Phosphorylation of T-cell antigen receptor-associated proteins: correlation with activation for killing and/or for gamma-interferon production by a cytolytic T-cell clone.
The activation-induced phosphorylation of T-cell antigen receptor (Ti)-associated proteins was investigated in order to analyse possible signal-transduction mechanisms leading to two distinct effector functions of a mouse cytolytic T-cell clone (KB5.C20): target cell killing (independent of protein synthesis) and de novo production of gamma interferon (gIFN; dependent on gIFN gene expression). Ti-associated T3-like proteins were first identified by immunoprecipitation of 125I-labelled cell surface proteins from 1% digitonin lysates of clone KB5.C20 by 1- and 2-dimensional (non-reduced (NR)/reduced (R)) gel electrophoresis. In addition to the alpha and beta chains of the Ti (NR: 80-Kd; R: 43 and 40 Kd), two doublets of 35-37 Kd (NR) and 32-34 Kd (NR) leading to bands of 25, 16 and 14 Kd (R) were identified, as well as three bands (25, 23 and 22 Kd (NR)) leading to 27-, 25- and 21-Kd bands (R). Activation of clone KB5.C20 (prelabelled with 32P-orthophosphate) with either anti-Ti mAb or exposure to both ionomycin and phorbol myristic acetate (PMA) induced the phosphorylation of 21- and 25-27-Kd (R) Ti-associated proteins, whereas exposure to either ionomycin or PMA alone induced only weak phosphorylation of 21-Kd (R) components. A weak phosphorylation of 32- and 34-Kd Ti-associated proteins was sometimes observed after stimulation with anti-Ti mAb. Functional studies suggested that activation for gIFN production was observed only when both the 21- and 25-27-Kd proteins were phosphorylated, whereas activation for killing (when measured by PMA-induced non-specific killing) could occur in conditions where no phosphorylation of the 25-27-Kd protein was detected.
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000502:physiology*; D017252:CD3 Complex; D002999:Clone Cells; D003602:Cytotoxicity, Immunologic; D004357:Drug Synergism; D004987:Ethers / Q000494:pharmacology; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D007467:Iodoproteins / Q000276:immunology; D015759:Ionomycin; D008213:Lymphocyte Activation; D046911:Macromolecular Substances; D008970:Molecular Weight; D010750:Phosphoproteins / Q000276:immunology; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D017252:CD3 Complex; D004987:Ethers; D007467:Iodoproteins; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D015759:Ionomycin; D007371:Interferon-gamma; D013755:Tetradecanoylphorbol Acetate