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Originally published In Press as doi:10.1074/jbc.M007042200 on August 11, 2000

J. Biol. Chem., Vol. 275, Issue 44, 34719-34727, November 3, 2000
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Protein Kinase C alpha  Controls Erythropoietin Receptor Signaling*

Marieke von LindernDagger §, Martine Parren-van AmelsvoortDagger , Thamar van DijkDagger , Evi Deiner, Emile van den AkkerDagger , Sjenet van Emst-de Vries||, Peter Willems||, Hartmut Beug, and Bob LöwenbergDagger

From the Dagger  Institute of Hematology, Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands, the  Institute of Molecular Pathology, Dr. Bohrgasse 7, A-1030 Vienna, Austria, and the || Department of Biochemistry, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands

Received for publication, August 4, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The erythropoietin receptor (EpoR)1 is essential for the regulation of proliferation, differentiation, and survival of erythroid cells (1, 2). It does not contain a kinase domain and depends on associated kinases for its function. Association of JAK2 with the EpoR is crucial for erythropoiesis (3, 4). In addition, Src family kinases such as LYN appear to function in EpoR signaling (5, 6). Following ligand binding the receptor conformation changes (7, 8), subsequently inducing phosphorylation and activation of associated kinases (9). Phosphorylation of cytoplasmic tyrosine residues of the EpoR creates docking sites for SH2 domain-containing signaling intermediates, which are in turn phosphorylated. Molecules recruited to the tyrosine-phosphorylated EpoR include SHP1, SHP2, Grb2, SHC, Gab-1, the p85 subunit of phosphoinositol 3-kinase (PI3K), and STAT5 (10-19).

Active PKC is important for the development of erythroid cells (20). In the presence of PKC inhibitors, Epo-induced mitogenesis is abrogated (21, 22), as is the ability of bone marrow cells to form erythroid colonies (colony-forming units-erythroid; Ref. 23). Inhibition of PKC also blocks Epo-induced activation of RAF1, ERK1 and -2, and AP-1 (24, 25) as well as up-regulation of Bcl-X, GATA-2, and c-Myc (26, 27). Therefore, PKC appears to have a major role in EpoR signal transduction. However, the signaling pathway(s) in which PKC is involved has not been established. Activation of PKC involves its translocation to the plasma membrane (28, 29), where it may come in close contact with receptor complexes. PKC has been implicated in functional regulation of several growth factor receptors. The tyrosine kinase receptors specific for SCF, epidermal growth factor, and insulin are phosphorylated on serine residues by activated PKC, which suppresses their tyrosine kinase activity (30, 31). PKC also affects signaling from the high affinity IgE receptor (Fcepsilon RI) on mast cells (32). This receptor recruits SYK, Src-like kinases, and the Tec family kinase Emt, which requires phosphorylation through PKC to be fully functional (33). Similarly, PKC phosphorylation of Bruton's tyrosine kinase, another Tec family kinase, is thought to be important for B-cell receptor signaling (34, 35).

PKC activation produces pleiotropic effects in cells, which is partly explained by the large variety of PKC isoforms. PKCalpha , -beta I, -beta II, and -gamma are classical, Ca2+-dependent kinases, while PKCdelta , -epsilon , -eta , -theta , and -µ function independent of Ca2+. PKC can be activated by phorbol ester, which results in degradation and depletion of certain isoforms (PKCalpha , -delta , and -epsilon ). The atypical PKC isoforms (PKCzeta , -lambda , and -iota ) function independent of phorbol esther (28). Certain isoforms are ubiquitously expressed (PKCalpha and -delta ); others are selectively expressed in specific cell types (28, 29). The large variety of PKC isoforms and the multitude of effects described have hampered studies concerning the biological role of PKC in proliferation and differentiation of hematopoietic cells.

We recently showed that primary, human erythroid progenitors have the capacity to undergo up to 20 renewal divisions in vitro in the presence of Epo, SCF, and dexamethasone resulting in a 106-fold increase of cell numbers (36). These erythroid precursors develop to mature erythrocytes during 3-5 cell divisions as soon as the "renewal factors" Epo, SCF, and dexamethasone are replaced by "differentiation factors" Epo and insulin. Renewal divisions are characterized by size control (maintenance of a constant cell size) and sustained low levels of hemoglobin. Differentiation divisions are characterized by a loss of size control, resulting in a ~4-fold reduction in cell volume and shortening of the G1 phase of the cell cycle, while cellular hemoglobin increases (37). Epo is required for both renewal and differentiation divisions, while SCF plus dexamethasone are essential to sustain renewal divisions with no or minimal differentiation (36, 38). Since PKC was reported to be essential for at least some effects of EpoR activation and appeared to inhibit SCF signaling, we set out to further define the role of PKC in erythropoiesis, particularly with respect to regulation of proliferation and differentiation of erythroid cells.

In this paper, we demonstrate that constitutive PKC activity is essential for phosphorylation and activation of the EpoR and multiple Epo-induced pathways. Our data suggest that PKC may control EpoR function directly or indirectly at the receptor level. In addition, LY294002, a specific inhibitor of phosphatidylinositide 3-kinase (PI3K), also reduced Epo-induced phosphorylation of multiple substrates, which could be partially reversed by activators of PKC.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Cell Culture-- Primary erythroid progenitors were grown from human bone marrow as described previously (36) in presence of recombinant human Epo (0.5 unit/ml, a kind gift from Janssen-Cilag, Tilburg, The Netherlands), recombinant human SCF (100 ng/ml, a kind gift from Amgen) and dexamethasone (5 × 10-7 M, Sigma). Cells were cultured at 1.5 - 3 × 106/ml through daily dilutions or medium changes with fresh medium containing factors. Cells were counted on an electronic cell counter (CASY-1, Schärfe-System, Germany). To induce terminal differentiation, cells were washed and transferred to medium with recombinant human Epo (5 units/ml), human insulin (1 unit/ml Actrapid, Bayer-Leverkusen) and a high concentration of iron-loaded transferrin (0.7 mg/ml).

