The thrombopoietin receptor can mediate proliferation without activation of the Jak-STAT pathway.

Cytokine receptors of the hematopoietic receptor superfamily lack intrinsic tyrosine kinase domains for the intracellular transmission of their signals. Instead all members of this family associate with Jak family nonreceptor tyrosine kinases. Upon ligand stimulation of the receptors, Jaks are activated to phosphorylate target substrates. These include STAT (signal transducers and activators of transcription) proteins, which after phosphorylation translocate to the nucleus and modulate gene expression. The exact role of the Jak-STAT pathway in conveying growth and differentiation signals remains unclear. Here we describe a deletion mutant of the thrombopoietin receptor (c-mpl) that has completely lost the capacity to activate Jaks and STATs but retains its ability to induce proliferation. This mutant still mediates TPO-induced phosphorylation of Shc, Vav, mitogen-activated protein kinase (MAPK) and Raf-1 as well as induction of c-fos and c-myc, although at somewhat reduced levels. Furthermore, we show that both wild-type and mutant receptors activate phosphatidylinositol (PI) 3-kinase upon thrombopoietin stimulation and that thrombopoietin-induced proliferation is inhibited in the presence of the PI 3-kinase inhibitor wortmannin. These results demonstrate that the Jak-STAT pathway is dispensable for the generation of mitogenic signals by a cytokine receptor.

Jak-STAT signaling induced by the v-abl oncogene.

The effect of the v-abl oncogene of the Abelson murine leukemia virus (A-MuLV) on the Jak-STAT pathway of cytokine signal transduction was investigated. In murine pre-B lymphocytes transformed with A-MuLV, the Janus kinases (Jaks) Jak1 and Jak3 exhibited constitutive tyrosine kinase activity, and the STAT proteins (signal transducers and activators of transcription) normally activated by interleukin-4 and interleukin-7 were tyrosine-phosphorylated in the absence of these cytokines. Coimmunoprecipitation experiments revealed that in these cells v-Abl was physically associated with Jak1 and Jak3. Inactivation of v-Abl tyrosine kinase in a pre-B cell line transformed with a temperature-sensitive mutant of v-abl resulted in abrogation of constitutive Jak-STAT signaling. A direct link may exist between transformation by v-abl and cytokine signal transduction.

Direct interaction of Jak1 and v-Abl is required for v-Abl-induced activation of STATs and proliferation.

In Abelson murine leukemia virus (A-MuLV)-transformed cells, members of the Janus kinase (Jak) family of non-receptor tyrosine kinases and the signal transducers and activators of transcription (STAT) family of signaling proteins are constitutively activated. In these cells, the v-Abl oncoprotein and the Jak proteins physically associate. To define the molecular mechanism of constitutive Jak-STAT signaling in these cells, the functional significance of the v-Abl-Jak association was examined. Mapping the Jak1 interaction domain in v-Abl demonstrates that amino acids 858 to 1080 within the carboxyl-terminal region of v-Abl bind Jak1 through a direct interaction. A mutant of v-Abl lacking this region exhibits a significant defect in Jak1 binding in vivo, fails to activate Jak1 and STAT proteins, and does not support either the proliferation or the survival of BAF/3 cells in the absence of cytokine. Cells expressing this v-Abl mutant show extended latency and decreased frequency in generating tumors in nude mice. In addition, inducible expression of a kinase-inactive mutant of Jak1 protein inhibits the ability of v-Abl to activate STATs and to induce cytokine-independent proliferation, indicating that an active Jak1 is required for these v-Abl-induced signaling pathways in vivo. We propose that Jak1 is a mediator of v-Abl-induced STAT activation and v-Abl induced proliferation in BAF/3 cells, and may be important for efficient transformation of immature B cells by the v-abl oncogene.

Transcriptional repression of Stat6-dependent interleukin-4-induced genes by BCL-6: specific regulation of iepsilon transcription and immunoglobulin E switching.

The BCL-6 proto-oncogene encodes a POZ/zinc-finger transcription factor that is expressed in B cells and a subset of CD4(+) T cells within germinal centers. Recent evidence suggests that BCL-6 can act as a sequence-specific repressor of transcription, but the target genes for this activity have not yet been identified. The binding site for BCL-6 shares striking homology to the sites that are the target sequence for the interleukin-4 (IL-4)-induced Stat6 (signal transducers and activators of transcription) signaling molecule. Electrophoretic mobility shift assays demonstrate that BCL-6 can bind, with different affinities, to several DNA elements recognized by Stat6. Expression of BCL-6 can repress the IL-4-dependent induction of immunoglobulin (Ig) germ line epsilon transcripts, but does not repress the IL-4 induction of CD23 transcripts. Consistent with the role of BCL-6 in modulating transcription from the germ line epsilon promoter, BCL-6(-/-) mice display an increased ability to class switch to IgE in response to IL-4 in vitro. These animals also exhibit a multiorgan inflammatory disease characterized by the presence of a large number of IgE(+) B cells. The apparent dysregulation of IgE production is abolished in BCL-6(-/-) Stat6(-/-) mice, indicating that BCL-6 regulation of Ig class switching is dependent upon Stat6 signaling. Thus, BCL-6 can modulate the transcription of selective Stat6-dependent IL-4 responses, including IgE class switching in B cells.

