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1.
Leuk Lymphoma ; 42(3): 445-55, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11699409

ABSTRACT

Recent experimental data suggest that one of the major effects of BCR-ABL gene expression in hematopoietic cells is the inhibition of apoptosis. Although the exact mechanisms of this phenomenon are not clear, it is thought to be related to the fact that BCR-ABL induces several signalling pathways also activated by growth factors. In order to determine the anti-apoptotic role of BCR-ABL in a hematopoietic cell line and to by-pass the influence of cytokine-dependence, BCR-ABL gene was expressed in the autonomously growing myelomonocytic U937 cell line using retroviral vectors. There was no resistance to apoptosis induced by either serum deprivation or different doses of etoposide in any U937 clones expressing BCR-ABL protein. In addition to serum deprivation and etoposide, BCR-ABL-expressing clones were not protected from apoptosis induced by TNF, ceramide-C2 and FAS-cross-linking. BCL2 expression was absent in U937 cells and BAX levels were identical between Neo and BCR-ABL clones. To further investigate the mechanisms of this phenomenon, band-shift assays were performed to detect activation of STAT molecules. No constitutive activation of STATs was detected in either NeoR or BCR-ABL-U937 cells, although both IFN-gamma and GM-CSF activated STAT1 and STAT5, respectively, with similar kinetics in both NeoR and BCR-ABL-U937 cells. In addition, the GM-CSF-induced-STAT5 activation was found to be weakened in all clones expressing BCR-ABL. In both control NeoR and BCR-ABL-transfected clones, band-shift assays revealed the presence of an abnormal truncated STAT5 recognized only by an anti-N-terminal but not by an anti-C-Terminal STAT5 antibody. These findings suggest a possible link between the absence of anti-apoptotic potential of BCR-ABL and abnormalities of the STAT5 pathway, including, absence of constitutive activation of STAT5, inhibition of GM-CSF-induced STAT5 activation and expression of a carboxyl-terminal-truncated STAT5.


Subject(s)
Apoptosis/physiology , DNA-Binding Proteins/genetics , Fusion Proteins, bcr-abl/genetics , Genes, abl , Milk Proteins , Trans-Activators/genetics , Cell Differentiation , Cell Division , Ceramides , Cloning, Molecular , DNA-Binding Proteins/metabolism , Etoposide/toxicity , Fusion Proteins, bcr-abl/metabolism , Humans , Phosphorylation , Recombinant Proteins/metabolism , STAT5 Transcription Factor , Sequence Deletion , Trans-Activators/metabolism , Transfection , Tumor Necrosis Factor-alpha/toxicity , U937 Cells , fas Receptor/physiology
2.
Oncogene ; 20(18): 2197-204, 2001 Apr 26.
Article in English | MEDLINE | ID: mdl-11402314

ABSTRACT

In this study, we show that upon thrombopoietin (Tpo) stimulation the two adapter proteins Gab1 and Gab2 are strongly tyrosine phosphorylated and associated with Shc, SHP2, PI 3-kinase and Grb2 in mpl-expressing UT7 cells. Although Gab1 and Gab2 seem to mediate overlapping biological signals in many cells, only Gab1 is expressed and phosphorylated in response to Tpo in primary human megakaryocytic progenitors; furthermore, it associates with the same proteins. Although a low level of tyrosine phosphorylated IRS-2 protein is also detected in PI 3-kinase immunoprecipitates, Gab proteins are the essential proteins associated with PI 3-kinase after Tpo stimulation. We demonstrate that, albeit no association is detected between the Tpo receptor mpl and Gab proteins, Y112 located in the C-terminal cytoplasmic domain of mpl is required for Gab1/2 tyrosine phosphorylation. Gab proteins are not tyrosine phosphorylated after Tpo stimulation of UT-7 and Ba/F3 cells expressing a mpl mutant lacking Y112. Moreover, no activation of the PI 3-kinase/Akt pathway is observed in cells expressing this mpl mutant. Finally, we show that this mutant does not allow cell proliferation, thereby confirming that PI 3-kinase activation is required for Tpo-induced cell proliferation.


Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/physiology , Protein Serine-Threonine Kinases , Thrombopoietin/pharmacology , Adaptor Proteins, Signal Transducing , Animals , Cell Division/drug effects , Cell Division/physiology , Enzyme Activation/drug effects , Fetal Blood/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/metabolism , Humans , Megakaryocytes/cytology , Megakaryocytes/drug effects , Megakaryocytes/metabolism , Mice , Phosphoproteins/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Rabbits , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology , Thrombopoietin/genetics , Tyrosine/metabolism
3.
Eur Cytokine Netw ; 12(2): 365-7, 2001.
Article in English | MEDLINE | ID: mdl-11399528

ABSTRACT

Inducible gene expression systems in mammalian cells have been shown to be valuable processes to study the specific function of a protein in differentiation, proliferation or survival/apoptosis. Usually, these systems use as inducible reagents, compounds that are thought to be neutral and devoid of physiological or biologically undesirable effects in mammalian cells. We recently used the ecdysone inducible gene expression system in hematopoietic cells and found that the two inducer analogs of ecdysone, muristerone A and ponasterone A, altered the signaling pathways induced by IL-3 in the pro-B cell-line, Ba/F3. Indeed, we showed that these two analogs potentiate the IL-3-dependent activation of the PI 3-kinase/Akt pathway, which could ultimately interfere with the growth, and/or survival of these cells.


