Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Red blood cell development is primarily controlled by erythropoietin (EPO). Several studies have revealed the importance of EPO-R Y343 and Y479 for erythroid cell growth, differentiation, and survival. In order to isolate critical signaling proteins that bind to EPO-R, we initiated a Cloning of Ligand Target (COLT) screen using a murine embryonic day 16 phage library and a biotinylated EPO-R Y343 phosphopeptide. One of the clones isolated encodes Phospholipase C (PLC)gamma1. PLCgamma1 is rapidly tyrosine phosphorylated upon EPO stimulation and associates with EPO-R in an SH2-domain-dependent manner. Although PLCgamma1 bound EPO-R Y343, Y401, Y429, Y431, and Y479 in the COLT screen, PLCgamma1 required Y479 for association with EPO-R in Ba/F3-EPO-R cells. Studies have identified EPO-R Y479 as important for ERK activation. Since PI3-kinase binds EPO-R Y479, one group has suggested that ERK activation downstream of PI3-kinase accounts for the importance of this residue in EPO signaling. However, we show that inhibition of PI3-kinase does not abolish ERK activation. Furthermore, we demonstrate interaction of PLCgamma1 with Grb2 and SOS2. Hence, we have identified a novel adapter function for PLCgamma1 in EPO signaling in which recruitment of PLCgamma1 to EPO-R may lead to activation of the ERK pathway.
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PMID:Erythropoietin receptor Y479 couples to ERK1/2 activation via recruitment of phospholipase Cgamma. 1595 1

Hematopoietic cytokines, including interleukin (IL)-3 and erythropoietin (Epo), regulate hematopoiesis by stimulating their receptors coupled with the Jak2 tyrosine kinase to induce receptor tyrosine phosphorylation and activate mainly the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways. Here we demonstrate that IL-3 or Epo induces a rapid and transient (peaking at 30 min) as well as late progressive increase in reactive oxygen species (ROS) in a hematopoietic progenitor model cell line, 32Dcl3, and its subclone expressing the Epo receptor (EpoR), 32D/EpoR-Wt. The cytokine-induced ROS generation was not affected in 32Dcl3 cells depleted of mitochondrial DNA. The antioxidant N-acetyl-L-cysteine (NAC) inhibited IL-3-induced tyrosine phosphorylation of Jak2, IL-3 receptor betac subunit (IL-3Rbetac), and STAT5 as well as activation-specific phosphorylation of Akt, MEK, and ERK, while treatment of cells with H2O2 activated these signaling events. NAC also inhibited the EpoR-induced transphosphorylation of IL-3Rbetac. Moreover, NAC treatment reduced the expression levels of c-Myc, Cyclin D2, and Cyclin E, and induced expression of p27, thus inhibiting the G1 to S phase transition of cells cultured with IL-3. Further studies have shown that the degradation of c-Myc was facilitated or inhibited by treatment of cells with NAC or H2O2, respectively. These data indicate that the rapid generation of ROS by cytokine stimulation, which is at least partly independent of mitochondria, may play a role in activation of Jak2 and the STAT5, PI3K/Akt, and Ras/MEK/ERK signaling pathways as well as in transactivation of cytokine receptors. The cytokine-induced ROS generation was also implicated in G1 to S progression, possibly through stabilization of c-Myc and induction of G1 phase Cyclin expression leading to suppression of p27.
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PMID:Reactive oxygen species generated by hematopoietic cytokines play roles in activation of receptor-mediated signaling and in cell cycle progression. 1598 52

