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ABSTRACT: Persistent tyrosine phosphorylation of Stat3 and Stat5 is associated with oncogenic activity. Phosphorylation of the conserved tyrosine residue (pTyr) was long believed to be the only essential prerequisite to promote activation and nuclear translocation of Stat proteins. It has become evident, however, that post-translational protein modifications like serine phosphorylation, acetylation, glycosylation as well as protein splicing and processing constitute further regulatory mechanisms to modulate Stat transcriptional activity and to provide an additional layer of specificity to Jak-Stat signal transduction. Significantly, most vertebrate Stat proteins contain one conserved serine phosphorylation site within their transactivation domains. This phosphorylation motif is located within a P(M)SP sequence. Stat transcription factor activity is negatively influenced by mutation of the serine to alanine. Moreover, it was shown for both Stat3 and Stat5 that their capacity to transform cells was diminished. This review addresses recent advances in understanding the regulation and the biochemical and biological consequences of Stat serine phosphorylation. In particular, we discuss their role in persistently activated Stat proteins for cancer research.
Frontiers in Bioscience 01/2011; 16:3043-56. · 3.52 Impact Factor
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Katrin Friedbichler,
Marc A Kerenyi,
Boris Kovacic, Geqiang Li,
Andrea Hoelbl,
Saliha Yahiaoui,
Veronika Sexl,
Ernst W Müllner,
Sabine Fajmann,
Sabine Cerny-Reiterer,
Peter Valent,
Hartmut Beug,
Fabrice Gouilleux,
Kevin D Bunting,
Richard Moriggl
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ABSTRACT: Stat5 transcription factors are essential gene regulators promoting proliferation, survival, and differentiation of all hematopoietic cell types. Mutations or fusions of oncogenic tyrosine kinases often result in constitutive Stat5 activation. We have modeled persistent Stat5 activity by using an oncogenic Stat5a variant (cS5). To analyze the hitherto unrecognized role of Stat5 serine phosphorylation in this context, we have generated cS5 constructs with mutated C-terminal serines 725 and 779, either alone or in combination. Genetic complementation assays in primary Stat5(null/null) mast cells and Stat5(DeltaN) T cells demonstrated reconstitution of proliferation with these mutants. Similarly, an in vivo reconstitution experiment of transduced Stat5(null/null) fetal liver cells transplanted into irradiated wild-type recipients revealed that these mutants exhibit biologic activity in lineage differentiation. By contrast, the leukemogenic potential of cS5 in bone marrow transplants decreased dramatically in cS5 single-serine mutants or was completely absent upon loss of both serine phosphorylation sites. Our data suggest that Stat5a serine phosphorylation is a prerequisite for cS5-mediated leukemogenesis. Hence, interference with Stat5a serine phosphorylation might provide a new therapeutic option for leukemia and myeloid dysplasias without affecting major functions of Stat5 in normal hematopoiesis.
Blood 09/2010; 116(9):1548-58. · 9.90 Impact Factor
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ABSTRACT: The antiangiogenic drug sunitinib is a receptor tyrosine kinase inhibitor with significant, yet not curative, therapeutic effects in metastatic renal cell carcinoma (RCC). Sunitinib is also an immunomodulator, potently reversing myeloid-derived suppressor cell (MDSC) accumulation and T-cell inhibition in the blood even of nonresponder RCC patients. We observed that sunitinib similarly prevented MDSC accumulation and restored normal T-cell function to the spleens of tumor-bearing mice, independent of the capacity of sunitinib to inhibit tumor progression (RENCA>CT26>4T1). Both monocytic and neutrophilic splenic MDSC were highly repressible by sunitinib. In contrast, MDSC within the microenvironment of 4T1 tumors or human RCC tumors proved highly resistant to sunitinib and ambient T-cell function remained suppressed. Proteomic analyses comparing tumor to peripheral compartments showed that granulocyte macrophage colony-stimulating factor (GM-CSF) predicted sunitinib resistance and recombinant GM-CSF conferred sunitinib resistance to MDSC in vivo and in vitro. MDSC conditioning with GM-CSF uniquely inhibited signal transducers and activators of transcription (STAT3) and promoted STAT5 activation. STAT5ab(null/null) MDSC were rendered sensitive to sunitinib in the presence of GM-CSF in vitro. We conclude that compartment-dependent GM-CSF exposure in resistant tumors may account for the regionalized effect of sunitinib upon host MDSC modulation and hypothesize that ancillary strategies to decrease such regionalized escape will enhance the potency of sunitinib as an immunomodulator and a cancer therapy.
