Anti-CD2-induced tyrosine phosphorylation of T cell polypeptides is independent of the PMA-induced modification of p56lck.
The molecular basis for T cell activation involves the phosphorylation of of polypeptides at both serines and tyrosines. We find that with human peripheral T cells the serine phosphorylation of p56lck is independent of the more rapid tyrosine phosphorylation of other polypeptides via stimulation of the CD2 receptor with anti-CD2 (anti-T11(2) and anti-T11(3) mAb's). Triton X-100 soluble polypeptides were analyzed by Western blotting with the subsequent immunodetection by anti-phosphotyrosine or anti-lck antibodies. While polypeptides from resting T cells showed very low levels of endogenous tyrosine phosphorylation, incubation with anti-CD2 for periods as short as 30 sec resulted in the tyrosine phosphorylation of a 75-kDa polypeptide (p75). Polypeptide bands were also observed at 27 and 54 kDa, but these were artifacts from the reaction of anti-CD2 with the horse anti-mouse secondary antibody used in our detection system. Preincubation of T cells with phenylarsine oxide amplified the anti-CD2-induced tyrosine phosphorylation of the p75 and revealed additional phosphotyrosine polypeptides of 120, 100, and 33 kDa. The mitogenic combination of phorbol 12-myristate 13-acetate (PMA) with anti-CD2 changed neither the intensity nor the pattern of the tyrosine phosphorylation observed with anti-CD2 alone. The tyrosine phosphorylation of the p75 was not induced by concanavalin A (Con A) or PMA. While PMA alone failed to stimulate tyrosine phosphorylation above resting levels, PMA induced the nearly complete conversion of p56lck into p60lck (the lck-shift) at 30 min; no lck-shift was observed at 30 and 90 sec. Neither anti-CD2 nor Con A induced the lck-shift. Whereas PMA with either anti-CD2 or Con A was required for mitogenesis, only anti-CD2 led to tyrosine phosphorylation, and only PMA induced the lck-shift.
Breithaupt
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TB
Department of Biochemistry, University of Osteopathic Medicine and Health Sciences, Des Moines, Iowa 50312.
Rivera-Quiñones
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Molina
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Báez
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Eylar
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EH
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D000945:Antigens, Differentiation, T-Lymphocyte / Q000276:immunology*; D001152:Arsenicals; D018801:CD2 Antigens; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008938:Mitosis / Q000187:drug effects; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011971:Receptors, Immunologic / Q000276:immunology*; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000945:Antigens, Differentiation, T-Lymphocyte; D001152:Arsenicals; D018801:CD2 Antigens; D010455:Peptides; D011971:Receptors, Immunologic; C029341:oxophenylarsine; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D013755:Tetradecanoylphorbol Acetate