Subject(s)
Ecdysterone/analogs & derivatives , Ecdysterone/pharmacology , Gene Expression Regulation/drug effects , Interleukin-3/metabolism , Protein Serine-Threonine Kinases , Signal Transduction/drug effects , Animals , Cell Line , Enzyme Activation , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt
4.
Oncogene ; 20(17): 2080-90, 2001 Apr 19.
Article in English | MEDLINE | ID: mdl-11360192

ABSTRACT

Signal Transducer and Activator of Transcription (STATs) are important mediators of cytokine and growth factor-induced signal transduction. STAT5A and STAT5B have been shown to play a role in survival and proliferation of hematopoietic cells both in vitro and in vivo and to contribute to the growth and viability of cells transformed by the TEL-JAK2 oncoprotein. In this study, we investigated the molecular mechanisms by which constitutively active STAT5 proteins induce cell proliferation and survival of Ba/F3 cell lines expressing either dominant positive STAT5A or STAT5B variants or TEL-JAK2 or TEL-ABL fusion proteins. Our results showed that active STAT5 constitutively interacted with p85, the regulatory subunit of the PI 3-kinase. A constitutive activity of the PI 3-kinase/Akt pathway was observed in these cells and required for their cell cycle progression. In contrast, while activity of the PI 3-kinase/Akt pathway was required for survival of Ba/F3 cells expressing the constitutively active forms of STAT5A or STAT5B, it was dispensable for cells transformed by TEL-JAK2 or TEL-ABL fusion proteins, suggesting that additional survival pathways take place in these transformed cells.


Subject(s)
DNA-Binding Proteins/physiology , Hematopoietic Stem Cells/cytology , Milk Proteins , Phosphatidylinositol 3-Kinases/physiology , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/physiology , Signal Transduction/physiology , Trans-Activators/physiology , Animals , Cell Cycle/physiology , Cell Division/physiology , Cell Line , Cell Survival/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , Erythropoietin/pharmacology , Fusion Proteins, bcr-abl/biosynthesis , Fusion Proteins, bcr-abl/physiology , Hematopoietic Stem Cells/enzymology , Humans , Mice , Oncogene Proteins, Fusion/biosynthesis , Oncogene Proteins, Fusion/physiology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein-Tyrosine Kinases , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , STAT5 Transcription Factor , Trans-Activators/genetics , Trans-Activators/metabolism , Transformation, Genetic , Tumor Suppressor Proteins
5.
FEBS Lett ; 497(2-3): 148-52, 2001 May 25.
Article in English | MEDLINE | ID: mdl-11377430

ABSTRACT

Constitutively active tyrosine kinases are frequently expressed in various types of human leukemias as the result of chromosomal translocations. The TEL-Jak2 fusion oncoprotein possesses transforming properties in both animal and cellular models, that are tightly dependent on Stat5 activation. In the IL-3-independent TEL-Jak2-transformed Ba/F3 cells, activation of the PI-3K/Akt pathway appears essential to cell proliferation. Here we report a sustained activation of NF-kappaB factors in Ba/F3 cells, which inhibition dramatically impairs cell viability, indicating that NF-kappaB signaling exerts a major role in the anti-apoptotic activities of TEL-Jak2 oncoprotein.


Subject(s)
Cell Transformation, Neoplastic/metabolism , I-kappa B Proteins , Leukemia/metabolism , NF-kappa B/metabolism , Oncogene Proteins, Fusion/metabolism , Protein Serine-Threonine Kinases , Animals , Cell Division/drug effects , Cell Line, Transformed , Cell Survival/drug effects , DNA/metabolism , DNA-Binding Proteins/metabolism , Enzyme Inhibitors/pharmacology , Interleukin-3/pharmacology , Leukemia/etiology , MAP Kinase Signaling System/drug effects , Mice , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NF-KappaB Inhibitor alpha , NF-kappa B/antagonists & inhibitors , Oncogene Proteins, Fusion/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Protein-Tyrosine Kinases , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , p38 Mitogen-Activated Protein Kinases
6.
Oncogene ; 20(7): 849-58, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11314018

