Novel tools to dissect the dynamic regulation of TCR signaling by the kinase Csk and the phosphatase CD45

Abstract

Although the biochemical events induced by T-cell receptor (TCR) triggering have been well studied, both the mediators and function of basal signaling in T cells remain poorly understood. Furthermore, the precise mechanisms by which MHC-peptide interaction with the TCR disrupt the basal equilibrium to induce downstream signaling are also unclear. Here we describe novel approaches to understand the basal state of T cells and the mechanisms of TCR triggering by perturbing regulation of the Src family kinases (SFKs). The SFKs are critical proximal mediators of TCR signaling that are in turn tightly regulated by the tyrosine kinase Csk and the receptor-like tyrosine phosphatase CD45. We have developed a small-molecule analog-sensitive allele of Csk and an allelic series of mice in which expression of CD45 is varied across a broad range. Our studies have unmasked contributions of Csk and CD45 to maintain the basal state of T cells and also suggest that dynamic regulation of Csk may be involved in TCR triggering.

Figure 1. SFK activity is reciprocally regulated by Csk and CD45

Phosphorylation of a c-terminal tail negative regulatory tyrosine of SFKs by Csk facilitates its interaction with its SH2 domain, resulting in a closed, catalytically inactive conformation. Dephosphorylation of this site by CD45 favors an open conformation. Phosphorylation of the catalytic site tyrosine is required for full kinase activity. CD45 and PEP can negatively regulate SFK activity by dephosphorylating the catalytic site tyrosine.

Figure 2. Highly specific inhibition of Csk^AS allele leads to SFK activation

A. The bulky gate keeper residue of wildtype (WT) Csk is mutated from threonine to glycine in the Csk^AS allele, generating a larger catalytic pocket that can bind a bulkier analog of PP1, 3-IB-PP1. B. Wildtype (WT) or Csk^AS BAC transgenic (AS) thymocytes were treated with DMSO or 10μM 3-IB-PP1 for 3 minutes, then lysed and analyzed by immunoblotting for SFK phosphorylation using Lck pY505 Ab and Src pY416 Ab (Cell Signaling) that recognize the inhibitory phospho-tyrosine of Lck and the activation loop phospho-tyrosine of the SFKs respectively (upper band = Fyn, lower band = Lck). Total Lck is probed as a loading control.

Figure 3. An allelic series of CD45 expressing mice unmasks positive and negative regulation of Lck

A. Surface CD45 expression assessed by FACS in double positive thymocytes from CD45 allelic series mice (L= lightning allele; H Tg= CD45 H transgene expressed in the context of nomal endogenous CD45 expression). B. Unstimulated thymocytes from allelic series mice are probed for SFK phosphorylation using Lck pY505 Ab and Src pY416 Ab (Cell signaling) that recognize the inhibitory phospho-tyrosine of Lck and the activation loop phospho-tyrosine of the SFKs respectively (upper band = Fyn, lower band = Lck). Total Lck is probed as a loading control.

Figure 4. Csk and CD45 regulate T cell basal and inducible signaling by controlling the dynamic equilibrium of Lck activity

A. In resting T cells, Csk is recruited to the plasma membrane by membrane-resident adaptors that have yet to be fully identified. Together with CD45, they control the phosphorylation status of the negative regulatory tyrosine of Lck, resulting in the generation of an equilibrium of distinct species of Lck with different levels of activity. It remains to be determined whether each of these Lck species have distinct localizations at the membrane, and whether they differentially associate with CD4/CD8 coreceptors. This equilibrium level of Lck activity sets the T cell basal signaling tone that may allow for peripheral survival, downregulation of RAG genes expression as well as increase TCR sensitivity to ligand. B. When the T cell encounters an antigen presenting cell (APC) bearing its cognate pMHC, the TCR is engaged, inducing the phosphorylation of the TCR ITAMs and ZAP-70 by active Lck. Precisely how this occurs remains poorly defined, and may involve an increase in the amount of active Lck due to a change in the relative activities of Csk and CD45 at the membrane, or a relocalization of preexisting active Lck to the proximity of the TCR. A change in the balance of Csk and CD45 activity may be achieved by relocalization of Csk off the membrane or away from Lck. The additional interaction of CD28 with B7 may initiate actin remodeling events that promote downstream propagation of the proximal TCR signal to the calcium and MAPK pathways, by enabling hydrolysis of PIP₂ by PLCγ1. Full activation of TCR signaling results in T cell proliferation and differentiation.