Engagement of multicomponent immunoreceptors such as the T cell antigen receptor 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 adapter protein. We have performed studies to characterize how LAT is phosphorylated and binds a number of critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes 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 and the enzyme pathways that are coupled to and activated at LAT 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, after diacylglycerol binding, 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 that leads 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. We have previously generated a mouse in which SOS1 can be deleted in T cells allowing us to study the role of this enzyme in activating Ras and subsequently Erk in these cells. This year we have discovered a novel third pathway leading to Erk activation in T cells. We previously showed that another adapter molecule, Bam32, is coupled to Erk activation in T cells, as loss of this molecule resulted in a decrease in activation of Erk, cellular proliferation and cytokine production. An extensive series of experiments was completed dedicated to determining the linkage of Bam32 to the Erk pathway. We discovered a novel protein complex comprised of Bam32, PLCgamma and Pak1, a protein serine kinase (PSK). The interaction, in this complex, of PLCgamma and Pak1 activates the latter enzyme, which in turn, activates a cascade of PSKs, Raf1, Mek1/2 and Erk. Details of the activation mechanism were explored and interestingly we showed that this pathway was independent of LAT and SLP-76. Additionally, we showed that the PLCgamma bound at this complex was inactive as an enzyme. The complexity of Erk activation was the topic of another study in the past year. Over a decade ago we generated a mouse in which all the LAT contains a mutation (Y136F) that results in the inability of PLCgamma to bind LAT and become activated. These mice developed a lymphoproliferative disease with abnormally expanding T cells, which is fatal by about six months. We expected that the failure of activation of PLCgamma in these mice would result in the absence of RasGrp activation and thus a defect in Ras and Erk activation, as indicated above. Surprisingly, we showed that abnormal Erk activation in the expanding T cell population of these mice is actually a hallmark of the disease. In the new study we showed that, despite the absence of activation via PLCgamma, RasGrp is responsible for Erk activation in the T cells of these mice. We found that the PTK Lck is hyperactivated and phosphorylates the PSK, protein kinase C-theta. This enzyme, in turn, activates RasGrp leading ultimately to the activation of Erk. The abnormal mice bearing the LAT Y136F mutation were the subject of another project this year. Many studies have indicated that cohorts of genes can be regulated by microRNAs (miRNAs). Numerous disease states are now characterized by abnormal expression of miRNAs. We tested whether the abnormal lymphoproliferative state observed in the LAT Y136F mice could be associated with alterations in miRNA expression. We also asked whether normal states of T cell proliferation in response to infection or adoptive transfer of T cells could be regulated by the same or different miRNAs. Using multiple assays we showed that the mutant T cells and T cells proliferating in response to infection or adoptive transfer demonstrate some similar and some unique alterations in expression of particular miRNAs. 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 and the fate of these clusters at the site of T cell activation. This year we completed experiments addressing a controversy in the field. As described above, the adapter LAT serves as the nucleation site for the generation of multiple signaling complexes needed for T cell activation. LAT is known to be expressed in the plasma membrane and most investigators in the field thought that signaling complexes formed at the plasma membrane were the site of initial T cell activation. Nonetheless two recently published studies indicated that vesicles containing intracellular LAT were the critical source of the LAT activated following TCR engagement. We developed constructs enabling expression of LAT molecules that could be used to unambiguously identify cell-surface LAT molecules. With this new reagent we confirmed the original hypothesis that initial LAT activation occurs at resident plasma membrane LAT. We suspect that minutes later LAT coming to the plasma membrane from intracellular vesicles becomes activated, and studies to demonstrate the entire fate of LAT protein are nearing completion.

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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 :
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
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
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
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
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
Sherman, Eilon; Barr, Valarie; Samelson, Lawrence E (2013) Super-resolution characterization of TCR-dependent signaling clusters. Immunol Rev 251:21-35
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
Coussens, Nathan P; Hayashi, Ryo; Brown, Patrick H et al. (2013) Multipoint binding of the SLP-76 SH2 domain to ADAP is critical for oligomerization of SLP-76 signaling complexes in stimulated T cells. Mol Cell Biol 33:4140-51
Sherman, Eilon; Barr, Valarie A; Samelson, Lawrence E (2013) Resolving multi-molecular protein interactions by photoactivated localization microscopy. Methods 59:261-9

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