scholarly journals Ubiquitination and degradation of the thrombopoietin receptor c-Mpl

Blood ◽  
2010 ◽  
Vol 115 (6) ◽  
pp. 1254-1263 ◽  
Author(s):  
Sebastian J. Saur ◽  
Veena Sangkhae ◽  
Amy E. Geddis ◽  
Kenneth Kaushansky ◽  
Ian S. Hitchcock
Keyword(s):  
Negative Regulation ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Cytokine Signaling ◽  
Hematopoietic Stem ◽  
Stem Cell Maintenance ◽  
Negative Regulatory Pathway ◽  
Thrombopoietin Receptor ◽  
Interfering Rna ◽  
Regulation Of Growth

Abstract Regulation of growth factor and cytokine signaling is essential for maintaining physiologic numbers of circulating hematopoietic cells. Thrombopoietin (Tpo), acting through its receptor c-Mpl, is required for hematopoietic stem cell maintenance and megakaryopoiesis. Therefore, the negative regulation of Tpo signaling is critical in many aspects of hematopoiesis. In this study, we determine the mechanisms of c-Mpl degradation in the negative regulation of Tpo signaling. We found that, after Tpo stimulation, c-Mpl is degraded by both the lysosomal and proteasomal pathways and c-Mpl is rapidly ubiquitinated. Using site-directed mutagenesis, we were able to determine that c-Mpl is ubiquitinated on both of its intracellular lysine (K) residues (K553 and K573). By mutating these residues to arginine, ubiquitination and degradation were significantly reduced and caused hyperproliferation in cell lines expressing these mutated receptors. Using short interfering RNA and dominant negative overexpression, we also found that c-Cbl, which is activated by Tpo, acts as an E3 ubiquitin ligase in the ubiquitination of c-Mpl. Our findings identify a previously unknown negative regulatory pathway for Tpo signaling that may significantly impact our understanding of the mechanisms affecting the growth and differentiation of hematopoietic stem cells and megakaryocytes.

scholarly journals YRRL motifs in the cytoplasmic domain of the thrombopoietin receptor regulate receptor internalization and degradation

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2222-2231 ◽  
Author(s):  
Ian S. Hitchcock ◽  
Maximus M. Chen ◽  
Jennifer R. King ◽  
Kenneth Kaushansky
Keyword(s):  
Point Mutations ◽  
Adaptor Protein ◽  
Receptor Internalization ◽  
Lysosomal Degradation ◽  
Hematopoietic Stem ◽  
Thrombopoietin Receptor ◽  
Stem And Progenitor Cells ◽  
Increased Strength ◽  
Interfering Rna ◽  
Stimulation Of

Abstract Thrombopoietin (Tpo), acting through the c-Mpl receptor, promotes the survival and proliferation of hematopoietic stem and progenitor cells and drives megakaryocyte differentiation. The proproliferation and survival signals activated by Tpo must therefore be tightly regulated to prevent uncontrolled cell growth. In this work, we determined the mechanisms that control Tpo-stimulated c-Mpl internalization and defined the processes leading to its degradation. Stimulation of BaF-Mpl cells with Tpo leads to rapid, clathrin-dependent endocytosis of the receptor. Using small interfering RNA (siRNA), we found that inhibition of adaptor protein 2 (AP2), which mediates endocytosis of transmembrane proteins, strongly attenuates Tpo-stimulated c-Mpl internalization. AP2 interacts with YXXΦ motifs and we identified 2 such motifs in c-Mpl (Y8RRL and Y78RRL) and investigated Tpo-stimulated internalization of receptors bearing point mutations at these sites. After Tpo stimulation, internalization was greatly reduced in c-Mpl Y78F and c-Mpl Y8+78F, and these cell lines also exhibited increased proliferation and increased strength and duration of Jak2, STAT5, AKT, and ERK1/2 activation in response to Tpo. We also found that the Y8RRL motif regulates Tpo-stimulated lysosomal degradation of c-Mpl. Our data establishes that c-Mpl cytoplasmic YRRL motifs are responsible for both Tpo-mediated internalization via interactions with AP2 and lysosomal targeting after endocytosis.

scholarly journals Cell confluency-induced Stat3 activation regulates NHE3 expression by recruiting Sp1 and Sp3 to the proximal NHE3 promoter region during epithelial dome formation

2009 ◽  
Vol 296 (1) ◽  
pp. C13-C24 ◽  
Author(s):  
Hsiao-Wen Su ◽  
Shainn-Wei Wang ◽  
Fayez K. Ghishan ◽  
Pawel R. Kiela ◽  
Ming-Jer Tang
Keyword(s):  
Cell Density ◽  
Mdck Cells ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Physical Interaction ◽  
Dome Formation ◽  
Nuclear Extracts ◽  
Sodium Hydrogen Exchanger ◽  
Interfering Rna ◽  
Transcription 3

