Association and activation of Jak-Tyk kinases by CNTF-LIF-OSM-IL-6 beta receptor components.

A recently defined family of cytokines, consisting of ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM), and interleukin-6 (IL-6), utilize the Jak-Tyk family of cytoplasmic tyrosine kinases. The beta receptor components for this cytokine family, gp130 and LIF receptor beta, constitutively associate with Jak-Tyk kinases. Activation of these kinases occurs as a result of ligand-induced dimerization of the receptor beta components. Unlike other cytokine receptors studied to date, the receptors for the CNTF cytokine family utilize all known members of the Jak-Tyk family, but induce distinct patterns of Jak-Tyk phosphorylation in different cell lines.

Signaling by the cytokine receptor superfamily: JAKs and STATs.

A variety of cytokines, lymphokines and growth factors function by interacting with receptors that are members of the cytokine receptor superfamily. These receptors share extracellular motifs and have limited similarity in their cytoplasmic domains. Although lacking catalytic domains, this family of receptors couples ligand binding with the induction of tyrosine phosphorylation. Recent studies have shown that this is mediated by members of the Janus kinase (JAK) family of cytoplasmic protein tyrosine kinases. JAKs physically associate with the membrane-proximal region of the ligand-bound receptor, leading to their tyrosine phosphorylation and activation. The activated JAKs phosphorylate the receptors as well as cytoplasmic proteins belonging to a family of transcription factors called the signal transducers and activators of transcription (STATs), providing a novel signaling pathway that is shared by all members of the cytokine receptor superfamily.

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.

Dna damage-induced G(1) arrest in hematopoietic cells is overridden following phosphatidylinositol 3-kinase-dependent activation of cyclin-dependent kinase 2.

Exposure of hematopoietic cells to DNA-damaging agents induces p53-independent cell cycle arrest at a G(1) checkpoint. Previously, we have shown that this growth arrest can be overridden by cytokine growth factors, such as erythropoietin or interleukin-3, through activation of a phosphatidylinositol 3-kinase (PI 3-kinase)/Akt-dependent signaling pathway. Here, we show that gamma-irradiated murine myeloid 32D cells arrest in G(1) with active cyclin D-cyclin-dependent kinase 4 (Cdk4) but with inactive cyclin E-Cdk2 kinases. The arrest was associated with elevated levels of the Cdk inhibitors p21(Cip1) and p27(Kip1), yet neither was associated with Cdk2. Instead, irradiation-induced inhibition of cyclin E-Cdk2 correlated with absence of the activating threonine-160 phosphorylation on Cdk2. Cytokine treatment of irradiated cells induced Cdk2 phosphorylation and activation, and cells entered into S phase despite sustained high-level expression of p21 and p27. Notably, the PI 3-kinase inhibitor, LY294002, completely blocked cytokine-induced Cdk2 activation and cell growth in irradiated 32D cells but not in nonirradiated cells. Together, these findings demonstrate a novel mechanism underlying the DNA damage-induced G(1) arrest of hematopoietic cells, that is, inhibition of Cdk2 phosphorylation and activation. These observations link PI 3-kinase signaling pathways with the regulation of Cdk2 activity.

Erythropoietin induces activation of Stat5 through association with specific tyrosines on the receptor that are not required for a mitogenic response.

The cytoplasmic domain of the erythropoietin receptor (EpoR) contains a membrane-distal region that is dispensable for mitogenesis but is required for the recruitment and tyrosine phosphorylation of a variety of signaling proteins. The membrane-proximal region of 96 amino acids is necessary and sufficient for mitogenesis as well as Jak2 activation, induction of c-fos, c-myc, cis, the T-cell receptor gamma locus (TCR-gamma), and c-pim-1. The studies presented here demonstrate that this region is also necessary and sufficient for the activation of Stat5A and Stat5B. The membrane-proximal domain contains a single tyrosine, Y-343, which when mutated eliminates the ability of the receptor to couple Epo binding to the activation of Stat5. Furthermore, peptide competitions demonstrate that this site, when phosphorylated, can disrupt Stat5 DNA binding activity, consistent with a role of Y-343 as a site of recruitment to the receptor. Cells expressing the truncated, Y343F mutant (a mutant with a Y-to-F alteration at position 343) proliferate in response to Epo in a manner comparable to that of the controls. However, in these cells, Epo stimulation does not induce the appearance of transcripts for cis, TCR-gamma, or c-fos, suggesting a role for Stat5 in their regulation.

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.

Stat4, a novel gamma interferon activation site-binding protein expressed in early myeloid differentiation.

