This project is designed to understand the role of tyrosine protein phosphorylation in the activation of B lymphocytes by antigen. The binding of antigen or anti-immunoglobulin antibody to immunoglobulin M or D on the surface of mature B cells induces the rapid phosphorylation of approximately 10 proteins on tyrosine. Surface immunoglobulin therefore must be coupled in some manner to one or more tyrosine protein kinases or phosphatases. Because an inhibitor of tyrosine phosphorylation renders B cells unresponsive to stimulation through the antigen receptor, tyrosine phosphorylation is apparently important in B cell activation. B cells that do not express the tyrosine protein phosphatase CD45 are unresponsive to the cross-linking of surface immunoglobulin. This insensitivity could be due to increased inhibitory phosphorylation of a tyrosine protein kinase. We will therefore study the phosphorylation of the several src-family tyrosine protein kinases found associated with the antigen receptor complex. Additionally, we will ask whether the insensitivity of CD45-negative B cells is reversed by introduction into them of a constitutively-activated version of a src-family kinase. We will also study the interaction of the src-family kinases with the antigen receptor complex. We will identify the domain of the src kinases responsible for their interaction by introduction into B cells of mutated src kinases. Additionally, we will ask whether the cytoplasmic domains of either or both of the immunoglobulin-associated proteins, Ig-alpha and Ig-beta, can interact with tyrosine kinases directly. If so, we will ask whether these domains can participate in signal transduction. Finally, we will exploit newly-isolated monoclonal antibodies to two novel tyrosine phosphorylated proteins in activated B cells. The 95K and 140K proteins that these antibodies recognize will be characterized and their genes cloned. These studies have the potential to reveal the identities of tyrosine protein kinases and substrates that are important in the regulation of the proliferation of normal B cells and may play a role in the unregulated growth of malignant B cells such as those transformed by Abelson murine leukemia virus.
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