Activation of signal transducer and activator of transcription-3 (Stat3) during cell confluency is related to its regulatory roles in cell growth arrest- or survival-related physiological or developmental processes. We previously demonstrated that this signaling event triggers epithelial dome formation by transcriptional augmentation of sodium hydrogen exchanger-3 (NHE3) expression. However, the detailed molecular mechanism remained unclear. By using serial deletions, site-directed mutagenesis, and EMSA analysis, we now demonstrate Stat3 binding to an atypical Stat3-response element in the rat proximal NHE3 promoter, located adjacent to a cluster of Sp cis-elements (SpA/B/C), within −77/−36 nt of the gene. SpB (−58/−55 nt) site was more effective than SpA (−72/−69 nt) site for cooperative binding of Sp1/Sp3. Increasing cell density had no effect on Sp1/Sp3 expression but resulted in their increased binding to the SpA/B/C probe along with Stat3 and concurrently with enhanced nuclear pTyr705-Stat3 level. Immunoprecipitation performed with the nuclear extracts demonstrated physical interaction of Stat3 and Sp1/Sp3 triggered by cell confluency. Stat3 inhibition by overexpression of dominant-negative Stat3-D mutant in MDCK cells or by small interfering RNA-mediated knockdown in Caco-2 cells resulted in inhibition of the cell density-induced NHE3 expression, Sp1/Sp3 binding, and NHE3 promoter activity and in decreased dome formation. Thus, during confluency, ligand-independent Stat3 activation leads to its interaction with Sp1/Sp3, their recruitment to the SpA/B/C cluster in a Stat3 DNA-binding domain-dependent fashion, increased transcription, and expression of NHE3, to coordinate cell density-mediated epithelial dome formation.

scholarly journals Thrombopoietin signaling to chromatin elicits rapid and pervasive epigenome remodeling within poised chromatin architectures

2017 ◽  
Author(s):  
Federico Comoglio ◽  
Hyun Jung Park ◽  
Stefan Schoenfelder ◽  
Iros Barozzi ◽  
Daniel Bode ◽  
...  
Keyword(s):  
Regulatory Elements ◽  
Cytokine Signaling ◽  
Regulatory Activity ◽  
Hematopoietic Stem ◽  
Chromatin Dynamics ◽  
Stem Cell Maintenance ◽  
Chromatin Looping ◽  
Regulatory Dynamics ◽  
Rapid Modulation ◽  
Regulatory Landscapes

AbstractThrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relation between cis-regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 minutes of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra‐ and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

scholarly journals Spred-2 Suppresses Aorta-Gonad-Mesonephros Hematopoiesis by Inhibiting MAP Kinase Activation

2004 ◽  
Vol 199 (5) ◽  
pp. 737-742 ◽  
Author(s):  
Ikuo Nobuhisa ◽  
Reiko Kato ◽  
Hirofumi Inoue ◽  
Makiko Takizawa ◽  
Keisuke Okita ◽  
...  
Keyword(s):  
Map Kinase ◽  
Specific Inhibitor ◽  
Cell Interaction ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Dominant Negative Mutant ◽  
Hematopoietic Stem ◽  
Map Kinase Pathway ◽  
Kinase Pathway

In midgestation mouse embryos, the aorta-gonad-mesonephros (AGM) region generates hematopoietic stem cells and definitive hematopoiesis is regulated by cell–cell interaction and signaling molecules. We showed that a Ras/mitogen-activated protein (MAP) kinase signaling-specific inhibitor and a dominant negative mutant Ras blocked the production of CD45+ hematopoietic cells in embryonic day 11.5 AGM culture, indicating an essential role for the MAP kinase pathway in AGM hematopoiesis. Overexpression of the Ras/MAP kinase pathway regulator, Spred-2, in the AGM culture significantly reduced the number of CD45+ cells. In contrast, production of CD45+ cells from the AGM region of Spred-2–null mice was up-regulated as compared with wild-type littermates. Furthermore, Spred-2–deficient mice exhibited elevated hematopoietic colony formation from vascular endothelial-cadherin+ cells. These data indicate that Spred-2 functions as a negative regulator of AGM hematopoiesis by inhibiting hematopoietic cytokine signaling.

scholarly journals Disruption of a Receptor-Mediated Mechanism for Intracellular Sorting of Proinsulin in Familial Hyperproinsulinemia

2003 ◽  
Vol 17 (9) ◽  
pp. 1856-1867 ◽  
Author(s):  
Savita Dhanvantari ◽  
Fu-Sheng Shen ◽  
Tiffany Adams ◽  
Christopher R. Snell ◽  
ChunFa Zhang ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Secretory Pathway ◽  
Secretory Granules ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Dominant Negative Mutant ◽  
Sorting Receptor ◽  
Intracellular Sorting ◽  
Regulated Secretory Pathway ◽  
Interfering Rna

Abstract In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a β-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.

scholarly journals Hematopoietic Stem Cell Transplantation Resolves the Immune Deficit Associated with STAT3-Dominant-Negative Hyper-IgE Syndrome

2021 ◽  
Author(s):  
Stephanie C. Harrison ◽  
Christo Tsilifis ◽  
Mary A. Slatter ◽  
Zohreh Nademi ◽  
Austen Worth ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Early Report ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Hematopoietic Stem ◽  
Hyper Ige Syndrome

AbstractAutosomal dominant hyper-IgE syndrome caused by dominant-negative loss-of-function mutations in signal transducer and activator of transcription factor 3 (STAT3) (STAT3-HIES) is a rare primary immunodeficiency with multisystem pathology. The quality of life in patients with STAT3-HIES is determined by not only the progressive, life-limiting pulmonary disease, but also significant skin disease including recurrent infections and abscesses requiring surgery. Our early report indicated that hematopoietic stem cell transplantation might not be effective in patients with STAT3-HIES, although a few subsequent reports have reported successful outcomes. We update on progress of our patient now with over 18 years of follow-up and report on an additional seven cases, all of whom have survived despite demonstrating significant disease-related pathology prior to transplant. We conclude that effective cure of the immunological aspects of the disease and stabilization of even severe lung involvement may be achieved by allogeneic hematopoietic stem cell transplantation. Recurrent skin infections and abscesses may be abolished. Donor TH17 cells may produce comparable levels of IL17A to healthy controls. The future challenge will be to determine which patients should best be offered this treatment and at what point in their disease history.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

Sign in / Sign up

Export Citation Format

Share Document