Erythropoietin induces the tyrosine phosphorylation of GAB1 and its association with SHC, SHP2, SHIP, and phosphatidylinositol 3-kinase

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.     

SH2-Containing protein tyrosine phosphatase-1 (SHP-1) association with Jak2 in UT-7/Epo cells

We have investigated the interaction of the SH2-containing protein tyrosine phosphatase-1 (SHP-1) and Jak2 in an erythropoietin (Epo)-dependent human leukemia cell line, UT-7/Epo, using reciprocal immunoprecipitation and immunoblotting. The Epo-induced kinetics and dose response on phosphorylated Jak2 in anti-SHP-1 precipitates of UT-7/Epo cell lysates were similar to those in direct anti-Jak2 precipitates, suggesting that Jak2 coprecipitated with SHP-1. Furthermore, immunoblotting with anti-Jak2 and anti-SHP-1 antibodies indicated that SHP-1 appeared to be constitutively associated with non-tyrosine-phosphorylated Jak2 in UT-7/Epo cells in the absence of Epo and without phosphorylation of the Epo receptor (EpoR). Competition studies with C-terminal SHP-1 and Jak2 peptides decreased the amounts of SHP-1 and Jak2 detected in immunoprecipitates supporting the specific coprecipitation of SHP-1 and Jak2. In the presence of a recombinant GST-fusion protein containing both the N-terminal and C-terminal SH2 domains of SHP-1, anti-GST precipitated the fusion protein but not cellular Jak2. These studies suggest that SHP-1 and Jak2 are constitutively associated in UT-7/EPO cells. The association is not dependent upon Epo and is not mediated via SHP-1 SH2 binding. Sequential double immunoprecipitation demonstrated that only a small portion of intracellular Jak2 and SHP-1 molecules are constitutively associated. This partial association pattern may allow a more flexible and diverse regulation of Jak2 and SHP-1 activities. Whether Jak2 and SHP-1 are directly associated with each other or are part of a larger complex needs further investigation.     

Erythroid gene expression is differentially regulated by erythropoietin, haemin and delta-aminolaevulinic acid in UT-7 cells

Erythropoietin (Epo) is essential for the later stages of erythropoiesis, acting to promote cell survival and proliferation, but its role in differentiation remains to be defined. The UT-7 cell line exhibits both erythroid and megakaryocytic characteristics and can be induced to differentiate along the erythroid pathway by Epo or the megakaryocytic pathway by phorbol myristic acetate. We have compared the effects of Epo and the chemical inducers, delta-aminolaevulinic acid (delta-ALA) and haemin on the differentiation capacity of UT-7 cells. Epo alone promoted relatively early events in erythroid maturation, without significant changes in haemoglobin production or morphology. GATA-2 and c-myb were down-regulated by Epo, and GATA-2 was further down-modulated by the inducers. Conversely, SCL expression was up-regulated by Epo and further increased by haemin and delta-ALA. Epo caused an increase in the proportion of cells expressing cell surface glycophorin A (GPA) and up-regulated beta- and gamma-globin by several fold. Both haemin and delta-ALA caused a de novo increase in alpha-globin expression as well as enhancing Epo-induced beta-globin expression, leading to a marked increase in haemoglobin production. These results suggest that haemoglobin production in UT-7 cells is limited by a deficiency of erythroid-specific aminolaevulinic acid synthase (ALAS-E) activity or globin synthesis as a consequence of their immaturity as a multipotential cell line.     

Association of the erythropoietin receptor with protein tyrosine kinase activity.

