Activation of STAT6 is not dependent on phosphotyrosine‐mediated docking to the interleukin‐4 receptor and can be blocked by dominant‐negative mutants of both receptor subunits

Moriggl, Richard; Erhardt, Ingrid; Kammer, Winfried; Berchtold, Susanne; Schnarr, Bernd; Lischke, Antje; Groner, Bernd; Friedrich, Karlheinz

doi:10.1046/j.1432-1327.1998.2510025.x

Cited by 16 publications

(20 citation statements)

References 48 publications

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“…This cell line has been widely used for studying proliferative responses to a range of cytokines, including IL-2, IL-3, IL-4, erythropoietin, and granulocytemacrophage colony-stimulating factor. [30][31][32][33][34][35][36] Previous studies with BaF-B03 cells have shown that there are at least 3 pathways that may cooperate to produce a proliferative signal. 37 The results of the present study emphasize the pivotal role of the cyclin:CDK-pRB: E2F-1 pathway in mediating the cellular proliferative response in BaF-B03 cells and suggest that multiple pathways converge at E2F-1.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of E2F-1 leads to cytokine-independent proliferation and survival in the hematopoietic cell line BaF-B03

Gala

Marreiros

Stewart

et al. 2001

Blood

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Cytokine receptors activate signals that regulate the transcription factor E2F-1, which then coordinates the expression of genes essential for DNA synthesis and cell cycle progression. Overexpression of E2F-1 most often induces S-phase entry followed by apoptosis, but in some cell types it leads to continuous proliferation and transformation. Here, it is shown that constitutive expression of E2F-1 promotes cytokine-independent proliferation in the murine pro-B cell line BaF-B03. There was no enhancement of apoptosis following cytokine withdrawal in these cells, despite the presence of intact p53-dependent apoptotic pathways. Notwithstanding the continuous presence of E2F-1, the cell cycle-dependent expression of cyclin A, cyclin B1, cyclin D1, cyclin E, and proliferating-cell nuclear antigen was restored with a pattern equivalent to that associated with cytokine stimulation. These findings provide evidence that, in the absence of cytokine, constitutive expression of E2F-1 can promote cell cycle progression and prevent apoptosis. (Blood. 2001;97:227-234)

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Section: Discussionmentioning

confidence: 99%

Overexpression of E2F-1 leads to cytokine-independent proliferation and survival in the hematopoietic cell line BaF-B03

Gala

Marreiros

Stewart

et al. 2001

Blood

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“…Cleared cell lysates were incubated with 1 l of rabbit serum-specific for STAT5a or STAT5b (51) for 3 h at 4°C. Precipitation conditions employed for other antibodies were as described (5,45,52). Immunocomplexes were collected from lysates with 25 l of protein A-Sepharose (Sigma) or anti-mouse IgG-agarose (Sigma) and analyzed by Western blot as described (45).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we showed that COS-7 cells do not express significant amounts of endogenous STAT5 or STAT6 but provide all components necessary for transiently expressed STATs to drive transcription of a suitable reporter gene construct (36,47,52,53). Therefore, we reconstituted the bipartite hIL-4 receptor in COS-7 cells by transient expression of both hIL-4R␣ and human ␥c and introduced a luciferase reporter gene under the control of the STAT5-responsive ␤-casein promoter (53).…”

Section: Recruitment Of ␥C and Jak3 Into The Il-4 Receptor Complex Ismentioning

confidence: 99%

“…Heterologous hIL-4R subunits, which were included as invariable constituents of all individual assays, could not be detected by Western blot analysis due to low expression and the small number of cells used in each transfection experiment. However, we can infer their expression indirectly, since the functional effects of dominantnegative mutants of both hIL-4R␣ and human ␥c can be readily measured under these experimental conditions (52).…”

Section: Fig 4 Hil-4-induced Translocation Of Stat5a To the Cell Numentioning

confidence: 99%

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The Interleukin-4 Receptor Activates STAT5 by a Mechanism That Relies upon Common γ-Chain

Lischke¹,

Moriggl²,

Brändlein³

et al. 1998

Journal of Biological Chemistry

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Interleukin (IL)-4,1 a pleiotropic modulator of the immune system (1), exerts its activity on target cells through the interleukin-4 receptor. Different cell types, including lymphoid and myeloid blood cells, express a bipartite IL-4 receptor (IL-4R), which consists of the IL-4R ␣-chain (IL-4R␣) (2) and the common ␥ receptor chain (␥c) (3). IL-4R␣ may also form a functional IL-4 receptor in conjunction with the IL-13 receptor instead of ␥c, especially in nonimmune cells (4). In vitro IL-4R␣ has also been shown to trigger intracellular signal transduction as a homodimer without participation of a heterologous receptor subunit (5, 6); however, the possible physiological relevance of IL-4R␣ homodimers is not yet known.IL-4-induced dimerization of receptor subunits results in the rapid onset of various cytoplasmic events. Janus kinases JAK1 and JAK3, which are associated with IL-4R␣ and ␥c, respectively (7, 8), become activated, probably by transphosphorylation, and as a result several other constituents of the activated receptor complex are phosphorylated. Tyrosine phosphorylation of IL-4R␣, IRS-2 (insulin receptor substrate 2, originally termed 4PS/IL-4-induced phosphorylation substrate), phosphatidylinositol 3-kinase, STAT6, and probably other proteins generate a network of protein-protein contacts mediated by interactions between phosphotyrosines and Src homology 2 domains (9). Despite structure-function studies by several laboratories (10 -16), it is only partially understood how these IL-4-induced molecular events lead to long term cellular processes such as suppression of apoptosis, proliferation, and differentiation.The aspect of IL-4R-triggered intracellular signal transduction that has been best characterized is the JAK-STAT pathway. STAT (signal transducer and activator of transcription) factors are central components of signaling cascades triggered by cytokine receptors and are phosphorylated in response to ligand stimulation through receptor-associated Janus kinases (JAKs). STAT factors then dimerize and translocate to the cell nucleus, where they interact with cognate DNA sequences of ␥-interferon-responsive sites (GASs) and modulate transcription of target genes (17).Following signal induction by the ligand, both the IL-4 receptor and the IL-13 receptor specifically mediate the activation of STAT6 (15, 18 -21). Gene regulation by STAT6 appears to be of central importance for IL-4-governed immune regulation, since STAT6 knockout mice are unable to develop Th2 cells; are impaired in cell surface expression of CD23, IL-4R␣, and major histocompatibility complex class II; and show a drastic defect in immunoglobulin class switching (22)(23)(24).Results obtained in the course of investigations on related cytokine receptors raised the question of whether STAT6 is the only STAT protein involved in IL-4-induced intracellular signaling. The IL-4 receptor shares the common ␥-subunit with the receptors for IL-2, , and all cytokine receptors utilizing the ␥c subunit, with the exception of the IL-4R, activate STAT5 ...

