 protein was not detectable and the proteins for PAK1 and PAK2 were only detectable in pregnant tissue. Moreover there was a large increase in the constitutively active p34 protein fragment of PAK2 in pregnant tissue. Protein expression of RhoA-activated protein kinases isoforms (ROK1 and ROK2) also increased during pregnancy. Stimulation of RhoA signaling in pregnant myometrial tissue with lysophosphatic acid (LPA) increased the level of myosin light chain (MLC20) phosphorylation. Preincubation of the tissue with C3 toxin inhibited LPA-stimulated MLC20 phosphorylation and lowered the basal phosphorylation level of MLC20. Thus ROKS and PAKS have the potential to regulate uterine contractility and/or load-bearing during human pregnancy., (Copyright 2000 Academic Press.)
- Published
- 2000
- Full Text
- View/download PDF
20. Regulation and function of WASp in platelets by the collagen receptor, glycoprotein VI.
- Author
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Gross BS, Wilde JI, Quek L, Chapel H, Nelson DL, and Watson SP
- Subjects
- Humans, Blood Platelets physiology, Platelet Aggregation physiology, Platelet Membrane Glycoproteins physiology, Wiskott-Aldrich Syndrome blood, Wiskott-Aldrich Syndrome physiopathology
- Abstract
Wiskott Aldrich syndrome (WAS) is an X-linked recessive disorder associated with abnormalities in platelets and lymphocytes giving rise to thrombocytopenia and immunodeficiency. WAS is caused by a mutation in the gene encoding the cytoskeletal protein (WASp). Despite its importance, the role of WASp in platelet function is not established. WASp was recently shown to undergo tyrosine phosphorylation in platelets after activation by collagen, suggesting that it may play a selective role in activation by the adhesion molecule. In the present study, we show that WASp is heavily tyrosine phosphorylated by a collagen-related peptide (CRP) that binds to the collagen receptor glycoprotein (GP) VI, but not to the integrin alpha2beta1. Tyrosine phosphorylation of WASp was blocked by Src family kinase inhibitors and reduced by treatment with wortmannin and in patients with X-linked agammaglobulinemia (XLA), a condition caused by a lack of functional expression of Btk. This indicates that Src kinases, phosphatidylinositol 3-kinase (PI 3-kinase), and Btk all contribute to the regulation of tyrosine phosphorylation of WASp. The functional importance of WASp was investigated in 2 WAS brothers who show no detectable expression of WASp. Platelet aggregation and secretion from dense granules induced by CRP and thrombin was slightly enhanced in the WAS platelets relative to controls. Furthermore, there was no apparent difference in morphology in WAS platelets after stimulation by these agonists. These observations suggest that WASp does not play a critical role in intracellular signaling downstream of tyrosine kinase-linked and G protein-coupled receptors in platelets.
- Published
- 1999
21. Dichotomous regulation of myosin phosphorylation and shape change by Rho-kinase and calcium in intact human platelets.
- Author
-
Bauer M, Retzer M, Wilde JI, Maschberger P, Essler M, Aepfelbacher M, Watson SP, and Siess W
- Subjects
- 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid pharmacology, Amides pharmacology, Blood Platelets ultrastructure, Cell Size drug effects, Enzyme Inhibitors pharmacology, Hemostatics pharmacology, Humans, Ligands, Phosphorylation, Pyridines pharmacology, Receptors, Thrombin agonists, Receptors, Thrombin metabolism, Thrombin pharmacology, Vasoconstrictor Agents pharmacology, rhoB GTP-Binding Protein, Blood Platelets metabolism, Calcium metabolism, GTP-Binding Proteins metabolism, Membrane Proteins metabolism, Myosin Light Chains metabolism
- Abstract
Both Rho-kinase and the Ca(2+)/calmodulin-dependent myosin light chain (MLC) kinase increase the phosphorylation of MLC. We show that upon thrombin receptor stimulation by low-dose thrombin or the peptide ligand YFLLRNP, or upon thromboxane receptor activation by U46619, shape change and MLC phosphorylation in human platelets proceed through a pathway that does not involve an increase in cytosolic Ca(2+). Under these conditions, Y-27632, a specific Rho-kinase inhibitor, prevented shape change and reduced the stimulation of MLC-phosphorylation. In contrast, Y-27632 barely affected shape change and MLC-phosphorylation by adenosine diphosphate (ADP), collagen-related peptide, and ionomycin that were associated with an increase in cytosolic Ca(2+) and inhibited by BAPTA-AM/EGTA treatment. Furthermore, C3 exoenzyme, which inactivates Rho, inhibited preferentially the shape change induced by YFLLRNP compared with ADP and ionomycin. The results indicate that the Rho/Rho-kinase pathway is pivotal in mediating the MLC phosphorylation and platelet shape change by low concentrations of certain G protein-coupled platelet receptors, independent of an increase in cytosolic Ca(2+). Our study defines 2 alternate pathways, Rho/Rho-kinase and Ca(2+)/calmodulin-regulated MLC-kinase, that lead independently of each other through stimulation of MLC-phosphorylation to the same physiological response in human platelets (ie, shape change).
- Published
- 1999
22. The ordered visual transduction complex of the squid photoreceptor membrane.
- Author
-
Lott JS, Wilde JI, Carne A, Evans N, and Findlay JB
- Subjects
- Amino Acid Sequence, Animals, Cell Membrane physiology, Decapodiformes, Models, Molecular, Molecular Sequence Data, Protein Conformation, TRPC Cation Channels, Calcium Channels chemistry, Calcium Channels physiology, Photoreceptor Cells, Invertebrate chemistry, Photoreceptor Cells, Invertebrate physiology, Vision, Ocular physiology
- Abstract
The study of visual transduction has given invaluable insight into the mechanisms of signal transduction by heptahelical receptors that act via guanine nucleotide binding proteins (G-proteins). However, the cyclic-GMP second messenger system seen in vertebrate photoreceptor cells is not widely used in other cell types. In contrast, the retina of higher invertebrates, such as squid, offers an equally accessible transduction system, which uses the widespread second messenger chemistry of an increase in cytosolic calcium caused by the production of inositol-(1,4,5)-trisphosphate (InsP3) by the enzyme phospholipase C, and which may be a model for store-operated calcium influx. In this article, we highlight some key aspects of invertebrate visual transduction as elucidated from the combination of biochemical techniques applied to cephalopods, genetic techniques applied to flies, and electrophysiology applied to the horseshoe crab. We discuss the importance and applicability of ideas drawn from these model systems to the understanding of some general processes in signal transduction, such as the integration of the cytoskeleton into the signal transduction process and the possible modes of regulation of store-operated calcium influx.
- Published
- 1999
- Full Text
- View/download PDF
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