Biomedical subjects

D M Wojchowski

Publications and source records attributed to D M Wojchowski.

At least 37 records · Page 2Linked to original sources

PubMed1996

Interactions among Janus kinases and the prolactin (PRL) receptor in the regulation of a PRL response element.

PRL regulates milk gene expression, at least in part, by activating JAK2 kinase and STAT5 (signal transducer and activator of transcription 5), initially termed mammary gland factor (MGF). These experiments were initiated to gain a better understanding of the mechanisms of transcriptional activation via PRL receptor (PRL-R) signaling. Binding of PRL to the recombinant pigeon PRL-R-activated transcription driven by a 2.8 kbp 5'-fragment of the rat beta-casein gene. PRL enhanced the expression of chimeric reporters containing the beta-casein PRL response element (PRE), but not the c-fos sis-inducible element, when the reporters were transfected into Chinese hamster ovary cells with the PRL-R. Wild type receptor, which contains a duplication of the entire extracellular ligand-binding domain, was only slightly more effective than a truncation mutant with a single extracellular domain. Transfection with either JAK1, JAK2, or JAK3 increased basal transcription through both the PRE and sis-inducible element. Coexpression of JAK2 with PRL-R resulted in amplification of the induction of the PRE by PRL, whereas JAKs 1 and 3 did not amplify the PRL effect. Overexpression of JAK2 mutants blocked PRE activation by PRL. Mutant JAK2 also interfered with PRE activation by JAK3 but did not affect JAK1's stimulatory effect.

Animals↗

PubMed1995

Erythropoietin-dependent inhibition of apoptosis is supported by carboxyl-truncated receptor forms and blocked by dominant-negative forms of Jak2.

Apoptosis, or programmed cell death (PCD), recently has emerged as an important homeostatic mechanism within several hematopoietic lineages. This process is subject to both positive and negative modulation by cytokines and within the erythroid lineage is inhibited by interleukin-3, stem cell factor, and erythropoietin (Epo). Through the expression of carboxyl-truncated Epo receptor mutants in FDC-P1 cells, a receptor form possessing 80 membrane-proximal cytoplasmic residues is shown to efficiently mediate Epo-dependent inhibition of PCD. This is in contrast to previous studies that attributed this activity to a distal carboxyl-terminal receptor subdomain (and/or heterodimerization of wild type Epo receptors with a truncated non-functional receptor form). Epo-dependent inhibition of PCD also is shown to be blocked by ectopic expression of kinase-deficient dominant-negative forms of Jak2 (Jak2 delta VIII and Jak2-829), further underlining a role of this membrane-proximal subdomain of the Epo receptor in the inhibition of PCD. To our knowledge, this comprises the first direct evidence for an essential role for a Jak tyrosine kinase (Jak2) in this apoptotic response pathway.

Amino Acid Sequence↗

PubMed1995

Erythropoietin-induced recruitment of Shc via a receptor phosphotyrosine-independent, Jak2-associated pathway.

Based on the recently implicated role of Shc as a signaling effector for type I cytokine receptors, factors which mediate the recruitment and phosphorylation of Shc in the erythropoietin receptor (EPOR) system have been studied. FDC-P1 cells stably expressing the wild type murine EPOR supported the EPO-induced association of Shc with Jak2 and its rapid tyrosine phosphorylation. However, this did not depend upon the presence of phosphotyrosine sites within the EPOR and was mediated by a mitogenically deficient receptor form (EPOR329) lacking cytoplasmic tyrosine residues. This was shown both by Western blotting of Shc and Jak2 co-immunoprecipitates and through the development of an in vitro assay for cytokine-induced Shc phosphorylation. The direct association of Shc with Jak2 also was observed and was shown to depend upon EPO-exposure and the SH2 subdomain of Shc. Together, these studies indicate that Jak2, in part, may mediate the EPO-induced phosphorylation of Shc.

Animals↗

PubMed1995

Erythropoietin-induced transcription at the murine beta maj-globin promoter. A central role for GATA-1.

