Lakhu Keshvara - Academia.edu (original) (raw)

Papers by Lakhu Keshvara

Research paper thumbnail of Dectin-1 mediates zymosan responses in microglia

The Faseb Journal, Apr 1, 2007

Research paper thumbnail of Post-transcriptional regulation of inducible nitric oxide synthase (iNOS) by Protein Interacting with Never in Mitosis Gene A-1 (PIN1) in murine aortic endothelial cells

The Faseb Journal, Mar 1, 2008

Research paper thumbnail of Inhibition of Signaling Through the B Cell Antigen Receptor by the Protooncogene Product, c-Cbl, Requires Syk Tyrosine 317 and the c-Cbl Phosphotyrosine-Binding Domain1

The Journal of Immunology, Dec 1, 1999

Research paper thumbnail of Cyclin dependent kinase 5 phosphorylation of disabled 1 protein

Research paper thumbnail of The Role of the PTB Domain in Regulation of DAB1 Phosphorylation

The Faseb Journal, Apr 1, 2007

Research paper thumbnail of Keshvara LM, Isaacson CC, Yankee TM, Sarac R, Harrison ML, Geahlen RL.. Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J Immunol 161: 5276-5283

The Journal of Immunology

The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pat... more The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-S...

Research paper thumbnail of Inhibition of signaling through the B cell antigen receptor by the protooncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain

Journal of immunology (Baltimore, Md. : 1950), 1999

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical... more The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indi...

Research paper thumbnail of Beta-glucan activates microglia without inducing cytokine production in Dectin-1-dependent manner

Journal of immunology (Baltimore, Md. : 1950), 2008

Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive... more Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive responses within the CNS. Like other immune cells, microglia recognize and are activated by various pathogen-associated molecular patterns. beta-glucans are pathogen-associated molecular patterns present within fungal cell walls that are known to trigger protective responses in a number of immune cells. In an effort to better understand microglial responses to beta-glucans and the underlying response pathways, we sought to determine whether Dectin-1, a major beta-glucan receptor recently identified in leukocytes, plays a similar role in beta-glucan-induced activation in microglia. In this study, we report that Dectin-1 is indeed expressed on the surface of murine primary microglia, and engagement of the receptor with particulate beta-glucan resulted in an increase in tyrosine phosphorylation of spleen tyrosine kinase, a hallmark feature of the Dectin-1 signaling pathway. Moreover, phago...

Research paper thumbnail of Vav1 and PI3K are required for phagocytosis of β-glucan and subsequent superoxide generation by microglia

Molecular Immunology, 2009

Microglia are the resident innate immune cells that are critical for innate and adaptive immune r... more Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. ␤-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by ␤-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of ␤-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with ␤-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to ␤-glucans.

Research paper thumbnail of Domain c-Cbl Phosphotyrosine-Binding Requires Syk Tyrosine 317 and the Protooncogene Product, c-Cbl, Cell Antigen Receptor by the Inhibition of Signaling Through the B

Research paper thumbnail of β-Glucan attenuates TLR2- and TLR4-mediated cytokine production by microglia

Neuroscience Letters, 2009

Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS i... more Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS injury, and their activation is critical for maintaining homeostasis within the CNS. However, during inflammatory conditions, sustained microglial activation results in damage to surrounding neuronal cells. ␤-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that ␤-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate ␤-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of ␤-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, ␤-glucan suppressed TLR-mediated NF-B activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that ␤-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions.

Research paper thumbnail of Rescue of Ataxia and Preplate Splitting by Ectopic Expression of Reelin in reeler Mice

