Supplementary Figure 7: Csk directly phosphorylates CD3ζ but retards CD3ζ phosphorylation in the context of Lck.
From: In vitro membrane reconstitution of the T-cell receptor proximal signaling network
(a) Left, a cartoon showing a liposome membrane reconstituted with Csk and CD3ζ (~500 mm-2 each). Phosphorylation of CD3ζ was monitored by FRET using SNAP505-tSH2 (omitted in the cartoon) as described in Fig. 1a. Right, the time course of SNAP505-tSH2 fluorescence, before and after the addition of ATP. (b) The cartoon on the left shows a liposome membrane reconstituted with equal densities of Csk, CD3ζ and Lck (WT, Y394F, or Y505F mutant). Phosphorylation of CD3ζ was again monitored by FRET. The red traces in the plots corresponds to the the time course of SNAP505-tSH2 fluorescence before and after the addition of ATP, when both Lck and Csk were membrane reconstituted with CD3ζ; the black traces corresponds to conditions in which Csk omitted; the grey traces corresponds to conditions in which Lck was omitted. (c) Kinetic traces of SNAP505-tSH2 fluorescence when experiments as shown in b were repeated at lower surface density (~50 μm-2) of Lck. Results shown in this figure were repeatable in an independent measurement.
