Modulation of protein tyrosine phosphorylation during G1/S transition in activated human T-lymphoblasts.
We report that in activated human T-lymphoblasts synchronized early in the G1-phase of the cell cycle, addition of interleukin 2 (IL-2) stimulates transition into S-phase in conjunction with specific and reproducible protein tyrosine phosphorylation events. Prominent among these was de novo phosphorylation of the p56lck tyrosine kinase, which appeared as a single polypeptide on SDS-polyacrylamide gel electrophoresis prior to the addition of IL-2, but as a doublet of approximately 56 and 59 kDa 3 h after IL-2 addition. Although the lck polypeptide doublet persisted into S-phase, the 59-kDa form was virtually undetectable in T-lymphoblasts in log phase growth. In T-lymphoblasts metabolically labeled with 32Pi, antiphosphotyrosine antisera identified a major 56-kDa tyrosine-phosphorylated protein, whereas antisera to lck showed that phosphorylation of lck occurred on both the 56- and 59-kDa forms. Phosphoamino acid analyses identified phosphoserine as the major phosphoamino acid on both of the lck polypeptides, although small amounts of phosphotyrosine were also detected on the 56-kDa form. Immune complex kinase assays revealed that only the lower band of the lck doublet exhibited autocatalytic activity. Furthermore, the 59-kDa form neither displayed autocatalytic activity, nor was it a substrate for phosphorylation by the 56-kDa form. We conclude that entry into S-phase in activated human T-lymphoblasts is associated with tyrosine phosphorylation of a limited number of proteins and suggest that p56lck activity is regulated during G1/S.
Churcher
Y
Y
Department of Physiology, University College of London, United Kingdom.
Moss
S E
SE
D000936:Antigen-Antibody Complex; D002478:Cells, Cultured; D016193:G1 Phase; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D016196:S Phase; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D007376:Interleukin-2; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)