ABSTRACT

The leukemia-associated TEL-Jak2 fusion protein possesses a constitutive tyrosine kinase activity and transforming properties in hematopoietic cell lines and animal models. In the murine pro-B Ba/F3 cell line, this fusion constitutively activates the Signal Transducer and Activator of Transcription 5 (Stat5) factors and, as a consequence, induces the sustained expression of various Stat5-target genes including the Cytokine Inducible SH2-containing protein (Cis) gene, which codes for a member of the Suppressor of Cytokine Signaling (Socs) protein family. In TEL-Jak2-transformed Ba/F3 cells, we also observed the upregulation of the Socs1 gene, whose product has been reported to negatively regulate the Jak kinase activity. In transient transfection experiments, Socs1 physically interacts with TEL-Jak2 and interferes with the TEL-Jak2-induced phosphorylation and activation of Stat5 factors, probably through the Socs1-induced proteasome-mediated degradation of the fusion protein. Interestingly, TEL-Jak2-expressing Ba/F3 cells were found to be resistant to the anti-proliferative activities of gamma interferon (IFN-gamma) seemingly as a consequence of Socs1 constitutive expression. These results indicate that the Socs1-dependent cytokine feedback loop, although active, is bypassed by the TEL-Jak2 fusion, but may play a role in the leukemogenic process by altering the cytokine responses of the leukemic cells. Our results also suggest that Socs1 plays a role in shutting down the signaling from the normally activated Jak2 kinase by inducing its proteasome-dependent degradation.


Subject(s)
Carrier Proteins/biosynthesis , Cytokines/biosynthesis , Oncogene Proteins, Fusion/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins , Repressor Proteins/biosynthesis , Animals , B-Lymphocytes/metabolism , Cell Line, Transformed , Cells, Cultured , Cysteine Endopeptidases/metabolism , Gene Expression Regulation, Leukemic , Hematopoietic Stem Cells/metabolism , Janus Kinase 2 , Mice , Multienzyme Complexes/metabolism , Proteasome Endopeptidase Complex , Protein Binding , Receptors, Interferon/metabolism , Signal Transduction , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins , Ubiquitins/metabolism , Interferon gamma Receptor
7.
Mol Cell Biol ; 21(8): 2659-70, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11283246

ABSTRACT

Thrombopoietin (TPO) regulates growth and differentiation of megakaryocytes. We previously showed that extracellular signal-regulated kinases (ERKs) are required for TPO-mediated full megakaryocytic maturation in both normal progenitors and a megakaryoblastic cell line (UT7) expressing the TPO receptor (Mpl). In these cells, intensity and duration of TPO-induced ERK signal are controlled by several regions of the cytoplasmic domain of Mpl. In this study, we explored the signaling pathways involved in this control. We show that the small GTPases Ras and Rap1 contribute together to TPO-induced ERK activation in UT7-Mpl cells and that they do so by activating different Raf kinases as downstream effectors: a Ras-Raf-1 pathway is required to initiate ERK activation while Rap1 sustains this signal through B-Raf. Indeed, (i) in cells expressing wild-type or mutant Mpl, TPO-induced Ras and Rap1 activation correlates with early and sustained phases of ERK signal, respectively; (ii) interfering mutants of Ras and Rap1 both inhibit ERK kinase activity and ERK-dependent Elk1 transcriptional activation in response to TPO; (iii) the kinetics of activation of Raf-1 and B-Raf by TPO follow those of Ras and Rap1, respectively; (iv) RasV12-mediated Elk1 activation was modulated by the wild type or interfering mutants of Raf-1 but not those of B-Raf; (v) Elk1 activation mediated by a constitutively active mutant of Rap1 (Rap1V12) is potentiated by B-Raf and inhibited by an interfering mutant of this kinase. UT7-Mpl cells represent the second cellular model in which Ras and Rap1 act in concert to modulate the duration of ERK signal in response to a growth factor and thereby the differentiation program. This is also, to our knowledge, the first evidence suggesting that Rap1 may play an active role in megakaryocytic maturation.


Subject(s)
Mitogen-Activated Protein Kinases/metabolism , Neoplasm Proteins , Proto-Oncogene Proteins c-raf/metabolism , Receptors, Cytokine , Thrombopoietin/pharmacology , rap1 GTP-Binding Proteins/metabolism , ras Proteins/metabolism , Animals , Cell Line , Enzyme Activation/drug effects , Humans , Mice , Mutation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-raf/genetics , Receptors, Thrombopoietin , Signal Transduction , rap1 GTP-Binding Proteins/genetics
8.
Cell ; 103(1): 63-74, 2000 Sep 29.
Article in English | MEDLINE | ID: mdl-11051548

ABSTRACT

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. We demonstrate that Wingless (Wg) and Decapentaplegic (Dpp) confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated (dTCF, Mad, and Pointed-functioning in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted (Twist and Tinman) transcription factors on a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development.


