The role of Csk in the dynamic negative regulation of T cell receptor signaling
- Author(s): Tan, Ying Xim
- Advisor(s): Weiss, Arthur
- Lowell, Clifford
- et al.
The cytoplasmic tyrosine kinase, C-terminal Src kinase (Csk), negatively regulates T cell receptor (TCR) signaling by inhibiting Src family kinase (SFK) activity. Its function in T cells is poorly defined since genetic ablation in the T lineage causes abnormal thymic development. To circumvent this, we have generated a novel allele of Csk, CskAS, that can be inhibited rapidly and specifically by an analog of the common kinase inhibitor, PP1. We further generated bacterial artificial chromosome (BAC) transgenic mice expressing the CskAS allele in the absence of endogenous Csk expression. Initial characterization of CskAS by ectopic expression in Jurkat T cells revealed that in the basal state, Csk actively restrains Lck activity. Inhibition of CskAS induced ligand-independent initiation of TCR signaling and downstream activation events, indicating the presence of a feedback circuit sensitive to the basal signaling state. Dok-1 was identified as a candidate protein involved in this feedback rewiring. In contrast, we observed a block in TCR signaling at the level of PLCγ1 hydrolysis of phosphatidylinositol-(4,5)-bisphsophate (PIP2) following inhibition of CskAS in the BAC transgenic murine thymocytes. This implies that in primary thymocytes, activation of SFKs alone is insufficient for full TCR signaling . We further demonstrate that actin remodeling, most likely mediated by CD28 costimulation, is necessary for downstream propogation of proximal TCR signals by PLCγ1 to the calcium and MAPK pathways.