Interferon regulation of gene expression is dependent on the tyrosine phosphorylation and activation of the DNA-binding activity of two related proteins of 91 kDa (STAT1) and/or 113 kDa (STAT2). Recent studies have suggested that these proteins are substrates of Janus kinases and that proteins related in STAT1 are involved in a number of signalling pathways, including those activated in myeloid cells by erythropoietin and interleukin-3 (IL-3). To clone STAT-related proteins from myeloid cells, degenerate oligonucleotides were used in PCRs to identify novel family members expressed in myeloid cells. This approach allowed the identification and cloning of the Stat4 gene, which is 52% identical to STAT1. Unlike STAT1, Stat4 expression is restricted but includes myeloid cells and spermatogonia. In the erythroid lineage, Stat4 expression is differentially regulated during differentiation. Functionally, Stat4 has the properties of other STAT family genes. In particular, cotransfection of expression constructs for Stat4 and Jak1 and Jak2 results in the tyrosine phosphorylation of Stat4 and the acquisition of the ability to bind to the gamma interferon (IFN-gamma)-activated sequence of the interferon regulatory factor 1 (IRF-1) gene. Stat4 is located on mouse chromosome 1 and is tightly linked to the Stat1 gene, suggesting that the genes arose by gene duplication. Unlike Stat1, neither IFN-alpha nor IFN-gamma activates Stat4. Nor is Stat4 activated in myeloid cells by a number of cytokines, including erythropoietin, IL-3, granulocyte colony-stimulating factor, stem cell factor, colon-stimulating factor 1, hepatocyte growth factor, IL-2, IL-4, and IL-6.

Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis.

By searching a database of expressed sequences, we identified a member of the signal transducers and activators of transcription (Stat) family of proteins. Human and murine full-length cDNA clones were obtained and sequenced. The sequence of the human cDNA was identical to the recently published sequence for interleukin-4 (IL-4)-Stat (J. Hou, U. Schindler, W.J. Henzel, T.C. Ho, M. Brasseur, and S. L. McKnight, Science 265:1701-1706, 1994), while the murine Stat6 amino acid and nucleotide sequences were 83 and 84% identical to the human sequences, respectively. Using Stat6-specific antiserum, we demonstrated that Stat6 is rapidly tyrosine phosphorylated following stimulation of appropriate cell lines with IL-4 or IL-3 but is not detectably phosphorylated following stimulation with IL-2, IL-12, or erythropoietin. In contrast, IL-2, IL-3, and erythropoietin induce the tyrosine phosphorylation of Stat5 while IL-12 uniquely induces the tyrosine phosphorylation of Stat4. Inducible tyrosine phosphorylation of Stat6 requires the membrane-distal region of the IL-4 receptor alpha chain. This region of the receptor is not required for cell growth, demonstrating that Stat6 tyrosine phosphorylation does not contribute to mitogenesis.

[3H]dexamethasone binding to plasma membrane-enriched fractions from liver of nonadrenalectomized rats.

Using liver from nonadrenalectomized adult male rats, binding sites for [3H]dexamethasone in particulate fractions are demonstrated. The binding is thermolabile, saturable, and specific for glucocorticoid. The apparent dissociation constant (Kdapp) for [3H]dexamethasone (0.48 +/- 0.084 microM) is 60-fold greater than that for cytosolic receptor (7.9 +/- 1.5 nM). The Kdapp for [3H]cortisol in particulate fractions is 2.5-fold lower than for [3H]dexamethasone (Kdapp = 0.18 microM). The binding capacities for particulate and cytosolic glucocorticoid-binding sites also differ significantly, with particulate sites at least 9.1-fold more concentrated than cytosolic sites in liver tissue. Particulate sites are determined in Percoll density gradients to have a density of 1.039 g/cc. Saturable [3H]dexamethasone radioactivity coelutes from these gradients with the plasma membrane marker enzyme 5'-nucleotidase. Adrenalectomy causes the complete loss of particulate binding sites by 6 days postadrenalectomy; however, these sites can be regenerated to two thirds of the nonadrenalectomy level by 20-30 days postadrenalectomy.

Thermal distortion of diffraction-limited optical elements.

Recent developments of laser materials have advanced the state of the art to a point where the optical quality of many of these materials is approaching the diffraction limit. Using such components in a laser does not necessarily guarantee the generation of diffraction-limited laser beams. One of the severe problems is thermal distortion introduced in the optical cavity by the flash lamps. Ruby and glass lasers require a minimum of 0.6 J of heat deposition per joule of population inversion. Typical figures are 4-6 J per joule of population inversion.(1) Nonuniformities in the deposition of this heat cause optical distortions which virtually preclude diffraction-limited laser operation even if the materials themselves are of diffraction-limited optical quality. This paper will investigate these thermal effects in detail, and the relative sensitivity of a variety of materials to nonuniform energy depositions will be discussed. Water close to its point of maximum density and a certain special type of glass known as Pockels glass will be shown to have properties of particular interest for use in diffraction-limited lasers.

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.

Signal transduction through the receptor for erythropoietin.

