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:2.7.10.2 (focal adhesion kinase)
44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein tyrosine phosphorylation has been hypothesized to play a key role in the growth signaling induced by erythropoietin (Epo), although the Epo receptor (EpoR), a member of the cytokine receptor superfamily, lacks a tyrosine kinase domain. Recently, the JAK2 tyrosine kinase was shown to be activated on Epo stimulation and to bind to the cytoplasmic domain of EpoR in vitro. To further explore the mechanisms of activation of JAK2 in EpoR-mediated signal transduction, we assessed the conditions for association of JAK2 with EpoR in vivo. Epo stimulation rapidly induced association of JAK2 with the EpoR in an interleukin 3 (IL-3)-dependent cell line transfected with the wild-type EpoR. On Epo stimulation JAK2 also associated with a truncated mutant EpoR (H-mutant), which is mitogenetically active but not tyrosine phosphorylated, indicating that association does not require receptor phosphorylation and occurs in the membrane proximal region. However, association was not detected with mutant receptors inactivated by an internal deletion or a point mutation, Trp282 to Arg, in a membrane-proximal cytoplasmic region (PB or PM4 mutant, respectively). Immune complex kinase assays of anti-EpoR immunoprecipitates also revealed that activated JAK2 associates with the EpoR in Epo-stimulated cells. By this approach, association also occurred with the mitogenically active H mutant but not with the mitogenically inactive PB or PM4 mutants. In the immune complex kinases assays, EpoR, JAK2, and a 150-kD protein were phosphorylated on tyrosine. Taken together, the results further support the hypothesis that, on Epo stimulation, JAK2 associates with the membrane-proximal cytoplasmic region of the EpoR to be activated and induces tyrosine phosphorylation of cellular substrates, including the EpoR, to transduce a growth signal.
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PMID:Erythropoietin induces association of the JAK2 protein tyrosine kinase with the erythropoietin receptor in vivo. 806 43

The vav proto-oncogene product (Vav), which is specifically expressed in hematopoietic cells, contains multiple structural motifs commonly used by intracellular signaling molecules. Although a variety of stimuli including erythropoietin (Epo) have been shown to tyrosine phosphorylate Vav, little is known about the Vav signal transduction pathway. Here, we have investigated the role of Vav in the Epo signaling pathway by characterizing its interaction with other proteins, using the human Epo-responsive cell line, F-36P. Immunoprecipitation and immunoblot analyses have demonstrated that Vav was associated with the Epo receptor (EpoR) in an Epo-independent manner and was tyrosine-phosphorylated after Epo stimulation. Furthermore, two phosphotyrosine proteins (pp70 and pp100) co-immunoprecipitated with the regulatory subunit of phosphatidylinositol 3-kinase (PI3-kinase) (p85) were identified as EpoR and Vav, respectively. The interaction between Vav and p85 was shown to be mediated through the SH2 domains of p85 by an in vitro binding assay and confirmed by the presence of in vitro PI3-kinase activity associated with Vav. Treatment of the cells with antisense-vav and -p85 abrogated Epo-induced cell proliferation and PI3-kinase activity. Finally, we found that JAK2 was associated with Vav in vivo and that Vav could be tyrosine-phosphorylated by activated JAK2 in vitro. These results suggest the possible role of JAK2 for tyrosine phosphorylation of Vav and involvement of Vav and PI3-kinase in Epo-induced proliferative signals.
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PMID:Role of the vav proto-oncogene product (Vav) in erythropoietin-mediated cell proliferation and phosphatidylinositol 3-kinase activity. 916 69