Neuron, 2002

et al., 1983). Remarkably, these anatomical fea-St. Jude Children's Research Hospital tures are p... more et al., 1983). Remarkably, these anatomical fea-St. Jude Children's Research Hospital tures are present in other strains of mutant mice with 332 North Lauderdale alterations in the disabled-1 (dab-1) gene (Howell et al., Memphis, Tennessee 38105 1997b; Sheldon et al., 1997) or disruption of both the very low density lipoprotein receptor (vldlr) and the apolipoprotein E receptor-2 (apoER2) genes (Trommsdorff Summary et al., 1999). It is now clear that the protein products of these genes function downstream of Reelin as compo-The gene mutated in reeler (reelin) encodes a protein nents of a signaling pathway that governs cell positionsecreted by neurons in the developing brain that coning during brain development (Rice and Curran, 2001). trols laminar positioning of migrating cells in the CNS Reelin is a large protein secreted by Cajal-Retzius by an unknown mechanism. To investigate Reelin cells into the marginal zone during formation of the cortifunction, we used the nestin promoter to express Reecal plate, and by granule cell precursors in the EGL of lin ectopically in the ventricular zone and other brain the cerebellum before formation of the Purkinje cell plate regions in transgenic mice. In the presence of the (D'Arcangelo et al., 1995; Ogawa et al., 1995). Reelin endogenous protein, ectopic Reelin did not alter cell binds to the transmembrane receptors ApoER2 and migration in the neocortex or the cerebellum. How-VLDLR present on migrating cortical neurons and Purever, in the reeler background, ectopic Reelin induced kinje cells (D'Arcangelo et al., 1999; Hiesberger et al., tyrosine phosphorylation of Dab-1 in the ventricular 1999). The intracellular protein encoded by dab-1 is also zone and rescued some, but not all, of the neuroanaexpressed in migrating neurons destined for the cortical tomic and behavioral abnormalities characteristic of plate and in migrating Purkinje cells in the cerebellum reeler. These results indicate that Reelin does not (Howell et al., 1997b; Rice et al., 1998). It exhibits several function simply as a positional signal. Rather, it apstructural features of adaptor proteins that function in pears to participate in multiple events critical for neuprotein kinase signaling cascades (Howell et al., 1997a). ronal migration and cell positioning. Dab-1 binds to a cytoplasmic region of the lipoprotein receptors, and other transmembrane proteins con-T. (1997). scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature 389, 730-733. Sheppard, A.M., and Pearlman, A.L. (1997). Abnormal reorganization of preplate neurons and their associated extracellular matrix: an early manifestation of altered neocortical development in the reeler mutant mouse.

Research paper thumbnail of Reelin Is a Ligand for Lipoprotein Receptors

Neuron, 1999

A mutant phenotype identical to that observed in reeler has also been described in mice carrying ... more A mutant phenotype identical to that observed in reeler has also been described in mice carrying muta-St. Jude Children's Research Hospital 332 N. Lauderdale tions in the disabled-1 (dab1) gene (Howell et al., 1997b; Sheldon et al., 1997). Dab1 is an intracellular adapter Memphis, Tennessee 38105 protein that is expressed in Reelin target cells (Howell et al., 1997a; Rice et al., 1998). Dab1 accumulates in the absence of Reelin, suggesting that Reelin may promote Summary degradation of Dab1 (Sheldon et al., 1997; Rice et al., 1998). The addition of Reelin to primary neuronal cul-A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 tures results in increased tyrosine phosphorylation of Dab1 (Howell et al . Thus, Dab1 is thought to (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin function downstream of Reelin in a signal transduction cascade that controls appropriate cell positioning in the binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and developing brain. Recently, reeler-like defects were described in mice apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of lacking two members of the low-density lipoprotein receptor (LDLR) family, the very low-density lipoprotein apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association receptor (VLDLR) and the apolipoprotein E receptor 2

Research paper thumbnail of Dectin-1 mediates zymosan responses in microglia

Journal of Neuropathology and Experimental Neurology, 2007

Research paper thumbnail of Protein Never in Mitosis Gene A Interacting-1 regulates calpain activity and the degradation of cyclooxygenase-2 in endothelial cells

Journal of Inflammation, 2009

The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates t... more The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates turnover of inducible nitric oxide synthase (iNOS) in murine aortic endothelial cells (MAEC) stimulated with E. coli endotoxin (LPS) and interferon-γ (IFN). Degradation of iNOS was reduced by a calpain inhibitor, suggesting that PIN1 may affect induction of other calpain-sensitive inflammatory proteins, such as cyclooxygenase (COX)-2, in MAEC.

