Supplementary Figure 4: Enzyme kinetics for Lck on membranes using CD3ζ as a substrate.

(a) Representative traces for fluorescence changes of SNAP₅₀₅-tSH2 at different CD3ζ concentrations. Phosphorylation assays were done essentially as described in Fig. 1a,b, using SNAP₅₀₅-tSH2 (1.2 μM) as the FRET reporter for ITAM phosphorylation. (b) Coomassie-stained SDS-PAGE gels showing the SNAP₅₀₅-tSH2 and CD3ζ in the total (liposome-bound + solution) and solution phase, upon the completion of FRET measurements (t = 100 min). Following FRET measurements, all samples were subjected to liposome sedimentation (278,000g, 20 min), and the supernatant fraction was collected. Equal fractions of the total and supernatant samples were subjected to SDS-PAGE. SNAP₅₀₅-tSH2 was depleted from the supernatant as CD3ζ concentration increased. This experiment also suggests that 100% binding of SNAP₅₀₅-tSH2 to pCD3ζ corresponds to ~28% fluorescence quenching. (c) The molar phosphorylation signal plotted as a function of time. The fluorescence signal in a was converted to molar phosphorylation signal, assuming each SNAP₅₀₅-tSH2 binding reports phosphorylation of a single ITAM (hence, phospohrylation of a pair of tyrosine residues). (d) The initial rate (v₀) of phosphorylation plotted as a function of CD3ζ concentration. v₀ was calculated as the slope of the first 10% increase of the phosphorylation signal for each condition. Note: Lck density (~2.5 μm^-2) is two orders of magnitude lower than in Fig. 2a, so that the effect of trans autophosphorylation on v₀ is minimal.