CD28 is a pivotal T-cell molecular the plays important roles in T-cell activation, HIV pathogenesis, and the induction and maintenance of peripheral tolerance and autoimmunity. The overall objective of this proposal is to investigate the CD28 signal transduction mechanisms during human T-lymphocyte activation. Recent evidence strongly indicates that both JNK1 (the c-Jun N-terminal kinase) and the Rel/NF-kappaB transcription factors/oncoproteins are involved in the IL-2/HIV gene activation by CD28 signaling. To date, JNK1 is the only kinase known to be activated exclusively by TCR/CD3 plus CD28 costimulation but not by TCR/CD3 signaling alone. The purpose of this study will be; (i) to examine the potential modifications of RelA/cRel or lkappaBalpha by JNK1, and (ii) to define additional transcription factors or enzymes that are upstream or downstream of jNK1 in the CD28 signaling pathway.
The specific aims are as follows: 1. Roles of JNK1 Kinase in CD28 Signaling Pathway. We will test our hypothesis to determine if JNK1 directly or indirectly phosphorylates lkappa Balpha or RelA/cRel which leads to nuclear translocation or functional activation of RelA/cRel, and subsequently activation of IL-2/HIV gene transcription via the CD28RE/kappaB enhancer. The experiments will include protein interaction assays, kinase assays, transfections, and site- directed mutagenesis. 2. Cloning of Novel JNK1-interacting Proteins. We will isolate the human cDNAs encoding novel transcription factors or kinases that interact with JNK1 kinase using the yeast two-hybrid system. The experiments will include library screening, isolation/cloning of full-length cDNAs, antibody production, and in vivo protein interaction assays. 3. Biochemical and Functional Characterizations of Novel JNK1-interacting proteins (JIPs). We will further characterize the novel JIPs cloned in AIM 2. We expect the JIPs will fall into the following categories; (i) transcription factors that are the targets/substrates of JNK1, (ii) kinases that are the targets/substrates of JNK1, (iii) kinases that are upstream of JNK1, (iv) phosphatases that are upstream of JNK1, and (v) other JNK1- binding proteins. Various biochemical and functional assays, as described in AIM-1, will be used to define the functions of the JIPs. These studies will provide fundamental understanding about (i) the distal signaling events during CD28 signal transduction, (ii) the molecular mechanisms of gene regulation/oncogenesis mediated by Rel/NF kappaB proteins, and (iii) the actions of JNK1 in the emerging/novel stress- activated signaling pathway. An understanding of the CD28-mediated activation of Rel/NF-kappaB functions should also provide insight about the interplay between CD28 signaling and HIV-1 gene regulation/replication in HIV-1 pathogenesis. Finally, the information gained in these studies will be of value in the future design of therapeutic agents for immunological disorders including autoimmunity, graft rejection and AIDS.
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