Research paper thumbnail of  -Glucan Activates Microglia without Inducing Cytokine Production in Dectin-1-Dependent Manner

The Journal of Immunology, 2008

Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive... more Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive responses within the CNS. Like other immune cells, microglia recognize and are activated by various pathogen-associated molecular patterns. ␤-glucans are pathogen-associated molecular patterns present within fungal cell walls that are known to trigger protective responses in a number of immune cells. In an effort to better understand microglial responses to ␤-glucans and the underlying response pathways, we sought to determine whether Dectin-1, a major ␤-glucan receptor recently identified in leukocytes, plays a similar role in ␤-glucaninduced activation in microglia. In this study, we report that Dectin-1 is indeed expressed on the surface of murine primary microglia, and engagement of the receptor with particulate ␤-glucan resulted in an increase in tyrosine phosphorylation of spleen tyrosine kinase, a hallmark feature of the Dectin-1 signaling pathway. Moreover, phagocytosis of ␤-glucan particles and subsequent intracellular production of reactive oxygen species were also mediated by Dectin-1. However, unlike in macrophages and dendritic cells, ␤-glucan-mediated microglial activation did not result in significant production of cytokines or chemokines; thus, the interaction of microglial Dectin-1 with glucan elicits a unique response. Our results suggest that the Dectin-1 pathway may play an important role in antifungal immunity in the CNS.

Research paper thumbnail of Crystal Structures of the Dab Homology Domains of Mouse Disabled 1 and 2

Journal of Biological Chemistry, 2003

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic medi... more Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.

Research paper thumbnail of Syk Activation and Dissociation from the B-cell Antigen Receptor Is Mediated by Phosphorylation of Tyrosine 130

Journal of Biological Chemistry, 1997

Syk (p72(syk)) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential compon... more Syk (p72(syk)) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor.

Research paper thumbnail of Phosphorylation- and Activation-independent Association of the Tyrosine Kinase Syk and the Tyrosine Kinase Substrates Cbl and Vav with Tubulin in B-Cells

Journal of Biological Chemistry, 1999

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyro... more Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

Research paper thumbnail of Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

Glycoconjugate Journal, 1992

ELISA assays have been developed for ~(1-3)N-acetylgalactosaminyltransferase (blood group A trans... more ELISA assays have been developed for ~(1-3)N-acetylgalactosaminyltransferase (blood group A transferase) and c~(1-3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc~l-2Galfl-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuccd-2(GalNAcel-3)Galfl-R-BSA or Fucel-2(Galel-3)Galfl-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.

Research paper thumbnail of Dectin-1 mediates zymosan responses in microglia

The Faseb Journal, Apr 1, 2007

Research paper thumbnail of Post-transcriptional regulation of inducible nitric oxide synthase (iNOS) by Protein Interacting with Never in Mitosis Gene A-1 (PIN1) in murine aortic endothelial cells

The Faseb Journal, Mar 1, 2008

Research paper thumbnail of Inhibition of Signaling Through the B Cell Antigen Receptor by the Protooncogene Product, c-Cbl, Requires Syk Tyrosine 317 and the c-Cbl Phosphotyrosine-Binding Domain1

The Journal of Immunology, Dec 1, 1999

Research paper thumbnail of Cyclin dependent kinase 5 phosphorylation of disabled 1 protein

Research paper thumbnail of The Role of the PTB Domain in Regulation of DAB1 Phosphorylation

The Faseb Journal, Apr 1, 2007

Research paper thumbnail of Keshvara LM, Isaacson CC, Yankee TM, Sarac R, Harrison ML, Geahlen RL.. Syk- and Lyn-dependent phosphorylation of Syk on multiple tyrosines following B cell activation includes a site that negatively regulates signaling. J Immunol 161: 5276-5283

