The JNK kinase cascade plays a pivotal role in T-cell proliferation and apoptosis. HPK1, a PAK/STE20-like kinase, is a novel hematopoietic- specific activator of the JNK signaling pathway. Our working hypothesis about the HPK1 signaling pathway is as follows: TCR, CD28 yields Adaptors yields HPK1 yields MEKK1, TAK1 yields MKK4, MKK7 yields JNK yields T-Cell Activation/Apoptosis. This proposed study will focus on the signaling events immediately upstream of HPK1 in T-cell activation and apoptosis. The approach is designed to (i) test our hypothesis of Crk (or other adaptors) as the upstream regulator of HPK1, (ii) understand the regulation, functions, and modifications of HPK1 in T- cell activation, and (iii) test our hypothesis that HPK1 is involved in T-cell apoptosis. Our future understanding of host factors involved in the HPK1-mediated T-cell signaling pathways will provide information fundamental to the discovery, design, and evaluation of effective intracellular therapeutic agents for AIDS, immunological disorders, and cancers.
The specific aims are: 1. Study of the Regulation of HPK1 Function by Adaptors during T-Cell Activation.
This aim will test our hypothesis that Crk (or other adaptors) is an upstream regulator of HPK1. We will study the physical interaction between HPK1 and adaptors as well as the potential regulation of the HPK1-adaptor complex in T-cell activation. We will further study the role of phosphorylation in HPK1-adaptor interactions, examine the potential regulation of HPK1 by adaptors, map the adaptor- binding domains of HPK1, and evaluate the role of the individual proline-rich domains in HPK1 signal transduction in T cells. 2. Study of the Regulation and Phosphorylation of HPK1 in T-Cell Costimulation. We will test the hypothesis that HPK1 kinase activity is regulated by phosphorylation and T-cell costimulatory signals. We will study potential complex formation between HPK1 and either TCR or CD28. We will also study the potential phosphorylation of HPK1 by TCR/CD28-associated tyrosine kinases. We will map the phosphorylation sites of HPK1 by phosphopeptide mapping/sequencing, and then determine their role in T-cell activation, kinase activity, subcellular localization, and protein-protein interactions. 3. Study of the Regulation and Function of HPK1 in T-Cell Apoptosis. Fas signaling causes the cleavage of HPK1 which may lead to the irreversible activation of HPK1. We will test the hypothesis that HPK1 is involved in T-cell apoptosis. The biological role of HPK1 in T-cell apoptosis will be studied using the wild-type, the kinase domain, or a dominant-negative mutant of HPK1. We will identify Fas-induced cleavage sites on HPK1 and the protease activities responsible for HPK1 cleavage. The function of this proteolytic cleavage will be studied by generating dominant-negative, uncleavable mutants of HPK1.
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