The murine erythroid cell line LK-I/112 was cultured in StemPro medium (Life Technologies, Inc.) supplemented with Epo (0.5 unit/ml), SCF (100 ng/ml), and dexamethasone (5 × 10-7 M). Low molecular weight inhibitors GF109203X, chelerythrine, calphostin C, LY294002, and rapamycin were purchased from Biomol, phorbol 12-myristate 13-acetate (PMA) was from Sigma, and AG490 was a kind gift of Dr. A. Levitsky (Hebrew University, Jerusalem, Israel). All inhibitors were dissolved in Me2SO and diluted at least 500-fold in medium to obtain the final concentrations as indicated in the results.

Determination of Hemoglobin Accumulation and Cell Morphology-- To determine hemoglobin accumulation, three 50-µl aliquots of the cultures were removed and processed for photometric determination of hemoglobin (39). To analyze cell morphology, cells were cytocentrifuged onto slides and stained with histological dyes and neutral benzidine for hemoglobin (40). Images were taken using a CCD camera and processed with Adobe Photoshop.

Preparation of Cell Lysates, Immunoprecipitation, and Western Blotting-- Cultured cells were washed once with PBS and incubated in plain Iscove's medium without FCS or growth factors for 4 h at a density of 4 × 106/ml. If cells were factor-depleted for more than 4 h, they were incubated in serum-free medium containing BSA (1% w/v) as described previously (41). Cells were concentrated to 20-40 × 106/ml, stimulated 10 min at 37 °C with either 10 units/ml rhEpo or 1 µg/ml SCF. To stop the reaction, 10 volumes of ice-cold phosphate-buffered saline (PBS) supplemented with 10 µM Na3VO4 were added. Cells were pelleted and lysed for 30 min at 4 °C in lysis buffer (20 mM Tris-HCl, pH 8.0, 137 mM NaCl, 10 mM EDTA, 100 mM NaF, 1% (v/v) Nonidet P-40, 10% (v/v) glycerol, 2 mM Na3VO4, 1 mM Pefabloc SC, 50 µg/ml aprotinin, 50 µg/ml leupeptin, 50 µg/ml bacitracin, and 50 µg/ml iodoacetamide). Lysates used for JAK2 immunoprecipitation were prepared in a JAK2 buffer (50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 0.5% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mM Na3VO4, 1 mM Pefabloc SC, 50 µg/ml aprotinin, 50 µg/ml leupeptin, 50 µg/ml bacitracin, and 50 µg/ml iodoacetamide). Lysates were cleared by centrifugation at 4 °C for 15 min at 15,000 × g. Immunoprecipitations and Western blots were performed as described previously (42). In some instances, membranes were stripped in 62.5 mM Tris-HCl, pH 6.7, 2% (w/v) SDS, and 100 mM beta -mercaptoethanol at 50 °C for 30 min; reblocked; washed; and reprobed. Antibodies used were mouse monoclonal antibodies recognizing Grb2, SHP1, SHP2, LYN, or SHC (Transduction Laboratories, Lexington, KY); rabbit antisera recognizing JAK2, EpoR, or Gab1(Upstate Biotechnology, Lake Placid, NY); rabbit antisera recognizing the mouse EpoR, c-Kit, or p44/p42 mitogen-activated protein kinase (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit antisera recognizing phospho-PKB (Ser473) or PKB, mouse monoclonal antibodies recognizing phospho-p44/p42 mitogen-activated protein kinase (Thr202/Tyr204; New England Biolabs, Beverly, MA); and the anti-phosphotyrosine mouse monoclonal antibody 4G10 (Upstate Biotechnology). STAT5 antiserum was a kind gift of Dr. T. Decker (University of Vienna, Vienna, Austria). Subtype-specific PKC antibodies and the peptides against which the antibodies were raised were obtained from Life Technologies, Inc. (Paisley, Scotland).

Detection of Membrane-bound EpoR-- Human recombinant Epo was biotinylated using biotin-aminocaproyl-hydrazide as described previously (43). Cultured cells were washed and incubated in plain Iscove's medium without FCS or growth factors for 4 h in presence or absence of LY294002 (30 µM) or GF109203X (20 µM), or incubated in serum-free medium with BSA for 12 h in presence or absence of PMA (50 nM). Subsequently cells were stimulated with Epo (5 units/ml) or left untreated. The EpoR was detected using biotinylated Epo essentially as described previously (44). Cells were washed with PBS containing 1% FCS and 0.02% sodium azide and incubated in PBS/FCS/azide plus biotinylated Epo for 1 h at room temperature in presence or absence of a 100-fold excess of unlabeled Epo. Cells were washed, incubated with phycoerythrin-labeled streptavidin, washed, incubated with biotin-labeled anti-streptavidin, washed, incubated again with phycoerythrin-labeled streptavidin, and washed. Subsequently cells were analyzed by flow cytometry using fluorescence-activated cell sorting. Excess unlabeled Epo fully suppressed the signal obtained with biotinylated Epo, indicating specific detection of the EpoR.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay-- Nuclear extracts and STAT5 mobility shift assay were performed as described (41). The oligonucleotide probe used in this study was the beta -casein probe (5'-AGATTTCTAGGAATTCAATCC; Ref. 45). The DNA-protein complexes were separated by electrophoresis on 5% polyacrylamide gels containing 5% glycerol in 0.25× TBE. The gels were dried analyzed by autoradiography, and shifted probe was quantified on the ImageQuant PhosphorImager.

