The growth and maturation of B lymphocytes is regulated by signals through surface immunoglobulin (sIg) antigen receptors, through cell- interaction receptors and through cytokine receptors. Antigen-specific signals through the sIgM B-cell-receptor (BCR) complex can lead to proliferation or conversely to apoptosis. Crosslinking the sIgM BCR leads to activation of protein tyrosine kinases (PTK), to mobilization of intracellular free calcium [Ca2+]i and to activation of ser/thr kinases. The characteristics and regulation of the signaling via the BCR complex still are not well understood. The sIgM BCR contains sIgM and heterodimers of Ig/alpha and Ig/Beta, which are necessary for expression and signaling through the BCR. Recently we and others have found that the sIgM BCR not only has src-family PTK such as p53/56lyn associated with it, but also has a B-cell-specific adhesion molecule, CD22, and a protein tyrosine kinase (PTK) designated Spleen Tyrosine Kinase or SYK associated with it. We plan to elucidate the roles that SYK and CD22 play in regulating B cell activation.
Our Specific Aims are: 1) To test the hypothesis that SYK kinase plays an essential role in signaling via the sIg receptor and other B cell-associated surface receptors. Given the similarities between SYK and its T-cell-associated cousin, ZAP-70, we postulate that just as ZAP-70 is recruited into the CD3/T cell receptor (TCR) complex after CD3/TCR crosslinking, SYK is recruited into the BCR complex after sIg-crosslinking; 2) To test the hypothesis that CD22 functions to regulate signaling via the sIg receptor by interacting with SYK and/or other protein kinases. We will define the intracellular structures with which CD22 interacts and the molecular basis and functional consequences of these interactions; 3) To test the hypothesis that extracellular CD22 regulates B cell signaling by interactions with sIgM or other B cell-associated surface molecules. We will characterize the B surface molecules bound by mCD22 and determine whether binding of CD22 recombinant globulins (Rgs) affect B cell activation or growth; and 4) Test the hypothesis that SYK and/or CD22 are required for normal B cell maturation and responses to foreign antigens. Constructs encoding the inactive SYK will be transfected into B cell lines to access the affect of expression """"""""dominant negative"""""""" SYK on B cell transduction pathways. Based on these results, transgenic mice expressing """"""""dominant negative"""""""" SYK will be developed to assess the effect of mutated SYK on B and T cell activation in vivo. We will use genomic clones for mouse CD22 and SYK to target and disrupt Cd22 and Syk genes and construct CD22 and SYK """"""""knockout mice."""""""" Immature and mature B and T cells from CD22- negative or SYK-negative vs. normal control mice will be examined for surface phenotype, potential maturation arrest and for their ability to respond to mitogens and specific-antigens. Understanding the role of SYK and CD22 in B cell maturation may provide new insights into how B cells are dysregulated in processes leading to B cell malignancies such as lymphomas and lymphocytic leukemias.
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