The Journal of Immunology

The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pat... more The Syk protein tyrosine kinase is an essential component of the B cell Ag receptor signaling pathway. Syk is phosphorylated on tyrosine following B cell activation. However, the sites that are modified and the kinases responsible for these modifications have yet to be determined. To approach this problem, we used a mapping strategy based on the electrophoretic separation of peptides on alkaline polyacrylamide gels to identify the tryptic phosphopeptides derived from metabolically labeled Syk. In this work, we report that Syk from activated B cells is phosphorylated principally on six tyrosines: one located between the tandem SH2 domains (Tyr130); three in the linker region (Tyr317, Tyr342, and Tyr346); and two in the catalytic domain (Tyr519 and Tyr520). The linker region sites are the primary targets of the Src family protein tyrosine kinase, Lyn, and include a site that negatively (Tyr317) regulates receptor signaling. Efficient phosphorylation of the catalytic domain and inter-S...

Research paper thumbnail of Inhibition of signaling through the B cell antigen receptor by the protooncogene product, c-Cbl, requires Syk tyrosine 317 and the c-Cbl phosphotyrosine-binding domain

Journal of immunology (Baltimore, Md. : 1950), 1999

The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical... more The Syk protein-tyrosine kinase couples the B cell Ag receptor (BCR) to intracellular biochemical pathways. Syk becomes phosphorylated on multiple tyrosine residues upon receptor cross-linking. Tyrosine 317 is a site of phosphorylation located within the linker region of Syk that separates the amino-terminal, tandem pair of SH2 domains from the carboxyl-terminal catalytic domain. The amino acid sequence surrounding phosphotyrosine 317 matches the consensus sequence for recognition by the phosphotyrosine-binding (PTB) domain of the protooncogene product, c-Cbl. The overexpression of c-Cbl in DT40 B cells inhibits Ag receptor-mediated activation of the NF-AT transcription factor. The ability of overexpressed c-Cbl to inhibit signaling requires both Syk tyrosine 317 and a functional c-Cbl PTB domain. Mutant forms of Syk lacking tyrosine 317 exhibit an enhanced ability to couple the BCR to pathways leading to the activation of both NF-AT and Elk-1. Coimmunoprecipitation experiments indi...

Research paper thumbnail of Beta-glucan activates microglia without inducing cytokine production in Dectin-1-dependent manner

Journal of immunology (Baltimore, Md. : 1950), 2008

Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive... more Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive responses within the CNS. Like other immune cells, microglia recognize and are activated by various pathogen-associated molecular patterns. beta-glucans are pathogen-associated molecular patterns present within fungal cell walls that are known to trigger protective responses in a number of immune cells. In an effort to better understand microglial responses to beta-glucans and the underlying response pathways, we sought to determine whether Dectin-1, a major beta-glucan receptor recently identified in leukocytes, plays a similar role in beta-glucan-induced activation in microglia. In this study, we report that Dectin-1 is indeed expressed on the surface of murine primary microglia, and engagement of the receptor with particulate beta-glucan resulted in an increase in tyrosine phosphorylation of spleen tyrosine kinase, a hallmark feature of the Dectin-1 signaling pathway. Moreover, phago...

Research paper thumbnail of Vav1 and PI3K are required for phagocytosis of β-glucan and subsequent superoxide generation by microglia

Molecular Immunology, 2009

Microglia are the resident innate immune cells that are critical for innate and adaptive immune r... more Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. ␤-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by ␤-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of ␤-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with ␤-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to ␤-glucans.

Research paper thumbnail of Domain c-Cbl Phosphotyrosine-Binding Requires Syk Tyrosine 317 and the Protooncogene Product, c-Cbl, Cell Antigen Receptor by the Inhibition of Signaling Through the B

Research paper thumbnail of β-Glucan attenuates TLR2- and TLR4-mediated cytokine production by microglia

Neuroscience Letters, 2009

Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS i... more Microglia, the resident immune cells of the brain, are activated in response to any kind of CNS injury, and their activation is critical for maintaining homeostasis within the CNS. However, during inflammatory conditions, sustained microglial activation results in damage to surrounding neuronal cells. ␤-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that ␤-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate ␤-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of ␤-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, ␤-glucan suppressed TLR-mediated NF-B activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that ␤-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions.

