G-protein-independent activation of Tyk2 by the platelet-activating factor receptor.
Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic 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-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2-4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand.
Lukashova
V
V
Immunology Division, Department of Pediatrics and the Department of Anatomy and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
Asselin
C
C
Krolewski
J J
JJ
Rola-Pleszczynski
M
M
Stanková
J
J
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D020962:Heterotrimeric GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D009000:Monocytes / Q000378:metabolism; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000378:metabolism*; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D053634:TYK2 Kinase; D015534:Trans-Activators / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D020298:U937 Cells
D004268:DNA-Binding Proteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D015534:Trans-Activators; C039351:platelet activating factor receptor; D011505:Protein-Tyrosine Kinases; D053634:TYK2 Kinase; C070139:TYK2 protein, human; D020962:Heterotrimeric GTP-Binding Proteins