Using J2E cells and the murine beta maj-globin promoter as a model, we have performed the first direct analyses of erythropoietin (EPO)-activated transcription from defined templates. The -346 to +26 beta maj promoter was shown to comprise a target for maximal activation. This included a positive role for a -346 to -107-base pair (bp) domain in J2E cells, but not in F-MEL cells. Mutagenesis of a -215-bp AGATAA element within this domain showed that this effect did not require GATA-1 binding. In contrast, a critical role for GATA-1 at a -60-bp (G)GATAG element was defined by mutagenesis (GGg-TAG and TGATAG), complementation with a synthetic TGATAA element, and the demonstrated specific binding of GATA-1. Proximal CCAAT (-75) and CACCC (-90) elements also were shown to contribute to transcriptional activation in J2E cells, yet exerted quantitatively distinct effects in the F-MEL system. Based on these results, minimal [TGATAA]4-TATA and TGATAA-CACCC-TATA promoters were constructed and assayed in each system. Remarkably, the [TGATAA]4-TATA promoter, but not the TGATAA-CACCC-TATA promoter, was induced efficiently by EPO in J2E cells, whereas the TGATAA-CACCC-TATA promoter was highly induced by Me2SO in F-MEL cells. These findings suggest that mechanisms of EPO-induced transcription in J2E cells involve GATA-1 and differ from chemically activated mechanisms studied previously in F-MEL cells. Globin induction in J2E cells was not associated with effects of EPO on levels or nuclear translocation of GATA-1. However, hemoglobinization was induced by okadaic acid, 8-Br-cAMP, and forskolin, a finding consistent with induction mechanisms that may involve modulated serine/threonine phosphorylation.

Animals↗

PubMed1994

Steel factor affects SCL expression during normal erythroid differentiation.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

Base Sequence↗

PubMed1994

Dominant negative effects of a carboxy-truncated Jak2 mutant on Epo-induced proliferation and Jak2 activation.

Members of the Janus family of protein tyrosine kinases are emerging as primary, receptor-associated transducing factors among numerous cytokine systems. However, little is understood regarding mechanisms of recruitment of these kinases to receptor complexes and their ligand-dependent activation. To initially address these questions, we have assessed effects of ectopically expressing a carboxy-truncated form of Jak2 (Jak2-829) in Epo-responsive DAER cells. Expression of this truncation mutant at low levels efficiently inhibited both Epo-dependent activation of endogenous Jak2 and Epo-induced mitogenesis (10% to 39% of parental DAER cells). These results suggest that amino-terminal domains of Jak2 may mediate the assembly of Jak2/Epo receptor complexes and that integration of Jak2-829 into receptor complexes may effectively inhibit the activity of oligomeric Jak2/receptor assemblages.

Animals↗

PubMed1994

Inhibition of erythropoietin-induced mitogenesis by a kinase-deficient form of Jak2.

Receptors for a variety of hematopoietins, interferons alpha/beta and gamma, and growth hormone have recently been shown to mediate rapid, ligand-dependent activation of the Janus-type cytosolic protein-tyrosine kinases Jak1, Jak2, and/or tyk-2. This finding extends relatedness among class I and II cytokine receptors to a functional context and provides an initially satisfying mechanistic analogy to protein-tyrosine kinase-encoding receptors of the epidermal growth factor/platelet-derived growth factor/insulin family. Through the construction and expression of a kinase-deficient form of Jak2 (JK2 delta VIII) in interleukin-3 (IL-3)/erythropoietin (Epo)-dependent DAER cells, we have tested whether activation of Jak2 is required for induced mitogenesis via these class I cytokine receptors. Ectopic expression of JK2 delta VIII inhibited Epo- and IL-3-induced activation of endogenous wild-type Jak2, transiently attenuated IL-3-dependent growth, and essentially abrogated Epo-induced proliferation in this model system. These dominant-negative effects provide the first direct experimental evidence for an essential role for Janus kinase activation in mitogenesis and suggest that distinct effectors may mediate IL-3-induced versus Epo-induced pathways.

