Investigation of the kinetics and order of tyrosine phosphorylation in the T‐cell receptor ζ chain by the protein tyrosine kinase Lck

Abstract

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor f chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the f chain. Preliminary 1 H-NMR studies of recombinant f chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32 P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six f chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor f chain. After in vitro phosphorylation by Lck, the partially phosphorylated f chain was digested with trypsin. Separation and identification of the f chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six f chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.

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

“…3a,b ), yielding k cat , K M and the Hill coefficients ( n H ) ( Table 1 ). The observed k cat values varied from 1-7 s −1 , in the same range as solution measurements on recombinant WT Lck using a peptide substrate corresponding to the autophosphorylation site of c-Src 26 , but orders of magnitude higher than a recent study using peptide substrates corresponding to individual tyrosines in CD3ζ 27 . The apparent 2-D K M values (200-500 μm −2 ) are similar to physiological densities of CD3ζ (100-360 μm −2 ), suggesting that Lck is operating in T cells at almost its maximal capacity while maintaining sensitivity to changes in the substrate concentration.…”

“…Our data reported here have revealed that cooperativity is at least partially encoded in the TCR proximal signaling architecture itself. A recent mathematical model by Mukhopadyay et al 12 suggested that the TCR proximal signaling ultrasensitivity arises from the multiple phosphoacceptor sites on CD3ζ, sequential phosphorylation of these sites by Lck 27 , and protection from dephosphorylation by ZAP-70. We have obtained experimental support for ZAP-70 protection against CD45 dephosphorylation ( Fig 4a,c ) but found that the ability of ZAP-70 to bind phospho-ITAMs did not alter the cooperativity of the kinase-phosphatase reaction ( Fig.…”

An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

“…This preference explains why Lck does not readily phosphorylate tyrosine residues in LAT and SLP-76 that contain an aspartate residue at the −1 position (Figure 3). It also explains why Lck phosphorylates the first tyrosine residue in each ITAM faster than the second one (Housden et al, 2003), as the first tyrosine is typically preceded by a leucine while the second is preceded by a smaller aliphatic or polar residue (Figure 3A). …”

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway

“…5, “ITAMs”). Several studies showed that Lck initially phosphorylates the N-terminal tyrosine of ITAMs and then later phosphorylates the C-terminal tyrosine (61, 69); however, the precise order of ITAM phosphorylation is still under investigation (60, 61, 63, 69). In the computational part of our study that focused on Lck, we found three possible scenarios for ordered ITAM phosphorylation that were consistent with the SILAC data (Fig.…”