): In the immune system, T lymphocytes utilize integrin receptors to interact with extracellular matrix proteins and other cells in the body. Integrin activity on T cells is regulated so that T cells can circulate when searching for antigen and can stop circulating when they become activated by a relevant antigen in tissue. While unactivated T cells express integrins that mediate low levels of adhesion, stimulation of several different T cell antigens (""""""""integrin regulators"""""""") upregulates integrin functional activity within minutes without corresponding increases in integrin expression. During the prior funding period, we demonstrated a central role for phosphatidylinositol 3-kinase (PI 3-K) in the regulation of beta1 integrin functional activity by the T cell antigens CD2, CD7, and CD28, as well as the antigen-specific CD3/T cell receptor (TCR) complex. This request for continuing support will address the intracellular signaling pathways triggered by CD3/TCR stimulation that mediate these rapid increases in beta1 integrin functional activity. We specifically propose that CD3/TCR signaling regulates beta1 integrin function by ZAP-70- and PI 2-K dependent recruitment of the Tec family tyrosine Itk to detergent-insoluble membrane microdomains and subsequent activation of Itk by the src family kinase lck.
In aim 1, we will use ZAP-70-deficient T cells to define the role of ZAP-70 in regulating CD3-TCR-induced PI-3K activation and beta1 integrin activation.
In aim 2, we will explore the role of lck in regulating beta1 integrin function by the CD4 co-receptor and define the role of ZAP-70 and the microdomain-localized adapter protein LAT in CD3/TCR mediated membrane localization of Itk. The role of Itk in regulating CD3/TCR induced activation of small G-proteins and actin reorganization will also be elucidated.
In aim 3, we will define a novel function for the serine-threonine kinase protein kinase D (PKD) in activating beta1 integrins following CD3/TCR or phorbol ester stimulation. We will specifically address a role for PKD in recruiting specific PKC isoforms to the membrane and the regulation of this recruitment by ZAP070, PI 3-K and Itk. These studies will identify the mechanisms by which structurally distinct families of tyrosine serine-threonine and lipid kinases interact with each other to initiate intracellular signaling events that activate beta1 integrin function. Identification of these complex intracellular signaling responses promises to lead to the development of therapeutics capable of specifically modulating integrin function.
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