B cell activation is initiated by the binding of the antigen to the B cell receptor (BCR), triggering signal cascades that result in the transcription of a variety of genes associated with B cell activation. Following the initiation of signaling the antigen-bound BCR enters the cell and trafficks to specialized MHC class II-containing intracellular compartments where the antigen is proteolytically cleaved and the resulting peptides bound to MHC class II molecules that are ultimately expressed on the B cell surface allowing for interaction with antigen-specific helper T cells. We recently determined that BCR signaling also triggers reorganization of the endocytic compartments, recruiting endosomes containing toll-like receptors to autophagosome compartments into which the BCR trafficks. We now understand that the BCR continues to signal as it enters the cell and that the correct intracellular trafficking of the BCR and its recruitment of the TLRs depend on these signals. The goal of this project is to understand where discrete steps in the BCR signaling cascade occur and how the spatial and temporal organization of signaling regulates the outcome of antigen binding to the BCR. Particular focus will be on the synergistic interaction of the BCR with the intracellular TLRs. Our recent studies provided evidence that components of the BCR signaling pathway are activated sequentially and in defined subcellular locations. We observed that the phosphorylated form of Syk kinase, pSyk, appeared on the plasma membrane immediately following BCR crosslinking, whereas the phosphorylated forms of the MAPKinases p38, ERK and JNK were not detected until the BCR had internalized from the plasma membrane and trafficked to autophagosome-like class II-containing compartments. Using a highly selective inhibitor of endocytosis we showed that blocking BCR internalization resulted in the recruitment of both proximal and downstream kinases to the plasma membrane where MAP kinases were hyper-phosphorylated and Akt and its downstream target Foxo were hypo-phosphorylated leading to the dysregulation of gene transcription controlled through these pathways. These studies are important in demonstrating that the cellular location of the BCR serves to compartmentalize kinase activation to regulate the outcome of signaling. Future studies aimed at defining the molecular composition of the intracellular BCR signaling sites may provide new targets for therapeutics to block BCR signaling in autoimmune disease and in BCR-dependent B cell tumors. We propose that as the BCR moves into the cell from the plasma membrane and trafficks intracellularly, the local microenvironments of the receptor changes and that in each microenvironment BCR signaling cascades are propagated and regulated differently. We are also interested in understanding how the intracellular trafficking of the BCR facilitates interaction with the intracellular TLRs. We showed that following the antigen binding and internalization the BCR signals for the recruitment of TLR9 from multiple small endosomes to an LC3-positive autophagosome into which the BCR trafficks antigen and where synergistic signaling to p38 and JNK activation occurs. The recruitment of TLR9 to the BCR was by a dynein-mediated, microtubule-network dependent process. TLR9 is responsive to DNA and the recruitment of TLR9 to the autophagosome-like compartment was necessary for B cell hyper-responses to DNA-containing antigens. We have now determined that BCR signaling also results in the recruitment of the intracellular TLRs, TLR7 and TLR3, to autophagosomes. Thus, the recruitment of TLRs to the autophagosomes into which the BCR trafficks appears to be a general feature of BCR-TLR interactions. Although a general feature, the molecular mechanisms by which recruitment is achieved does not appear universal. For example, TLR9 and 7 signaling is not required for the BCR-induced movement of these receptors to the autophagosomes as evidenced by the observation that BCR-induced recruitment of TLR9 and 7 occurs in MyD88-deficient B cells. For TLR3 the case appears different in that BCR-induced phosphorylation of the cytoplasmic domain of TLR3 by Lyn kinase and thus the activation of TLR3 appears to play a role in its recruitment. Over the last year we initiated studies to use atomic force microscopy to describe the morphological changes that occur in B cells following activation through the BCR or TLRs. Our initial results provided evidence for remarkably dynamic rapid changes in morphology within seconds of B cell activation involving large extension of the plasma membrane. These changes are distinctively different for B cells activated through the BCR versus through TLRs. TLRs induce blebbing of the plasma and BCRs induce membrane ruffling. Our future work in this area will focus on understanding the function of the observed morphological changes and the use of electron tomography to characterize the molecular structure of these membrane extensions. During the last year we initiated a collaboration with Dr. Joshua Milner, LAD, NIAID, to characterize the effect of mutant phosphatase Cgamma2 in the trafficking of the BCR in human B cells. Dr. Milner recently described PLCgamma2-exon deletions in individuals in three families with dominantly inherited complex of cold-induced urticarial, antibody deficiencies and susceptibility to infection and autoimmune disease. We would like to know if the antibody deficiencies in these individuals are due to alterations in BCR signaling and in trafficking. Our initial results indicate the internalization and entry into early and late endosomal compartments is accelerated(?) in these patients and that signaling through ERK is diminished. We plan to further characterize signaling, trafficking and gene expression in these cells.
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