In Vitro Kinase Assay-- Sepharose beads with immune complexes were washed twice in lysis buffer and once in kinase buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM MgCl, 5 mM MnCl2, 0.5 mM dithiothreitol) at 4 °C. Beads were resuspended in 100 µl of kinase buffer supplemented with protease inhibitors (see above), 1 mM ATP (unlabeled), and 10 µCi of [gamma -32P]ATP (>5000 Ci/mmol, Amersham Pharmacia Biotech). The kinase reaction was incubated for 20 min at 22 °C. The reaction was stopped with an excess of ice-cold lysis buffer, and beads were washed twice in lysis buffer and once in PBS at 4 °C. Immune complexes were eluted by boiling for 5 min in sodium dodecyl sulfate (SDS) sample buffer. Following SDS-polyacrylamide gel electrophoresis, gels were dried and analyzed by autoradiography.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inhibition of PKC Blocks Epo-induced STAT5 DNA Binding-- To investigate whether inhibition of PKC impairs EpoR signaling, we first tested the effect of various PKC inhibitors on Epo-induced STAT5 DNA binding. Primary erythroid progenitors were factor-deprived in the presence of increasing concentrations of the PKC inhibitors GF109203X (blocking the ATP binding site; Ref. 46), chelerythrine (blocking the catalytic site; Ref. 47), calphostin C (blocking the regulatory site; Ref. 48), and Gö6976 (specific for Ca2+-dependent PKC subtypes; Ref. 49). Cells were stimulated with Epo, and nuclear extracts were tested for Epo-induced STAT5 DNA binding. All four inhibitors completely blocked Epo-induced STAT5 DNA binding in a dose-dependent manner (Fig. 1A). In control incubations the addition of solvent, Me2SO, had no effect on Epo-induced STAT5 DNA binding (data not shown). Cytological analysis indicated that incubation of the cells with the inhibitors did not induce any apparent cell death.


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  		Fig. 1.   Active PKC is required for Epo-induced STAT5 DNA binding. Human primary erythroid cells were factor-depleted for 4 h in the presence of various PKC inhibitors (A), the PI3K inhibitor LY294002, rapamycin (B), or the JAK2 inhibitor AG490 (C) at the concentrations indicated and stimulated 10 min. with Epo. Nuclear extracts were prepared and tested for STAT5 DNA binding by electrophoretic mobility shift assay, using a radioactively labeled probe from the beta -casein promoter. Only the slow migrating protein-DNA complex is shown.

We then tested the contribution of other pathways to Epo-induced Stat5 phosphorylation. First, PKC activity is often associated with PI3K activity. Addition of the PI3K inhibitor LY294002 inhibited Epo-induced STAT5 DNA binding as well (Fig. 1B). This was not due to inhibition of TOR (Target of Rapamycin) kinases (50), as addition of rapamycin had no effect on Epo-induced STAT5 DNA binding (Fig. 1B). Second, Epo-induced STAT5 phosphorylation was shown to depend on active JAK2 (45). Addition of the specific JAK2 inhibitor AG490 (51) resulted in concentration-dependent inhibition of STAT5 activation to an extent comparable to that induced by the PKC inhibitors (Fig. 1C).

PMA is a pleiotropic modulator of PKC activity. PMA activates PKC, but activation is followed by a rapid cleavage and depletion of certain PKC subtypes (29). We therefore evaluated the Epo response upon increasing exposure of the cells to PMA. Erythroid progenitors were factor-depleted for 8 h and subsequently stimulated with Epo. At various time points during starvation, GF109203X (20 µM) or PMA (50 nM) were added. Addition of GF109203 1-2 h before Epo stimulation of the cells largely inhibited Epo-induced STAT5 DNA binding (Fig. 2A). In contrast, short term exposure of the cells to PMA enhanced Epo-induced STAT5 DNA binding to 150% of control values. Upon longer exposure of the cells to PMA, Epo-induced STAT5 DNA binding was suppressed (Fig. 2A).


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  		Fig. 2.   Inhibition of Epo-induced STAT5 DNA binding is reversible and PI3K and PKC act in concert to control EpoR signaling. A, the effects of the PKC-activator PMA and the inhibitor GF109203X on Epo-induced STAT5 DNA binding were measured as a function of incubation time. The drugs were added to the human erythroid cells at the indicated times before stimulation of the cells with Epo. All cell samples were factor-depleted for 8 h. B and C, human erythroid cells were incubated with 20 µM chelerythrine (chel, B) or 30 µM LY294002 (LY, C). After 4 h, cells were washed to remove the inhibitors and recovery of Epo-induced STAT5 DNA binding was assessed (B and C, open circles). For this purpose, cells were stimulated with Epo at successive intervals following removal of the inhibitor. A parallel incubation with chelerythrine, re-added after washing, showed that the inhibition of Epo-induced STAT5 activation was maintained during the experiment (B). Addition of bryostatin (100 nM) in the continuing presence of LY294002 appeared to recover Epo-induced STAT5 DNA binding (C, closed symbols) The radioactive bands reflecting the gel-shifted probe were quantified on a ImageQuant PhosphorImager; results are displayed as percentage of probe shifted in absence of inhibitors.

In conclusion, four different inhibitors that block PKC activity via distinct mechanisms all prevented Epo-induced STAT5 DNA binding, while activation of PKC promoted Epo-induced STAT5 DNA binding followed by down-modulation. Addition of the PI3K inhibitor LY294002 or the JAK2 inhibitor AG490 also inhibited Epo-induced STAT5 DNA binding.

Abrogation of Epo-induced STAT5 DNA Binding by PKC Inhibitors Is Reversible, and PI3K and PKC May Act in Concert-- To verify that the inhibitors had no subtle effects on cell viability, we examined whether the inhibition of Epo-induced STAT5 DNA binding was reversible. Erythroid progenitors were factor-depleted in the presence of 20 µM chelerythrine or LY294002. Subsequently, cells were washed three times and re-seeded in medium lacking both inhibitor and Epo. At successive intervals, Epo-induced STAT5 DNA binding was assessed (Fig. 2, B and C). At 2-4 h after washing, Epo-induced STAT5 DNA binding appeared to be fully recoverable.