Cancer Research 05/2010; 70(9):3526-36. · 7.86 Impact Factor
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ABSTRACT: Grb2-associated binding (Gab) adapter proteins play major roles in coordinating signaling downstream of hematopoietic cytokine receptors. In hematopoietic cells, Gab2 can modulate phosphatidylinositol-3 kinase and mitogen associated protein kinase activities and regulate the long-term multilineage competitive repopulating activity of hematopoietic stem cells (HSCs). Gab2 may also act in a linear pathway upstream or downstream of signal transducer and activator of transcription-5 (STAT5), a major positive regulator of HSC function. Therefore, we aimed to determine whether Gab2 and STAT5 function in hematopoiesis in a redundant or non-redundant manner.
To do this we generated Gab2 mutant mice with heterozygous and homozygous deletions of STAT5. In heterozygous STAT5 mutant mice, deficiencies in HSC/multipotent progenitors were reflected by decreased long-term repopulating activity. This reduction in repopulation function was mirrored in the reduced growth response to early-acting cytokines from sorted double mutant c-Kit(+)Lin(-)Sca-1(+) (KLS) cells. Importantly, in non-ablated newborn mice, the host steady-state engraftment ability was impaired by loss of Gab2 in heterozygous STAT5 mutant background. Fetal liver cells isolated from homozygous STAT5 mutant mice lacking Gab2 showed significant reduction in HSC number (KLS CD150(+)CD48(-)), reduced HSC survival, and dramatic loss of self-renewal potential as measured by serial transplantation.
These data demonstrate new functions for Gab2 in hematopoiesis in a manner that is non-redundant with STAT5. Furthermore, important synergy between STAT5 and Gab2 was observed in HSC self-renewal, which might be exploited to optimize stem cell-based therapeutics.
PLoS ONE 01/2010; 5(2):e9152. · 4.09 Impact Factor
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ABSTRACT: Phosphorylated signal transducer and activator of transcription 5 (STAT5) is a biomarker and potential molecular target for hematologic malignancies. We have shown previously that lethal myeloproliferative disease (MPD) in mice mediated by persistently activated STAT5 (STAT5a(S711F)) requires the N-domain, but the mechanism was not defined. We now demonstrate by retrovirally complementing STAT5ab(null/null) primary mast cells that relative to wild-type STAT5a, STAT5a lacking the N-domain (STAT5aDeltaN) ineffectively protected against cytokine withdrawal-induced cell death. Both STAT5a and STAT5aDeltaN bound to a site in the bcl-2 gene and both bound near the microRNA 15b/16 cluster. However, only STAT5a could effectively induce bcl-2 mRNA and reciprocally suppress miR15b/16 leading to maintained bcl-2 protein levels. After retroviral complementation of STAT5ab(null/null) fetal liver cells and transplantation, persistently active STAT5a(S711F) lacking the N-domain (STAT5aDeltaN(S711F)) was insufficient to protect c-Kit(+)Lin(-)Sca-1(+) (KLS) cells from apoptosis and unable to induce bcl-2 expression, whereas STAT5a(S711F) caused robust KLS cell expansion, induction of bcl-2, and lethal MPD. Severe attenuation of MPD by STAT5aDeltaN(S711F) was reversed by H2k/bcl-2 transgenic expression. Overall, these studies define N-domain-dependent survival signaling as an Achilles heel of persistent STAT5 activation and highlight the potential therapeutic importance of targeting STAT5 N-domain-mediated regulation of bcl-2 family members.
Blood 12/2009; 115(7):1416-24. · 9.90 Impact Factor
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ABSTRACT: Currently, there is a major need in hematopoietic stem cell (HSC) transplantation to develop reduced-intensity regimens that do not cause DNA damage and associated toxicities and that allow a wider range of patients to receive therapy. Cytokine receptor signals through c-Kit and c-Mpl can modulate HSC quiescence and engraftment, but the intracellular signals and transcription factors that mediate these effects during transplantation have not been defined. Here we show that loss of one allele of signal transducer and activator of transcription 5 (STAT5) in nonablated adult mutant mice permitted engraftment with wild-type HSC. Conditional deletion of STAT5 using Mx1-Cre caused maximal reduction in STAT5 mRNA (> 97%) and rapidly decreased quiescence-associated c-Mpl downstream targets (Tie-2, p57), increased HSC cycling, and gradually reduced survival and depleted the long-term HSC pool. Host deletion of STAT5 was persistent and permitted efficient donor long-term HSC engraftment in primary and secondary hosts in the absence of ablative conditioning. Overall, these studies establish proof of principle for targeting of STAT5 as novel transplantation conditioning and demonstrate, for the first time, that STAT5, a mitogenic factor in most cell types, including hematopoietic progenitors, is a key transcriptional regulator that maintains quiescence of HSC during steady-state hematopoiesis.
