DIFFERENTIAL UTILIZATION OF JANUS KINASE-SIGNAL TRANSDUCER AND 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 an d 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, whic h formed IFN-gamma-activated sequence-binding complexes by itself and with STAT3. Although IL-2 and IFN-alpha activated STAT1 alpha and STAT 5, IL-2 predominantly activated STAT5, while IFN-alpha predominantly a ctivated STAT1 alpha. IL-2 induced less STAT1 alpha activation and IFN -alpha induced greater STAT5 activation in NK3.3 cells compared with p reactivated 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 conta ining STAT3 and STAT1 alpha, while in preactivated primary NK cells, i t preferentially induced complexes containing STAT3 and STAT1 alpha. T hus, signaling in NK3.3 cells is not always identical with that in pri mary NK cells. In contrast to IL-2 and IFN-alpha, IL-12 induced strong tyrosine phosphorylation of STAT4 and variable weak phosphorylation o f STAT3. However, supershift analyses using the c-fos promoter sis-ind ucible element probe showed that IL-12 activated STAT4, STAT1 alpha, a nd STAT3, acid induced complexes containing STAT4 only, STAT4 with STA T1 alpha, STAT3 with STAT1 alpha, or STAT1 alpha only in preactivated primary NK cells. STAT1 alpha activation by IL-12 correlated with incr eased phosphorylation of serine, but not tyrosine. Finally, IL-2 induc ed tyrosine phosphorylation of JAK1 and JAK3, while IL-12 induced phos phorylation of JAK2 and TYK2 in both preactivated primary NK and NK3.3 cells. Differential phosphorylation and consequent differential activ ation of both separate and overlapping STAT proteins by IL-2, IL-12, a nd IFN-alpha may provide a molecular basis for the similarities and di fferences in the actions of these cytokines on NK cells.