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Papers by michael cooke

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PLOS ONE, 2015

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of an... more Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/ Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca 2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

Advances in Experimental Medicine and Biology, 1992

PloS one, 2014

Chemokines promote T cell migration by transmitting signals that induce T cell polarization and i... more Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of Mst1-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. Mst1 deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the ant...

Proceedings of the National Academy of Sciences, 2004

The tissue-specific pattern of mRNA expression can indicate important clues about gene function. ... more The tissue-specific pattern of mRNA expression can indicate important clues about gene function. High-density oligonucleotide arrays offer the opportunity to examine patterns of gene expression on a genome scale. Toward this end, we have designed custom arrays that interrogate the expression of the vast majority of protein-encoding human and mouse genes and have used them to profile a panel of 79 human and 61 mouse tissues. The resulting data set provides the expression patterns for thousands of predicted genes, as well as known and poorly characterized genes, from mice and humans. We have explored this data set for global trends in gene expression, evaluated commonly used lines of evidence in gene prediction methodologies, and investigated patterns indicative of chromosomal organization of transcription. We describe hundreds of regions of correlated transcription and show that some are subject to both tissue and parental allele-specific expression, suggesting a link between spatial...

Nature Reviews Immunology, 2010

The membrane lipid phosphatidylinositol(3,4,5)trisphosphate (PtdInsP 3) regulates membrane recept... more The membrane lipid phosphatidylinositol(3,4,5)trisphosphate (PtdInsP 3) regulates membrane receptor signalling in many cells, including immunoreceptor signalling. Here, we review recent data that have indicated essential functions for the soluble PtdInsP 3 analogue inositol(1,3,4,5)tetrakisphosphate (InsP 4) in T cell, B cell and neutrophil development and function. Decreased InsP 4 production in immunocytes causes immunodeficiency in mice and might contribute to inflammatory vasculitis in Kawasaki disease in humans. InsP 4-producing kinases could therefore provide attractive drug targets for inflammatory and infectious diseases. Because it can be combinatorially phosphorylated on six hydroxyl groups, the cyclic polyalcohol D-myo-inositol is an ideal cellular information carrier. Its many derivatives form an "inositol code", whose most prominent members are soluble inositol phosphates (InsPs) [G] and their membrane-lipid counterparts, the phosphoinositides [G] 1, 2 (Fig. 1 , Box 1). In 1983, the identification of inositol(1,4,5)trisphosphate [Ins(1,4,5)P 3 , InsP 3 here] as a second-messenger [G] that mediates the receptor-induced Ca 2+ mobilization in most mammalian cells first demonstrated physiological importance for soluble InsPs 3. More recently, intriguing functions for several higher-order InsPs have been identified (Box 1). Moreover, the phosphoinositide-lipids phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P 2) and PtdIns(3,4,5)P 3 (PtdInsP 3) regulate signalling by many receptors in many cell types 4-9. PtdInsP 3 is generated by phosphoinositide 3-kinase (PI3K) and metabolized by lipid-phosphatases, including phosphatase-and-tensin-homologue (PTEN), SH2-domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) and SHIP2 10. The key to phosphoinositide function is the phosphorylation status of their cytosol-exposed InsP headgroups. These bind to specific domains in signalling or cytoskeletal proteins to control their membrane recruitment. Defects in phosphoinositide metabolism or function contribute

Nature Immunology, 2007

In the version of this article initially published, two labels in the key to Figure 6c are revers... more In the version of this article initially published, two labels in the key to Figure 6c are reversed. The blue line is "Ins(1,3,4,5)P4 (300 µM)" and the green line is "Ins(1,4,5,6)P4 (300 µM)." The error has been corrected in the HTML and PDF versions of the article.

Nature Genetics, 2005

We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that... more We combined large-scale mRNA expression analysis and gene mapping to identify genes and loci that control hematopoietic stem cell (HSC) function. We measured mRNA expression levels in purified HSCs isolated from a panel of densely genotyped recombinant inbred mouse strains. We mapped quantitative trait loci (QTLs) associated with variation in expression of thousands of transcripts. By comparing the physical transcript position with the location of the controlling QTL, we identified polymorphic cis-acting stem cell genes. We also identified multiple transacting control loci that modify expression of large numbers of genes. These groups of coregulated transcripts identify pathways that specify variation in stem cells. We illustrate this concept with the identification of candidate genes involved with HSC turnover. We compared expression QTLs in HSCs and brain from the same mice and identified both shared and tissue-specific QTLs. Our data are accessible through WebQTL, a web-based interface that allows custom genetic linkage analysis and identification of coregulated transcripts.