Subject(s)
Bacterial Proteins , Body Patterning/genetics , Cell Lineage/genetics , Drosophila Proteins , Signal Transduction/genetics , Transcription Factors/genetics , ras Proteins/genetics , ras Proteins/metabolism , Animals , Binding Sites/genetics , DNA-Binding Proteins , Drosophila/embryology , Drosophila/genetics , Drosophila/metabolism , Enhancer Elements, Genetic/genetics , Eye Proteins/genetics , Eye Proteins/metabolism , Gene Expression Regulation, Developmental/genetics , Heart/embryology , Homeodomain Proteins/genetics , Insect Proteins/genetics , Insect Proteins/metabolism , Mesoderm/metabolism , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/metabolism , Myocardium/cytology , Myocardium/metabolism , Nerve Tissue Proteins , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Wnt1 Protein
9.
J Biol Chem ; 275(24): 18375-81, 2000 Jun 16.
Article in English | MEDLINE | ID: mdl-10849444

ABSTRACT

The binding of erythropoietin (Epo) to its receptor leads to the transient phosphorylation of the Epo receptor (EpoR) and the activation of intracellular signaling pathways. Inactivation mechanisms are simultaneously turned on, and Epo-induced signaling pathways return to nearly basal levels after 30-60 min of stimulation. We show that proteasomes control these inactivation mechanisms. In cells treated with the proteasome inhibitors N-Ac-Leu-Leu-norleucinal (LLnL) or lactacystin, EpoR tyrosine phosphorylation and activation of intracellular signaling pathways (Jak2, STAT5, phosphatidylinositol 3-kinase) were sustained for at least 2 h. We show that this effect was due to the continuous replenishment of the cell surface pool of EpoRs in cells treated with proteasome inhibitors. Proteasome inhibitors did not modify the internalization and degradation of Epo.EpoR complexes, but they allowed the continuous replacement of the internalized receptors by newly synthesized receptors. Proteasome inhibitors did not modify the synthesis of EpoRs, but they allowed their transport to the cell surface. N-Ac-Leu-Leu-norleucinal, but not lactacystin, also inhibited the degradation of internalized Epo.EpoR complexes, most probably through cathepsin inhibition. The internalized EpoRs were not tyrosine-phosphorylated, and they did not activate intracellular signaling pathways. Our results show that the proteasome controls the down-regulation of EpoRs in Epo-stimulated cells by inhibiting the cell surface replacement of internalized EpoRs.


Subject(s)
Cysteine Endopeptidases/metabolism , Down-Regulation , Multienzyme Complexes/metabolism , Receptors, Cell Surface/metabolism , Receptors, Erythropoietin/metabolism , Cell Membrane/metabolism , Cells, Cultured , Cysteine Proteinase Inhibitors/pharmacology , Down-Regulation/drug effects , Erythropoietin/physiology , Humans , Phosphorylation , Proteasome Endopeptidase Complex , Recombinant Proteins/metabolism , Signal Transduction , Tyrosine/metabolism
10.
Oncogene ; 19(16): 2033-42, 2000 Apr 13.
Article in English | MEDLINE | ID: mdl-10803464

ABSTRACT

Mpl is the receptor for thrombopoietin, the primary regulator of platelet production by megakaryocytes. Upon stimulation by its ligand, Mpl receptor induces proliferation and differentiation of hematopoietic cell lines of various origins. In this paper, we show that Mpl is also able to transform FRE rat fibroblasts in the presence of MGDF (pegylated Megakaryocyte Growth and Development Factor), a modified form of its ligand. We also demonstrate that upon MGDF stimulation Mpl receptor activates the classical transduction pathways described for hematopoietic cell lines in FRE cells. Introduction of Mpl deletion mutants in FRE cells allowed us to demonstrate that the C-terminal region of the Mpl intracytoplasmic domain, which is involved in hematopoietic differentiation, is necessary for the transformation process. Within that region, site-directed mutagenesis showed that the Y112 residue, which is required for Shc phosphorylation, is essential for rat fibroblast transformation by Mpl/MGDF, suggesting the involvement of Shc in Mpl-mediated transformation. Interestingly, we showed that transformation correlated with strong and sustained MAPK activation. Neither Jak2, Stat3 nor Stat5 phosphorylation was sufficient to induce the transformation process. Taken altogether, our results suggest the oncogenicity of Mpl in fibroblastic cells in the presence of its ligand.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Cell Transformation, Neoplastic , Milk Proteins , Neoplasm Proteins , Proto-Oncogene Proteins/metabolism , Receptors, Cytokine , 3T3 Cells/metabolism , 3T3 Cells/virology , Animals , Cytoplasm/metabolism , DNA-Binding Proteins/metabolism , Fibroblasts/drug effects , Fibroblasts/pathology , Genetic Vectors/genetics , Janus Kinase 2 , Leukemia Virus, Murine/genetics , Ligands , MAP Kinase Signaling System , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Mutation , Protein-Tyrosine Kinases/metabolism , Proteins/metabolism , Proto-Oncogene Proteins/genetics , Rats , Rats, Inbred Strains , Receptors, Thrombopoietin , STAT3 Transcription Factor , STAT5 Transcription Factor , Shc Signaling Adaptor Proteins , Signal Transduction , Src Homology 2 Domain-Containing, Transforming Protein 1 , Thrombopoietin/metabolism , Thrombopoietin/pharmacology , Trans-Activators/metabolism
11.
Blood ; 95(6): 2076-83, 2000 Mar 15.
Article in English | MEDLINE | ID: mdl-10706877

