The broad, long-term objective of this proposal is to characterize a recently identified serine kinase activity that is responsible for phosphorylating seven autoantigens during apoptosis. Recent studies have demonstrated that some autoantigens undergo posttranslational modifications such as proteolytic cleavage and phosphorylation during apoptosis, and it is hypothesized that these and other modifications allow these proteins to bypass or overcome normal mechanisms of tolerance, contributing to the production of autoantibodies. Kinase cascades play critical roles in important cellular functions such as cell cycle regulation and receptor-mediated signalling, and protein phosphorylation has been directly implicated in the regulation of apoptosis. There are two specific aims of this proposal: i.) To identify autoantigens that are phosphorylated during apoptosis using combined biochemical and antibody affinity purification techniques; and ii.) To characterize and identify the kinase that phosphorylates the substrates identified in the first specific aim. In the later years of the proposal, cDNAs encoding the kinase will be expressed in mammalian cells, and the effects of the kinase on programmed cell death pathways using several complementary assays will be addressed. Once achieved, this system will be utilized to screen known serine kinase inhibitors for their ability to block autoantigen phosphorylation, with the long- term goal to better understand the mechanisms underlying autoantibody production. The results of this proposal may prove useful in drug screening and rational drug design for treatment of diseases including SLE, Sjogren's disease and scleroderma. This award will be instrumental in preparing the candidate for a career as a clinician-scientist in the field of autoimmune disease. The proposal includes attendance at several basic science courses directly related to the field, and acquisition of expertise in several new techniques including molecular cloning, library screening, biochemical purification, and protein expression. Dr. Anderson and members of an Advisory Committee will provide hands-on training in performance of the techniques and interpretation of the results. The commitment of Dr. Anderson and the Division to the applicant's development as a fully independent investigator, together with the rich intellectual environment provided by Harvard Medical School and Brigham and Women's Hospital, make this an ideal environment in which to complete this proposal.
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