We have examined the signal transduction mechanism of the hematopoietic growth factor erythropoietin (Epo). Epo stimulation of Ba/F3 cells transfected with the Epo receptor resulted in increases in tyrosine phosphorylation of proteins of 97, 75, and 55 kDa. Epo-induced increases in tyrosine phosphorylation of a 97-kDa protein were also detected within the Epo receptor complex, suggesting that a protein tyrosine kinase is associated with the Epo receptor. Protein tyrosine kinase activity was found within the Epo receptor complex and modulation of this activity was observed after treatment of cells with Epo. Furthermore, constitutively high amounts of protein kinase activity were observed in Epo receptor complexes isolated from autonomously growing cells coexpressing the Epo receptor and the leukemogenic glycoprotein gp55. The dominant phosphotyrosylprotein found associated with the Epo receptor was 97 kDa. An Epo receptor-associated protein of identical molecular mass was also found to bind ATP, a characteristic critical for protein kinases. Collectively, these data demonstrate that the Epo receptor is associated with protein tyrosine kinase activity and further suggest that a 97-kDa phosphotyrosylprotein associated with the Epo receptor is a protein tyrosine kinase involved in Epo-mediated signal transduction.     

Tyrosine phosphorylation of the erythropoietin receptor: role for differentiation and mitogenic signal transduction

The erythropoietin (Epo) receptor belongs to the cytokine receptor superfamily. Although the cytokine receptors do not possess a tyrosine kinase consensus sequence in the intracellular domain, rapid stimulation of a tyrosine kinase activity occurs after activation by the ligand. We and others have shown that Epo induces the tyrosine phosphorylation of its cognate receptor as well as phosphorylation of other proteins. In this report, we examined the role of the receptor tyrosine residues in signal transduction. Eight tyrosine residues are located within the intracellular domain of the murine Epo receptor. A single tyrosine residue is present in the region previously shown to be sufficient for proliferative signal transduction. This tyrosine (Tyr 343) was mutated to phenylalanine. Moreover, mutant receptors were also generated with either a tyrosine residue or a phenylalanine residue at position 343 and with a COOH terminal truncation that removed the 7 other tyrosine residues. Expression vectors carrying these mutated receptors were transfected into the interleukin-3-dependent murine cell line Ba/F3. Epo-induced growth was sustained efficiently by all these receptors, although receptors without any tyrosine residues conferred a significantly reduced mitogenic activity. Moreover, all receptors were able to mediate Epo-dependant accumulation of beta-globin mRNA. The mutated receptors all induced the tyrosine phosphorylation of several cellular proteins after Epo stimulation. However, the truncated receptors induced the phosphorylation of a reduced number of proteins, suggesting that phosphorylated tyrosines of the receptor could have a role in the recruitment either of a tyrosine kinase or of tyrosine kinase substrate proteins. The receptors were all able to mediate Epo- induced activation of phosphatidylinositol 3-kinase, although truncated receptors no longer bound phosphatidylinositol 3-kinase.     

Autocrine stimulation by erythropoietin (Epo) requires Epo secretion

Erythropoietin (Epo) autocrine stimulation has been implicated in erythroblastic leukemia. To examine whether this stimulation could occur intracellularly, we developed Epo autocrine models of stimulation in the human pluripotent UT-7 cell line. Retroviral expression of Epo totally abolished the growth factor requirement of UT-7 cells. Autonomous proliferation was not cell density-dependent and occurred at a unicellular level, showing a genuine autocrine mode of stimulation. Total blockage of Epo secretion induced by the endoplasmic reticulum- retention amino acids Lys-Asp-Glu-Leu (KDEL) signals in 11 lines prevented autonomous proliferation, whereas a leaky retention system, observed in 3 other lines, resulted in limited autocrine stimulation without true long-term autonomous proliferation. Production of Epo, in contrast to KDEL-modified Epo, induced reductions in Epo binding, Epo receptor (EpoR) mRNA, and phosphorylation levels similar to those induced by the addition of exogenous Epo to the parental cell line. In addition, autonomous growth and survival were inhibited by the addition of Epo-neutralizing antibodies, affording evidence that autocrine stimulation through EpoR activation takes place on the cell surface. Finally, phenotypic analysis of the virus-infected clones indicated that Epo production did not change the differentiative capacities of UT- 7 cells. All these data show that Epo autocrine stimulation is dependent on Epo secretion and takes place on the cell surface. From all analyzed parameters, the effects of Epo autocrine stimulation and those of exogenously added Epo appear to be identical.     