ABSTRACT

The involvement of the cytokine signaling pathway in oncogenesis has long been postulated. Recently, rearrangements of the gene encoding the tyrosine Janus kinase 2 (JAK2) have been reported in human leukemias indicating a direct JAK-signal transduction and activator of transcription (STAT)-mediated leukemic process. The leukemia-associated TEL-JAK2 fusion protein is formed by the oligomerization domain of the translocated ets leukemia (TEL) protein fused to the catalytic domain of JAK2. TEL-mediated oligomerization results in a constitutive tyrosine kinase activity that, in turn, is able to confer growth factor independence to the murine hematopoietic interleukin-3 (IL-3)-dependent Ba/F3 cell line. Results of the present study indicate that fusion proteins containing the oligomerization domain of TEL and the tyrosine kinase domains of Jak1, Jak2, JAK3, or TYK2 share similar properties and are able to efficiently substitute for the survival and mitogenic signals controlled by IL-3, without concomitant activation of the IL-3 receptor. Electrophoretic mobility shift assays demonstrated Stat5 as the only activated Stat factor in TEL-Jak2- and TEL-Jak1-expressing cells, whereas other Stats, namely Stat1 and Stat3, could be detected in TEL-JAK3-, TEL-TYK2-, and also in TEL-ABL-expressing Ba/F3 cells. High levels of expression of the Stat5-target genes pim-1, osm, and Cis were observed in all the cytokine-independent cell lines. Furthermore, the expression of a dominant negative form of Stat5A markedly interfered with the growth factor independence process mediated by TEL-Jak2 in Ba/F3 cells. Because the BCR-ABL and TEL-PDGFbetaR oncoproteins also activate Stat5, activation of this factor should be a crucial step in activated tyrosine kinase-mediated leukemogenesis. (Blood. 2000;95:2076-2083)


Subject(s)
Cell Transformation, Neoplastic/metabolism , DNA-Binding Proteins/metabolism , Oncogene Proteins, Fusion/metabolism , Protein-Tyrosine Kinases/metabolism , Recombinant Fusion Proteins/metabolism , Repressor Proteins , Transcription Factors/metabolism , Animals , Blotting, Northern , Catalytic Domain , Cell Division , Cell Line , Cell Nucleus/enzymology , Cell Nucleus/metabolism , Cytokines/metabolism , DNA-Binding Proteins/chemistry , Enzyme Activation , Humans , Interleukin-3/metabolism , Leukemia/enzymology , Leukemia/metabolism , Mice , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins c-ets , Time Factors , Transcription Factors/chemistry , Transfection , ETS Translocation Variant 6 Protein
12.
Oncogene ; 19(9): 1164-72, 2000 Feb 24.
Article in English | MEDLINE | ID: mdl-10713704

ABSTRACT

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.


Subject(s)
Bone Marrow Cells/cytology , DNA-Binding Proteins/physiology , Interleukin-3/physiology , MAP Kinase Kinase Kinase 1 , Milk Proteins , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins , Trans-Activators/physiology , Animals , Bone Marrow Cells/enzymology , Carrier Proteins/metabolism , Cell Cycle , Cell Division/genetics , Cell Line , Cell Survival/genetics , Cell Survival/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , Mice , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Proto-Oncogene Proteins c-akt , Proto-Oncogene Proteins c-bcl-2/biosynthesis , STAT5 Transcription Factor , Sequence Deletion , Signal Transduction/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , bcl-Associated Death Protein , bcl-X Protein
13.
Oncogene ; 19(52): 5997-6006, 2000 Dec 07.
Article in English | MEDLINE | ID: mdl-11146551

ABSTRACT

SIAH-1, a human homologue of the Drosophila seven in absentia (Sina), has been implicated in ubiquitin-mediated proteolysis of different target proteins through its N-terminal RING finger domain. SIAH-1 is also induced during p53-mediated apoptosis. Furthermore, SIAH-1-transfected breast cancer cell line MCF-7 exhibits an altered mitotic process resulting in multinucleated giant cells. Now, using the two-hybrid system, we identified two new SIAH interacting proteins: Kid (kinesin like DNA binding protein) and alpha-tubulin. We demonstrate that SIAH is involved in the degradation of Kid via the ubiquitin-proteasome pathway. Our results suggest that SIAH-1 but not its N-terminal deletion mutant, affects the mitosis by an enhanced reduction of kinesin levels. Our results imply, for the first time, SIAH-1 in regulating the degradation of proteins directly implicated in the mitotic process.


