Two Discrete Regions of Interleukin-2 (IL2) Receptor β Independently Mediate IL2 Activation of a PD98059/Rapamycin/Wortmannin-insensitive Stat5a/b Serine Kinase

Abstract

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 ␤ (IL2R␤) that could independently restore the ability of a truncated IL2R␤ mutant to mediate Stat5a/b phosphorylation and DNA binding to the ␥-activated site of the ␤-casein gene promoter. These observations demonstrated that there is no strict requirement for one particular IL2R␤ 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-beidentified serine kinase mediates Stat5a/b activation.Interleukin-2 (IL2) 1 is a key regulator of normal immune function and acts on a variety of lymphoid cell types including T lymphocytes, B lymphocytes, and natural killer cells (1). IL2-induced effects are mediated by heterodimerization of two related transmembrane proteins of the hematopoietin receptor family that are designated IL2 receptor ␤-and ␥-chains (IL2R␤ and ILR2␥) (2-4). In addition to this pair of essential receptor subunits, a third non-conforming protein with a short cytoplasmic domain (TAC or IL2R␣) represents an accessory receptor subunit that can serve as a positive affinity modulator through its regulated expression (5-7). IL2-induced dimerization of IL2R␤ and IL2R␥ results in stimulation of the receptor-associated Janus kinases (JAK) JAK1 and/or JAK3 through intermolecular transphosphorylation (8, 9

Induction of erythroid differentiation by inhibition of Ras/ERK pathway in a Friend murine leukemia cell line

“…Consistent with this hypothesis, a speci®c (Dudley et al, 1995;Dumont et al, 1998;Lu et al, 1998;Kirken et al, 1997), was found to induce erythroid di erentiation in dose-dependent manners not only in SKT6 cells but also in two other Friend leukemia-derived cell lines, TL200 and 1-2 (Figure 5a,b,c). In these experiments, growth-suppressing e ects of the drug became evident at the concentrations e ective in inducing di erentiation.…”

“…On the other hand, wortmannin (Kimura et al, 1994;Damen et al, 1995;Kirken et al, 1997), an inhibitor of PI3K, did not induce di erentiation even at the concentrations that suppress cell growth to a comparable degree (Figure 5e), suggesting that growthsuppression alone was not su cient for inducing di erentiation. This ®nding also suggests that PI3K, a key component of another signaling pathway downstream of Ras, does not play a major role in the Rasmediated inhibition of erythroid di erentiation observed in this system.…”

Naturally occurring dominant-negative Stat5 suppresses transcriptional activity of estrogen receptors and induces apoptosis in T47D breast cancer cells

“…Equal amounts of total protein from whole cell lysates were prepared and used for SDS-PAGE. For immunoprecipitation from clarified cell lysates, polyclonal rabbit antisera (2 ml/ml) specific to peptides corresponding to the unique COOH-termini of Stat5a or Stat5b (Kirken et al, 1997a), and rabbit polyclonal antibodies of ERa (H-184, Santa Cruz Biotechnology) or ERb (H-150, Santa Cruz Biotechnology) were used and captured after incubation with protein A-Sepharose beads (Pharmacia Biotech, Cat. # 17-0780-01).…”

Serine/threonine phosphorylation in cytokine signal transduction

“…[114][115][116] Unlike other STAT proteins, STAT5a does not contain a classical MAPK recognition sequence, and is not phosphorylated by this kinase. Thus, the serine/threonine kinase, which phosphorylates STAT5a, remains unknown.…”