UCSF

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, Csk^AS, 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 Csk^AS allele in the absence of endogenous Csk expression. Initial characterization of Csk^AS by ectopic expression in Jurkat T cells revealed that in the basal state, Csk actively restrains Lck activity. Inhibition of Csk^AS 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 Csk^AS 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.