Erythropoietin (EPO) supports the proliferation and differentiation of erythroid lineage cells. The receptor for erythropoietin is a member of the cytokine receptor family. Introduction of EPOR into IL-3 dependent cells confers the ability to proliferate in response to EPO. Associated with this, EPO induces the expression of a number of immediate-early response genes. Mutagenesis studies have addressed the function role of various motifs and domains in receptor function and established essential roles for the conserved cysteines and the WSXWS motif. The signal transducing pathways activated by EPOR include induction of tyrosine phosphorylation. Within the cytoplasmic domain a relatively small membrane proximal region is essential for induction of tyrosine phosphorylation, expression of immediate early genes and for mitogenesis. The role of various kinases in this response is discussed as well as an assessment of potential substrates of tyrosine phosphorylation.

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.

Cytokine receptors and signal transduction.

The past few years have seen an explosion in the identification, cloning and characterization of cytokines and their receptors. The pleiotropic effects of many of the growth factors and the considerable redundancy in the actions of growth factors have contributed to a mass of descriptive literature that often seems to defy summary. Only recently have common concepts begun to emerge. First, cytokines mediate their effects through a large family of receptors that have evolved from a common progenitor and retain structural and functional similarities. Within the haematopoietic system, the cytokines are not usually instructive in differentiation, but rather supportive, and may contribute to some differentiation-specific responses. The patterns of expression of cytokine receptors are therefore a product of differentiation and provide for changes in physiological regulation. The second important concept that is emerging is that the cytokines mediate their mitogenic effects through a common signal-transducing pathway involving tyrosine phosphorylation. Thus, although the cytokine receptor superfamily members do not have intrinsic protein tyrosine kinase activity, by coupling to activation of tyrosine phosphorylation they may affect cell growth by pathways that are common with the large family of growth factor receptors that contain intrinsic protein tyrosine kinase activity. The coupling of cytokine binding to tyrosine phosphorylation and mitogenesis requires a relatively small membrane-proximal domain of the receptors. This region has limited sequence similarity which may be required for the association of individual receptors with an appropriate kinase. Activation of kinase activity results from the dimerization or oligomerization of receptor homodimers or heterodimers. Again this requirement is similar to that seen with the growth factor receptors which have intrinsic protein tyrosine kinase activity. The protein tyrosine kinases that couple cytokine binding to tyrosine phosphorylation are members of the Jak family of kinases. The ubiquitous expression of these kinases provides a common cellular background on which the cytokine receptors can function and on which unique functionally distinct receptors have evolved. In particular, tyk2 is required for the responses initiated by IFN-alpha while Jak2 has been implicated in the responses to G-CSF, IL-3, EPO, growth hormone, prolactin and IFN-gamma.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA damage-induced cell-cycle arrest of hematopoietic cells is overridden by activation of the PI-3 kinase/Akt signaling pathway.

Exposure of hematopoietic cells to DNA-damaging agents induces cell-cycle arrest at G1 and G2/M checkpoints. Previously, it was shown that DNA damage-induced growth arrest of hematopoietic cells can be overridden by treatment with cytokine growth factors, such as erythropoietin (EPO) or interleukin-3 (IL-3). Here, the cytokine-activated signaling pathways required to override G1 and G2/M checkpoints induced by gamma-irradiation (gamma-IR) are characterized. Using factor-dependent myeloid cells stably expressing EPO receptor (EPO-R) mutants, it is shown that removal of a minimal domain required for PI-3K signaling abrogated the ability of EPO to override gamma-IR-induced cell-cycle arrest. Similarly, the ability of cytokines to override gamma-IR-induced arrest was abolished by an inhibitor of PI-3K (LY294002) or by overexpression of dominant-negative Akt. Moreover, the ability of EPO to override these checkpoints in cells expressing defective EPO-R mutants could be restored by overexpression of a constitutively active Akt. Thus, activation of a PI-3K/Akt signaling pathway is required for cytokine-dependent suppression of DNA-damage induced checkpoints. Together, these findings suggest a novel role for PI-3K/Akt pathways in the modulation of growth arrest responses to DNA damage in hematopoietic cells. (Blood. 2001;98:834-841)

High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector.