Amino Acid Sequence↗

PubMed1994

The extended box 2 subdomain of erythropoietin receptor is nonessential for Jak2 activation yet critical for efficient mitogenesis in FDC-ER cells.

The development of erythroid progenitor cells depends upon exposure to the glycoprotein hormone, erythropoietin (EPO). Binding of EPO to its transmembrane receptor leads to the rapid tyrosine phosphorylation of several cellular targets including Shc, Raf-1, Gap120, the cloned EPO receptor (EPOR), pp100/97, and a M(r) 130,000 EPO-activated receptor-associated Janus protein tyrosine kinase, Jak2. A membrane-proximal cytosolic region of the EPOR recently has been shown to be essential for the activation of Jak2 and sufficient for EPO-induced mitogenesis. This cytosolic region includes 8-12 amino acid box 1 and box 2 subdomains, which are conserved in certain class I receptors as well as a more distal 10-40 amino acid subdomain (extended box 2 subdomain, ExBx2), which likewise is implicated in mitogenic signaling. Through the expression of EPOR carboxyl-terminal truncation mutants in FDC-P1 cells, we presently show that an EPOR form truncated within the ExBx2 domain efficiently activates Jak2, yet is deficient in mitogenesis. Efficient expression of this mutant receptor at the cell surface and its ability to activate Jak2 indicate that poor mitogenic activity does not result from aberrant transport or folding. Rather, failure of this mutant to support proliferation above nominal rates underlines an apparent role for the EPOR ExBx2 subdomain in the activation of a distinct primary mitogenic effector.

Amino Acid Sequence↗

PubMed1994

In vitro transcription of erythroid promoters using baculoviral-expressed human GATA-1: purification, physicochemistry, and activities.

GATA-1 is a cys-2/cys-2 zinc finger transcriptional activator that is required for erythrocyte development in chimeric mice and contributes to the expression of all erythroid genes studied to date, including the erythropoietin receptor, glycophorin B, and porphobilinogen deaminase genes. Transactivation by GATA-1 is mediated by either an amino-terminal acidic domain, R1, or an independent adjacent domain, R2, and may involve the coordinate action of cofactors (NF-E2, EKLF, and Sp1) which bind adjacent cis-elements. To directly assess mechanisms of transactivation, we have developed an efficient cell-free transcription system using recombinant human GATA-1 (rhGATA-1) expressed in SF9 cells. Levels of baculoviral expression of GATA-1 were > or = 200-fold higher than endogenous levels in erythroid K562 cells. Factors from each source were essentially equivalent in molecular weight and DNA binding properties, and highly similar in phosphotryptic peptide composition. Notably, DNA binding was inhibited following treatment with alkaline phosphatase. In both SF9 and K562 cells, GATA-1 occurred largely as heterogeneous multimers, thus complicating its isolation by standard procedures. However, significant purification of this factor (> or = 100-fold; > or = 75% purity) was accomplished via DNA affinity chromatography. In cell-free assays, this rhGATA-1 was shown to be remarkably active in transactivating model erythroid promoters. This work establishes an efficient in vitro system for direct analyses of mechanisms, cofactors, and functional domains of GATA-1 which regulate transcription at defined proximal promoters.

Baculoviridae↗

PubMed1993

Association of the p85 regulatory subunit of phosphatidylinositol 3-kinase with an essential erythropoietin receptor subdomain.