Blood 03/2009; 113(20):4856-65. · 9.90 Impact Factor
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ABSTRACT: The aim of this study was to identify and characterize the causative mutation in the thrombocytopenic mouse strain HLB219 that was generated at the Jackson Laboratory as part of a large-scale N-ethyl-N-nitrosourea mutagenesis screen.
The HLB219 mutation was identified by interval mapping of F2 mice generated from intercross breeding of HLB219 to both BALB/cByJ (BALB) and 129/SvImJ (129/Sv). Mpl was identified as a candidate gene and sequenced. The mutation was characterized in vivo in mouse hematopoietic stem/progenitor cell assays and in cell culture by expression in Ba/F3 cells.
A novel mutation in the thrombopoietin (TPO) receptor Mpl in HLB219 mice caused a Cys-->Arg substitution at codon 40 in the extracellular region of the receptor. Mice homozygous for the Mpl(hlb219) mutation had an 80% decrease in the number of platelets in comparison to the wild-type C57BL/6J strain, low numbers of bone marrow megakaryocytes, high TPO levels, and decreased competitive repopulating ability, consistent with a loss-of-function mutation in the receptor. Mice heterozygous for Mpl(hlb219) however, showed an overdominance effect with a significant increase in platelet number. Functional analysis in vitro demonstrated that Ba/F3 cells expressing the mutant MPL(hlb219) protein failed to activate extracellular signal-regulated kinase and signal transducers and activators of transcription 5, but proliferated in the absence of TPO and required constitutive phosphorylation of RAC-alpha serine/threonine protein kinase (AKT) for cytokine-independent growth.
Thrombocytopenia in HLB219 mice is caused by a recessive mutation in Mpl that abrogates mitogen-activated protein kinase-extracellular signal regulated kinase and janus kinase-signal transducers and activators of transcription signaling.
Experimental hematology 01/2009; 37(2):276-84. · 3.11 Impact Factor
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Geqiang Li,
Zhengqi Wang,
Yi Zhang,
Zizhen Kang,
Eleonora Haviernikova,
Yongzhi Cui,
Lothar Hennighausen,
Richard Moriggl,
Demin Wang,
William Tse,
Kevin D Bunting
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ABSTRACT: Signal transducer and activator of transcription 5 (STAT5) is a critical regulator of hematopoietic development and its impaired activation is associated with hematopoietic and immune cell defects. However, much of this information has been learned from knockout mice that still retain the potential for expression of STAT5 proteins that are N-terminally truncated due to alternative internal translation initiation codons. The goal of these studies was to use transplantation-based assays to analyze the degree of STAT5 deltaN activity in hematopoietic stem cells (HSC) and throughout lymphomyeloid development.
We have directly compared E14.5 fetal liver cells from mice with potential to express STAT5ab deltaN (STAT5ab(deltaN/deltaN)) with mice completely lacking STAT5a and STAT5b (STAT5abnull/null). We have also utilized retroviral complementation of STAT5abnull/null fetal liver HSC to enforce expression of full-length STAT5a or STAT5a lacking the first 136 amino acids (STAT5a deltaN).
We report that STAT5 is required for HSC, lymphocyte, and erythrocyte development. We demonstrate that restored expression of STAT5a in STAT5abnull/null HSC provides a strong selective advantage, correcting T- and B-lymphocyte and erythrocyte development. Interestingly, Gr-1(+) blood cells were inversely correlated with B lymphocytes and both were normalized by STAT5a expression. In contrast, transduction of STAT5a deltaN only provided partial B-lymphocyte development.
These studies define the role of STAT5 in maintaining normal lymphoid vs myeloid balance during hematopoiesis and highlight a major role for the N-domain in HSC function. The platform of retroviral complementation described here will be particularly useful for future studies to subdefine the N-domain regions that are critical for hematopoiesis.
