G-protein-independent activation of Tyk2 by the platelet-activating factorreceptor

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activi ty through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed th at PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the m onocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunop recipitated and co-localized with PAFR, independently of ligand binding. De letion mutants of Tyk2 indicated that the N terminus of the kinase was impo rtant for the binding to PAFR. Activation of Tyk2 was followed by a time-de pendent 2-4-fold increase in the level of tyrosine phosphorylation of signa l transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 a nd a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In Mono Mac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stim ulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cel ls were transfected with PAFR and constructs containing PAFR promoter 1, co upled to the luciferase reporter gene, PAF induced a 3.6-fold increase in p romoter activation in the presence of Tyk2. Finally, PAFR mutants that coul d not couple to G-proteins were found to effectively mediate Tyk2 activatio n and signaling. Taken together, these findings suggest an important role f or the Janus kinase/STAT pathway in PAFR signaling, independent of G-protei ns, and in the regulation of PAF receptor expression by its ligand.