title: Gene transfer investigations of p56-LCK function in IL-2-dependent T-cell lines: implications for mechanisms of IL-2-signal transduction.
abstract: Recently, the SRC-like non-receptor protein tyrosine kinase p56-LCK has been shown to physically associate with the interleukin-2 receptor (IL-2-R) complex and to undergo rapid elevations in its tyrosine kinase activity upon stimulation of T lymphocytes with IL-2. The functional significance of p56-LCK kinase activation for IL-2-mediated lymphocyte responses, however, has never been directly assessed. Using gene transfer approaches, we have achieved markedly elevated levels of p56-LCK kinase activity in the IL-2-dependent cytolytic T-cell line CTLL-2 and the helper line HT-2. CTLL-2 and HT-2 cells that were stably transfected with expression plasmids encoding either the normal human p56-LCK or a constitutively active version of the mouse p56-LCK kinase (LCK[Y505]) contained striking elevations in the levels of tyrosine phosphorylation on several proteins (34-36, 50-60, 62-68, 77-78, 104-110 kDa), as determined by immunoblot analysis using anti-phosphotyrosine antibodies. CTLL-2 and HT-2 LCK- and LCK(Y505F)-transfected cells remained dependent on IL-2 for their growth and survival in culture despite the findings that (i) IL-2 specifically stimulated elevations in the activity of the endogenous p56-LCK in untransfected CTLL-2 cells without affecting the activities of the other SRC-like kinases in these cells (p59-FYN, p62-YES) and that (ii) IL-2-mediated regulation of p56-LCK correlated with IL-2-driven proliferation of these T cells. Specifically, no elevation in the proliferation (DNA synthesis) or growth of these T cells was found at any of the concentrations of IL-2 examined (0.01-25 U/ml), relative to untransfected and control transfected cells. Furthermore, when cultured in the absence of IL-2, transfected T cells whose relative levels of p56-LCK activity were elevated by approximately 20-50-fold died with the same kinetics as control cells and underwent apoptosis, as defined by uptake of trypan blue dye and DNA fragmentation assays, respectively. Taken together, these data indicate that while IL-2 can up-regulate the enzymatic activity of p56-LCK, elevated levels of p56-LCK tyrosine kinase activity are insufficient to stimulate IL-2-mediated pathways required for T-cell growth and survival. These findings thus imply the existence of other signal-transducing molecules, besides p56-LCK, that physically participate in IL-2R complexes and that are necessary for initiation of the biochemical events ultimately responsible for IL-2's pleiotropic actions on lymphocytes.
authors.affiliation: University of Pennsylvania, Department of Pathology, Philadelphia 19104-6082.
authors.forename: R S
authors.initials: RS
authors.lastname: Taichman
authors.forename: T
authors.initials: T
authors.lastname: Torigoe
authors.forename: S
authors.initials: S
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Miyashita
authors.forename: J C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D008213:Lymphocyte Activation; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D051379:Mice; D010957:Plasmids; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000201:enzymology / Q000276:immunology*; D014162:Transfection
chemical_list: D007376:Interleukin-2; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)