Selective and regulatable expansion of transduced cells could augment gene therapy for many disorders. The activation of modified growth factor receptors via synthetic chemical inducers of dimerization allows for the coordinated growth of transduced cells. This system can also provide information on specific receptor-mediated signaling without interference from other family members. Although several receptor subunits have been investigated in this context, little is known about the precise molecular events associated with dimerizer-initiated signaling. We have constructed and expressed an AP20187-regulated KDR chimeric receptor in human TF1 cells and analyzed activation of this gene switch using functional, biochemical, and microarray analyses. When deprived of natural ligands, GM-CSF, interleukin-3, or erythropoietin, AP20187 prevented apoptosis of transduced TF1 cells, induced dose-dependent proliferation, and supported long-term growth. In addition, AP20187 stimulation activated the signaling molecules associated with mitogen-activated protein kinase and phosphatidyl-inositol 3-kinase/Akt pathways. Microarray analysis determined that a number of transcripts involved in a variety of cellular processes were differentially expressed. Notably, mRNAs affiliated with heat stress, including Hsp70 and Hsp105, were up-regulated. Functional assays showed that Hsp70 and Hsp105 protected transduced TF1 cells from apoptosis and premature senescence, in part through regulation of Akt. These observations delineate specific roles for kinase insert domain-containing receptor, or KDR, signaling and suggest strategies to endow genetically modified cells with a survival advantage enabling the generation of adequate cell numbers for therapeutic outcomes.
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PMID:Specific pharmacological dimerization of KDR in lentivirally transduced human hematopoietic cells activates anti-apoptotic and proliferative mechanisms. 1607 62

Alternative splicing of signaling proteins can contribute to the complexity of signaling networks. We find that expression of mouse RON, but not human RON, results in constitutive receptor autophosphorylation, ligand-independent activation of the mitogen-activated protein kinase pathway, and association of the receptor with c-Src. Using chimeric receptors, we mapped the region for this difference in signaling capacity of mouse and human RON to the juxtamembrane domain. Expression of these receptors in primary erythroid progenitor cells also demonstrated a functional difference in the ability of mouse and human RON to support erythropoietin-independent colony formation that mapped to the juxtamembrane domain. Splicing of the mouse RON receptor tyrosine kinase transcript results in the constitutive deletion of an exon used by all other known RON orthologs that encodes part of the juxtamembrane domain of the receptor. Mutational analysis indicated that the two tyrosines present in this region in human RON, one of which has been previously shown to be a c-Cbl binding site, are not responsible for this difference. However, deletion of this region in the context of human RON enhanced receptor phosphorylation, activation of mitogen-activated protein kinase, and association of c-Src at levels comparable with those observed with mouse RON. These data provide direct evidence that the divergence of exon usage among different species can generate a protein with novel activity and subsequently add to the complexity of cellular signaling regulation.
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PMID:Altered exon usage in the juxtamembrane domain of mouse and human RON regulates receptor activity and signaling specificity. 1616 96

Although it is almost certain that alpha(+)-thalassemia protects against malaria, the mechanisms for that are still unknown. It has been suggested that an increased number of young circulating red blood cells in alpha(+)-thalassemic children, as a result of some degree of ineffective erythropoiesis, could be related to the high frequencies of the alpha(+)-thalassemic allele in malaria endemic areas. Reticulocyte evaluation in this condition, however, has been poorly performed so far. Our objective was to determine the reticulocyte number and maturation degree, in addition to the soluble transferrin receptor and serum erythropoietin levels, in alpha(+)-thalassemia heterozygotes, comparing them with normal alpha-genotype controls. One hundred twenty-one alpha(+)-thalassemia carriers (-alpha(3.7)/alphaalpha) and 249 controls (alphaalpha/alphaalpha), all of them with normal serum ferritin levels, were subclassified according to age (1-5, 6-10, 11-15, 16-20, and over 20 years old). Reticulocyte analyzes were carried out by flow cytometry and sTfR and s-Epo levels determined by immunonephelometry and chemiluminescence, respectively. The comparisons did not show any significant difference between thalassemics and controls regarding the reticulocyte parameters [percentages and absolute values, P = 0.2643 and 0.5421; high, medium, and low maturation degree, P = 0.2579, 0.2196, and 0.4192; RET maturity index (RMI), P = 0.2471, respectively], as well as the s-Epo levels (P = 0.5711). The sTfR concentrations were higher in the thalassemic group (P = 0.0001), but statistical significance was due only to the 1-5 and over 20 subgroups (P = 0.0082 and 0.0436, respectively). The results found here are compatible with a compensated erythropoiesis and do not confirm the hypothesis mentioned above.
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PMID:Reticulocyte evaluation in alpha(+)-thalassemia. 1636 54