Using an active, HAI epitope-tagged form of the murine erythropoietin (EPO) receptor and via direct coimmunoprecipitation, the p85 regulatory subunit of phosphatidyl inositol-3 kinase (p85/PI3-K) is shown to associate with the EPO receptor in transfected FDC-P1 cell lines. Coimmunoprecipitation of p85 with epitope-tagged EPO receptors was observed initially in FDC-HER cells labeled metabolically with [32P]orthophosphate, and association of these factors was confirmed by Western analyses of receptor immunoprecipitates using p85 antiserum. Interestingly, this association occurred in the absence of ligand, and exposure of FDC-HER cells to EPO did not detectably affect levels of receptor-associated p85 or overall levels of p85 phosphorylation. However, EPO was observed to stimulated the rapid formation of phosphatidylinositol 32P-phosphate in FDC-HER and FDC-ER cells. Through baculovirus-mediated expression of epitope-tagged EPO receptor forms in SF9 cells, domains for p85 association were mapped. Analyses of receptor forms with cytosolic truncations and deletions delineated a candidate subdomain for p85 binding to an essential extended box-2 region (P329-E374; including a putative motif for SH2 binding, Y343LVL). These findings extend a mechanistic alignment between the EPO receptor and protein tyrosine kinase-encoding receptors that likewise activate PI3-K, and expand the importance of further defining pathways to PI3-K activation.

Amino Acid Sequence↗

PubMed1993

Receptor-targeted transfection using stable maleimido-transferrin/thio-poly-L-lysine conjugates.

Cell surface ligand-receptor systems provide favorable routes for DNA transfection due to target cell specificity, transfer efficiency, and low toxicity. Using the transferrin receptor system as a model, an approach to transfection is developed here within which involves the complexing of DNA to stable maleimido-transferrin/thio-poly-L-lysine conjugates. These studies establish the importance of precise stoichiometry for activity of ligand:poly-L-lysine conjugates, as well as a chemistry for their controlled conjugation. Also considered quantitatively are effects of the following related parameters on the efficiency of receptor-mediated transfection: lysine polymer length, conjugate concentration, DNA:conjugate ratio, and treatment of target cells with chloroquine and desferrioxamine. Compared directly to standard procedures (electroporation, modified DEAE-dextran, lipofection, and modified Ca2PO4 protocols), transfection via this transferrin receptor-mediated system was > or = 10-fold more efficient, and essentially nontoxic to erythroleukemic F-MEL and J2E cells. Following transfection these cells retained the physiological capacity to undergo induced differentiation in response to dimethyl sulfoxide (F-MEL cells), or to erythropoietin (J2E cells), the natural hormonal regulator of erythropoiesis. Thus, an optimized approach to transferrin receptor-mediated transfection is developed which should be widely applicable for alternate cells and ligand-receptor systems both in vitro and in vivo.

Animals↗

PubMed1993

Signal transduction in the erythropoietin receptor system.

Developing erythroid cells require the glycoprotein hormone, erythropoietin (EPO) as an activator of the rapid proliferation of early proerythroblasts (colony forming units-erythroid [CFU-e]), and subsequently as an activator of late erythroid gene expression. Activation of these growth and differentiation events proceeds from the binding of EPO at its transmembrane receptor (Class I cytokine receptor), to the engagement of a complex set of signaling pathways. Studies of reconstituted activities of the cloned EPO receptor in transfected hematopoietic cell lines have served well in identifying receptor domains and downstream mediators involved in proliferative signaling. Extracellular domains have been defined which contribute to ligand binding, receptor processing and transport, and possible dimerization. Cytosolic regions have been delineated which mediate induced mitogenesis, early gene transcription, activated protein tyrosine phosphorylation, down modulation of EPO- and granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced proliferation, and direct association with PI3- and JAK-2 kinases. These newly defined properties begin to align the EPO receptor mechanistically with growth factor receptors (GFR) which encode, or likewise associate with, regulated protein tyrosine kinases including the Class II cytokine receptors for interferons alpha/beta and gamma. An improved understanding of factors which mediate EPO-induced late erythroid gene activation also is emerging. These factors and pathways may be distinct from those associated with EPO-induced proliferation and may involve induced increases in cellular Ca++, cAMP and arachidonic acid, as well as the modulation of GATA-1, and/or SCL. Attributes of model systems used in studies of the role of EPO in late erythroid differentiation also are considered.

Animals↗

PubMed1992

Cytokine-induced phosphorylation of pp100 in FDC-ER cells is at tyrosine residues.

Using FDC-P1 cells stably transfected with a murine erythropoietin receptor cDNA as a model, we recently have shown that erythropoietin (EPO), IL-3 and GM-CSF each induce the rapid phosphorylation of a common cytosolic target, i.e., a M(r) 100,000 phosphoprotein "pp100". Presently, we demonstrate that cytokine-induced phosphorylation of pp100 is primarily at tyrosine residues. This is shown by Western blotting with the anti-phosphotyrosine antibody PY20, and by the resistance of [32P]-pp100 to hydroxide-mediated hydrolysis of phosphates. These data, together with the recent observation by Linnekin et al. that pp100/p97 apparently associates directly with EPO receptors, suggest that pp100 may comprise an immediate common component in the signal transduction pathways of EPO, IL-3, GM-CSF and possibly other type I/II cytokine receptors. Additional analyses suggest that pp100 is distinct from a previously described M(r) 100,000 cytosolic target which is tyrosine phosphorylated in hematopoietic cells upon activation of T-cell receptors.

Animals↗

PubMed1992

Interleukin 3, granulocyte-macrophage colony-stimulating factor, and transfected erythropoietin receptors mediate tyrosine phosphorylation of a common cytosolic protein (pp100) in FDC-ER cells.

Receptors for the hematopoietic growth factors erythropoietin, interleukin 3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) are members of a structurally related receptor superfamily. Interestingly, while none of these receptors encode tyrosine kinase activities, induced tyrosine phosphorylation has been observed in various responsive cells stimulated with each factor. Toward defining possible common transduction pathways which are activated by these three cytokines, we have studied induced protein phosphorylation in murine myeloid FDC-P1 cells stably transfected with an erythropoietin receptor cDNA (FDC-ER cells). FDC-ER cells proliferate in response to erythropoietin (Quelle, D. E., and Wojchowski, D. M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 4801-4805), and presently are shown to rapidly phosphorylate a M(r) 100,000 cytosolic protein (pp100) at tyrosine residues in response to this factor. Phosphorylation of pp100 also is induced in FDC-P1 and FDC-ER cells in response to IL-3 or GM-CSF. Importantly, quantitative analyses showed identical concentration dependencies for factor-induced pp100 phosphorylation and induced cell proliferation. Moreover, a selective loss of proliferative responsiveness to GM-CSF in FDC-ER cells was associated with a reduced capacity of GM-CSF to induce pp100 phosphorylation. Finally, limited differences in tryptic phosphopeptide maps of pp100 as isolated following exposure to erythropoietin, IL-3, or GM-CSF were observed, suggesting that these factors also may preferentially induce phosphorylation of pp100 at distinct sites. These findings are consistent with a role for pp100 as a common cytosolic transducer in the apparently convergent pathways of erythropoietin-, IL-3-, and GM-CSF-induced proliferation of myeloid progenitor cells.

Animals↗

PubMed1992

Phosphorylatable and epitope-tagged human erythropoietins: utility and purification of native baculovirus-derived forms.

The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.

Amino Acid Sequence↗

PubMed1992

Mutations in the WSAWSE and cytosolic domains of the erythropoietin receptor affect signal transduction and ligand binding and internalization.