The purpose of this review is to give an update of the recent progress in research on erythropoietin (Epo), the hormone that regulates red blood cell production. Epo is a glycoprotein with a molecular mass of approx 30 kDa, which circulates in plasma of the human with 165 amino acids with three N-linked and one O-linked acidic oligosaccharide side chains in the molecule. Both the alpha (39% CHO) and beta (24% CHO) forms are available for clinical use, and there does not appear to be any difference in the pharmacokinetics of these two forms of Epo. Radioimmunoassays and enzyme-linked immunoabsorbant (ELISA) assays are available in a kit form. Serum levels of Epo in normal human subjects range between 1 and 27 mmu/ml or approx 5 pmol/l. It seems clear that the cells in the adult mammalian kidney which produce Epo are the interstitial cells in the peritubular capillary bed and the perivenous hepatocytes in the liver. Expression of the human Epo gene sequences that direct expression in the kidney are located 6-14 kilobases 5' to the gene; whereas the sequences that control hepatocyte-specific expression are located within 0.7 KS to the 3'-flanking region and 0.5 KS to the 5'-flanking region. The signal transduction pathways postulated to be involved in the expression of Epo are: kinases A, G and C; both a constitutive factor and a second hypoxia-inducible factor-1 (HIF-1) located in the 5' end of an hypoxia inducible enhancer region of the Epo gene; and reactive oxygen species. The primary target cell in the bone marrow acted on by Epo is the colony-forming unit erythroid (CFU-E) which has the highest number of Epo receptors. It has been postulated that Epo decreases the rate which Epo-dependent progenitor cells undergo programed cell death (apoptosis). There are two major signal transduction pathways activated by the Epo receptor: the JAK2-STAT5 pathway and the ras pathway. Both pathways involve tyrosine phosphorylation. The approved clinical uses of Epo are the anemias associated with end-stage renal disease, cancer chemotherapeutic agents, and patients with HIV infection receiving AZT. Other anemias reported to respond to Epo therapy are anemia of prematurity, rheumatoid arthritis, and myelodysplasia. Other uses of Epo under investigation are in perioperative surgery and preoperative autologous blood donation.
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PMID:Erythropoietin: physiologic and pharmacologic aspects. 940 40

In humans, studies of the erythroid cell lineage are hampered by difficulties in obtaining sufficient numbers of erythroid progenitors. In fact, these progenitors in bone marrow or peripheral blood are scarce and no specific antibodies are available. We describe a new method which allows proliferation in liquid culture of large numbers of pure normal human erythroid progenitors. CD34+ cells were cultured for 7 d in serum-free conditions with the cytokine mixture interleukin (IL)-3/IL-6/stem cell factor (SCF). This resulted in cell expansion and the appearance of a high proportion of CD36+ cells which were purified on day 7. Methylcellulose clones from these cells were composed of 96.6% late BFU-E and 3.4% CFU-GM. These CD36+ cells could be recultured with the same cytokine mixture plus or minus erythropoietin (Epo) for a further 2-7 d. In both conditions further amplification of CD36+ cells was observed, but Epo induced a more dramatic cell expansion. Glycophorin-positive mature cells appeared only in the presence of Epo, and terminal red cell differentiation was observed after 7 d of secondary culture. Cells obtained from adult CD34+ progenitors mostly contained adult haemoglobin, whereas cord blood-derived cells contained equal proportions of adult and fetal haemoglobin. Activation of STAT5 and tyrosine phosphorylation of the Epo receptor and JAK2 were observed after Epo stimulation of these cells. This new method represents a straightforward alternative to the procedures previously described for the purification of normal erythroid progenitors and is useful in the study of erythropoietic regulation.
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PMID:Purification, amplification and characterization of a population of human erythroid progenitors. 1051 92

Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo(-/-) and EpoR(-/-) mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210(BCR-ABL) and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR(-/-) mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of fibroblasts or bone marrow cells can fully support normal erythroid development. These results demonstrate that activated tyrosine kinase oncoproteins implicated in tumorigenesis and human leukemia can functionally complement for cytokine receptor signaling pathways to support normal erythropoiesis in EpoR-deficient cells. Moreover, terminal differentiation of erythroid cells requires generic signals provided by activated protein tyrosine kinases and does not require a specific signal unique to a cytokine receptor.
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PMID:BCR-ABL and v-SRC tyrosine kinase oncoproteins support normal erythroid development in erythropoietin receptor-deficient progenitor cells. 1055 95

Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors with distinct modes of action on EpoR signaling in primary human erythroblasts and in a recently established murine erythroid cell line. Active PKC appeared essential for Epo-induced phosphorylation of the Epo receptor itself, STAT5, Gab1, Erk1/2, AKT, and other downstream targets. Under the same conditions, stem cell factor-induced signal transduction was not impaired. LY294002, a specific inhibitor of phosphoinositol 3-kinase, also suppressed Epo-induced signal transduction, which could be partially relieved by activators of PKC. PKC inhibitors or LY294002 did not affect membrane expression of the EpoR, the association of JAK2 with the EpoR, or the in vitro kinase activity of JAK2. The data suggest that PKC controls EpoR signaling instead of being a downstream effector. PKC and phosphoinositol 3-kinase may act in concert to regulate association of the EpoR complex such that it is responsive to ligand stimulation. Reduced PKC-activity inhibited Epo-dependent differentiation, although it did not effect Epo-dependent "renewal divisions" induced in the presence of Epo, stem cell factor, and dexamethasone.
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PMID:Protein kinase C alpha controls erythropoietin receptor signaling. 1094 Mar 12