The hematopoietic cytokine erythropoietin (Epo) prevents neuronal death during ischemic events in the brain and in neurodegenerative diseases, presumably through its antiapoptotic effects. To explore the role of different signaling pathways in Epo-mediated antiapoptotic effects in differentiated human neuroblastoma SH-SY5Y cells, we employed a prolactin receptor (PrlR)/erythropoietin receptor (EpoR) chimera system, in which binding of prolactin (Prl) to the extracellular domain activates EpoR signaling in the cytosol. On induction of apoptosis by staurosporine, Prl supports survival of the SH-SY5Y cells expressing the wild-type PrlR/EpoR chimera. In these cells Prl treatment strongly activates the STAT5, AKT, and MAPK signaling pathways and induces weak activation of the p65 NF-kappaB factor. Selective mutation of the eight tyrosine residues of the EpoR cytoplasmic domain results in impaired or absent activation of either STAT5 (mutation of Tyr(343)) or AKT (mutation of Tyr(479)) or both (mutation of all eight tyrosine residues). Most interestingly, Prl treatment does not prevent apoptosis in cells expressing mutant PrlR/EpoR chimeras in which either the STAT5 or the AKT signaling pathways are not activated. In contrast, ERK 1/2 is fully activated by all mutant PrlR/EpoR chimeras, comparable with the level seen with the wild-type PrlR/EpoR chimera, implying that activation of the MAPK signaling pathway per se is not sufficient for antiapoptotic activity. Therefore, the antiapoptotic effects of Epo in neuronal cells require the combinatorial activation of multiple signaling pathways, including STAT5, AKT, and potentially MAPK as well, in a manner similar to that observed in hematopoietic cells.
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PMID:Antiapoptotic effects of erythropoietin in differentiated neuroblastoma SH-SY5Y cells require activation of both the STAT5 and AKT signaling pathways. 1640 71

In this study we confirmed the presence of the erythropoietin (EPO) receptor on both cultured cortical neurons and PC12 cells and showed that EPO can induce changes in p38, ERK, and JNK signaling molecules in these cells. We induced EPO preconditioning in cortical neuronal cultures that protected neurons from a subsequent in vitro ischemic insult (transient oxygen-glucose deprivation). To investigate downstream changes in protein expression in EPO-preconditioned cortical neuronal cultures, we used two-dimensional gel electrophoresis. Overall, EPO preconditioning resulted in protein up-regulation, and, from 84 of the most differentially expressed proteins selected for identification, the proteins or tentative proteins were identified in 57 cases, representing 40 different proteins. Different protein spots representing the same or closely related protein(s) occurred for 13 of the identified proteins and are likely to represent posttranslational modifications or proteolytic fragments of the protein. Two proteins (78-kD glucose-regulated protein and tropomyosin, fibroblast isoform 1) were detected in control neuronal cultures, but not following EPO preconditioning treatment, whereas one protein (40S ribosomal protein SA) was detected only following EPO preconditioning. Most of the other proteins identified had not previously been associated with EPO preconditioning and will aid in the understanding of EPO's neuroprotective response and possibly the development of new therapeutic interventions to inhibit neuronal death in acute and chronic neurodegenerative diseases.
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PMID:Erythropoietin preconditioning in neuronal cultures: signaling, protection from in vitro ischemia, and proteomic analysis. 1643 92

The number and properties of endothelial progenitor cells (EPC) in disease states is of considerable interest due to the importance attributed to this distinct cell population. However, there has been no study comparing each of the methods employed in the same sampled individuals. Herein, we performed an analysis of several methods used for circulating EPC assessment and correlated them with humoral factors known to influence their numbers. Thirty-eight individuals (mean age of 34 +/- 9 years) were tested. Peripheral blood mononuclear cells were obtained and stained for FACS analysis with antibodies to CD34, CD45, CD133, and KDR and the remaining cells grown under endothelial cell conditions for assessment of colony-forming unit (CFU) numbers and adhesive properties. Levels of circulating vascular endothelial growth factor (VEGF), erythropoietin (EPO), and C-reactive protein (CRP) were determined and correlated with each of the EPC markers. CFU numbers did not correlate with CD34/KDR or CD34/CD133/KDR and negatively correlated with CD34/ CD133 numbers. CD34/KDR numbers correlated with CD34/CD133/KDR, but not with CD34/ CD133. Only CD34/KDR and CD34/CD133/KDR correlated with VEGF serum levels. The number of EPC adhering to fibronectin and endothelial cells correlated with CFU numbers and not with either of the EPC membrane markers. Current methods for quantitatively assessing numbers of circulating EPC are not correlated. VEGF serum levels are associated only with CD34/KDR and CD34/ CD133/KDR, whereas CFU numbers correlate with EPC functional properties. These findings may suggest that CD34/KDR is more appropriate for the definition of circulating EPC, whereas CFU numbers are more likely to reflect their ability to proliferate.
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PMID:Comparative analysis of methods for assessment of circulating endothelial progenitor cells. 1654 91