Molecular Cell, 2009

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we s... more The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATPcompetitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn À/À mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

Mammalian Genome, 2006

The mouse N-ethyl-N-nitrosourea (ENU) mutagenesis program at the Genomics Institute of the Novart... more The mouse N-ethyl-N-nitrosourea (ENU) mutagenesis program at the Genomics Institute of the Novartis Research Foundation (GNF) uses MouseTRACS to analyze phenotype screens and manage animal husbandry. MouseTRACS is a Web-based laboratory informatics system that electronically records and organizes mouse colony operations, prints cage cards, tracks inventory, manages requests, and reports Institutional Animal Care and Use Committee (IACUC) protocol usage. For efficient phenotype screening, MouseTRACS identifies mutants, visualizes data, and maps mutations. It displays and integrates phenotype and genotype data using likelihood odds ratio (LOD) plots of genetic linkage between genotype and phenotype. More detailed mapping intervals show individual single nucleotide polymorphism (SNP) markers in the context of phenotype. In addition, dynamically generated pedigree diagrams and inventory reports linked to screening results summarize the inheritance pattern and the degree of penetrance. MouseTRACS displays screening data in tables and uses standard charts such as box plots, histograms, scatter plots, and customized charts looking at clustered mice or cross pedigree comparisons. In summary, MouseTRACS enables the efficient screening, analysis, and management of thousands of animals to find mutant mice and identify novel gene functions. MouseTR-ACS is available under an open source license at http://mousetracs.sourceforge.net.

The Journal of Immunology, 2009

Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) converts inositol 1,4,5-trisphosphate to inosi... more Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) converts inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate upon Ag receptor activation and controls the fate and function of lymphocytes. To determine the role of Itpkb in B cell tolerance, Itpkb−/− mice were crossed to transgenic mice that express a BCR specific for hen egg lysozyme (IgHEL). B cells from Itpkb−/− IgHEL mice possess an anergic phenotype, hypoproliferate in response to cognate Ag, and yet they exhibit enhanced Ag-induced calcium signaling. In IgHEL transgenic mice that also express soluble HEL, lack of Itpkb converts anergy induction to deletion. These data establish Itpkb as a negative regulator of BCR signaling that controls the fate of developing B cells and tolerance induction.

Journal of Experimental Medicine, 2012

B cell development requires tight regulation to allow for the generation of a diverse repertoire ... more B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)–induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell–dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatm...

The Journal of experimental medicine, 1994

Successful antibody production in vivo depends on a number of cellular events, one of the most im... more Successful antibody production in vivo depends on a number of cellular events, one of the most important of these being cognate B cell-T cell interaction. To examine this phenomenon in vitro, homogeneous populations of hen egg lysozyme (HEL)-specific small resting B cells and naive CD4+ HEL-specific T cells (derived from immunoglobulin [Ig] and T cell receptor transgenic mice, respectively) were cultured together. On addition of intact HEL protein. HEL-specific B cells increase their expression of activation molecules, including a B7-related protein and CD44, and enlarge into blast cells. Within the same cultures, HEL-specific CD4+ T cells also increase expression of the activation markers CD69 and CD44, enlarge, secrete lymphokines, and proliferate. This response is radiation sensitive, supporting the conclusion that HEL-specific B cells present antigen to and activate the naive T cells. By contrast, when a synthetic peptide fragment of HEL is used to bypass B cell antigen-receptor...

International Immunology, 2003

IgE plus antigen-stimulated mast cells degranulate, synthesize leukotrienes and secrete cytokines... more IgE plus antigen-stimulated mast cells degranulate, synthesize leukotrienes and secrete cytokines. According to the coalescence model this process is initiated in speci®c membrane compartments termed rafts. There, enhanced levels of glycosphingolipids and cholesterol stabilize the interaction of FceRI and Lyn, and thus facilitate the ®rst steps of signal transduction. Enforced changes in raft architecture by cholesterol deprivation and exogenous application of glycosphingolipids in¯uence these early events by loss of tyrosine kinase activity or receptor-independent signal initiation respectively. Here we show that exogenously added cholesterol accumulates in rafts and activates mast cells. An investigation of the signaling events reveals that in contrast to IgE plus antigenmediated stimulation, cholesterol triggers p38 mitogen-activated protein kinase and preferentially induces expression of FosB. Consequently, a comparative large-scale microarray analysis demonstrates that a number of IgE plus antigen-induced immediate early genes (peak expression at 30 min after induction) are repressed by cholesterol. These changes further translate into altered expression levels and time kinetics of a number of early genes (peak expression at 2 h after stimulation). As the most prominent example for cholesterol-dependent genes, we identi®ed PAI1 (plasminogen activator inhibitor 1), a protein regarded as a risk factor for atherosclerosis.