Conditions presently have been established for the high-level expression and simplified purification of recombinant human erythropoietin produced in Spodoptera frugiperda cells. Expression, as mediated by infection with a recombinant baculovirus, was accomplished in suspension culture using reduced levels of serum and media supplements experimentally determined to provide optimum levels of factor production (500,000 U/L). Purification of this recombinant human erythropoietin to virtual homogeneity (greater than or equal to 99%) was accomplished via a simple three-step procedure involving isocratic elution from DEAE-Sephacel, reverse-phase high performance liquid chromatography (HPLC) on a C4 medium, and the single-step elution of purified hormone from concanavalin A agarose. Overall, an 890-fold purification was accomplished with a recovery of 80% as assayed in vitro. Biologically, this purified erythropoietin is highly active, possessing a specific activity in vitro of 200,000 U/mg protein. Chemically, this erythropoietin (molecular weight [mol wt] 26,200) appears exceptionally uniform in its oligosaccharide constitution (30%) as contrasted with heterogeneously glycosylated erythropoietins derived from mammalian cells (mol wt 30,000 to 38,000; 40% to 50% complex-type oligosaccharide). Thus, human erythropoietin as presently produced in an insect cell line comprises not only an abundant source of highly active, readily purified hormone for studies of its mechanism of action and cell surface receptor, but also represents a uniquely homogeneous form that should prove advantageous for direct structural analyses.

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.

Signaling through the hematopoietic cytokine receptors.

Hematopoiesis is regulated through the interaction of a variety of growth factors with specific receptors of the cytokine receptor superfamily. Although lacking catalytic domains, all the receptors couple ligand binding to the rapid induction of protein tyrosine phosphorylation. This is mediated through a novel family of protein tyrosine kinases termed the Janus kinases (Jaks) which associate with the receptors and are activated following ligand binding. Depending upon the cytokine/receptor system, one or more of the four known Jaks (Jak1, Jak2, Jak3, Tyk2) is/are involved. The activated Jaks phosphorylate both themselves and the receptor subunits, creating docking sites for SH2-containing proteins including SHC, which couples receptor engagement to activation of the ras pathway, and HCP, a protein tyrosine phosphatase which negatively affects the response. In addition, the Jaks phosphorylate one or more of a family of signal transducers and activators of transcription (Stats). Phosphorylation of Stats induces their nuclear translocation and DNA-binding activity. Activation of Stats is independent of activation of the ras pathway and represents a novel signaling pathway correlated with mitogenesis.

Jaks and Stats in cytokine signaling.

Hematopoiesis is regulated through the binding of cytokines to receptors of the cytokine receptor superfamily. Although lacking catalytic domains, members of the cytokine receptor superfamily mediate ligand-dependent activation of protein tyrosine phosphorylation through their association and activation of members of the Janus kinase (Jak) family of protein tyrosine kinases. The activated Jaks phosphorylate the receptors which creates docking sites for SH2-containing signaling proteins which are tyrosine phosphorylated following their association with the complex. Among the substrates of tyrosine phosphorylation are members of the signal transducers and activators of the transcription family of proteins (Stats). Various cytokines induce the tyrosine phosphorylation and activation of one or more of the seven family members. The pattern of Stat activation provides a level of cytokine individuality that is not observed in the activation of other signaling pathways. The role of various Stats in the biological responses to cytokines has been assessed through the analysis of receptor mutations which disrupt Stat activation and more recently by disruption of the genes in mice. Our results have demonstrated that the activation of Stat5a and Stat5b by erythropoietin is critical for the activation of a number of immediate early genes but is not required for a mitogenic response. Mice in which the genes for Stat4 and Stat6 are disrupted are viable but lack functions that are mediated by interleukin 12 (IL-12) or IL-4, respectively, suggesting that these Stats perform very specific functions in immune responses.

Induction of tyrosine phosphorylation of Vav and expression of Pim-1 correlates with Jak2-mediated growth signaling from the erythropoietin receptor.

The receptor for erythropoietin (Epo) belongs to the cytokine receptor family and lacks a tyrosine kinase domain. However, it has been hypothesized that a tyrosine kinase, Jak2, associates with the membrane proximal cytoplasmic region of Epo receptor (EpoR) and mediates the growth signaling from the receptor through tyrosine phosphorylation of cellular substrates. To explore the growth signaling pathways from the EpoR, we analyzed substrates of tyrosine phosphorylation induced by Epo stimulation in cells expressing various mutant EpoRs. The vav proto-oncogene product was found to be tyrosine phosphorylated after Epo stimulation in cells expressing the wild-type EpoR or a truncated receptor, H mutant, that retains the growth signaling function. In these cells, Epo also induced the expression of a serine/threonine kinase, Pim-1. However, Epo stimulation did not have any effect on Vav or Pim-1 in cells expressing a mutant EpoR, PM4 mutant, inactivated by a point mutation, Trp282 to Arg, in the membrane proximal region, which abrogates the interaction with Jak2. On the other hand, both tyrosine phosphorylation of Vav and expression of Pim-1 were observed constitutively in cells expressing a mutant EpoR that is constitutively activated by a point mutation, Arg 129 to Cys, in the extracellular domain. Jak2 was also constitutively tyrosine phosphorylated and activated in cells expressing this mutant, which confirms the crucial role of Jak2 in growth signaling from the EpoR. Taken together, these observations suggest that the tyrosine phosphorylation of Vav and the expression of Pim-1 may play important roles in growth signaling from the EpoR.