Signal transducer and activator of transcription (STAT) proteins are phosphorylated and activated by Janus kinases (JAKs). Recently, several groups identified a recurrent somatic point mutation constitutively activating the hematopoietic growth factor receptor-associated JAK2 tyrosine kinase in diverse chronic myeloid disorders - most commonly classic myeloproliferative disorders (MPD), especially polycythemia vera. We hypothesized that the JAK2 V617F mutation might also be present in samples from patients with acute myeloid leukemia (AML), especially erythroleukemia (AML-M6) or megakaryoblastic leukemia (AML-M7), where it might mimic erythropoietin or thrombopoietin signaling. First, we documented STAT3 activation by immunoblotting in AML-M6 and other AML subtypes. Immunoperoxidase staining confirmed phosphorylated STAT3 in malignant myeloblasts (21% of cases, including all AML-M3 samples tested). We then analyzed genomic DNA from 162 AML, 30 B-cell lymphoma, and 10 chronic lymphocytic leukemia (CLL) samples for JAK2 mutations, and assayed a subset for SOCS1 and FLT3 mutations. Janus kinase2 V617F was present in 13/162 AML samples (8%): 10/13 transformed MPD, and three apparent de novo AML (one of 12 AML-M6, one of 24 AML-M7, and one AML-M2 - all mixed clonality). FLT3 mutations were present in 5/32 (16%), while SOCS1 mutations were totally absent. Lymphoproliferative disorder samples were both JAK2 and SOCS1 wild type. Thus, while JAK2 V617F is uncommon in de novo AML and probably does not occur in lymphoid malignancy, unexplained STAT3 activation is common in AML. Janus kinase2 extrinsic regulators and other proteins in the JAK-STAT pathway should be interrogated to explain frequent STAT activation in AML.
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PMID:JAK2 V617F is a rare finding in de novo acute myeloid leukemia, but STAT3 activation is common and remains unexplained. 1659 6

The mechanisms through which hematopoietic cytokines accelerate revascularization are unknown. Here, we show that the magnitude of cytokine-mediated release of SDF-1 from platelets and the recruitment of nonendothelial CXCR4+ VEGFR1+ hematopoietic progenitors, 'hemangiocytes,' constitute the major determinant of revascularization. Soluble Kit-ligand (sKitL), thrombopoietin (TPO, encoded by Thpo) and, to a lesser extent, erythropoietin (EPO) and granulocyte-macrophage colony-stimulating factor (GM-CSF) induced the release of SDF-1 from platelets, enhancing neovascularization through mobilization of CXCR4+ VEGFR1+ hemangiocytes. Although revascularization of ischemic hindlimbs was partially diminished in mice deficient in both GM-CSF and G-CSF (Csf2-/- Csf3-/-), profound impairment in neovascularization was detected in sKitL-deficient Mmp9-/- as well as thrombocytopenic Thpo-/- and TPO receptor-deficient (Mpl-/-) mice. SDF-1-mediated mobilization and incorporation of hemangiocytes into ischemic limbs were impaired in Thpo-/-, Mpl-/- and Mmp9-/- mice. Transplantation of CXCR4+ VEGFR1+ hemangiocytes into Mmp9-/- mice restored revascularization, whereas inhibition of CXCR4 abrogated cytokine- and VEGF-A-mediated mobilization of CXCR4+ VEGFR1+ cells and suppressed angiogenesis. In conclusion, hematopoietic cytokines, through graded deployment of SDF-1 from platelets, support mobilization and recruitment of CXCR4+ VEGFR1+ hemangiocytes, whereas VEGFR1 is essential for their angiogenic competency for augmenting revascularization. Delivery of SDF-1 may be effective in restoring angiogenesis in individuals with vasculopathies.
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PMID:Cytokine-mediated deployment of SDF-1 induces revascularization through recruitment of CXCR4+ hemangiocytes. 1664 59


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