In vitro membrane reconstitution of the T-cell receptor proximal signaling network

Abstract

T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45, and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over a ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that coclustering TCR-Lck or detaching Csk from the membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs.

Phosphatase CD45 Both Positively and Negatively Regulates T Cell Receptor Phosphorylation in Reconstituted Membrane Protein Clusters

“…Conversely, autophosphorylation on Tyr 394 , a positive regulatory site, was observed under all conditions tested, and may play a key role in regulating catalytic activity of diffuse Lck that lacks Tyr 505 autophosphorylation. This observation is largely consistent with previous analyses of c-Src and Lck autophosphorylation (24,35 (35).…”

“…However, CD3 phosphorylation levels were sharply decreased in the presence of only 50 -100 m Ϫ2 CD45 molecules, which is significantly lower than the physiological CD45 density in T cell membranes (ϳ1000 m Ϫ2 ) (34). This trend is in good agreement with previous measurements in solution phase or on liposomes (6,24). CD3 phosphorylation in the absence of CD45 appeared to saturate at lower Lck density (ϳ100 m Ϫ2 ) in homogeneous membranes, whereas that in His 10 -protein clusters increased with Lck density (100 -400 m Ϫ2 ), suggesting a slower reaction rate in the clusters (Fig.…”

“…1D). This time scale is comparable with that reported recently on liposomes (24). Note that there could be a delay in the detection of phosphorylation because antibody binding is not instantaneous, and antibody binding to pre-phosphorylated CD3 on supported bilayers was nearly 90% complete within 30 s (Fig.…”

“…1E). We observed a positive allosteric effect (Hill coefficient 4.23), as reported previously (24). The k cat measured in our system (0.21 s Ϫ1 ) is orders of magnitude higher than the value obtained in solution phase but is smaller than that obtained on liposomes (Table 1) (24 -26).…”

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells

“…For instance, in response to very weak interactions with self-pMHC, the TCR signaling pathway found in naive T cells, rather than being silent, delivers low-intensity signals that imprint on naive T cells a heightened reactivity to foreign pMHC (2). A network of PTKs, protein tyrosine phosphatases (PTPases), and transmembrane adaptors dynamically control the activity of LCK in the ground state (3)(4)(5)(6)(7)(8). In the ground state, the levels of active LCK are set to a point ensuring that maximal CD3 ITAM and ZAP-70 phosphorylation only occurs upon engagement of the TCR with agonist pMHC ligands.…”