ITAM phosphorylation and binding in the T-cell receptor chain
ITAM phosphorylation and binding in the T-cell receptor chain
T-cells play a crucial role in the adaptive immune response, detecting and destroying infectious agents. The T-cell receptor complex (TCR) is responsible for recognising foreign antigens and for transducing an appropriate activation signal towards the T-cell nucleus. Following recognition of an appropriate antigen, Src-family protein tyrosine kinases such as Lck phosphorylate tyrosines located within Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in the cytoplasmic regions of TCR γ, δ, ε and ζ chains.
The studies described in this thesis examine the role of Lck in the potential ordered phosphorylation of TCRζ, and the subsequent binding interaction of the resultant phosphorylated ITAMs with the tandem SH2 domain of ZAP-70.
Assaying the incorporation of 32P-phosphate into peptides based on ITAM sequences, Lck was found to phosphorylate the six ITAM tyrosines with different efficiencies, which suggested that phosphorylation would occur in the order 1N (first), 3N, 3C, 2N, 1C, 2C (last). This potential for ordered phosphorylation by Lck was explored using on-line HPLC-MS to analyse the phosphorylation status of the entire cytosolic ζ chain protein, following digestion with trypsin, which confirmed tyrosine 1N as the first to the phosphorylated.
The binding of the phosphorylated ITAM peptide mimics to the tandem SH2 domain from ZAP-70 (tSH2) was then explored using a variety of spectroscopic techniques. The decrease of tSH2 fluorescence upon binding to peptide ligands was used to measure Kds for the complex under various conditions. The presence of sodium chloride or potassium phosphate was shown to diminish the interaction, demonstrating the importance of electrostatic interactions in the binding, which was confirmed by isothermal calorimetry studies showing that the reaction is enthalpically-driven. Stopped flow fluorescence studies revealed that the rate of the bimolecular interaction between tSH2 and the ligand becomes limited by a unimolecular process at higher ligand concentrations, which is thought to be the conformational change detected using far UV circular dichroism. These results led to the proposal of a binding model, in which a conformational rearrangement is responsible for the fluorescence signal change. This may occur either before or after the binding of the ITAM. Parallel experiments showed the existence of two conformers of tSH2 and suggested that only one of these can bind to the peptide ligand.
University of Southampton
Street, Hazel R
573a3056-ee7e-48a7-afe6-ff417d523f03
2002
Street, Hazel R
573a3056-ee7e-48a7-afe6-ff417d523f03
Street, Hazel R
(2002)
ITAM phosphorylation and binding in the T-cell receptor chain.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
T-cells play a crucial role in the adaptive immune response, detecting and destroying infectious agents. The T-cell receptor complex (TCR) is responsible for recognising foreign antigens and for transducing an appropriate activation signal towards the T-cell nucleus. Following recognition of an appropriate antigen, Src-family protein tyrosine kinases such as Lck phosphorylate tyrosines located within Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in the cytoplasmic regions of TCR γ, δ, ε and ζ chains.
The studies described in this thesis examine the role of Lck in the potential ordered phosphorylation of TCRζ, and the subsequent binding interaction of the resultant phosphorylated ITAMs with the tandem SH2 domain of ZAP-70.
Assaying the incorporation of 32P-phosphate into peptides based on ITAM sequences, Lck was found to phosphorylate the six ITAM tyrosines with different efficiencies, which suggested that phosphorylation would occur in the order 1N (first), 3N, 3C, 2N, 1C, 2C (last). This potential for ordered phosphorylation by Lck was explored using on-line HPLC-MS to analyse the phosphorylation status of the entire cytosolic ζ chain protein, following digestion with trypsin, which confirmed tyrosine 1N as the first to the phosphorylated.
The binding of the phosphorylated ITAM peptide mimics to the tandem SH2 domain from ZAP-70 (tSH2) was then explored using a variety of spectroscopic techniques. The decrease of tSH2 fluorescence upon binding to peptide ligands was used to measure Kds for the complex under various conditions. The presence of sodium chloride or potassium phosphate was shown to diminish the interaction, demonstrating the importance of electrostatic interactions in the binding, which was confirmed by isothermal calorimetry studies showing that the reaction is enthalpically-driven. Stopped flow fluorescence studies revealed that the rate of the bimolecular interaction between tSH2 and the ligand becomes limited by a unimolecular process at higher ligand concentrations, which is thought to be the conformational change detected using far UV circular dichroism. These results led to the proposal of a binding model, in which a conformational rearrangement is responsible for the fluorescence signal change. This may occur either before or after the binding of the ITAM. Parallel experiments showed the existence of two conformers of tSH2 and suggested that only one of these can bind to the peptide ligand.
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Published date: 2002
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Local EPrints ID: 464680
URI: http://eprints.soton.ac.uk/id/eprint/464680
PURE UUID: 9c687584-7fab-4fdf-a58b-37d771702a14
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Date deposited: 04 Jul 2022 23:56
Last modified: 16 Mar 2024 19:41
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Author:
Hazel R Street
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