Immunology Letters, 2011

Light chain receptor editing is an important mechanism that prevents B cell self-reactivity. We h... more Light chain receptor editing is an important mechanism that prevents B cell self-reactivity. We have previously shown that tonic signaling through the BCR represses RAG expression at the immature B cell stage, and that initiation of light chain rearrangements occurs in the absence of these tonic signals in an in vitro model of B cell development. To further test our hypothesis we studied the effect of itpkb deficiency (itpkb-/mice) or Raf hyper-activation (Raf-CAAX transgenic mice), two mutations that enhance BCR signaling, on receptor editing in an in vivo model. This model relies on transferring bone marrow from wild-type or mutant mice into mice expressing an anti-kappa light chain transgene. The anti-kappa transgene induces receptor editing of all kappa light chain expressing B cells, leading to a high frequency of lambda light chain expressing B cells. Anti-κ transgenic recipients of bone marrow from itpkb-/or Raf-CAAX mice showed lower levels of editing to λ light chain than did non-transgenic control recipients. These results provide evidence in an in vivo model that enhanced BCR signaling at the immature B cell stage of development suppresses light chain receptor editing.

Immunology & Cell Biology, 2007

Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent ... more Selection of B cells subjected to hypermutation in germinal centres (GC) during T cell-dependent (TD) antibody responses yields memory cells and long-lived plasma cells that produce high affinity antibodies biased to foreign antigens rather than self-antigens. GC also form in T-independent (TI) responses to polysaccharide antigens but failed selection results in GC involution and memory cells are not generated. To date there are no markers that allow phenotypic distinction of T-dependent and TI germinal centre B cells. We compared the global gene expression of GC B cells purified from mice immunized with either TD or TI antigens and identified eighty genes that are differentially expressed in TD GC. Significantly, the largest cluster comprises genes involved in growth and guidance of neuron axons such as Plexin B2, Basp1, Nelf, Shh, Sc4mol and Sult4a. This is consistent with formation of long neurite (axon and dendrite)-like structures by mouse and human GC B cells, which may facilitate T:B cell interactions within GC, affinity maturation and B cell memory formation. Expression of BASP1 and PLEXIN B2 protein is very low or undetectable in resting and TI GC B cells, but markedly upregulated in GC B cells induced in the presence of T cell help. Finally we show some of the axon growth genes upregulated in TD-GC B cells including Basp1, Shh, Sult4a, Sc4mol are also preferentially expressed in post-GC B cell neoplasms.

Blood, 2007

Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of ... more Platelet glycoprotein VI (GPVI) is a key receptor for collagens that mediates the propagation of platelet attachment and activation. Targeted disruption of the murine gene Gp6 on a mixed 129 × 1/SvJ × C57BL/6J background causes the expected defects in collagen-dependent platelet responses in vitro. The extent of this dysfunction in all Gp6−/− mice is uniform and is not affected by genetic background. However, the same Gp6−/− mice exhibit 2 diametrically opposed phenotypes in vivo. In some mice, tail bleeding times are extremely prolonged, and thrombus formation in an in vivo carotid artery ferric chloride-injury model is significantly impaired. In other littermates, tail bleeding times are within the range of wild-type mice, and in vivo thrombus formation is indistinguishable from that of control mice. Directed intercrosses revealed that these phenotypes are heritable, and a genome-wide single-nucleotide polymorphism scan revealed the most significant linkage to a single locus (8 me...

Cell Cycle, 2008

Nearly 25 years ago the first function of an inositol phosphate, namely Ins(1,4,5)P3, was reporte... more Nearly 25 years ago the first function of an inositol phosphate, namely Ins(1,4,5)P3, was reported to act as a "second messenger" to mobilize calcium from the endoplasmic reticulum (ER). Since this discovery, many other inositol phosphates and the kinases and phosphatases that generate these inositol phosphates have subsequently been discovered. However, the function of these "higher order" inositol phosphates in biological processes, if any, has remained a mystery. Interest in higher order inositol phosphates, such as Ins(1,3,4,5)P4, was renewed this year following reports of novel roles for these molecules in distinct processes within the immune system ranging from T cell development, B cell development and tolerance induction, as well as neutrophil and mast cell function. In this review, we will touch upon recent advances in inositol phosphate function in mammalian cells. More specifically, we will highlight new studies that have identified novel functions for specific higher order inositol phosphates, such as Ins(1,3,4,5)P4, in the immune system.