Investigation of the kinetics and order of tyrosine phosphorylation in the T-cell receptor zeta chain by the protein tyrosine kinase Lck.
We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor zeta chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the zeta chain. Preliminary 1H-NMR studies of recombinant zeta 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 32P 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 zeta 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 zeta chain. After in vitro phosphorylation by Lck, the partially phosphorylated zeta chain was digested with trypsin. Separation and identification of the zeta 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 zeta 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.
Housden
Hazel R
HR
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, UK.
Skipp
Paul J S
PJ
Crump
Matthew P
MP
Broadbridge
Robert J
RJ
Crabbe
Tom
T
Perry
Martin J
MJ
Gore
Michael G
MG
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry* / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D008565:Membrane Proteins; D010455:Peptides; D011948:Receptors, Antigen, T-Cell; C054979:antigen T cell receptor, zeta chain; D014443:Tyrosine; D005982:Glutathione Transferase; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)