Subject(s)
Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/metabolism , Kinesins/metabolism , Mitosis , Multienzyme Complexes/metabolism , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Tubulin/metabolism , Cell Cycle , DNA-Binding Proteins/genetics , Fluorescent Antibody Technique , Gene Expression Regulation , Humans , Kinesins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Precipitin Tests , Proteasome Endopeptidase Complex , Protein Binding , Protein Structure, Tertiary , Sequence Deletion/genetics , Substrate Specificity , Transfection , Tubulin/genetics , Tumor Cells, Cultured , Two-Hybrid System Techniques , Ubiquitin-Protein Ligases , Ubiquitins/metabolism
14.
Eur Cytokine Netw ; 10(4): 463-70, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10586112

ABSTRACT

This review describes the main properties of a new family of cytokine-inducible proteins which interfere with the Jak/Stat transduction pathway and negatively regulate the duration of cytokine-induced signal activation. These proteins act not only as negative feedback regulators but also inhibit response to cytokines different from those used to induce their expression. These proteins are potentially important regulators of inflammatory and immune responses of hematopoiesis and hormone response.


Subject(s)
Carrier Proteins/metabolism , Cytokines/pharmacology , Immediate-Early Proteins/metabolism , Intracellular Signaling Peptides and Proteins , Milk Proteins , Proteins/metabolism , Repressor Proteins , Signal Transduction/drug effects , Animals , Carrier Proteins/chemistry , Carrier Proteins/genetics , Cytokines/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Elongin , Humans , Immediate-Early Proteins/chemistry , Immediate-Early Proteins/genetics , Protein Structure, Tertiary/physiology , Proteins/chemistry , Proteins/genetics , STAT5 Transcription Factor , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling 3 Protein , Suppressor of Cytokine Signaling Proteins , Trans-Activators/metabolism , Transcription Factors/metabolism , Up-Regulation
15.
Blood ; 94(5): 1601-13, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10477685

ABSTRACT

In several erythroleukemia cell lines, activation of mitogen-activated protein kinases (MAPK) by phorbol esters or megakaryocyte growth and development factor (MGDF) is required for induction of megakaryocytic phenotype and growth arrest. To support this model, we have examined the effect of a specific inhibitor of this pathway (PD98059) on human CD34(+) hematopoietic progenitors isolated from cord blood (CB), induced to differentiate along the megakaryocytic lineage in liquid cultures supplemented with rhuMGDF. RhuMGDF induced a sustained activation of MAPK in megakaryocytes and this activation was completely inhibited in the presence of low concentrations of PD98059 (6 to 10 micromol/L). At this concentration, PD98059 induced an increase in cell proliferation, resulting in accumulation of viable cells and a prolongation of the life time of the cultures. This increase correlated with an increase in DNA synthesis rather than with a reduction in apoptosis. This effect was combined with developmental changes indicative of delayed megakaryocytic differentiation: (1) PD98059-treated cells tended to retain markers of immature progenitors as shown by the increased proportion of both CD34(+) and CD41(+)CD34(+) cells. (2) PD98059-treated cultures were greatly enriched in immature blasts cells. (3) PD98059 increased megakaryocytic progenitors able to form colonies in semisolid assays. Thus, the MAPK pathway, although not required for megakaryocyte formation, seems to be involved in the transition from proliferation to maturation in megakaryocytes. Inhibition of MAPK activation also led to an increase in the number and size of erythroid colonies without affecting granulocyte/macrophage progenitor numbers suggesting that, in addition to the megakaryocytic lineage, the MAPK pathway could play a role in erythroid lineage differentiation.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/physiology , Hematopoietic Stem Cells/physiology , Signal Transduction/drug effects , Thrombopoietin/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cell Division/drug effects , Cell Division/physiology , Cells, Cultured , Enzyme Inhibitors/pharmacology , Fetal Blood/cytology , Flavonoids/pharmacology , Hematopoietic Stem Cells/cytology , Humans
16.
Oncogene ; 18(29): 4191-9, 1999 Jul 22.
Article in English | MEDLINE | ID: mdl-10435632

