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(Journal of Leukocyte Biology. 2000;68:293-300.)
© 2000 by Society for Leukocyte Biology

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

Charaf E. Ahnadi^*,, Patrick Giguère, Serge Gravel^*, Danièle Gagné, Anne-Christine Goulet, Tamàs Fülöp, Jr, Marcel D. Payet and Gilles Dupuis^*

^* Departments of Biochemistry and
Physiology and Biophysics,
Clinical Research Center and
Centre de Recherche en Gérontologie et Gériatrie, Faculty of Medicine, University of Sherbrooke, Quebec, Canada

Correspondence: Dr. Gilles Dupuis, Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada, J1H 5N4. E-mail: gdupui01{at}courrier.usherb.ca

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 ß-subunit of the Ti heterodimer or the /-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC , ßI, ßII, and were reduced by 90% or more in PMA-treated cells, whereas the expression of PKC decreased by 30%. In contrast, the chronic treatment with PMA increased the expression of PKC and PKC. 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.

Key Words: protein kinase C • down-regulation • phorbol myristyl acetate • signal transduction • protein tyrosine kinase

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