Engagement of multicomponent immunoreceptors such as the T cell antigen receptor (TCR) results in rapid activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate a number of enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane protein. LAT is a critical transmembrane adaptor protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing these other adaptor molecules and enzymes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, genetic and microscopic techniques are currently employed to study the characteristics of LAT-based signaling complexes. A critical pathway activated after TCR engagement and primarily dependent on the LAT molecule is the ERK enzymatic pathway. A number of our older studies demonstrated how interaction of the enzyme phospholipase C gamma (PLCgamma) with a particular phosphorylated tyrosine of LAT results in activation of an enzyme cascade leading to ERK activation. In this pathway PLCgamma activation leads to breakdown of phosphoinositide lipids leading to diacylglycerol production. This product has a number of targets, one of which is the enzyme RasGRP, which activates the small G protein Ras and thereby controls a cascade leading to ERK activation. This route to ERK activation is not the only pathway coupled to LAT leading to ERK activation. The adapter molecule Grb2 also binds phosphorylated LAT and brings the Ras activator molecule SOS1 to LAT as well. In many cell types SOS1 is the central Ras activator. The significance of SOS1 in T cells has been poorly studied. To address SOS function we have successfully generated a mouse in which SOS1 can be deleted in T cells. We have conducted and continue a series of experiments designed to test the role of SOS1 in thymic development and peripheral T cell function. Our published study indicates that SOS1 is required for the normal development and expansion of immature T cells during intrathymic development (the double negative to double positive phase). At a later stage in intrathymic development during the period when appropriate TCRs are selected (the double positive phase), SOS1 seems less important. We also found that SOS1 is most abundant in the earliest of thymocytes (the double negative phase) and it decreases over the course of thymocyte maturation. Studies continue to determine the relative importance of another SOS isoform, SOS2, along with SOS1 and RasGRP. The mice we have made will also prove to be an important reagent for many scientists as they allow for targeted SOS deletion in any cell or tissue of interest. In addition to biochemical and genetic studies of signaling molecules the laboratory has developed new methods of visualizing T cell activation using confocal microscopy. Many of the signaling molecules involved in the early TCR-coupled activation process have been tagged with fluorescent markers and expressed in T cells. The group has used these methods to observe the process of the assembly of signaling molecules into signaling clusters at the site of T cell activation using confocal and high-resolution microscopy. In studies published this year we have studied the fate of LAT molecules. We have demonstrated that LAT is ubiquitinylated following T cell activation. Inhibition of this post-translational change results in prolonged LAT half-life and enhanced T cell signaling. These findings are under investigation in hopes of understanding how LAT ubiquitinylation may regulate T cell responses both in cell lines and in vivo in mouse models.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010304-13
Application #
8348968
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2011
Total Cost
$2,013,204
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Balagopalan, Lakshmi; Yi, Jason; Nguyen, Tiffany et al. (2018) Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation. Nat Commun 9:2013
Guittard, Geoffrey; Gallardo, Devorah L; Li, Wenmei et al. (2017) Unexpected Cartilage Phenotype in CD4-Cre-Conditional SOS-Deficient Mice. Front Immunol 8:343
Yi, Jason C; Samelson, Lawrence E (2016) Microvilli set the stage for T-cell activation. Proc Natl Acad Sci U S A 113:11061-11062
Palmer, Douglas C; Guittard, Geoffrey C; Franco, Zulmarie et al. (2015) Cish actively silences TCR signaling in CD8+ T cells to maintain tumor tolerance. J Exp Med 212:2095-113
Hui, King Lam; Balagopalan, Lakshmi; Samelson, Lawrence E et al. (2015) Cytoskeletal forces during signaling activation in Jurkat T-cells. Mol Biol Cell 26:685-95
Rodriguez-Peña, A B; Gomez-Rodriguez, J; Kortum, R L et al. (2015) Enhanced T-cell activation and differentiation in lymphocytes from transgenic mice expressing ubiquitination-resistant 2KR LAT molecules. Gene Ther :
Rouquette-Jazdanian, Alexandre K; Kortum, Robert L; Li, Wenmei et al. (2015) miR-155 Controls Lymphoproliferation in LAT Mutant Mice by Restraining T-Cell Apoptosis via SHIP-1/mTOR and PAK1/FOXO3/BIM Pathways. PLoS One 10:e0131823
Guittard, Geoffrey; Kortum, Robert L; Balagopalan, Lakshmi et al. (2015) Absence of both Sos-1 and Sos-2 in peripheral CD4(+) T cells leads to PI3K pathway activation and defects in migration. Eur J Immunol 45:2389-95
Kunii, Naoki; Zhao, Yangbing; Jiang, Shuguang et al. (2013) Enhanced function of redirected human T cells expressing linker for activation of T cells that is resistant to ubiquitylation. Hum Gene Ther 24:27-37
Sommers, Connie L; Rouquette-Jazdanian, Alexandre K; Robles, Ana I et al. (2013) miRNA signature of mouse helper T cell hyper-proliferation. PLoS One 8:e66709

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