ZAP-70 tyrosines 315 and 492 transmit non-genomic glucocorticoid (GC) effects in T cells

► Y315 and Y492 in ZAP-70 kinase are involved in non-genomic GC signaling. ► Short time high-dose DX treatment led to LAT, SLP-76 and Cbl Y-phosphorylation. ► The DX-induced Y-phosphorylation of SLP-76 and Cbl was abolished in F315-, F492-ZAP-70 cells. ► Combined treatment with anti-CD3 and DX cause...

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Published in:	Molecular immunology Vol. 53; no. 1-2; pp. 111 - 117
Main Authors:	Boldizsar, F., Szabo, M., Kvell, K., Czompoly, T., Talaber, G., Bjorkan, J., Bartis, D., Nemeth, P., Berki, T.
Format:	Journal Article
Language:	English
Published:	England Elsevier Ltd 01-01-2013
Subjects:	Blotting, Western Cbl Cbl protein CD3 antigen Dexamethasone - immunology Dexamethasone - pharmacology Flow Cytometry Glucocorticoids Glucocorticoids - immunology Glucocorticoids - pharmacology Humans Immunoprecipitation Jurkat Cells Lymphocytes T Non-genomic GC action Phenylalanine Phosphorylation Signal transduction Signal Transduction - drug effects Signal Transduction - immunology SLP-76 SLP-76 protein T-cell receptor T-Lymphocytes - drug effects T-Lymphocytes - immunology T-Lymphocytes - metabolism TcR signaling Tyrosine Tyrosine - immunology Tyrosine - metabolism ZAP-70 ZAP-70 protein ZAP-70 Protein-Tyrosine Kinase - drug effects ZAP-70 Protein-Tyrosine Kinase - immunology Itk RT DMSO F WB Cbl DP ZAP-70 DX Fluo-3AM Y Hsp-90 GC Th1 Non-genomic GC action WT Th2 CD TcR SLP-76 Syk PVDF, RPMI ATPase PKB IP TcR signaling PKC PLCγ GR MAPK ITIM IL2 Lck SH2 LAT
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Summary:	► Y315 and Y492 in ZAP-70 kinase are involved in non-genomic GC signaling. ► Short time high-dose DX treatment led to LAT, SLP-76 and Cbl Y-phosphorylation. ► The DX-induced Y-phosphorylation of SLP-76 and Cbl was abolished in F315-, F492-ZAP-70 cells. ► Combined treatment with anti-CD3 and DX caused elevated Y-phosphorylation of LAT and Cbl. ► DX partly inhibited the anti-CD3-elicited Ca2+-signal and the Y-phosphorylation of SLP-76. 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 Ca2+-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.
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ISSN:	0161-5890 1872-9142
DOI:	10.1016/j.molimm.2012.07.007