Erythroid homeostasis depends critically upon erythropoietin (Epo) and stem cell factor cosignaling in late progenitor cells. Epo bioresponses are relayed efficiently by minimal receptor forms that retain a single Tyr-343 site for STAT5 binding, while forms that lack all cytoplasmic Tyr(P) sites activate JAK2 and the transcription of c-Myc plus presumed additional target genes. In FDCER cell lines, which express endogenous c-Kit, the signaling capacities of such minimal Epo receptor forms (ER-HY343 and ER-HY343F) have been dissected to reveal: 1) that Epo-dependent mitogenesis, survival, and bcl-x gene expression via ER-HY343 depend upon the intactness of the Tyr-343 STAT5 binding site; 2) that ER-HY343-dependent bcl-x(L) gene transcription is enhanced markedly via c-Kit; 3) that socs-3, plfap, dpp-1, and cacy-bp gene transcription is induced via ER-HY343, whereas dpp-1 and cacy-bp gene expression is also supported by ER-HY343F; 4) that ectopically expressed SOCS-3 suppresses proliferative signaling by not only ER-HY343 but also c-Kit; and 5) that in FDCER and primary erythroid cells, c-Kit appears to provide the primary route to MAPK activation. Thus, integration circuits exist in only select downstream pathways within Epo and stem call factor receptor signaling.
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PMID:Integrative signaling by minimal erythropoietin receptor forms and c-Kit. 1112 55

Previous reports have shown a direct effect of erythropoietin (Epo) on vascular smooth muscle cells (VSMCs). Our aim was to assess expression of the Epo receptor (EpoR) on VSMCs and to study the activation of two major signaling cascades activated by Epo, namely JAK2/STAT5 and MAPK pathways. All experiments were performed in parallel using the Epo-responsive UT7 cell line. From semiquantitative RT-PCR experiments, VSMCs were estimated to express approximately 30-fold less EpoR mRNA than UT7 cells. Epo-induced phosphorylation of proteins involved in the EpoR/JAK2/STAT5 cascade could not be detected in VSMCs, even using pharmacological doses of Epo (250 IU/ml). In contrast, a strong activation of MAP kinase pathway was detected with as low as 10 IU/ml Epo. We suggest that MAPK activation reflects a physiologically relevant effect of Epo on VSMCs that may be correlated to cell proliferation.
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PMID:Low doses of EPO activate MAP kinases but not JAK2-STAT5 in rat vascular smooth muscle cells. 1140 98

Regulation of survival, expansion, and differentiation of erythroid progenitors requires the well-controlled activity of signaling pathways induced by erythropoietin (Epo) and stem cell factor (SCF). In addition to qualitative regulation of signaling pathways, quantitative control may be essential to control appropriate cell numbers in peripheral blood. We demonstrate that Bruton's tyrosine kinase (Btk) is able to associate with the Epo receptor (EpoR) and Jak2, and is a substrate of Jak2. Deficiency of Btk results in reduced and delayed phosphorylation of the EpoR, Jak2, and downstream signaling molecules such as Stat5 and PLCgamma1 as well as in decreased responsiveness to Epo. As a result, expansion of erythroid progenitors lacking Btk is impaired at limiting concentrations of Epo and SCF. In addition, we show that SCF induces Btk to interact with TNF-related apoptosis-inducing ligand (TRAIL)-receptor 1 and that lack of Btk results in increased sensitivity to TRAIL-induced apoptosis. Together, our results indicate that Btk is a novel, quantitative regulator of Epo/SCF-dependent expansion and survival in erythropoiesis.
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PMID:Btk is required for an efficient response to erythropoietin and for SCF-controlled protection against TRAIL in erythroid progenitors. 1500 95

Activation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.
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PMID:Both proteasomes and lysosomes degrade the activated erythropoietin receptor. 1535 19


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