Since PKC inhibitors and LY294002 both resulted in a virtually complete suppression of EpoR signaling (Fig. 1), PI3K is likely to act within the same pathway as PKC. To examine whether PI3K would act up- or downstream of PKC, cells treated with LY294002 were subsequently stimulated with the PKC activators PMA and bryostatin (52). Both PKC activators restored Epo-induced STAT5 DNA binding in the continuing presence of LY294002 to near 100% of control values (Fig. 2C). However, in the same experiment, the PKC activators boosted Epo-induced STAT5 DNA binding to 180% of control levels in the absence of LY294002 (data not shown). Thus, activation of PKC can partially compensate a lack of PI3K activity. This would suggest that PKC and PI3K may act in concert to control Epo responsiveness.

PKC Inhibitors Do Not Alter the Kinetics of Epo-induced STAT5 Activation-- We next examined whether the PKC inhibitors altered the time kinetics of Epo-induced STAT5 activation. Cells were pretreated with 20 µM GF109203X or 100 nM Gö6976 and Epo-induced STAT5 DNA binding was analyzed at successive time intervals. In the presence of PKC inhibitor, the increment of STAT5 DNA binding was reduced as compared with untreated control cells. In both cases, maximal activation was reached at 20 min following the onset of Epo stimulation, indicating an effect on the magnitude rather than on the duration of the response (Fig. 3). In addition we examined whether the tyrosine phosphatase inhibitor sodium orthovanadate (Na3VO4) would restore Epo-induced STAT5 activation. Although Na3VO4 caused a general increase of DNA-bound STAT5 both in the presence and absence of PKC inhibitors, 10 µM Na3VO4 did not relieve suppression of Epo-induced STAT5 by PKC inhibitors (data not shown). Thus, reduction of Epo-induced STAT5 activation following inhibition of PKC does not result from accelerated down-regulation of EpoR signaling.


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  		Fig. 3.   PKC does not change the kinetics of Epo-induced STAT5 DNA binding in time. Human erythroid cells were incubated with the PKC inhibitors GF109203X (20 µM) and Gö6976 (100 nM), subsequently stimulated with Epo for 1, 2, 5, 10, 20, 40, and 60 min and STAT5 DNA binding was assessed in nuclear extracts. Shifted probe was quantified on a ImageQuant PhosphorImager, and results are displayed as relative units.

PKC Activity Is Required for the Activation of Multiple Signal Transduction Routes Initiated upon Binding of Epo to Its Receptor-- Inhibition of PKC could specifically affect Epo-induced STAT5 activation, or it could interfere with signaling from the EpoR in general. Human erythroid progenitors were preincubated with the PKC inhibitor GF109203X (20 µM), which suppressed Epo-induced phosphorylation of STAT5, the EpoR, and JAK2 in response to Epo (Fig. 4A). GF109203X had no effects in absence of Epo.


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  		Fig. 4.   Inhibition of PKC also impairs phosphorylation of the EpoR, Gab1, and ERK1/2. A, human erythroid cells were exposed to the PKC inhibitor GF109203X (GF, 20 µM) and subsequently stimulated with Epo. EpoR, STAT5, and JAK2 were immunoprecipitated (IP) from cell lysates, and phosphorylation on tyrosine was demonstrated on Western blots of the immune complexes, using the anti-phosphotyrosine antibody 4G10. The Western blots were reprobed with the antibodies used for the immunoprecipitation, to verify the identity of the tyrosine-phosphorylated proteins and to control equal loading. Total EpoR is just visible above the immunoglobulin band. B, in similar experiments murine erythroblasts (LK-I/11) were incubated with the inhibitors AG490 (AG, 50 µM), GF109203X (GF, 20 µM) or LY294002 (LY, 30 µM). The EpoR, JAK2, and Gab1 were immunoprecipitated and tested for tyrosine phosphorylation. Whole cell lysates were evaluated directly on Western blots for phosphorylated ERK1/2 (P-ERK) or total ERK1/2 (ERK) as a control. Whole cell lysates were also screened with the anti-phosphotyrosine antibody 4G10. The indicated tyrosine-phosphorylated proteins are the EpoR (#) and STAT5 (Dagger ). The position of size markers on the Western blot is indicated in kDa.

Similar to erythroid progenitors from human bone marrow, murine erythroid progenitors from fetal liver can be expanded in presence of Epo, SCF, and dexamethasone. These cells are grown in serum-free medium and are a much more reproducible and clean system for signal transduction studies in erythroid progenitors. Moreover, using p53-/- fetal liver cells, indefinite cultures of erythroblasts could be established reproducibly. Like human erythroblasts, the cells retain the capacity to differentiate in presence of Epo plus insulin.2 In a cloned culture of these mouse erythroid progenitors, LK-I/11, STAT5 was similarly inhibited by PKC inhibitors (data not shown). To examine which pathways are controlled by PKC and PI3K, the cells were pre-incubated with the JAK2 inhibitor AG490 (50 µM), GF109203X (20 µM), or LY294002 (30 µM). All three inhibitors suppressed Epo-induced tyrosine phosphorylation of the EpoR, STAT5, and Gab1 (Fig. 4B; data not shown). Furthermore, the inhibitors suppressed Epo-induced phosphorylation of ERK on Thr202/Tyr204 and Epo-induced tyrosine phosphorylation of several proteins detected in whole cell lysate (Fig. 4B). Exceptionally, Epo-induced JAK2 phosphorylation was reduced to a lesser extent (Fig. 4B).

In conclusion, active PKC is required not only for Epo-induced STAT5 DNA binding, but also for Epo-induced phosphorylation of the EpoR, and downstream targets both in human and mouse erythroid progenitors.

PKC Inhibitors Do Not Affect c-Kit Signal Transduction, while LY294002 Shows Partial but Distinct Control of c-Kit Function in Erythroid Progenitors-- Subsequently, we wanted to examine whether other receptors present in the same cells are similarly controlled by PKC. Murine erythroid progenitors were preincubated with increasing concentrations of the PKC inhibitor Gö6976 and subsequently stimulated with Epo or SCF. Ligand-induced phosphorylation of ERK1/2 and PKB was analyzed using phosphospecific antibodies on Western blots. Gö6976 inhibited Epo-induced phosphorylation of ERK and PKB, but it did not affect SCF-induced phosphorylation of these proteins (Fig. 5A). Similarly, Epo- and SCF-induced phosphorylation of ERK1/2 and PKB was assessed in LK-I/11 cells preincubated with increasing concentrations of the PI3K inhibitor LY294002. At 30 µM, LY294002 suppressed phosphorylation of ERK by Epo, while it did not affect SCF-induced ERK phosphorylation (Fig. 5B). Thus, active PKC and PI3K are required to render the EpoR permissive for ligand-induced signaling, but they do not control c-Kit signaling.