Establishment and characterization of an erythropoietin-dependent subline, UT-7/Epo, derived from human leukemia cell line, UT-7

UT-7 is a human leukemic cell line capable of growing in interleukin-3 (IL-3), granulocyte/macrophage colony-stimulating factor (GM-CSF), or erythropoietin (Epo) (Komatsu et al, Cancer Res 51:341, 1991). To study the effect of Epo on proliferation and differentiation of UT-7, we maintained the UT-7 cell culture for more than 6 months in the presence of Epo. As a result, a subline, UT-7/Epo, was established. The growth of UT-7/Epo could be supported by Epo but not by GM-CSF or IL-3. UT- 7/Epo showed a greater level of heme content and ratio of benzidine- positive staining cells than did UT-7. Butyric acid promoted the synthesis of hemoglobin in UT-7/Epo, but not UT-7. Further, the mRNA concentrations of the c-myb oncogene and GM-CSF receptor beta-subunit were decreased substantially in UT-7/Epo cells. These findings showed that UT-7/Epo cells had progressed further in erythroid development than UT-7 cells, and suggested that long-term culture in Epo had promoted this differentiation. Whereas availability of the Epo receptor (Epo-R) for binding of Epo was reduced in UT-7/Epo cells compared with UT-7 cells, the Epo-R showed a similar affinity for Epo. This observation suggested that change(s) in postreceptor signaling step might be involved in the establishment and maintenance of the UT-7/Epo phenotype.     

Critical role for PI 3-kinase in the control of erythropoietin-induced erythroid progenitor proliferation

The production of red blood cells is tightly regulated by erythropoietin (Epo). The phosphoinositide 3-kinase (PI 3-kinase) pathway was previously shown to be activated in response to Epo. We studied the role of this pathway in the control of Epo-induced survival and proliferation of primary human erythroid progenitors. We show that phosphoinositide 3 (PI 3)-kinase associates with 4 tyrosine-phosphorylated proteins in primary human erythroid progenitors, namely insulin receptor substrate-2 (IRS2), Src homology 2 domain-containing inositol 5'-phosphatase (SHIP), Grb2-associated binder-1 (Gab1), and the Epo receptor (EpoR). Using different in vitro systems, we demonstrate that 3 alternative pathways independently lead to Epo-induced activation of PI 3-kinase and phosphorylation of its downstream effectors, Akt, FKHRL1, and P70S6 kinase: through direct association of PI 3-kinase with the last tyrosine residue (Tyr479) of the Epo receptor (EpoR), through recruitment and phosphorylation of Gab proteins via either Tyr343 or Tyr401 of the EpoR, or through phosphorylation of IRS2 adaptor protein. The mitogen-activated protein (MAP) kinase pathway was also activated by Epo in erythroid progenitors, but we found that this process is independent of PI 3-kinase activation. In erythroid progenitors, the functional role of PI 3-kinase was both to prevent apoptosis and to stimulate cell proliferation in response to Epo stimulation. Finally, our results show that PI 3-kinase-mediated proliferation of erythroid progenitors in response to Epo occurs mainly through modulation of the E3 ligase SCF(SKP2), which, in turn, down-regulates p27(Kip1) cyclin-dependent kinase (CDK) inhibitor via proteasome degradation.     

Granulocyte-macrophage colony-stimulating factor and erythropoietin act competitively to induce two different programs of differentiation in the human pluripotent cell line UT-7.

