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

Abstract

Abstract 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 β-subunit of the Ti heterodimer or the ɛ/δ-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC α, βI, βII, and δ were reduced by 90% or more in PMA-treated cells, whereas the expression of PKCθ decreased by ∼30%. In contrast, the chronic treatment with PMA increased the expression of PKCɛ and PKCζ. 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 p59fyn or ZAP-70 were not changed in PMA-treated cells but p56Ick migrated as a large band of Mr 60–62 kDa. The decreased mobility of p56Ick was related to a state of hyperphosphorylation. The activity of modified p56Ick 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.