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  		Fig. 5.   The effects of PKC and PI3K inhibitors are not observed following SCF stimulation of erythroid cells. Murine LK-I/11 cells were exposed to increasing concentrations of the PKC inhibitor Gö6976 (A) or the PI3K inhibitor LY294002 (B) and subsequently stimulated with either Epo or SCF. Whole cell lysates were subjected to Western blot analysis and probed with antibodies specific for Thr202/Tyr204-phosphorylated ERK1/2 (P-ERK) or total ERK1/2, for Ser473 phosphorylated PKB (P-PKB) or total PKB.

Phosphorylation of PKB by Epo or SCF was completely suppressed at 7.5 µM LY294002. This efficient inhibition of Epo- and SCF-induced phosphorylation of PKB represents inhibition of Epo- or SCF-induced PI3K activity, as previously documented (10, 13, 14, 53). Apparently, the LY294002 concentration required to suppress EpoR activity is at least 5-fold higher than that required to inhibit PKB phosphorylation through either the EpoR or c-Kit. This suggests that inhibition of EpoR signaling may require a complete inhibition of all PI3K activity and not only inhibition of class Ialpha PI3K activated by the EpoR and c-Kit.

Expression of PKC Subtypes in Erythroid Progenitors-- Epo responses were blocked by Gö6976, a specific inhibitor of Ca2+-dependent PKC subtypes, implicating PKCalpha , -beta , or -gamma as the isoforms involved. We used isotype-specific antibodies to investigate which PKC subtypes are expressed in human primary erythroblasts. Of the Ca2+-dependent PKCs, only PKCalpha was expressed. Of the Ca2+-independent novel PKC subtypes, PKCepsilon , PKCeta , and PKCtheta were expressed as well as the atypical subtype PKCzeta (Table I). This suggests that PKCalpha may mediate transregulatory control of the EpoR complex.

                              
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Table I
Expression of PKC subtypes in human erythroid progenitors grown from bone marrow
PKC isotypes were assessed in primary erythroid progenitors grown from human bone marrow, using isotype-specific antibodies on a Western blot. As a control, the antibodies were pretreated with the peptides against which they were raised, which abrogated the specific signal.

Inhibition of PKC Suppresses Mainly Erythroid Differentiation-- Although PKC appears to be required for EpoR signaling, it has been shown to attenuate signaling by c-Kit (30). Erythroid "renewal conditions" require a low concentration of Epo plus SCF, whereas, instead, differentiation is induced at high concentrations of Epo in the absence of SCF. The use of mass cultures of primary erythroid progenitors allowed us to study two distinct biological end points of EpoR activation: (i) induction of sustained proliferation in co-operation with SCF plus dexamethasone, and (ii) induction of terminal differentiation in co-operation with insulin (36). We exposed human erythroid progenitors to these distinct conditions in the presence of GF109203X or calphostin C and examined whether inhibitors of PKC could affect the balance between erythroid proliferation and differentiation. The inhibitors were tested in independent experiments; because of some heterogeneity between distinct cultures of primary progenitors, these experiments have different time kinetics. We did not consider LY294002, since this inhibitor interferes both with EpoR signaling and activation of PI3K and PKB by Epo and SCF.

Addition of 4 µM GF109203X resulted in rapid decline of cell numbers under all conditions. At 2 and 1 µM GF109203, immature cells divided under renewal conditions. Total cell numbers increased, although the increase was less than the control culture (Fig. 6A) and morphological analysis revealed a significant proportion of blast cells showing mitosis (Fig. 6E). Under differentiation conditions, the same concentration of GF109203X fully inhibited differentiation and suppressed hemoglobin accumulation (Fig. 6B). Cytospins of control cultures under differentiation conditions reveal hemoglobinized and enucleating cells (Fig. 6E). In the presence of PKC inhibitors, exclusively small dead cells are detected under differentiation conditions (Fig. 6E). Addition of the PKC inhibitor calphostin C had effects similar to those of GF109203X. Cell death occurred under all conditions at 2 µM calphostin C. However, at 0.5-1 µM calphostin C, the number of immature cells increased under renewal conditions (Fig. 6C) and mitotic cells were detected in cytospins (Fig. 6E). Under differentiation conditions, 0.5-1 µM calphostin C prevented differentiation and hemoglobinization of the cells (Fig. 6, D and E). Cultures of primary erythroid progenitors always show some differentiating cells under renewal conditions (Fig. 6E, @). Since the PKC inhibitors prevent differentiation and cause cell death instead, under renewal conditions there will also be some dead cells in cultures exposed to these inhibitors (Fig. 6E, #).


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  		Fig. 6.   Partial inhibition of PKC interferes with erythroid differentiation, but not with proliferation. Human erythroid cells were transferred to colony-forming units-erythroid medium supplemented with Epo/SCF/dexamethasone or with Epo/insulin. GF109203X (GF, 1 or 2 µM; A, B, and E), or calphostin C (CalC, 1 or 0.5 µM, C-E) were added. A and C, to assess proliferation, cells were counted daily, kept at 1.5 to 3 × 106 cells/ml and total cell numbers were calculated (open symbols, renewal conditions, Epo, SCF, dexamethasone; closed symbols, differentiation conditions, Epo, insulin). B and D, hemoglobin levels per cell volume were determined at days 4 and 7 (R, renewal conditions, Epo, SCF, dexamethasone); D, differentiation conditions, Epo, insulin. E, to distinguish renewing, differentiating, and dead cells, cell samples from the indicated experiments were cytocentrifuged onto slides at day 4 and stained with histological dyes and neutral benzidine to reveal hemoglobin and histological details. Arrows indicate the various cell types. Maturing cells accumulate hemoglobin, visible by a brown staining of cytoplasma (@). Enucleation (@1) results in erythrocytes (@2). Actively dividing cells (*) are present under proliferation conditions. Dead cells (#) are evident from their small size, lack of hemoglobin, and condensed chromatin.

