Analysis of protein kinase C - theta function

Research project P 14394 Analysis of Protein Kinase C - Theta (PKCq ) function Gottfried BAIER 06.03.2000 Supra molecular complex (SNIAC) /immunological synapse (i-synapse) consists of accessory molecules together with the TCR/CD3 complex and is functionally involved in cytoskeletal remodeling and ultimately cell adhesion. Current hypotheses implicate functional key players such as TCR/CD3, CD28, Lck, ZAP-70, CD45, PLC71, PI3K, PKCO, GTPases, actin cytoskeleton, and LFA-1. Here wepropose a comprehensive investigation of PKCO`s role in T cell function, e.g. its contribution in membrane-proximal signaling complexes at the point of interaction of T cell and APC. The specific objectives of this study are to: (1) Determination of the changes in PKCo downstream effectors that occur in response to formation of specialized adhesion/signaling domains and biochen-dcal characterization of novel components of the PKC6 associated signaling pathways including of cytoskeleton dynamics. (2) Investigation of the morphology of T cell antigen recognition by 3D reconstruction of fixed T cell-APC conjugates stained by mAbs specific for PKCO and physically or functionally interacting signaling molecules and time laps video recording of living cells transfected with these GFP-fused signaling components. (3) Discovery of PKCO- regulated transcription factors and target genes employing gene chips/filter array technology. Finally, the use of PKCo KO mice will provide a scientific model for the "in vivo" study and to proof the physiological relevance of the working hypothesis. The results provided by this study will allow to understand how subtle modifications in the cell-cell stimulatory context are translated by the complex interaction of signaling molecules and the cytoskeleton. The definition of PKCO`s exact role in these basic concepts of T cell activation will provide new insights of how signals are transmitted downstream from the SMAC to regulate versatile biological T cell responses in vivo.

The human immune system controls and modulates major diseases that attract the highest healthcare costs in developed countries, e.g. (i) infectious disease and protective vaccination, (ii) inflammatory diseases e.g. arthritis, chronic obstructive pulmonary disease, asthma, (iii) cancer and autoimmune diseases, (iv) cardiovascular disorders e.g. arteriosclerosis, (v) body`s rejection of donated organs or bone marrow in transplantation, including future virus-based gene therapy approaches. Importantly, the delicate balance of attacking foreign pathogens and human chronic disease-associated immune dysfunctions (that cannot anymore be turned off) is regulated primarily by T lymphocytes. Thereby, lymphoid homeostasis is maintained by the dynamic regulation of lymphocyte activation, proliferation, programmed cell death, and tissue specific homing. The protein kinase C (PKC) family of serinehreonine protein kinase gene family has been extensively studied for over a decade and it is widely accepted in the scientific community that PKC function is crucial in T cell biology. Nevertheless, no systematic comparative studies of the cellular functions attributed to the PKC gene module in T cells (e.g. highly T cell expressed PKC isotypes) are available. Here, the physiological T cell functions of representative as well as most critical PKC family members are proposed to be investigated employing our ongoing mouse genetic (e.g. genetic elimination of T cell expressed PKC genes by homologous recombination) and complementary molecular T cell biology studies. Given the central role of T lymphocytes in immune responses, a more complete understanding of the complex (patho)physiological cellular functions of the PKC gene module may advance our fundamental understanding of cell-to-cell communication, as well as may lead to the discovery of unique aspects of T lymphocyte cell proliferation and differentiation. There are no doubts that the analysis of such higher levels of PKC gene function description uses, may implicate an enormous potential (in cooperation with pharmaceutical industry) for the discovery of innovative therapeutic drugs targeting the PKC module and its surrogate effectors with improved efficacy and selectivity.