Supplementary Figure 3: Kinase activities of WT and mutant LCK* proteins on ZAP70 phosphorylation and recruitment to plasma membrane after uncaging.

(a) Plot of ZAP70 Y319 phosphorylation kinetics by wildtype LCK* from two identical sets of experiments with different numbers of time points collected, as indicated within the figure. (b) Plot of the kinetics of ZAP70 Y319 phosphorylation by wildtype or Y394F LCK* after uncaging, in the presence or absence of dominant negative version of CSK (K222R). Data in a and b are relative to final time point (15 min) of wildtype LCK* in each experiment and were fit using a 3-parameter logistic function, and are presented as mean ± s.e.m. from independent experiments (n = 4 in a [22 time points], n = 12 in a [6 time points] and n = 3 in b). (c,d) Quantification of the activities of wildtype and mutant LCK* kinases derived from the phosphorylation and recruitment kinetic curves. (c) Initial reaction rates (V₀) of ZAP70 Y319 phosphorylation by LCK* mutants relative to WT LCK* after uncaging (related to Fig. 2b-d and 3a-d). (d) Time to achieve half-maximal recruitment of ZAP70 to plasma membrane by WT LCK* or indicated mutants after UV illumination at 5 mW/cm² (related to Fig. 2f-h). For this measure of LCK* reaction rate, a lower value indicates more efficient kinase activity, which was not detectable (n.d.) for the Y394F mutant. Data are presented as mean ± s.e.m. from independent experiments (n = 4). (e) Identification of proline in the PxxP motif in the linker region of HCK (PDB ID: 1QCF) that binds intramolecularly to the SH3 domain. The amino acid numbering used here is based on human LCK sequence.