Thymocytes and peripheral T cells express two types of Ras Guanine nucleotide Exchange Factors (RasGEFs), SOS and RasGRP, which can activate Ras and Ras downstream MAP kinase (MAPK) pathways in response to T cell receptor (TCR) ligation. Distinct activation patterns of MAPK s as well as the subcellular localization of MAPK activation appear to regulate the discrimination between positive and negative selection of thymocytes. The molecular and biochemical mechanisms that establish the right amplitude, duration, pattern, as well as the correct cellular localization of the Ras-MAPK signals during thymocyte selection are not known. In peripheral T cells distinct MAPK activation patterns can also be detected. It is to be determined what exact roles these different patterns play in T cells. Here we take a molecular and biochemical approach to understand novel regulatory mechanisms that control the characteristics of T lymphocyte Ras-MAPK signals. Our recent publications reported that RasGRP1 signals to Ras in an analog manner while SOS signals to Ras in a digital manner. Our computational models predicted that (i) regulated, analog RasGRP1-Ras signals are the basis for positive selection and that (ii) high RasGRP1 levels might aberrantly signal to Ras. These findings indicate that RasGRP1 function and activity must be controlled. The molecular and biochemical mechanisms of RasGRP1 regulation and the potential impact on the Ras-MAPK signals and on thymocyte or T cell function are unknown. Our preliminary data indicate that RasGRP1 is regulated by three novel mechanisms: induced degradation, regulation by calcium, and dimerization. On the basis of the phenotypes of RasGRP1- overexpressing cancers and our novel Rasgrp1ANAEF mouse model, we hypothesize that RasGRP1 function and activity is controlled via three regulatory mechanisms to warrant high fidelity Ras-MAP kinase signals in thymocytes and peripheral T cells.
The specific aims test these hypotheses.
In aim 1, a largely biochemical approach will be taken to identify the mechanism of TCR-induced RasGRP1 degradation. We will use chemical inhibitors and lentiviral shRNA to analyze the signals that are required for RasGRP1 degradation and we will test our hypothesis that RasGRP1 is regulated via ubiquitination. We will evaluate how RasGRP1 degradation impacts the activation characteristics of the Ras-ERK MAPK pathway in cell line reconstitution- and in ex vivo thymocyte- experiments, and we will eventually test how RasGRP1 degradation influences thymocyte development.
In aim 2, molecular-, biochemical-, and in vivo- approaches are combined to investigate how RasGRP1 s calcium-binding EF hands regulate RasGRP1 function. We will determine the mechanism of RasGRP1 regulation via its EF hands and we will continue our analysis of thymocyte selection and peripheral T cell function in a novel ENU mouse model with a point mutation in RasGRP1 s C-terminal EF hand. We will determine how this mutation affects the amplitude, duration, pattern, as well as the cellular localization of the Ras-MAPK signals.
In aim 3, molecular and biochemical assays will examine the mechanism of RasGRP1 dimerization and the role of dimer formation in regulating RasGRP1 function. We will establish the details and dynamics of RasGRP1 dimer formation in cell line reconstitution- and in ex vivo thymocyte- experiments. We will determine how dimer formation affects the characteristics of TCR-induced Ras-MAPK signals, eventually in in vivo experiments. Successful completion of the proposed studies will lead to a detailed understanding of the complex signaling characteristics (amplitude, duration, pattern, cellular localization) of TCR-induced Ras-MAPK signals in developing T cells and in peripheral T cells. The studies will have implications for human autoimmune diseases and T cell lymphoma. Our findings will also impact research on different immune cells, like B cells, and on various cancer cells types that are reported to express RasGRP1. Our studies will also lead to the generation of novel tools (mouse models and antibodies) that will benefit the scientific (Immunology) research community.
T cells demonstrate a variety of specific Ras-MAP kinase activation patterns, either during development in the thymus or when performing their function in peripheral lymphoid organs. The goal of this project is to determine the molecular and biochemical mechanisms that underlie the generation of these specific ERK-, P38-, and JNK- MAP kinase activation patterns, downstream of the small GTPase Ras and how these mechanisms relate to T cell function. Results obtained from the proposed research will provide mechanistic insights into the biochemical cues that drive T cell selection and activation, will be significant to other immune cells and cancer as well, and will be of relevance to the mission of the NIH and to a broad audience of researchers studying the Ras-MAPK pathways in various biological processes.
| Limnander, Andre; Zikherman, Julie; Lau, Tannia et al. (2014) Protein kinase C? promotes transitional B cell-negative selection and limits proximal B cell receptor signaling to enforce tolerance. Mol Cell Biol 34:1474-85 |
| Ksionda, Olga; Limnander, Andre; Roose, Jeroen P (2013) RasGRP Ras guanine nucleotide exchange factors in cancer. Front Biol (Beijing) 8:508-532 |
| Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul et al. (2013) Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1. Elife 2:e00813 |
| Limnander, Andre; Depeille, Philippe; Freedman, Tanya S et al. (2011) STIM1, PKC-? and RasGRP set a threshold for proapoptotic Erk signaling during B cell development. Nat Immunol 12:425-33 |