The UT-7 cell line was established from a patient with a megakaryoblastic leukemia (Komatsu et al, Cancer Res 51: 341, 1991). Its proliferation is strictly dependent on the presence of hematopoietic growth factors including erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-3 (IL-3). We investigated the differentiation capacities of this cell line under the action of several growth factors, using immunomarkers, flow cytometry, and ultrastructural techniques. In the presence of GM-CSF and IL-3, eosinophil and basophil promyelocytes were detected, as well as a few cells with erythroid and megakaryocytic (MK) differentiation features. In contrast, Epo induced a marked erythroid differentiation with an increase of glycophorin A expression, accompanied by a few hemoglobinized cells. Differentiation induced by the growth factors took 24 to 48 hours to begin, and increased with cell passages to a plateau at 2 weeks of culture. However, this was not only due to a cell selection because the differential effects of Epo and GM-CSF were observed from a single cell clone and the phenotype could be reversed by opposite growth factors, even after a long period of culture. We subsequently investigated the phenotype of UT-7 in the presence of combinations of Epo, IL-3, and GM-CSF, and showed that GM-CSF and IL-3 act predominantly over Epo. This effect was mediated by a rapid downmodulation of Epo receptors by GM-CSF at messenger RNA and binding sites levels, without a change in receptor affinities. On the other hand, Epo had no effect on number and affinity of GM-CSF receptors. This study shows that UT-7 is a growth factor-dependent pluripotent cell line in which commitment may be directed by a hierarchical action of growth factors through an early and rapid transmodulation of growth factor receptors.     

HCV蛋白NS5A对PI3K/Akt通路的分子调节作用

目的研究丙肝病毒（HCV）蛋白NS5A对PI3K/Akt信号的调节机制及其意义。方法 HepG2细胞分别转染NS5A质粒和对照载体。提取总蛋白,用Western blotting法分析PI3K信号Akt磷酸化水平,并用免疫沉淀法检测p85酪氨酸磷酸化水平及p85与p110蛋白间的相互作用。结果 NS5A转染细胞p-Akt蛋白水平上调,同时p85酪氨酸磷酸化水平显著提高,但催化亚基p110与调节亚基p85的结合作用没有明显变化。结论丙肝病毒（HCV）蛋白NS5A可以和PI3Kp85亚基结合而调节PI3K/Akt信号通路,但其机制可能有p85/p110以外的机制。这可能为临床丙肝IFN敏感性的诊断与治疗提供依据。     

Rapid protein tyrosine phosphorylation selectively induced in murine responsive ELM-I-1 cells by erythropoietin.

We investigated whether protein tyrosine phosphorylation was induced by erythropoietin (Epo) in two murine Epo-responsive cell lines (ELM-I-1, which proliferates autonomously and is induced to differentiate by Epo, and DA-1ER, which grows in a manner dependent on Epo or interleukin-3 (IL-3) without differentiation). In ELM-I-1, Epo induced the tyrosine phosphorylation of a protein of about 80-85 kDa (py80) which appeared in the Triton-X soluble fraction of the cell lysate in a time- and concentration-dependent manner. Maximal levels of phosphorylation were obtained within 5-10 min at Epo concentrations above 0.1 U/ml. IL-3 is known to promote the proliferation of this cell line, but it did not induce py80 phosphorylation. In DA-1ER, tyrosine phosphorylation of py80 was not induced by either Epo or IL-3. These findings suggest that there are multiple pathways of Epo signaling and that one of them could be via tyrosine kinase activation. Furthermore, it is possible that the tyrosine phosphorylation of py80 is involved in the pathway leading only to erythroid differentiation but not to cellular proliferation.     

Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells

The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth- factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G- protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces proliferative signals, in part, through downstream activation of p21 RAS. Furthermore, p21 RAS activity is linked to pathways that regulate c-jun and c-fos expression.     

G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein.

The mechanism of mitogen-activated protein (MAP) kinase activation by pertussis toxin-sensitive Gi-coupled receptors is known to involve the beta gamma subunits of heterotrimeric G proteins (G beta gamma), p21ras activation, and an as-yet-unidentified tyrosine kinase. To investigate the mechanism of G beta gamma-stimulated p21ras activation, G beta gamma-mediated tyrosine phosphorylation was examined by overexpressing G beta gamma or alpha 2-C10 adrenergic receptors (ARs) that couple to Gi in COS-7 cells. Immunoprecipitation of phosphotyrosine-containing proteins revealed a 2- to 3-fold increase in the phosphorylation of two proteins of approximately 50 kDa (designated as p52) in G beta gamma-transfected cells or in alpha 2-C10 AR-transfected cells stimulated with the agonist UK-14304. The latter response was pertussis toxin sensitive. These proteins (p52) were also specifically immunoprecipitated with anti-Shc antibodies and comigrated with two Shc proteins, 46 and 52 kDa. The G beta gamma- or alpha 2-C10 AR-stimulated p52 (Shc) phosphorylation was inhibited by coexpression of the carboxyl terminus of beta-adrenergic receptor kinase (a G beta gamma-binding pleckstrin homology domain peptide) or by the tyrosine kinase inhibitors genistein and herbimycin A, but not by a dominant negative mutant of p21ras. Worthmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) inhibited phosphorylation of p52 (Shc), implying involvement of PI3K. These results suggest that G beta gamma-stimulated Shc phosphorylation represents an early step in the pathway leading to p21ras activation, similar to the mechanism utilized by growth factor tyrosine kinase receptors.     

Lnk inhibits erythropoiesis and Epo-dependent JAK2 activation and downstream signaling pathways

Erythropoietin (Epo), along with its receptor EpoR, is the principal regulator of red cell development. Upon Epo addition, the EpoR signaling through the Janus kinase 2 (JAK2) activates multiple pathways including Stat5, phosphoinositide-3 kinase (PI-3K)/Akt, and p42/44 mitogen-activated protein kinase (MAPK). The adaptor protein Lnk is implicated in cytokine receptor signaling. Here, we showed that Lnk-deficient mice have elevated numbers of erythroid progenitors, and that splenic erythroid colony-forming unit (CFU-e) progenitors are hypersensitive to Epo. Lnk(-/-) mice also exhibit superior recovery after erythropoietic stress. In addition, Lnk deficiency resulted in enhanced Epo-induced signaling pathways in splenic erythroid progenitors. Conversely, Lnk overexpression inhibits Epo-induced cell growth in 32D/EpoR cells. In primary culture of fetal liver cells, Lnk overexpression inhibits Epo-dependent erythroblast differentiation and induces apoptosis. Lnk blocks 3 major signaling pathways, Stat5, Akt, and MAPK, induced by Epo in primary erythroblasts. In addition, the Lnk Src homology 2 (SH2) domain is essential for its inhibitory function, whereas the conserved tyrosine near the C-terminus and the pleckstrin homology (PH) domain of Lnk are not critical. Furthermore, wild-type Lnk, but not the Lnk SH2 mutant, becomes tyrosine-phosphorylated following Epo administration and inhibits EpoR phosphorylation and JAK2 activation. Hence, Lnk, through its SH2 domain, negatively modulates EpoR signaling by attenuating JAK2 activation, and regulates Epo-mediated erythropoiesis.     

Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.     

TGF-beta1 drives and accelerates erythroid differentiation in the epo-dependent UT-7 cell line even in the absence of erythropoietin

OBJECTIVE: TGF-beta1 is a powerful inhibitor of erythropoiesis. However, its mechanisms of action are not fully elucidated yet at the cellular level. In this work we have studied the effects of TGF-beta on UT-7 cell survival, proliferation and differentiation. MATERIALS AND METHODS: UT-7 cell line is strictly dependent on growth factors for cell survival, growth, and differentiation. Epo (2 U/mL) induces erythroid differentiation as assessed by up regulation of glycophorin A and the presence of 5%-10% benzidine positive cells (BPC). In contrast, even in the presence of Epo (2 U/mL), GM-CSF (1 ng/mL) inhibits erythroid differentiation. RESULTS: When UT-7 cells were switched from GM-CSF to Epo, TGF-beta1 (2 ng/mL) induced a rapid (3 days [Epo+TGF-beta1] vs 8 days [Epo]) and marked erythroid differentiation (80% [Epo+TGF-beta1] vs 10% [Epo] BPC) including Hemoglobin A synthesis (HbA/HbF ratio of 1 [Epo] vs 4 [Epo+TGF-beta1]). In the presence of GM-CSF, although to a lesser extent, TGF-beta1 induced erythroid differentiation (40% BPC). This effect was not a consequence of TGF-beta1-induced apoptosis because, in the presence of Epo or GM-CSF, apoptosis occurred only at day 8 or 10, respectively. Moreover, although SCF inhibited apoptotic effect of TGF-beta1, SCF+TGF-beta1+Epo was the best combination to give rise to the highest number of hemoglobinized cells. We further demonstrated that induction of erythroid differentiation by TGF-beta1 was not due to an autocrine loop involving Epo/Epo-R or to a prolongation of the G1 phase of the cell cycle. CONCLUSION: Taken together, these data suggest that TGF-beta1 is an inducer of erythroid differentiation, even stronger than Epo at the cellular level.     

