T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols

William F Hawse,Richard T Cattley

doi:10.1074/jbc.ra118.006524

William F Hawse, Richard T Cattley

Open Access

https://doi.org/10.1074/jbc.ra118.006524

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Abstract

T-cell receptor (TCR) signaling strength is a dominant factor regulating T-cell differentiation, thymic development, and cytokine signaling. The molecular mechanisms by which TCR signal strength is transduced to downstream signaling networks remains ill-defined. Using computational modeling, biochemical assays, and imaging flow cytometry, we found here that TCR signal strength differentially generates phosphatidylinositol species. Weak TCR signals generated elevated phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and reduced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) levels, whereas strong TCR signals reduced PI(4,5)P2 and elevated PIP3 levels. A proteomics screen revealed that focal adhesion kinase bound PI(4,5)P2, biochemical assays disclosed that focal adhesion kinase is preferentially activated by weak TCR signals and is required for optimal Treg induction, and further biochemical experiments revealed how TCR signaling strength regulates AKT activation. Low PIP3 levels generated by weak TCR signals were sufficient to activate phosphoinositide-dependent kinase-1 to phosphorylate AKT on Thr-308 but insufficient to activate mTOR complex 2 (mTORC2), whereas elevated PIP3 levels generated by a strong TCR signal were required to activate mTORC2 to phosphorylate Ser-473 on AKT. Our results provide support for a model that links TCR signaling to mTORC2 activation via phosphoinositide 3-kinase signaling. Together, the findings in this work establish that T cells measure TCR signal strength by generating different levels of phosphatidylinositol species that engage alternate signaling networks to control cell fate decisions.

Highlights

T-cell receptor (TCR) signaling strength is a dominant factor regulating T-cell differentiation, thymic development, and cytokine signaling
The following assumptions were included in the model (Fig. 1A) [2, 5, 11, 21]: 1) ligation of peptide-MHC (p-MHC) to a TCR and stimulation through CD28 activates multiple kinases that phosphorylate phosphatidylinositols, including PI3K, which converts PI[4,5]P2 to PIP3; 2) increased TCR signal strength promotes PTEN degradation as previously demonstrated [5]; and 3) PDK1 is activated by lower levels of PIP3 [23] (Kd of 1.6 nM) than mTOR complex 2 (mTORC2) [24, 25] (Kd of 141 nM via SIN1 component [26])
PTEN reduction resulting from a strong TCR signal diminished PI[4,5]P2 accumulation and allowed for elevated PIP3 generation. These simulations predicted that the regulation of PTEN abundance by TCR signal strength results in a redistribution of phosphatidylinositol species generated

Summary

ARTICLE cro

T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols. Cattley From the Department of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Edited by Peter Cresswell

Results

TCR signal strength regulates the extent and duration of TCR capping

Discussion

Analysis of cells by imaging flow cytometry

Western blotting