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“…Depending on the receptor studied, and sometimes even on the experimental system used to analyze receptor activity, Stat docking sites were either found to be an absolute requirement, necessary to increase the potential to phosphorylate Stats, or of no importance at all (12)(13)(14). For several receptors, the requirement for receptor docking varied with the cell type or experimental situation under study (15)(16)(17)(18)(19)(20)(21)(22)(23)(24). The observation of Stat activation independent of receptor binding fueled the search for alternative ways by which cytokine receptors cause Stat phosphorylation.…”

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confidence: 99%

Jak2-Stat5 Interactions Analyzed in Yeast

Barahmand‐pour

Meinke

Groner³

et al. 1998

Journal of Biological Chemistry

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Many cytokine receptors employ Janus protein tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats) for nuclear signaling. Here, we have established yeast strains in which an autoactivated Jak2 kinase induces tyrosine phosphorylation, dimerization, nuclear translocation, and DNA binding of a concomitantly expressed Stat5 protein. Transcriptional activity of Stat5 on a stably integrated, Stat-dependent reporter gene required the C-terminal fusion of the VP16 transactivation domain. In such yeast strains, the interaction between Jak2 and Stat5 was analyzed without interference by other mammalian proteins involved in regulating Jak-Stat signaling, and mutant versions of both proteins were analyzed for their ability to productively interact. Complexes between Jak2 and Stat5 were found to be stable under stringent co-immunoprecipitation conditions. Deletion of the Jak homology regions 2-7 (JH2-JH7) of Jak2, leaving only the kinase domain (JH1) intact, reduced the ability of the kinase to phosphorylate Stat5, whereas deletion of the JH2 domain caused an increased enzymatic activity. A site-directed R618K mutation in the Stat5 SH2 domain abolished the phosphorylation by Jak2, while deletion of the C terminus led to Stat5 hyperphosphorylation. A single phosphotyrosine-SH2 domain interaction was sufficient for the dimerization of Stat5, but such dimers bound to DNA very inefficiently. Together, our data show that yeast cells are appropriate tools for studying Jak-Stat or Stat-Stat interactions. Our mutational analysis suggests that the Stat5 SH2 domain is essential for the interaction with Jak2 and that the kinase domain of Jak2 is sufficient for Jak2-Stat5 interaction. Therefore, the Jak kinase domain may be all that is needed to cause Stat phosphorylation in situations where receptor docking is dispensable.The biological activity of cytokines generally requires changes in the pattern of gene expression. Signal transduction to the cell nucleus is therefore an essential component in a cytokine response. Many cytokine receptors contain amino acid motifs mediating constitutive binding with Jak protein tyrosine kinases (Jaks 1 ; Refs. 1 and 2). After ligand-receptor interaction receptor chains cluster, and this most likely enables Jaks on neighboring chains to cross-phosphorylate and activate each other. Important substrates of activated Jaks are the signal transducer and activator of transcription (Stat) proteins (2-5). A distinctive feature of these transcription factors is the presence of an SH2 domain. The Stat SH2 domain can be instrumental in binding phosphotyrosines on ligand-activated receptors. Receptor-associated Stats are then phosphorylated by Jaks on a single tyrosine residue. In the context of the interferon-␥ receptor, the ␤-chain-associated Jak2 kinase could be exchanged for another Jak kinase without consequences for Stat activation or biological activity (6). Similarly, an erythropoietin receptor box 2 motif conferred Jak2 binding upon an interleukin-2 receptor ␤-chain, and ...

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Activation of STAT6 is not dependent on phosphotyrosine‐mediated docking to the interleukin‐4 receptor and can be blocked by dominant‐negative mutants of both receptor subunits

Cited by 16 publications

References 48 publications

Overexpression of E2F-1 leads to cytokine-independent proliferation and survival in the hematopoietic cell line BaF-B03

Overexpression of E2F-1 leads to cytokine-independent proliferation and survival in the hematopoietic cell line BaF-B03

The Interleukin-4 Receptor Activates STAT5 by a Mechanism That Relies upon Common γ-Chain

Jak2-Stat5 Interactions Analyzed in Yeast

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