Research paper thumbnail of Rescue of Ataxia and Preplate Splitting by Ectopic Expression of Reelin in reeler Mice

Neuron, 2002

et al., 1983). Remarkably, these anatomical fea-St. Jude Children's Research Hospital tures are p... more et al., 1983). Remarkably, these anatomical fea-St. Jude Children's Research Hospital tures are present in other strains of mutant mice with 332 North Lauderdale alterations in the disabled-1 (dab-1) gene (Howell et al., Memphis, Tennessee 38105 1997b; Sheldon et al., 1997) or disruption of both the very low density lipoprotein receptor (vldlr) and the apolipoprotein E receptor-2 (apoER2) genes (Trommsdorff Summary et al., 1999). It is now clear that the protein products of these genes function downstream of Reelin as compo-The gene mutated in reeler (reelin) encodes a protein nents of a signaling pathway that governs cell positionsecreted by neurons in the developing brain that coning during brain development (Rice and Curran, 2001). trols laminar positioning of migrating cells in the CNS Reelin is a large protein secreted by Cajal-Retzius by an unknown mechanism. To investigate Reelin cells into the marginal zone during formation of the cortifunction, we used the nestin promoter to express Reecal plate, and by granule cell precursors in the EGL of lin ectopically in the ventricular zone and other brain the cerebellum before formation of the Purkinje cell plate regions in transgenic mice. In the presence of the (D'Arcangelo et al., 1995; Ogawa et al., 1995). Reelin endogenous protein, ectopic Reelin did not alter cell binds to the transmembrane receptors ApoER2 and migration in the neocortex or the cerebellum. How-VLDLR present on migrating cortical neurons and Purever, in the reeler background, ectopic Reelin induced kinje cells (D'Arcangelo et al., 1999; Hiesberger et al., tyrosine phosphorylation of Dab-1 in the ventricular 1999). The intracellular protein encoded by dab-1 is also zone and rescued some, but not all, of the neuroanaexpressed in migrating neurons destined for the cortical tomic and behavioral abnormalities characteristic of plate and in migrating Purkinje cells in the cerebellum reeler. These results indicate that Reelin does not (Howell et al., 1997b; Rice et al., 1998). It exhibits several function simply as a positional signal. Rather, it apstructural features of adaptor proteins that function in pears to participate in multiple events critical for neuprotein kinase signaling cascades (Howell et al., 1997a). ronal migration and cell positioning. Dab-1 binds to a cytoplasmic region of the lipoprotein receptors, and other transmembrane proteins con-T. (1997). scrambler and yotari disrupt the disabled gene and produce a reeler-like phenotype in mice. Nature 389, 730-733. Sheppard, A.M., and Pearlman, A.L. (1997). Abnormal reorganization of preplate neurons and their associated extracellular matrix: an early manifestation of altered neocortical development in the reeler mutant mouse.

Research paper thumbnail of Reelin Is a Ligand for Lipoprotein Receptors

Neuron, 1999

A mutant phenotype identical to that observed in reeler has also been described in mice carrying ... more A mutant phenotype identical to that observed in reeler has also been described in mice carrying muta-St. Jude Children's Research Hospital 332 N. Lauderdale tions in the disabled-1 (dab1) gene (Howell et al., 1997b; Sheldon et al., 1997). Dab1 is an intracellular adapter Memphis, Tennessee 38105 protein that is expressed in Reelin target cells (Howell et al., 1997a; Rice et al., 1998). Dab1 accumulates in the absence of Reelin, suggesting that Reelin may promote Summary degradation of Dab1 (Sheldon et al., 1997; Rice et al., 1998). The addition of Reelin to primary neuronal cul-A signaling pathway involving the extracellular protein Reelin and the intracellular adaptor protein Disabled-1 tures results in increased tyrosine phosphorylation of Dab1 (Howell et al . Thus, Dab1 is thought to (Dab1) controls cell positioning during mammalian brain development. Here, we demonstrate that Reelin function downstream of Reelin in a signal transduction cascade that controls appropriate cell positioning in the binds directly to lipoprotein receptors, preferably the very low-density lipoprotein receptor (VLDLR) and developing brain. Recently, reeler-like defects were described in mice apolipoprotein E receptor 2 (ApoER2). Binding requires calcium, and it is inhibited in the presence of lacking two members of the low-density lipoprotein receptor (LDLR) family, the very low-density lipoprotein apoE. Furthermore, the CR-50 monoclonal antibody, which inhibits Reelin function, blocks the association receptor (VLDLR) and the apolipoprotein E receptor 2

