Regulation of the dynamic binding of PLC-γ1 to LAT

The activation of Phospholipase C-γ1 (PLC-γ1) plays a crucial role in T cell function. The current model is that following engagement of the T-cell antigen receptor (TCR), PLC-γ1 is recruited to the LAT complex where it becomes phosphorylated and activated. PLC-γ1 then stays at LAT to mediate calcium influx and activate PKC. Preliminary data from our lab show that PLC-γ1 requires the phosphorylation of LAT tyrosine 132 for activation but the overall phosphorylation kinetics of PLC-γ1 tyrosine 783 are more rapid than LAT tyrosine 132. Furthermore, we determined that PLC-γ1 has a substantially weaker interaction with LAT compared to Grb2. These findings suggest that PLC-γ1 has a transient and dynamic interaction with LAT during its activation. However, the cellular localization of PLC-γ1 upon phosphorylation at LAT remains unknown. To address this knowledge gap, Total Internal Reflection Microscopy (TIRF) will be used to determine the co-localization of tyrosine 783 phosphorylated PLC-γ1 with TCR or LAT clusters after TCR stimulation of human T cells. In addition, biochemical methods will be used to further understand the interaction of phosphorylated PLC-γ1 with LAT. Collectively, our data will provide a better understanding of the mechanisms that regulate the activation and function of PLC-γ1 in human T cells.

Vacaflores A, Freedman SN, Chapman NM, Houtman JC. Pretreatment of activated human CD8 T cells with IL-12 leads to enhanced TCR-induced signaling and cytokine production. Mol Immunol. 2017 Jan;81:1-15. doi: 10.1016/j.molimm.2016.11.008. PubMed PMID: 27883938; PubMed Central PMCID: PMC5201458.

Vacaflores A, Chapman NM, Harty JT, Richer MJ, Houtman JC. Exposure of Human CD4 T Cells to IL-12 Results in Enhanced TCR-Induced Cytokine Production, Altered TCR Signaling, and Increased Oxidative Metabolism. PLoS One. 2016 Jun 9;11(6):e0157175. doi: 10.1371/journal.pone.0157175. eCollection 2016. PubMed PMID: 27280403; PubMed Central PMCID: PMC4900534.

Bartelt RR, Light J, Vacaflores A, Butcher A, Pandian M, Nash P, Houtman JC. Regions outside of conserved PxxPxR motifs drive the high affinity interaction of GRB2 with SH3 domain ligands. Biochim Biophys Acta. 2015 Oct;1853(10 Pt A):2560-9. doi: 10.1016/j.bbamcr.2015.06.002. Epub 2015 Jun 12. PubMed PMID: 26079855; PubMed Central PMCID: PMC4558342.

Bilal MY, Vacaflores A, Houtman JC. Optimization of methods for the genetic modification of human T cells. Immunol Cell Biol. 2015 Nov;93(10):896-908. doi:10.1038/icb.2015.59. Epub 2015 Jun 1. PubMed PMID: 26027856; PubMed Central PMCID: PMC4659746.

Cadra S, Gucciardi A, Valignat MP, Theodoly O, Vacaflores A, Houtman JC, Lellouch AC. ROZA-XL, an improved FRET based biosensor with an increased dynamic range for visualizing zeta associated protein 70 kD (ZAP-70) tyrosine kinase activity in live T cells. Biochem Biophys Res Commun. 2015 Apr 10;459(3):405-10. doi: 10.1016/j.bbrc.2015.02.117. Epub 2015 Feb 28. PubMed PMID: 25735979.

Cruz-Orcutt N, Vacaflores A, Connolly SF, Bunnell SC, Houtman JC. Activated PLC-γ1 is catalytically induced at LAT but activated PLC-γ1 is localized at both LAT- and TCR-containing complexes. Cell Signal. 2014 Apr;26(4):797-805. doi:10.1016/j.cellsig.2013.12.022. Epub 2014 Jan 8. PubMed PMID: 24412752; PubMed Central PMCID: PMC3935424.