In conclusion, an intermediate PKC activity permits renewal divisions, which require low concentrations of Epo and full activity of c-Kit, while the same intermediate PKC activity is not sufficient to allow full activation of the EpoR, strong enough to promote erythroid differentiation. Thus, both the biochemical and the biological data are in accord with the idea that reduction of PKC to a certain threshold level specifically attenuates EpoR signaling, which inhibits differentiation but does not affect proliferation induced by cooperative SCF/Epo signaling.

PKC Affects Neither in Vitro JAK2 Kinase Activity Nor Its Association with the EpoR-- Inhibitors of PKC and PI3K suppressed Epo-induced tyrosine phosphorylation of the EpoR and JAK2, the earliest events in activation of the EpoR·JAK2 complex (Fig. 4B and data not shown). Using biotinylated Epo, we could demonstrate that inhibition of PKC and PI3K does not affect EpoR membrane expression (see "Experimental Procedures"; data not shown). This prompted us to investigate whether inhibition of PI3K and PKC would affect JAK2 kinase activity per se or the association of JAK2 with the EpoR. Primary human erythroid progenitors and murine erythroid cells (LK I/11) were preincubated with specific inhibitors: the JAK2 inhibitor AG490 (50 µM), the PKC inhibitor Gö6976 (500 nM), and the PI3K inhibitor LY294002 (30 µM). Subsequently, cells were stimulated with Epo, and JAK2 was immunoprecipitated from cell lysates. One half of the immune complex was tested on Western blot to verify that equal amounts of JAK2 were precipitated (data not shown), and the other half was used to assess in vitro kinase activity. Inhibitors were either used to preincubate the cells (in vivo treatment) or added during the JAK2 in vitro kinase assay, using lysates of Epo-stimulated, non-pretreated cells (in vitro treatment). In contrast to JAK2 tyrosine phosphorylation in vivo (measured by phosphotyrosine antibodies), which was strongly inducible by Epo (Fig. 4), JAK2 in vitro autophosphorylation was maximal in the presence or absence of Epo. Both in vivo and in vitro addition of AG490 inhibited JAK2 kinase activity. We assume that AG490 added in vivo remains associated with JAK2 during the immunoprecipitations. Significantly, the PKC and PI3K inhibitors had no effect on the in vitro kinase activity of JAK2, irrespective whether they were added in vivo or in vitro (Fig. 7A). The same lysates were examined for Epo-induced tyrosine phosphorylation of the EpoR and JAK2 and for tyrosine-phosphorylated proteins on a Western blot to verify that the inhibitors had been effective in these experiments (data conform to those shown in Fig. 4B). The experimental results indicate that PKC and PI3K inhibitors prevent Epo-induced phosphorylation of the EpoR, but that they do not affect JAK2 kinase activity per se, as determined in in vitro kinase assays.


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  		Fig. 7.   Inhibition of PKC or PI3K neither impairs JAK2 in vitro kinase activity per se, nor the association of JAK2 with the EpoR. A, JAK2 was immunoprecipitated from lysates of Epo-stimulated mouse erythroid cells (LK-I/11). Cells were incubated with AG490 (AG, 50 µM), Gö6976 (Go, 500 nM), or LY294002 (LY, 30 µM) during factor depletion, indicated as in vivo treatment. Immunocomplexes were collected by Sepharose beads and subjected to an in vitro kinase assay. As controls, Sepharose beads without antibody were incubated with cell lysates (no ab) to control for unspecific kinase activity. AG490 (10 µM), Gö6976 (100 nM), or LY294002 (6 µM) were also added during the kinase reaction of immunocomplexes of Epo-stimulated, non-pretreated cells, indicated as in vitro treatment. B, immune complexes precipitated by anti-EpoR antibodies from a similar set of lysates were subjected to an in vitro kinase assay according to the same protocol as panel A.

To analyze whether PKC inhibitors interfere with the association of the EpoR and JAK2, we determined JAK2 kinase activity co-immunoprecipitating with the EpoR from lysates of cells treated with the various inhibitors as described above. Immunocomplexes containing the EpoR were subjected to an in vitro kinase assay, which resulted in incorporation of radioactively labeled phosphate mainly into a 130-kDa protein co-migrating with JAK2. Addition of AG490 in vitro inhibited phosphorylation of this protein (Fig. 7B). Together, these results indicate that active JAK2 kinase is co-immunoprecipitated with the EpoR. Preincubation of the cells with AG490 also inhibited phosphorylation of JAK2. Since it is very well possible that AG490 remains bound to JAK2 (Fig. 7A), we cannot conclude that AG490 inhibits association of JAK2 with the EpoR. Significantly, the PKC and PI3K inhibitors had no effect on the in vitro kinase activity of JAK2 co-immunoprecipitating with the EpoR, suggesting that they do not interfere with the association of JAK2 with the EpoR (Fig. 7B). In parallel experiments, the tyrosine kinases LYN, SYK, and TEC were immunoprecipitated and their kinase activity was determined in the presence or absence of inhibitors. AG490 did not inhibit the in vitro kinase activity of these kinases (data not shown), confirming the in vitro specificity of AG490 with regard to tyrosine kinases as described previously (51).

