Janus Kinase 2 Activation by the Platelet-Activating Factor Receptor (PAFR): Roles of Tyk2 and PAFR C Terminus

• Published in: The Journal of immunology (1950) 2003-10, Vol.171 (7), p.3794-3800
• Main Authors: Lukashova, Viktoria, Chen, Zhangguo, Duhe, Roy J, Rola-Pleszczynski, Marek, Stankova, Jana
• Format: Article
• Language: English
• Subjects: Animals; Catalysis; Cell Line; Cercopithecus aethiops; COS Cells; DNA, Complementary - genetics; Enzyme Activation - physiology; Humans; Janus Kinase 2; Monocytes - enzymology; Monocytes - metabolism; Peptide Fragments - physiology; Phosphorylation; Platelet Activating Factor - metabolism; Platelet Membrane Glycoproteins - genetics; Platelet Membrane Glycoproteins - physiology; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; Protein-Tyrosine Kinases - physiology; Proteins - genetics; Proteins - physiology; Proto-Oncogene Proteins; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - physiology; Sequence Deletion; Signal Transduction - physiology; Transfection; TYK2 Kinase
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.171.7.3794

Platelet-activating factor (PAF) is a phospholipid with multiple physiological and pathological actions. The PAF receptor (PAFR) belongs to the G protein-coupled, heptahelical receptor superfamily. Recently, we have shown that PAF signals through the Janus kinase (Jak)/STAT pathway and that Tyk2 plays an essential role in PAF-induced PAFR promoter 1 activation. In the present study we found that PAF stimulated Jak2 tyrosine phosphorylation in the monocytic cell line MonoMac-1 as well as in COS-7 cells transfected with PAFR and Jak2 cDNAs. The use of a G protein-uncoupled PAFR (D289A) mutant indicated that Jak2 activation was G protein independent. Interestingly, following PAF stimulation, Jak2 coimmunoprecipitated with PAFR in the presence of active Tyk2, but not with a kinase-inactive Tyk2 mutant, K930I. Moreover, Tyk2-K930I completely blocked PAF-stimulated Jak2 phosphorylation. Gradual deletion of C-terminal residues of the PAFR resulted in progressively decreased Jak2 activation. Deletion of 12 C-terminal residues in mutant V330Stop diminished Jak2 tyrosine phosphorylation by 17%. Further deletions of 25-37 residues from the PAFR C-tail (C317Stop, M311Stop, and T305Stop) resulted in a 50% decrease in Jak2 phosphorylation compared with the wild-type receptor. Complete removal of the C tail resulted in a mutant (K298Stop) that failed to activate Jak2, suggesting that the receptor C-terminal region contains important domains for Jak2 activation. Finally, the coexpression of a minigene encoding the C terminus of PAFR partially inhibited PAF-induced kinase activation. Taken together, our results indicate that PAF activates Jak2 and that Tyk2 and the C-terminal tail of PAFR are of critical importance for PAF-induced Jak2 activation.