Janus kinase 2 activation by the platelet-activating factor receptor (PAFR): roles of Tyk2 and PAFR C terminus.
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.
D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D053614:Janus Kinase 2; D009000:Monocytes / Q000201:enzymology / Q000378:metabolism; D010446:Peptide Fragments / Q000502:physiology*; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000378:metabolism*; D010980:Platelet Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D015398:Signal Transduction / Q000502:physiology; D053634:TYK2 Kinase; D014162:Transfection
D018076:DNA, Complementary; D010446:Peptide Fragments; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D053634:TYK2 Kinase; C070139:TYK2 protein, human