Two discrete regions of interleukin-2 (IL2) receptor beta independently mediate 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 factors. For full transcriptional activation, Stat1 and Stat3 also require phosphorylation of a conserved serine residue within a mitogen-activated protein kinase phosphorylation consensus site. On the other hand, two recently identified and highly homologous Stat5a and Stat5b proteins 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 serine kinase. We now report that IL2 stimulated marked phosphorylation of serine and tyrosine residues of both Stat5a and Stat5b in human T lymphocytes and in several IL2-responsive lymphocytic cell lines. No Stat5a/b phosphothreonine was detected. Phosphoamino acid analysis also revealed that Stat5a/b phosphotyrosine levels were maximized within 1-5 min of IL2 stimulation, whereas serine phosphorylation kinetics were slower. Interestingly, IL2-induced serine phosphorylation of Stat5a differed quantitatively and temporally from that of Stat5b with Stat5a serine phosphorylation leveling off after 10 min and the more pronounced Stat5b response continuing to rise for at least 60 min of IL2 stimulation. Furthermore, we identified two discrete domains of IL2 receptor beta (IL2Rbeta) that could independently restore the ability of a truncated IL2Rbeta mutant to mediate Stat5a/b phosphorylation and DNA binding to the gamma-activated site of the beta-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2Rbeta region for Stat5 phosphorylation. Finally, we established that the IL2-activated Stat5a/b serine kinase is insensitive to several selective inhibitors of known IL2-stimulated kinases including MEK1/MEK2 (PD98059), mTOR (rapamycin), and phosphatidylinositol 3-kinase (wortmannin) as determined by phosphoamino acid and DNA binding analysis, thus suggesting that a yet-to-be-identified serine kinase mediates Stat5a/b activation.
Kirken
R A
RA
Intramural Research Support Program, Science Applications International Corporation Frederick, Frederick, Maryland, 21702-1201, USA.Kirken@mail.ncifcrf.gov
Malabarba
M G
MG
Xu
J
J
DaSilva
L
L
Erwin
R A
RA
Liu
X
X
Hennighausen
L
L
Rui
H
H
Farrar
W L
WL
D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology*; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D007700:Kinetics; D010766:Phosphorylation; D011090:Polyenes / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D015375:Receptors, Interleukin-2 / Q000502:physiology*; D012694:Serine / Q000378:metabolism; D020123:Sirolimus; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007376:Interleukin-2; D011090:Polyenes; D015375:Receptors, Interleukin-2; D014443:Tyrosine; D012694:Serine; C106612:Stat5b serine kinase; D017346:Protein Serine-Threonine Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D020123:Sirolimus; D000077191:Wortmannin