Efficient thymocyte selection is critical to generate T lymphocytes with a very broad spectrum of specific T cell receptors (TCR) that recognize foreign antigens, while avoiding development of autoimmunity. The exact molecular mechanisms underlying thymocyte positive versus negative selection are not know, but different patterns of Ras-MAP kinase (MAPK) activation have long been postulated to be involved. MAPK are tunable signaling molecules that lie downstream of active Ras and that regulate gene expression. We hypothesize that the choice of Ras Guanine nucleotide Exchange Factors usage, namely RasGRP or SOS + RasGRP, governs distinct Ras activation characteristics and thymocyte selection. This project will focus on understanding the characteristics of Ras-MAPK signaling by RasGRP1 and SOS1 and how this affects thymocyte fate decisions by combining the biophysical, computational, and cellular-biochemical expertise of the Kuriyan (UCB), Groves (UCB), Chakraborty (MIT), and Roose (UCSF) labs. Combination of: 1) purified recombinant proteins;2) a model two dimensional lipid bilayer system containing defined quantities of recombinant proteins;3) computational modeling;and 4) model systems of the Jurkat T cell, DT40 B cell, and mutant cell lines and mouse model studies provide an unique approach that has not been taken previously. The overall goal of project #2 is to understand the distinct characteristics of Ras-MAPK activation via RasGRP1 or SOS1, downstream of LAT, and how these signaling events impact thymocyte selection.
In Aim 1, we will characterize RasGRP1 RasGEF function utilizing the three scientific disciplines in iterative approaches.
In Aim 2, we will define the requirement of allosteric regulation of the RasGEF SOS1.
In Aim 3. we will define the molecular basis of RasGRP1-SOS1 synergy.
In Aim 4, we will Characterize the role of LAT in digital Ras-MAPK signaling.
In Aim 5. we will define the role of SOS-mediated Ras activation in thymocyte selection. Understanding the specific nature of Ras activation and the downstream activation of the ERK, P38, and JNK MAPK will be the endpoint goals for this project; specifically we will focus on the role of the RasGEF characteristics of RasGRP1 and SOS1 as well as on phospho-LAT, which could serve as a point of bifurcation between analog and digital Ras signaling. We expect that we will gain insights from both concurrence and contradiction between the three disciplines. By building step-wise to increasingly complex models we aim to understand the molecular mechanism of selective Ras-MAPK activation patterns and how these impact thymocyte selection.

Public Health Relevance

Despite many years of excellent research, the molecular details that discriminate positive and negative selection of thymocyte are still unknown. Our collaborative approach provides unique opportunities to understand how reliable selection exactly occurs and will help understand how development of autoreactive T cells can occur, which can cause development of autoimmune diseases in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program Projects (P01)
Project #
5P01AI091580-03
Application #
8503586
Study Section
Special Emphasis Panel (ZAI1-RRS-I)
Project Start
2013-07-01
Project End
2016-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
3
Fiscal Year
2013
Total Cost
$694,676
Indirect Cost
$103,543
Name
University of California San Francisco
Department
Type
DUNS #
094878337
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Tan, Ying Xim; Manz, Boryana N; Freedman, Tanya S et al. (2014) Inhibition of the kinase Csk in thymocytes reveals a requirement for actin remodeling in the initiation of full TCR signaling. Nat Immunol 15:186-94
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Ross, Jenny O; Melichar, Heather J; Au-Yeung, Byron B et al. (2014) Distinct phases in the positive selection of CD8+ T cells distinguished by intrathymic migration and T-cell receptor signaling patterns. Proc Natl Acad Sci U S A 111:E2550-8
Chakraborty, Arup K; Weiss, Arthur (2014) Insights into the initiation of TCR signaling. Nat Immunol 15:798-807
Lin, Wan-Chen; Iversen, Lars; Tu, Hsiung-Lin et al. (2014) H-Ras forms dimers on membrane surfaces via a protein-protein interface. Proc Natl Acad Sci U S A 111:2996-3001

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