Anti-CD3 and phorbol ester induce distinct phosphorylated sites in the SH2 domain of p56lck.
P56lck is a protein tyrosine kinase of the Src family specifically expressed in T lymphocytes. Triggering of T cells with anti-CD3 or with phorbol 12-myristate 13-acetate (PMA) results in the appearance of slower migrating forms (shift) of p56lck. To investigate the phosphorylation sites on the shifted forms of p56lck and to assess the role of protein kinase C in this phosphorylation, Jurkat cells were treated with a selective inhibitor of this kinase (GF 109203X). This inhibitor completely reversed the shift induced by PMA but only partially reversed the one induced after triggering with anti-CD3. To analyze the shift further, p56lck was immunoprecipitated from in vivo labeled cells treated either with anti-CD3 or with PMA. Tryptic phosphopeptides were generated and analyzed by using a combination of thin layer chromatography, high reticulation polyacrylamide gel electrophoresis, reverse phase chromatography, and phosphopeptide sequencing. We identified serine 158 as a newly phosphorylated site after PMA treatment and tyrosine 192 and serine 194 in the major tryptic phosphopeptide obtained after anti-CD3 triggering. The three sites identified are located in the SH2 domain of p56lck; this suggests that their phosphorylation may regulate the interaction with other proteins or with other internal domains in p56lck.
Soula
M
M
Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 332, Paris, France.
Rothhut
B
B
Camoin
L
L
Guillaume
J L
JL
Strosberg
D
D
Vorherr
T
T
Burn
P
P
Meggio
F
F
Fischer
S
S
Fagard
R
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D017252:CD3 Complex / Q000276:immunology*; D002478:Cells, Cultured; D007211:Indoles / Q000494:pharmacology; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008301:Maleimides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014357:Trypsin
D017252:CD3 Complex; D007211:Indoles; D008301:Maleimides; D012694:Serine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D011493:Protein Kinase C; D014357:Trypsin; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate