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

© 2009 Liu et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Characterization of the in vivo phosphorylation sites on Syk including a tyrosine in the inter-SH2 domain that modulates the interaction of Syk with the B cell antigen receptor

Syk is a non-receptor protein-tyrosine kinase that plays a critical role in the antigen receptor-mediated signaling in B cells. Aggregation of the B cell antigen receptor (BCR) results in recruitment of Syk to the receptor. This association is required for the subsequent activation and tyrosine phosphorylation of Syk. Although both Syk activation and tyrosine phosphorylation are thought to be essential for the activation of downstream signaling pathways, the sites of in vivo phosphorylation have not been reported. To determine sites of in vivo phosphorylation, Myc epitope-tagged Syk was overexpressed in Syk-deficient DT40 chicken B cells and metabolically labeled with (\sp{32}P) orthophosphate. Modified tyrosines were identified by comparing the migration pattern of the resulting tryptic phosphopeptides on a 40% polyacrylamide gel with similar maps of previously identified phosphopeptides derived from in vitro autophosphorylated Syk. The results indicate that at least six tyrosines are modified in response to stimulation of B cells. Our results suggest that phosphorylation of a tyrosine (Tyr-130) located between the two SH2 domains causes Syk to dissociate from the antigen receptor. Substitution of this tyrosine with a phenylalanine, which prevents phosphorylation at this site, increased the affinity of Syk for the receptor and enhanced BCR-mediated signaling. In contrast, a glutamate substitution greatly reduced the interaction and resulted in reduced signaling. We also identified another tyrosine (Tyr-317), located in the hinge region between the SH2 domains and the kinase domain, that plays a role in modulating the interaction of Syk with the BCR. Replacement of this tyrosine with a phenylalanine also resulted in increased affinity for the receptor and enhanced signaling. These results reveal a novel mechanism by which Syk function is negatively regulated by factors that modulate its interaction with the receptor

Β-Glucan Attenuates TLR2- and TLR4-Mediated Cytokine Production by Microglia

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. β-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that β-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate β-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of β-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, β-glucan suppressed TLR-mediated NF-κB activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that β-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions

Vav1 and PI3k Are Required for Phagocytosis of β-Glucan and Subsequent Superoxide Generation by Microglia

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. β-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by β-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of β-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 β-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 β-glucans

Β-Glucan Activates Microglia Without Inducing Cytokine Production in Dectin-1-Dependent Manner

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. β-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 β-glucans and the underlying response pathways, we sought to determine whether Dectin-1, a major β-glucan receptor recently identified in leukocytes, plays a similar role in β-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 β-glucan resulted in an increase in tyrosine phosphorylation of spleen tyrosine kinase, a hallmark feature of the Dectin-1 signaling pathway. Moreover, phagocytosis of β-glucan particles and subsequent intracellular production of reactive oxygen species were also mediated by Dectin-1. However, unlike in macrophages and dendritic cells, β-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

β-Glucan attenuates TLR2- and TLR4-mediated cytokine production by microglia

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. β-Glucans are widely recognized immunomodulators, but the molecular mechanisms underlying their immunomodulatory actions have not been fully explored. We previously reported that β-glucans activate microglia through Dectin-1 without inducing significant amount of cytokines and chemokines. Here, we show that particulate β-glucans attenuate cytokine production in response to TLR stimulation; this inhibitory activity of β-glucan is mediated by Dectin-1 and does not require particle internalization. At the molecular level, β-glucan suppressed TLR-mediated NF-κB activation, which may be responsible for the diminished capacity of microglia to produce cytokines in response to TLR stimulation. Overall, these results suggest that β-glucans may be used to prevent or treat excessive microglial activation during chronic inflammatory conditions