JAK-STAT signaling activated by Abl oncogenes.

The Abl oncoproteins v-Abl and BCR-Abl can activate member of the signal transducers and activators of transcription (STAT) family of signaling proteins. The mechanisms by which these oncoproteins activate STATs appear to differ. In cells transformed by v-Abl, Janus kinase (JAK) tyrosine kinases are constitutively activated. In these cells, the v-Abl oncoprotein and the JAK kinases physically associate. Mapping of the JAK interaction domain in v-Abl demonstrates that amino acids within the carboxyl terminal region of v-Abl bind JAKs through a direct interaction. A mutant of v-Abl lacking this region does not bind or activate JAK 1 in vivo, fails to activate STAT proteins, does not induce cellular proliferation, and is less efficient in cellular transformation. Kinase inactive mutants of JAK 1 inhibit the ability of v-Abl to activate STATs, to induce cytokine-independent proliferation, and to transform bone marrow cells. Interestingly, these effects correlate with defects in the activation of several pathways by v-Abl including Akt, PI3-kinase, STATs, and Ras. These data suggest that Jak kinases may play an important role in v-Abl induced transformation. Oncogene (2000).

BAD: undertaker by night, candyman by day.

The BH3-only pro-apoptotic proteins are upstream sensors of cellular damage that selectively respond to specific, proximal death and survival signals. Genetic models and biochemical studies indicate that these molecules are latent killers until activated through transcriptional or post-translational mechanisms in a tissue-restricted and signal-specific manner. The large number of BH3-only proteins, their unique subcellular localization, protein-interaction network and diverse modes of activation suggest specialization of their damage-sensing function, ensuring that the core apoptotic machinery is poised to receive input from a wide range of cellular stress signals. The apoptotic response initiated by the activation of BH3-only proteins ultimately culminates in allosteric activation of pro-apoptotic BAX and BAK, the gateway proteins to the mitochondrial pathway of apoptosis. From activation of BH3-only proteins to oligomerization of BAX and BAK and mitochondrial outer membrane permeabilization, an intricate network of interactions between the pro- and anti-apoptotic members of the BCL-2 family orchestrates the decision to undergo apoptosis. Beyond regulation of apoptosis, multiple BCL-2 proteins have recently emerged as active components of select homeostatic pathways carrying other cellular functions. This review focuses on BAD, which was the first BH3-only protein linked to proximal survival signals through phosphorylation by survival kinases. In addition to findings that delineated the physiological role of BAD in apoptosis and its dynamic regulation by phosphorylation, studies pointing to new roles for this protein in other physiological pathways, such as glucose metabolism, are highlighted. By executing its 'day' and 'night' jobs in metabolism and apoptosis, respectively, BAD helps coordinate mitochondrial fuel metabolism and the apoptotic machinery.

A thrombopoietin receptor mutant deficient in Jak-STAT activation mediates proliferation but not differentiation in UT-7 cells.

Thrombopoietin (TPO) stimulates proliferation and differentiation of cells of the megakaryocytic lineage. It exerts its function by binding and activating c-mpl, a member of the hematopoietic receptor superfamily. Upon binding of TPO to its receptor, numerous signaling events are triggered. These include activation of the Jak-STAT (signal transducers and activators of transcription) pathway, mitogen-activated protein kinase (MAPK), Tec, and phospatidylinositol (PI) 3-kinase and phosphorylation of Shc and Vav. The contribution of different signaling pathways to the induction of specific cellular processes such as proliferation and differentiation is incompletely understood. We have previously described a mutant of c-mpl that fails to activate the Jak-STAT pathway but nevertheless retains its ability to mediate proliferation and activation of most signaling events in the murine hematopoietic precursor cell lines BAF/3 and 32D. We confirm here the ability of this mutant to mediate proliferation in the absence of Jak-STAT activation in the human cell line UT-7 and further show that this mutant fails to mediate TPO-induced megakaryocytic differentiation. Comparison of the signaling capacity of this mutant in UT-7 and BAF/3 cells shows considerable cell-type-specific differences. Whereas in BAF/3 cells the mutant still mediates activation of Shc, MAPK, Vav, and PI 3-kinase at levels comparable to the wild-type receptor, these events are strongly diminished in UT-7 cells expressing the mutant. Furthermore, we show that the C-terminal 25 amino acid residues of the receptor mutant are crucial for the mitogenic response in UT-7 cells.