Tyrosine phosphorylation of a 66,000 M(r) soluble protein in lectin-activated human peripheral blood T lymphocytes

H. J. Wedner, G. Bass

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Protein phosphorylation was studied in human T lymphocytes stimulated with the mitogenic lectins phytohemagglutinin (PHA) and concanavalin A (Con A). The T lymphocytes were prepared from the venous blood of normal volunteers, their intracellular ATP pools were labeled with [32P]orthophosphate, and protein phosphorylation was assayed in the soluble fraction by two-dimensional gel electrophoresis and autoradiography. When lymphocytes stimulated with PHA or Con A were compared to unstimulated control cells, there was a general increase in protein phosphorylation and the specific phosphorylation of a soluble protein with M(r) = 64.9 to 69 KD and pI = 5.6 to 5.8. Phosphorylation of this protein, designated TPP-66, was observed as early as 2 min after the addition of lectin with a gradual increase in the level of phosphorylation over the next 120 min. In the majority of experiments, there was no phosphorylation seen in the unstimulated lymphocytes; however, in some experiments, there was appreciable phosphorylation, which was seen beginning 60 min after the labeling period. When the TPP-66 spot from stimulated lymphocytes was excised from gels, was eluted, and was subjected to limited base hydrolysis followed by single-dimensional high voltage electrophoresis, the major phosphorylated residue migrated with phosphotyrosine. In some experiments, there was phosphorylation of serine residues in both the stimulated and control cells; tyrosine phosphorylation was never seen in the unstimulated cell population. These data suggest that, like other stimulae for cell growth, the induction of lymphocyte growth by lectins in associated with the activation of a tyrosine-specific kinase. Thus, tyrosine phosphorylation may play a key role in the transmission of the signal for lymphocyte growth from the exterior to the interior of the cell.

Original languageEnglish
Pages (from-to)4226-4231
Number of pages6
JournalJournal of Immunology
Issue number11
StatePublished - Jan 1 1986


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