Figure 1. Generation and characterization of the Csk^AS system.

(A) Mutation of the conserved gatekeeper residue of Csk (T266G) permits access to larger analogs of PP1. The eleven amino-terminal residues of Lck were fused to Csk^AS to enforce its localization to lipid rafts in the plasma membrane. (B) Structure of 3-IB-PP1. (C) Jurkat T cells were transiently cotransfected with either cytoplasmic- or membrane-Csk^AS and a GFP cotransfection marker. Cells were serum-starved, then stimulated with anti-TCR for 5 min in the presence of DMSO or 3-IBPP1 at doses shown, and analyzed for phospho-ERK. Data show % of live, GFP⁻ untransfected cells or live GFP⁺ transfected cells that are phospho-ERK⁺. Data are representative of 3 independent experiments.