2 DISCRETE REGIONS OF INTERLEUKIN-2 (IL2) RECEPTOR-BETA INDEPENDENTLYMEDIATE IL2 ACTIVATION OF A PD98059 RAPAMYCIN/WORTMANNIN-INSENSITIVE STAT5A/B SERINE KINASE/

Many cytokines, hormones, and growth factors activate Janus kinases to tyrosine phosphorylate select members of the Stat transcription facto rs, For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activat ed protein kinase phosphorylation consensus site. On the other hand, t wo recently identified and highly homologous Stat5a and Stat5b protein s lack this putative mitogen-activated protein kinase phosphorylation site, The present study set out to establish whether Stat5a and Stat5b are under the control of an interleukin-2 (IL2)-activated Stat5 serin e kinase, We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymp hocytes and in several IL2-responsive lymphocytic cell lines, No Stat5 a/b phosphothreonine was detected. Phosphoamino acid analysis also rev ealed that Stat5a/b phosphotyrosine levels were maximized within 1-5 m in of IL2 stimulation, whereas serine phosphorylation kinetics were sl ower, Interestingly, IL2-induced serine phosphorylation of Stat5a diff ered quantitatively and temporally from that of Stat5b with Stat5a ser ine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulat ion, Furthermore, we identified two discrete domains of IL2 receptor b eta (IL2R beta) that could independently restore the ability of a trun cated IL2R beta mutant to mediate Stat5a/b phosphorylation and DNA bin ding to the gamma-activated site of the beta-casein gene promoter, The se observations demonstrated that there is no strict requirement for o ne particular IL2R beta region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensiti ve to several selective inhibitors of known IL2-stimulated kinases inc luding MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3 kinase (wortmannin) as determined by phosphoamino acid and DNA bind ing analysis, thus suggesting that a yet-to-be-identified serine kinas e mediates Stat5a/b activation.