The terminal development of erythroid progenitor cells is promoted in part through the interaction of erythropoietin (EPO) with its cell surface receptor. This receptor and a growing family of related cytokine receptors share homologous extracellular features, including a well-conserved WSXWS motif. To explore the functional significance of this motif in the murine EPO receptor, five WSAWSE mutants were prepared and their signal-transducing, ligand binding, and endocytotic properties were compared. EPO receptors mutated at tryptophan residues (W-232, W-235----G; W-235----G; W-235----F) failed to mediate EPO-induced growth or pp100 phosphorylation, while S-236----T and E-237----K mutants exhibited partial to full activity (50 to 100% of wild-type growth and induced phosphorylation). Ligand affinity was reduced for mutant receptors (two- to fivefold), yet expression at the cell surface for all receptors was nearly equivalent. Also, the ability of mutated receptors to internalize ligand was either markedly reduced or abolished (W-235----F), indicating a role for the WSAWSE region in hormone internalization. Interestingly, receptor forms lacking 97% of the cytosolic domain (no signal-transducing capacity; binding affinity reduced two- to threefold) internalized EPO efficiently. This and all WSAWSE receptor forms studied also mediated specific cross-linking of 125I-EPO to three accessory membrane proteins (M(r)s, 120,000, 105,000, and 93,000). These findings suggest that the WSAWSE domain of the EPO receptor is important for EPO-induced signal transduction and ligand internalization. In contrast, although the cytosolic domain is required for growth signaling, it appears nonessential for efficient endocytosis.

Amino Acid Sequence↗

PubMed1991

Localized cytosolic domains of the erythropoietin receptor regulate growth signaling and down-modulate responsiveness to granulocyte-macrophage colony-stimulating factor.

Erythrocyte development in mammals depends in part upon the interaction of the glycopeptide hormone erythropoietin (EPO) with cell surface receptors on committed erythroid progenitor cells. Both this factor and an EPO receptor polypeptide previously have been cloned, yet little is presently understood concerning molecular mechanisms of receptor activation and signal transduction. To identify cytosolic receptor domains necessary for signaling, we have compared the activities of a series of deletionally mutated EPO receptor constructs by their expression in interleukin 3-dependent, myeloid FDC-P1 cells. EPO-induced growth was transduced efficiently in these cells by the full-length receptor (507 amino acids), and no measurable loss in activity resulted from the deletion of up to 111 carboxyl-terminal residues. In contrast, the deletion of 44 additional residues led to a dramatic loss (86.3% +/- 7.8%; mean +/- SD) in the ability of this receptor to mediate EPO-induced growth, thus indicating that residues between Gly-352 and Met-396 constitute a functionally critical cytosolic subdomain. Interestingly, the expression of full-length EPO receptors in FDC-P1 cells also led to a selective inhibition of normal proliferative responsiveness to the alternative hematopoietic factor granulocyte-macrophage colony-stimulating factor. Moreover, this inhibition was progressively reversed in forms of the EPO receptor in which distal cytosolic residues were sequentially deleted. These results suggest that EPO receptors normally may trans-modulate components in the pathway of granulocyte-macrophage colony-stimulating factor-induced proliferation and that this down-modulation, as exerted by intact EPO receptors, may play a role in promoting erythroid commitment during myeloid blood cell development.

Animals↗

PubMed1991

Proliferative action of erythropoietin is associated with rapid protein tyrosine phosphorylation in responsive B6SUt.EP cells.

Erythropoietin is a prime regulator of the growth and terminal differentiation of erythroid blood cells. However, little is understood concerning its molecular mechanism of action. Presently it is shown in the responsive, factor-dependent murine cell line B6SUt.EP that erythropoietin induces the tyrosine phosphorylation of six plasma membrane-associated proteins in a time- and concentration-dependent fashion (i.e. phosphoproteins PY153, PY140, PY100, PY93, PY74, and PY54). Among these, PY153 was prominent. For all proteins, maximal levels of phosphorylation were induced within 3-7 min at low factor concentrations (100-500 pM). These findings establish tyrosine kinase activation as a novel candidate pathway of erythropoietin-induced proliferation. In addition, the tyrosine phosphorylation of six proteins with identical Mr, as well as a Mr 104,000 protein, was induced in B6SUt.EP cells by interleukin 3. In contrast, no induced tyrosine phosphorylation was detectable in the erythropoietin-responsive, leukemic erythroid cell line. Rauscher Red 1, yet proteins of Mr 153,000 and 54,000 were shown to be phosphorylated constitutively at relative levels greater than those observed in B6SUt.EP cells. A possible role for these phosphoproteins in hematopoietic cell transformation is considered.

Animals↗