Taken together, active PKC and PI3K are required for Epo-induced tyrosine phosphorylation of the EpoR in vivo. However, respective in vitro kinase assays neither reveal an effect of PKC and PI3K on JAK2 in vitro kinase activity per se nor an effect of PKC and PI3 on EpoR-associated JAK2 in vitro kinase activity. This suggests that PKC and PI3K may affect the assembly of the EpoR complex required for full activity in vivo, but which is not required for JAK2 in vitro autophosphorylation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

PKC appears to be an important player in Epo-induced phosphorylation of the EpoR itself and downstream targets like STAT5, Gab1, ERK, and PKB. This was shown using various inhibitors and activators of PKC. We assume that the relevant PKC-subtype involved could be PKCalpha , since Gö6976, an inhibitor of Ca2+-dependent PKC subtypes, efficiently blocked this PKC effect and PKCalpha appeared to be the only Ca2+-dependent PKC subtype expressed in primary erythroid cells. A recent study also identifies specifically PKCalpha as the PKC subtype involved in Epo-induced erythroid differentiation (20).

Small molecular inhibitors are easy to use on large numbers of cells without interfering with the cell's physiology. Transient transfections in primary erythroid cells are highly inefficient, or only applicable to cell numbers too small to perform signaling studies (55). Stable expression of mutated proteins may be incompatible with expansion of erythroid progenitors, or it could affect the formation of protein complexes such that the effect extends the function of the protein under study. However, the risk of inhibitors is their potential aspecific effects. Using PKC inhibitors it also must be considered that some compounds only block a subset of PKC isotypes efficiently. Depending on the cell type, distinct isotypes may have the same function. As a result, certain inhibitors can be active in some cell types, but not in others (56, 57). To eliminate all these risks, we used inhibitors that block the ATP binding site (GF109203X), the catalytic site (chelerythrine), or the regulatory domain (calphostin C), plus an inhibitor specific for Ca2+-dependent PKC-isotypes and an inhibitor that depletes PKC from the cell by proteolysis (PMA > 4 h). These inhibitors yielded consistent results at relevant concentrations. Moreover, activation of PKC by two distinct activators (bryostatin and PMA) yielded opposite results. Therefore, we judge the obtained results to be reliable.

PKC and PI3K Act in Concert to Control EpoR Signaling-- Although inhibitors of PKC block EpoR signaling completely, average Epo-induced STAT5 DNA binding is still 25% of control levels in the presence of LY294002. Activation of PKC in the presence of LY294002 restores EpoR signaling to the level of untreated cells, but does not enhance Epo-induced STAT5 DNA binding to levels obtained by bryostatin or PMA in the absence of LY294002.

Activation of PI3K family members generates phospholipids that have an important role in membrane localization of certain proteins, particularly through interaction with pleckstrin homology domains (58, 59). Upon activation, PKCalpha is also recruited to the plasma membrane. Thus, a putative target of PI3K and PKC may be recruited to the membrane by phospholipids, resulting in proximity of the target and PKCalpha and subsequent phosphorylation of the target by PKCalpha . Such a model could very well explain the observed results; although phosphorylation of the target is crucial for EpoR signaling, impaired recruitment to the membrane may be overcome partially by enhanced activation of PKC. Therefore, we propose that PKC and PI3K act in concert to render the EpoR permissive for ligand-induced signal transduction.

The concentration of LY294002 required to suppress Epo-induced phosphorylation of JAK2 and the EpoR was 5-10-fold more than the concentration required to inhibit Epo- and SCF-induced phosphorylation of the PI3K target protein kinase B (PKB, AKT). However, PKB is activated specifically by class Ialpha PI3K activated upon stimulation of the EpoR or c-KIT. In contrast, to deplete the membrane from 3-phosphoinositides, PI3K activity needs to be suppressed more widely and some PI3K family members are less sensitive to LY294002 (60, 61).

PKC May Act Upstream of the EpoR-- Inhibition of PKC has been reported to interfere with Epo-induced phosphorylation of RAF and ERK (25) and with transcriptional activation of BclX, GATA-2, c-MYC, and c-FOS (24, 26, 27). Epo-induced up-regulation of c-MYC required PKCepsilon activation (26). The data presented in this paper indicate that inhibition of PKC interferes with phosphorylation of the EpoR itself. In addition, we observed marked inhibition of EpoR activation by Gö6976, specific for Ca2+-dependent PKC subtypes, of which only PKCalpha is expressed. Therefore, we hypothesize that PKCalpha may act upstream of the EpoR, similar to costimulation of the B-cell receptor (35) and T-cell receptor (62, 63) in lymphoid cells, which may involve PKCalpha or beta  rather than PKCepsilon . This does not exclude an Epo-dependent activation of PKCepsilon and subsequent up-regulation of c-MYC as one of the signaling pathways triggered by the EpoR. The requirement for co-stimulation in EpoR signaling has not been described so far. However, the failure of Epo to induce STAT5 activation in erythroid progenitors derived from marrow of patients with myelodysplastic syndrome suggests that such co-stimulatory pathways may exist and that impaired erythropoiesis as found in myelodysplastic syndrome could possibly result from a failure of these pathways (41).

Potential Targets of PKC in the EpoR Signaling Complex-- Intriguingly, PKC is required for Epo-induced phosphorylation of the EpoR, whereas it affects JAK2 phosphorylation to a much lesser extent. This suggests that JAK2 has to interact with additional proteins to allow ligand-induced EpoR phosphorylation. Close examination of the JAK2 sequence reveals five putative PKC target sites (Ser299, Ser411, Ser605, Ser759, Ser1115; Ref. 29). Phosphorylation of these sites may be required for the recruitment of other proteins, essential for phosphorylation of the EpoR and downstream signaling intermediates. Alternatively, it could be the interacting proteins that require phosphorylation to interact. Interesting candidate targets in this respect are TEC family members, recruited to the membrane by their pleckstrin homology domain, able to associate with PKC (64) and implicated in the organization of a proper signaling complex around the T- and B-cell receptors (34, 35). TEC itself is mainly expressed in hematopoietic cells and was reported to bind and phosphorylate JAK2 (65). The Src family kinase LYN is also a potential target. In the murine erythroid cell line J2E, LYN deficiency results in largely reduced ligand-induced EpoR phosphorylation and terminal differentiation (5, 66). It is also possible that PKC, together with tyrosine kinases complementing JAK2, is required to maintain a signaling structure, the so-called "signalosome." Therefore, potential scaffolding proteins containing multiple protein-protein interaction domains like the PDZ domain also must be considered as potential targets for PKC (67, 68).

