PNU156804 inhibits the signal 3 mediator Jak3 but not the signal 1 transducer p56Lck.
(A) Antiphosphotyrosine (αPY) immunoblot of PHA-activated T cells stimulated with anti-CD3 in the presence of PNU156804. T cells (5.0 × 107 cells/lane) were treated with DMSO or with 20 μM PNU156804 for 16 hours and then were stimulated in the presence or absence of 5 μg anti-CD3 for 5 minutes and subjected to cell lysis and clarification. Total cell lysates were separated by SDS-PAGE (lanes a-d) or were immunoprecipitated with antibodies to p56Lck (lanes e-h) and separated on 10% SDS-PAGE and then subjected to antiphosphotyrosine Western blot (WB) analysis. The blot was then stripped and reblotted with anti-Lck to verify equivalent loading (indicated in the lower panel). Arrow denotes migration of p56Lck. (B) PHA-activated human T cells were treated with DMSO or increasing concentrations of PNU156804 (0-10 μM; lanes a-j) or inactive control PNU159744 (lanes k-n) and were stimulated in the absence (−) or presence (+) of IL-2 (100 nM) for 10 minutes at 37°C. Cells were lysed, clarified, and immunoprecipitated (IP) with anti-Jak3 (αJak3), separated on 10% SDS-PAGE, transferred to PVDF membrane, and Western blotted with antiphosphotyrosine. The blot was stripped and reprobed with Jak3 antibody. (C) For antiphosphotyrosine immunoblot of Jak3 autokinase assay, PHA-activated T cells were stimulated for 10 minutes with (+) or without (−) 100 nM IL-2 and then immunoprecipitated with anti-Jak3 and treated for 15 minutes on ice with DMSO control (lanes a-d) or 10 μM PNU156 804 (lanes e-h). Jak3 was incubated for 20 minutes at 37°C in the absence (−) or presence (+) of 15 μM unlabeled ATP, separated by SDS-PAGE, transferred to PVDF membrane, and Western blotted with antiphosphotyrosine antibodies (upper panel). The same blots were reblotted with α-Jak3 to verify equal loading of enzyme (lower panel). Ratio of Tyr-phosphorylated Jak3 to total Jak3 protein was analyzed by densitometry and plotted as the stimulatory index (SI) value in arbitrary units.