ABSTRACT

Activation of the Jak/STAT pathway by cytokines has been shown to regulate differentiation, proliferation or apoptosis in hematopoeitic cells. Among the Stat proteins, STAT5 is activated by a broad range of cytokines. In order to study the role of STAT5 in hematopoietic cells, we stably expressed a dominant negative form of STAT5 (STAT5A delta749) in the IL-3 dependent bone marrow derived Ba/F3 cell line. Ba/F3 cells expressing STAT5A delta749 were found to be more sensitive to apoptosis than parental or control Ba/F3 cells after IL-3 withdrawal. Analysis of the expression of the cell death regulators, Bcl-2 and Bcl-x, revealed that the level of Bcl-x was lower in Ba/F3 cells expressing STAT5A delta749 than in control cells. IL-3 regulation of Bcl-x expression at protein and mRNA levels was impaired in these cells while that of Bcl-2 expression was unaffected. We further demonstrated that the Bcl-x gene promoter contained a proximal STAT consensus sequence that bound STAT5. Transactivation of a Bcl-x gene promoter reporter construct by STAT5 was observed in Ba/F3 cells. Introduction of a mutation in the STAT binding site abolished this transactivation. These data indicate that Bcl-x is probably a STAT5 target gene. They also support the involvement of STAT5 in hematopoietic cell survival.


Subject(s)
DNA-Binding Proteins/physiology , Gene Expression Regulation/drug effects , Hematopoietic Stem Cells/drug effects , Interleukin-3/pharmacology , Milk Proteins , Proto-Oncogene Proteins c-bcl-2/genetics , Trans-Activators/physiology , Animals , Apoptosis , Cell Line , DNA-Binding Proteins/genetics , Genes, Dominant , Genes, Reporter , Genes, bcl-2 , Hematopoietic Stem Cells/metabolism , Mice , Promoter Regions, Genetic , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Recombinant Fusion Proteins/physiology , STAT5 Transcription Factor , Sequence Deletion , Trans-Activators/genetics , Transcription, Genetic , bcl-X Protein
17.
Br J Haematol ; 106(2): 464-73, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10460607

ABSTRACT

Ineffective erythropoiesis in myelodysplasia is characterized by a defect in erythroid progenitor growth and by abnormal erythroid differentiation. Increased apoptosis of erythroid, granulocytic and megakaryocytic lineages is thought to account for cytopenias. Erythropoietin (Epo)-induced BFU-E and CFU-E growth was studied in 25 myelodysplastic syndrome (MDS) marrow specimens and found to be drastically diminished. To investigate the functionality of Epo-R in MDS marrow, we focused on Epo-induced STAT5 activation. Epo was able to stimulate STAT5 DNA binding activity in all normal and 12/24 MDS marrows tested, with no correlation between the level of STAT5 activation and the development of erythroid colonies in response to Epo. In contrast, impaired proliferation of erythroid progenitors was related to an increased expression of the transmembrane mediator of apoptotic cell death Fas/CD95 on the glycophorin A+ subpopulation. Therefore we conclude that the stimulation of pro-apoptotic signals rather than the defect of anti-apoptotic pathways resulting from Epo-stimulated Jak2-STAT5 pathway, predominantly accounts for ineffective erythropoiesis in myelodysplasia.


Subject(s)
Erythropoiesis/physiology , Myelodysplastic Syndromes/blood , Receptors, Erythropoietin/metabolism , Signal Transduction/physiology , fas Receptor/metabolism , Adult , Aged , Aged, 80 and over , Antigens, CD34/metabolism , Apoptosis , Cysteine Endopeptidases/metabolism , Erythroid Precursor Cells , Female , Flow Cytometry , Glycophorins/metabolism , Humans , Male , Middle Aged , Multienzyme Complexes/metabolism , Myelodysplastic Syndromes/metabolism , Proteasome Endopeptidase Complex , Tumor Cells, Cultured
18.
Mol Cell Biol ; 19(5): 3798-807, 1999 May.
Article in English | MEDLINE | ID: mdl-10207103

ABSTRACT

The hematopoietic proto-oncogene vav has been characterized as a Rac1-GDP/GTP exchanger protein which regulates cytoskeletal reorganization as well as signaling pathways leading to the activation of stress-activated protein kinases (SAPK/JNKs). Furthermore, vav overexpression enhances basal and T-cell receptor (TCR)-mediated stimulation of the nuclear factor of activated T cells (NFAT). We report here the interaction between Vav and hSiah2, a mammalian homolog of Drosophila Seven in absentia (Sina) that has been implicated in R7 photoreceptor cell formation during Drosophila eye development via the proteasome degradation pathway. Vav and hSiah2 interact in vitro and in vivo and colocalize in the cytoplasm of hematopoietic cells. The Src homology domain of Vav and the C-terminal region of hSiah2 are required for this interaction. We provide evidence for a negative regulation by hSiah2 of Vav-induced basal and TCR-mediated NFAT-dependent transcription. Overexpression of hSiah2 also inhibits the onco-Vav-induced JNK activation. Although the Vav-interacting domain is located in the C-terminal portion of hSiah2, the N-terminal region of hSiah2 is necessary for the inhibitory role that seems to be independent of the proteasome degradation.