Effect of PKC on Erythroid Proliferation and Differentiation-- We showed that active PKC is required for EpoR signaling, while it was shown to attenuate c-Kit signaling (30). Renewal divisions of primary erythroblasts require low Epo levels while high Epo levels are required for terminal erythroid differentiation. Thus, PKC could control a balance between proliferation and differentiation of erythroid cells. Decreased PKC activity is expected to result in reduced EpoR, maximal c-Kit signaling, while activation of PKC would result in maximal EpoR, reduced c-Kit signaling. In accordance, we observed that inhibition of PKC allowed "renewal divisions" in the presence of Epo, SCF, and dexamethasone, while Epo-induced terminal differentiation was completely inhibited. In the cultures used, the immature cells lack glycophorin A expression, while induction of differentiation is accompanied by increased glycophorin A expression. Thus, our data comply with recently published data demonstrating the requirement of PKCalpha for the development of CD34+ progenitors into GPA-positive erythroid cells (20). In this study, however, no discrimination between expansion and differentiation could be made.

What could be the primary signals regulating PKC activity in erythroid cells? Obvious candidates are ligands for seven-transmembrane-spanning receptors, which activate PKC via Galpha q subunits of heterotrimeric G proteins. Erythroid progenitors express a large number of such PKCalpha -activating serpentine receptors, such as the receptors for thrombin, adenosine (P2Y), and acetylcholine (69), all representing possible candidates warranting further study.

    ACKNOWLEDGEMENTS

We thank Drs. Oosterhuis and Kruithof (Janssen-Cilag B.V., Tilburg, The Netherlands) for their generous supply of recombinant human Epo, Dr. T. Decker for a generous gift of anti-STAT5 antiserum, Dr. A. Levitsky for the JAK2 inhibitor AG490, and Drs. Ivo Touw and Alister Ward for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by Dutch Cancer Society Grants EUR 95-1021 and EUR 99-2064, European Community Grant BMH4-CT 96 1355, and a fellowship from the Dutch Academy for Arts and Sciences (to M. v. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Inst. of Hematology, Erasmus University, P. O. Box 1738, 3000 DR Rotterdam, The Netherlands. Tel.: 31-10-408-7961; Fax: 31-10-408-9470; E-mail: vonlindern@hema.fgg.eur.nl.

Published, JBC Papers in Press, August 11, 2000, DOI 10.1074/jbc.M007042200

2 E. Deiner, M. von Lindern, and H. Beug, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: EpoR, erythropoietin receptor; Epo, erythropoietin; SCF, stem cell factor; PKC, protein kinase C; PI3K, phosphoinositol 3-kinase; SH, Src homology; STAT, signal transducer and activator of transcription; ERK, extracellular signal-regulated kinase; PKB, protein kinase B; PMA, phorbol 12-myristate 13-acetate; PBS, phosphate-buffered saline; FCS, fetal calf serum.

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ABSTRACT
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DISCUSSION
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E. van den Akker, T. van Dijk, M. Parren-van Amelsvoort, K. S. Grossmann, U. Schaeper, K. Toney-Earley, S. E. Waltz, B. Lowenberg, and M. von Lindern
Tyrosine kinase receptor RON functions downstream of the erythropoietin receptor to induce expansion of erythroid progenitors
Blood, June 15, 2004; 103(12): 4457 - 4465.
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U. Schmidt, E. van den Akker, M. Parren-van Amelsvoort, G. Litos, M. de Bruijn, L. Gutierrez, R. W. Hendriks, W. Ellmeier, B. Lowenberg, H. Beug, et al.
Btk Is Required for an Efficient Response to Erythropoietin and for SCF-controlled Protection against TRAIL in Erythroid Progenitors
J. Exp. Med., March 15, 2004; 199(6): 785 - 795.
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I. Lebigot, P. Gardellin, L. Lefebvre, H. Beug, J. Ghysdael, and C. T. Quang
Up-regulation of SLAP in FLI-1-transformed erythroblasts interferes with EpoR signaling
Blood, December 15, 2003; 102(13): 4555 - 4562.
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K. Li, C. Miller, S. Hegde, and D. Wojchowski
Roles for an Epo Receptor Tyr-343 Stat5 Pathway in Proliferative Co-signaling with Kit
J. Biol. Chem., October 17, 2003; 278(42): 40702 - 40709.
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A. D. Friedman, D. Nimbalkar, and F. W. Quelle
Erythropoietin Receptors Associate with a Ubiquitin Ligase, p33RUL, and Require Its Activity for Erythropoietin-induced Proliferation
J. Biol. Chem., July 11, 2003; 278(29): 26851 - 26861.
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J. W. Fisher
Erythropoietin: Physiology and Pharmacology Update
Experimental Biology and Medicine, January 1, 2003; 228(1): 1 - 14.
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C. Ito, E. Kusano, Y. Furukawa, H. Yamamoto, S.-I. Takeda, T. Akimoto, O. Iimura, Y. Ando, and Y. Asano
Modulation of the erythropoietin-induced proliferative pathway by cAMP in vascular smooth muscle cells
Am J Physiol Cell Physiol, December 1, 2002; 283(6): C1715 - C1721.
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R. L. Darley, L. Pearn, N. Omidvar, M. Sweeney, J. Fisher, S. Phillips, T. Hoy, and A. K. Burnett
Protein kinase C mediates mutant N-Ras-induced developmental abnormalities in normal human erythroid cells
Blood, December 1, 2002; 100(12): 4185 - 4192.
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Y. Saito, Y. Hojo, T. Tanimoto, J.-i. Abe, and B. C. Berk
Protein Kinase C-alpha and Protein Kinase C-epsilon Are Required for Grb2-associated Binder-1 Tyrosine Phosphorylation in Response to Platelet-derived Growth Factor
J. Biol. Chem., June 21, 2002; 277(26): 23216 - 23222.
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