Experimental Hematology 12/2007; 35(11):1684-94. · 2.90 Impact Factor
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ABSTRACT: The two closely related Stat5 (Stat5A and Stat5B) proteins are activated by a broad spectrum of cytokines. However, with the complication of the involvement of Stat5A/5B in stem cell function, the role of Stat5A/5B in the development and function of lymphocytes, especially B cells, is not fully understood. In this study, we demonstrated that Stat5A/5B(-/-) fetal liver cells had severe diminution of B cell progenitors but clearly had myeloid progenitors. Consistently, the mutant fetal liver cells could give rise to hemopoietic progenitors and myeloid cells but not B cells beyond pro-B cell progenitors in lethally irradiated wild-type or Jak3(-/-) mice. Deletion of Stat5A/5B in vitro directly impaired IL-7-mediated B cell expansion. Of note, reintroduction of Stat5A back into Stat5A/5B(-/-) fetal liver cells restored their abilities to develop B cells. Importantly, CD19-Cre-mediated deletion of Stat5A/5B in the B cell compartment specifically impaired early B cell development but not late B cell maturation. Moreover, the B cell-specific deletion of Stat5A/5B did not impair splenic B cell survival, proliferation, and Ig production. Taken together, these data demonstrate that Stat5A/5B directly control IL-7-mediated early B cell development but are not required for B cell maturation and Ig production.
The Journal of Immunology 08/2007; 179(2):1068-79. · 5.79 Impact Factor
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Yi Zhang,
Ernesto Diaz-Flores, Geqiang Li,
Zhengqi Wang,
Zizhen Kang,
Eleonora Haviernikova,
Sara Rowe,
Cheng-Kui Qu,
William Tse,
Kevin M Shannon,
Kevin D Bunting
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ABSTRACT: Gab2 is an important adapter molecule for cytokine signaling. Despite its major role in signaling by receptors associated with hematopoiesis, the role of Gab2 in hematopoiesis has not been addressed. We report that despite normal numbers of peripheral blood cells, bone marrow cells, and c-Kit(+)Lin(-)Sca-1(+) (KLS) cells, Gab2-deficient hematopoietic cells are deficient in cytokine responsiveness. Significant reductions in the number of colony-forming units in culture (CFU-C) in the presence of limiting cytokine concentrations were observed, and these defects could be completely corrected by retroviral complementation. In earlier hematopoiesis, Gab2-deficient KLS cells isolated in vitro responded poorly to hematopoietic growth factors, resulting in an up to 11-fold reduction in response to a cocktail of stem cell factor, flt3 ligand, and thrombopoietin. Gab2-deficient c-Kit(+)Lin(-) cells also demonstrate impaired activation of extracellular signal-regulated kinase (ERK) and S6 in response to IL-3, which supports defects in activating the phosphatidylinositol-3 kinase (PI-3K) and mitogen-associated protein kinase (MAPK) signaling cascades. Associated with the early defects in cytokine response, competitive transplantation of Gab2(-/-) bone marrow cells resulted in defective long-term multilineage repopulation. Therefore, we demonstrate that Gab2 adapter function is intrinsically required for hematopoietic cell response to early-acting cytokines, resulting in defective hematopoiesis in Gab2-deficient mice.
Blood 08/2007; 110(1):116-24. · 9.90 Impact Factor
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ABSTRACT: The mast cell (MC) inflammatory response is now linked not only to atopy, but also to arthritis, multiple sclerosis, heart disease, and resistance to bacterial infection. In the current study, we demonstrate that the signal transducer and activator of transcription 5 (Stat5) is rapidly activated by IgE cross-linkage, and that its expression is critical to the MC response. Stat5-deficient (Stat5KO) MC demonstrated a significant decrease in IgE-mediated degranulation, leukotriene B4 production, cytokine secretion, and survival signals. The defect in cytokine production may be caused by decreased cytokine mRNA stability. Stat5KO MC-induced cytokine mRNAs normally following IgE cross-linkage, but these mRNAs were not sustained over time and were degraded at twice the rate observed in WT cells. Interestingly, the RNA destabilizing protein tristetraprolin was induced following IgE cross-linkage in Stat5KO but not wild-type cells. Moreover, reducing tristetraprolin expression via short hairpin RNA transfection significantly increased IL-13 production in Stat5KO MC. Our work demonstrates that Stat5 is a critical factor in IgE-induced MC activation, acting in part via posttranscriptional control of cytokine mRNA stability. These data have a direct impact on MC-associated inflammatory and autoimmune diseases.
The Journal of Immunology 10/2006; 177(5):3421-6. · 5.79 Impact Factor