Research paper thumbnail of Dectin-1 mediates zymosan responses in microglia

Journal of Neuropathology and Experimental Neurology, 2007

Research paper thumbnail of Protein Never in Mitosis Gene A Interacting-1 regulates calpain activity and the degradation of cyclooxygenase-2 in endothelial cells

Journal of Inflammation, 2009

The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates t... more The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates turnover of inducible nitric oxide synthase (iNOS) in murine aortic endothelial cells (MAEC) stimulated with E. coli endotoxin (LPS) and interferon-γ (IFN). Degradation of iNOS was reduced by a calpain inhibitor, suggesting that PIN1 may affect induction of other calpain-sensitive inflammatory proteins, such as cyclooxygenase (COX)-2, in MAEC.

Research paper thumbnail of  -Glucan Activates Microglia without Inducing Cytokine Production in Dectin-1-Dependent Manner

The Journal of Immunology, 2008

Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive... more Microglia are the resident mononuclear phagocytic cells that are critical for innate and adaptive responses within the CNS. Like other immune cells, microglia recognize and are activated by various pathogen-associated molecular patterns. ␤-glucans are pathogen-associated molecular patterns present within fungal cell walls that are known to trigger protective responses in a number of immune cells. In an effort to better understand microglial responses to ␤-glucans and the underlying response pathways, we sought to determine whether Dectin-1, a major ␤-glucan receptor recently identified in leukocytes, plays a similar role in ␤-glucaninduced activation in microglia. In this study, we report that Dectin-1 is indeed expressed on the surface of murine primary microglia, and engagement of the receptor with particulate ␤-glucan resulted in an increase in tyrosine phosphorylation of spleen tyrosine kinase, a hallmark feature of the Dectin-1 signaling pathway. Moreover, phagocytosis of ␤-glucan particles and subsequent intracellular production of reactive oxygen species were also mediated by Dectin-1. However, unlike in macrophages and dendritic cells, ␤-glucan-mediated microglial activation did not result in significant production of cytokines or chemokines; thus, the interaction of microglial Dectin-1 with glucan elicits a unique response. Our results suggest that the Dectin-1 pathway may play an important role in antifungal immunity in the CNS.

Research paper thumbnail of Crystal Structures of the Dab Homology Domains of Mouse Disabled 1 and 2

Journal of Biological Chemistry, 2003

Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic medi... more Disabled (Dab) 1 and 2 are mammalian homologues of Drosophila DAB. Dab1 is a key cytoplasmic mediator in Reelin signaling that controls cell positioning in the developing central nervous system, whereas Dab2 is an adapter protein that plays a role in endocytosis. DAB family proteins possess an amino-terminal DAB homology (DH) domain that is similar to the phosphotyrosine binding/phosphotyrosine interaction (PTB/PI) domain. We have solved the structures of the DH domains of Dab2 (Dab2-DH) and Dab1 (Dab1-DH) in three different ligand forms, ligand-free Dab2-DH, the binary complex of Dab2-DH with the Asn-Pro-X-Tyr (NPXY) peptide of amyloid precursor protein (APP), and the ternary complex of Dab1-DH with the APP peptide and inositol 1,4,5-trisphosphate (Ins-1,4,5-P3, the head group of phosphatidylinositol-4,5-diphosphate (PtdIns-4,5-P2)). The similarity of these structures suggests that the rigid Dab DH domain maintains two independent pockets for binding of the APP/lipoprotein receptors and phosphoinositides. Mutagenesis confirmed the structural determinants specific for the NPXY sequence and PtdIns-4,5-P2 binding. NMR spectroscopy confirmed that the DH domain binds to Ins-1,4,5-P3 independent of the NPXY peptides. These findings suggest that simultaneous interaction of the rigid DH domain with the NPXY sequence and PtdIns-4,5-P2 plays a role in the attachment of Dab proteins to the APP/lipoprotein receptors and phosphoinositide-rich membranes.