p21ras activation via hemopoietin receptors and c-kit requires tyrosine kinase activity but not tyrosine phosphorylation of p21ras GTPase-activating protein.

Products of the ras gene family, termed p21ras, are GTP-binding proteins that have been implicated in signal transduction via receptors encoding tyrosine kinase domains. Recent findings have defined a superfamily of hemopoietin receptors that includes receptors for a number of interleukins and colony-stimulating factors. The intracellular portions of these receptors show only restricted homologies, have no tyrosine kinase domain, and provide no clues to the mode of signal transduction. However, in most cases the factors stimulate tyrosine phosphorylation. We demonstrate here that ligand-induced activation of the interleukin (IL)-2, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptors resulted in activation of p21ras in various hemopoietic cell lines. The only cytokine tested that binds to a hemopoietin receptor and that did not activate p21ras was IL-4. Activation of p21ras was also observed in response to Steel factor, which stimulates the endogenous tyrosine kinase activity of the c-kit receptor, as well as with phorbol esters, which activate protein kinase C. Experiments with protein kinase inhibitors implicated tyrosine kinase activity, but not protein kinase C activity, as the upstream signal in p21ras activation via these growth factor receptors. Attempts to demonstrate tyrosine phosphorylation of the p21ras GTPase-activating protein (GAP) were negative, suggesting that phosphorylation of GAP may not be the major mechanism for regulation of p21ras activity by tyrosine kinases.     

Comparative analysis of signaling pathways between mast cell growth factor (c-kit ligand) and granulocyte-macrophage colony-stimulating factor in a human factor-dependent myeloid cell line involves phosphorylation of Raf-1, GTPase-activating protein and mitogen-activated protein kinase.

Mast cell growth factor (MGF, the ligand for c-kit receptor) can stimulate proliferation of factor dependent myeloid cell line, M07e, and MGF synergizes with granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-3 in this effect. The effect of MGF on protein tyrosine kinase activity in M07e cells was investigated by immunoblotting with anti-phosphotyrosine mAb and this was compared with effects of GM-CSF. MGF stimulation rapidly induced or enhanced at least 12 tyrosine phosphorylated bands. Major bands had molecular weights of 145, 120, 110, 98, 62, 55 and 42 kD. P145, the most prominent phosphorylated protein, was identified as c-kit product using anti-c-kit-mAb (YB5.B8), suggesting ligand-dependent receptor autophosphorylation. Five of six tyrosine phosphorylated bands induced or enhanced by GM-CSF stimulation comigrated with those tyrosine phosphorylated by MGF (138, 120, 76, 55 and 42 kD). P42 was identified, at least in part, as mitogen-activated protein (MAP) kinase. MGF induced tyrosine phosphorylation of a complex of GTPase-activating protein (GAP, 120 kD) and GAP associated proteins (p62/p190) as detected by anti-GAP Ab immunoprecipitation followed by immunoblotting with anti-phosphotyrosine mAb. GM-CSF also stimulated slightly but consistently tyrosine phosphorylation of GAP and p190 but not p62. Both MGF and GM-CSF enhanced Raf-1 phosphorylation and increased Raf-1 associated kinase activity in vitro. Phosphoamino acid analysis revealed Raf-1 phosphorylation by these two growth factors occurred almost exclusively on serine residues. No tyrosine phosphorylation of Raf-1 protein was detected. These data suggest shared and unshared components of signaling pathways of both factors, which may be involved in cell proliferation.