ZAP-70 tyrosines 315 and 492 transmit non-genomic glucocorticoid (GC) effects in T cells.
ZAP-70 kinase is a key regulator of early T-cell signaling; moreover, it also participates in non-genomic glucocorticoid (GC) signaling. Short-term high-dose GC-analogue treatment induces the phosphorylation of the kinase, and its association with the GC receptor (GR). In the present work, first, we identified those tyrosine (Y) residues of the ZAP-70 kinase which were involved in non-genomic GC signaling using an array of P116 cells (ZAP-70-deficient Jurkat subclone) lentivirally-transfected with wild type or point-mutated ZAP-70 constructs where Y-residues were replaced with phenylalanine (F) at positions 069, 126, 178, 238, 292, 315, 492 or 493. Then, we characterized the GC-analogue-induced Y-phosphorylation of 3 key substrates of the ZAP-70 kinase: SLP-76, LAT and Cbl. Finally, we studied the cross talk between the non-genomic GC- and TcR/CD3 signaling pathways. Y-F mutations at positions 315 or 492 abolished the short high-dose Dexamethasone (DX) treatment-induced ZAP-70 phosphorylation suggesting that these Y-residues were involved in ZAP-70-mediated non-genomic GC actions. DX treatment alone induced Y-phosphorylation of LAT, SLP-76 and Cbl; moreover, in F315- and F492-ZAP-70 mutated cells decreased DX-induced Y-phosphorylation of SLP-76 and Cbl was observed indicating that these molecules might transmit downstream non-genomic GC signals in a ZAP-70 dependent manner. Short, high dose DX treatment influenced significantly the anti-CD3-induced signaling events: we observed alterations in LAT, SLP-76 and Cbl Y-phosphorylation and a decreased Ca(2+)-signal. These results confirm that ZAP-70 represents an important link between the non-genomic GC and TcR/CD3 signaling pathways. Importantly, the DX-induced effects on resting and activated T-cells are differentially mediated. These fine molecular details help to better understand the complex mechanism of non-genomic GC effects in T-cells.
University of Pecs, Department of Immunology and Biotechnology, H-7624, Pecs, Szigeti ut 12, Hungary. ferenc.boldizsar@aok.pte.hu
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F
Boldizsar
M
M
Szabo
K
K
Kvell
T
T
Czompoly
G
G
Talaber
J
J
Bjorkan
D
D
Bartis
P
P
Nemeth
T
T
Berki
D015153:Blotting, Western; D003907:Dexamethasone / Q000276:immunology* / Q000494:pharmacology; D005434:Flow Cytometry; D005938:Glucocorticoids / Q000276:immunology* / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology*; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology* / Q000378:metabolism; D014443:Tyrosine / Q000276:immunology / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000187:drug effects / Q000276:immunology*
D005938:Glucocorticoids; D014443:Tyrosine; D003907:Dexamethasone; D051746:ZAP-70 Protein-Tyrosine Kinase