Research paper thumbnail of Syk Activation and Dissociation from the B-cell Antigen Receptor Is Mediated by Phosphorylation of Tyrosine 130

Journal of Biological Chemistry, 1997

Syk (p72(syk)) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential compon... more Syk (p72(syk)) is a 72-kDa cytoplasmic protein-tyrosine kinase that serves as an essential component of the signal transduction machinery coupled to the B-cell antigen receptor. Syk is recruited to the receptor when it is cross-linked and, in response, becomes tyrosine-phosphorylated and activated before it dissociates from the receptor and appears in the cytoplasm. To begin to explore how tyrosine phosphorylation affects Syk activation and receptor binding, Tyr-130, which is localized within the Syk inter-Src homology 2 domain region, was substituted with Phe or Glu. Substitution of Tyr-130 with Phe enhanced the binding of Syk to the receptor and increased receptor-mediated protein tyrosine phosphorylation, while substitution with Glu greatly reduced this interaction. Replacement of Tyr-130 with Glu also increased the basal activity of the kinase, while replacement with Phe decreased its activity and uncoupled kinase activation from receptor engagement. These data suggest that the phosphorylation of Tyr-130 normally plays an important role in mediating both the activation of Syk and its release from the antigen receptor.

Research paper thumbnail of Phosphorylation- and Activation-independent Association of the Tyrosine Kinase Syk and the Tyrosine Kinase Substrates Cbl and Vav with Tubulin in B-Cells

Journal of Biological Chemistry, 1999

Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyro... more Aggregation of the B-cell antigen receptor leads to the activation of the 72-kDa Syk protein-tyrosine kinase and the phosphorylation of tubulin on tyrosine. To explore the requirement of Syk catalytic activity for tubulin phosphorylation, tubulin was isolated from cytosolic fractions from anti-IgM-activated B-cells (DT40) that lacked endogenous Syk and immunoblotted with anti-phosphotyrosine antibodies. Tubulin was not tyrosine-phosphorylated in Syk- B-cells. Phosphorylation could be restored by the expression of wild-type, but not catalytically inactive, Syk. However, both catalytically inactive and wild-type Syk were capable of constitutive association with tubulin, indicating that tubulin phosphorylation is not required for this interaction. Anti-phosphotyrosine antibody immunoblotting of proteins adsorbed to colchicine-agarose revealed the presence of three major tubulin-associated phosphoproteins of 110, 90, and 74 kDa, the phosphorylation of which was dependent on Syk expression. The proteins of 110 and 90 kDa were identified as Cbl and Vav, two proto-oncogene products known to become prominently phosphorylated following receptor engagement. Both proteins were shown to be constitutively associated with tubulin.

Research paper thumbnail of Enzyme-linked immunosorbent assays for the measurement of blood group A and B glycosyltransferase activities

Glycoconjugate Journal, 1992

ELISA assays have been developed for ~(1-3)N-acetylgalactosaminyltransferase (blood group A trans... more ELISA assays have been developed for ~(1-3)N-acetylgalactosaminyltransferase (blood group A transferase) and c~(1-3)galactosyltransferase (blood group B transferase) activities. In these assays, microtitre plates coated with the bovine serum albumin conjugate of a synthetic Fuc~l-2Galfl-R acceptor substrate are incubated with the appropriate nucleotide donor (UDP-GalNAc or UDP-Gal) and human serum as the enzyme source. The resulting trisaccharide products Fuccd-2(GalNAcel-3)Galfl-R-BSA or Fucel-2(Galel-3)Galfl-R-BSA are detected and quantified with monoclonal antibodies selected not to cross-react with the substrate structure. With less than a microliter of human serum, product formation is proportional to enzyme concentration and to time of incubation of up to 90 min.