Subject(s)
Mitogen-Activated Protein Kinases , Nuclear Proteins/metabolism , Oncogene Proteins/metabolism , Animals , COS Cells , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enzyme Activation , Fluorescent Antibody Technique , Genes, Reporter/genetics , Humans , JNK Mitogen-Activated Protein Kinases , Jurkat Cells , NFATC Transcription Factors , Proto-Oncogene Mas , Proto-Oncogene Proteins c-vav , Receptors, Antigen, T-Cell/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Ubiquitin-Protein Ligases
19.
Blood ; 93(8): 2578-85, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10194437

ABSTRACT

Five tyrosine-phosphorylated proteins with molecular masses of 180, 145, 116, 100, and 70 kD are associated with phosphatidylinositol 3-kinase (PI 3-kinase) in erythropoietin (Epo)-stimulated UT-7 cells. The 180- and 70-kD proteins have been previously shown to be IRS2 and the Epo receptor. In this report, we show that the 116-kD protein is the IRS2-related molecular adapter, GAB1. Indeed, Epo induced the transient tyrosine phosphorylation of GAB1 in UT-7 cells. Both kinetics and Epo dose-response experiments showed that GAB1 tyrosine phosphorylation was a direct consequence of Epo receptor activation. After tyrosine phosphorylation, GAB1 associated with the PI 3-kinase, the phosphotyrosine phosphatase SHP2, the phosphatidylinositol 3,4,5 trisphosphate 5-phosphatase SHIP, and the molecular adapter SHC. GAB1 was also associated with the molecular adapter GRB2 in unstimulated cells, and this association dramatically increased after Epo stimulation. Thus, GAB1 could be a scaffold protein able to couple the Epo receptor activation with the stimulation of several intracellular signaling pathways. Epo-induced tyrosine phosphorylation of GAB1 was also observed in normal human erythroid progenitors isolated from cord blood. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and thrombopoietin (TPO) also induced the tyrosine phosphorylation of GAB1 in UT-7 cells, indicating that this molecule participates in the signal transduction of several cytokine receptors.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , Erythropoietin/pharmacology , Hematopoietic Stem Cells/physiology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Protein Tyrosine Phosphatases/metabolism , Proteins/metabolism , Amino Acid Sequence , Fetal Blood , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Intracellular Signaling Peptides and Proteins , Leukemia , Molecular Weight , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphorylation , Phosphotyrosine/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Recombinant Proteins/metabolism , SH2 Domain-Containing Protein Tyrosine Phosphatases , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Transfection , Tumor Cells, Cultured , src Homology Domains
20.
Biochem Biophys Res Commun ; 256(3): 685-91, 1999 Mar 24.
Article in English | MEDLINE | ID: mdl-10080960

ABSTRACT

To study the role of the cytoplasmic domain and particularly the tyrosine residues of the erythropoietin receptor (EpoR) in erythroid differentiation of human primary stem cells, we infected cord blood-derived CD34+ cells with retroviruses encoding chimeric receptors containing the extracellular domain of the prolactin receptor (PRLR) and the cytoplasmic domain of either the normal EpoR or a truncated EpoR devoid of tyrosine residues. Erythroid differentiation of the infected progenitors could thus be studied after stimulation by PRL. The complete PRLR was used to assess its ability to substitute for EpoR in erythroid differentiation. Typical erythroid day-14 colonies were observed from CD34+ cells grown in PRL when infected with any of the three viral constructs. These results demonstrate that: (i) the activation of the virally transduced PRLR leads to erythroid colony formation showing that erythroid terminal differentiation can be induced by a non-erythroid receptor in human progenitors; (ii) a chimeric receptor PRLR/EpoR is able to transduce a signal leading to terminal erythroid differentiation of human CD34+ cells; (iii) in contrast to results previously reported in murine models, tyrosine residues of the EpoR are not required for growth and terminal differentiation of human erythroid progenitors.


Subject(s)
Antigens, CD34/analysis , Erythrocytes/cytology , Erythroid Precursor Cells/cytology , Receptors, Erythropoietin/metabolism , Tyrosine/metabolism , Cell Differentiation/drug effects , Cell Division/drug effects , Erythrocytes/drug effects , Erythrocytes/metabolism , Erythroid Precursor Cells/drug effects , Erythroid Precursor Cells/metabolism , Fetal Blood/cytology , Genetic Vectors/genetics , Growth Substances/pharmacology , Humans , Phosphorylation/drug effects , Precipitin Tests , Prolactin/pharmacology , RNA, Viral/analysis , Receptors, Erythropoietin/chemistry , Receptors, Erythropoietin/genetics , Receptors, Prolactin/chemistry , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retroviridae/genetics , Sequence Deletion , Signal Transduction/drug effects , Transduction, Genetic , Tumor Cells, Cultured , Tyrosine/genetics
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