Differential utilization of Janus kinase-signal transducer activator of transcription signaling pathways in the stimulation of human natural killer cells by IL-2, IL-12, and IFN-alpha.
IL-2-, IL-12-, and IFN-alpha-mediated signaling pathways were analyzed in primary NK cells and in the NK3.3 cell line. Gel mobility shift and immunoprecipitation analyses revealed that in addition to activating STAT3 (signal transducer and activator of transcription-3) and STAT5, IL-2 induced tyrosine and serine phosphorylation of STAT1 alpha, which formed IFN-gamma-activated sequence-binding complexes by itself and with STAT3. Although IL-2 and IFN-alpha activated STAT1 alpha and STAT5, IL-2 predominantly activated STAT5, while IFN-alpha predominantly activated STAT1 alpha. IL-2 induced less STAT1 alpha activation and IFN-alpha induced greater STAT5 activation in NK3.3 cells compared with preactivated primary NK cells. In NK3.3 cells, IL-2 induced comparable formation of c-fos promoter sis-inducible element IFN-gamma-activated sequence-binding complexes containing STAT3 alone with complexes containing STAT3 and STAT1 alpha, while in preactivated primary NK cells, it preferentially induced complexes containing STAT3 and STAT1 alpha. Thus, signaling in NK3.3 cells is not always identical with that in primary NK cells. In contrast to IL-2 and IFN-alpha, IL-12 induced strong tyrosine phosphorylation of STAT4 and variable weak phosphorylation of STAT3. However, supershift analyses using the c-fos promoter sis-inducible element probe showed that IL-12 activated STAT4, STAT1 alpha, and STAT3, and induced complexes containing STAT4 only, STAT4 with STAT1 alpha, STAT3 with STAT1 alpha, or STAT1 alpha only in preactivated primary NK cells. STAT1 alpha activation by IL-12 correlated with increased phosphorylation of serine, but not tyrosine. Finally, IL-2 induced tyrosine phosphorylation of JAK1 and JAK3, while IL-12 induced phosphorylation of JAK2 and TYK2 in both preactivated primary NK and NK3.3 cells. Differential phosphorylation and consequent differential activation of both separate and overlapping STAT proteins by IL-2, IL-12, and IFN-alpha may provide a molecular basis for the similarities and differences in the actions of these cytokines on NK cells.
Yu
C R
CR
Laboratory of Cellular Immunology, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.
Lin
J X
JX
Fink
D W
DW
Akira
S
S
Bloom
E T
ET
Yamauchi
A
A
D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D018664:Interleukin-12 / Q000494:pharmacology*; D007376:Interleukin-2 / Q000378:metabolism / Q000494:pharmacology*; D053613:Janus Kinase 1; D007694:Killer Cells, Natural / Q000187:drug effects*; D008213:Lymphocyte Activation / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D050794:STAT1 Transcription Factor; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics* / Q000276:immunology*; D015534:Trans-Activators / Q000096:biosynthesis / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects / Q000276:immunology*; D015533:Transcriptional Activation / Q000187:drug effects / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism / Q000494:pharmacology*; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D014443:Tyrosine; D012694:Serine; D011505:Protein-Tyrosine Kinases; C507904:JAK1 protein, human; D053613:Janus Kinase 1