Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibition of CD3- but not Ti-dependent antibody-triggered Ca2+ signaling.
We have treated Jurkat T lymphocytes with a concentration (160 nM) of phorbol myristyl acetate (PMA) that down-regulates conventional and novel protein kinase C (PKC) isozymes and we have investigated the effects on Ca2+ signaling and protein tyrosine phosphorylation using mAb (C305) directed against the beta-subunit of the Ti heterodimer or the epsilon/delta-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC alpha, betaI, betaII, and delta were reduced by 90% or more in PMA-treated cells, whereas the expression of PKCtheta decreased by approximately 30%. In contrast, the chronic treatment with PMA increased the expression of PKCepsilon and PKCzeta. There was a lack of Ca2+ response and myo-inositol trisphosphate (IP3) production in PMA-treated cells when they were exposed to mAb Leu 4 but the cells responded to mAb C305. The treatment with PMA did not affect the surface expression of Ti or CD3. The overall levels of tyrosine-phosphorylated proteins were markedly reduced in PMA-treated cells. We investigated whether these observations were related to defects in signal transduction related to protein tyrosine kinase (PTK) of the src and syk families. The electrophoretic mobilities of p59(fyn) or ZAP-70 were not changed in PMA-treated cells but p56(Ick) migrated as a large band of M(r) 60-62 kDa. The decreased mobility of p56(Ick) was related to a state of hyperphosphorylation. The activity of modified p56(Ick) was not up-regulated in activated Jurkat cells. Our data suggest that clonotypic Ti can trigger Ca2+ mobilization independently of conventional PKC isoforms. Our observations further suggest that conventional PKC isoforms are involved early in the cascade of events associated with Jurkat T lymphocyte activation.
Ahnadi
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CE
Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Giguère
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P
Gravel
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S
Gagné
D
D
Goulet
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AC
Fülöp
T
T
Payet
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MD
Dupuis
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G
D017252:CD3 Complex / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D002273:Carcinogens / Q000494:pharmacology*; D006801:Humans; D019169:Jurkat Cells; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014443:Tyrosine
D017252:CD3 Complex; D002273:Carcinogens; D011948:Receptors, Antigen, T-Cell; D014443:Tyrosine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium