Vol. 168, No. 2, 1990 April 30, 1990
RAPID
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 490-497
ACTIVATION BY ERYTHROPOIETIN OF PROTEIN KINASE NUCLEI OF ERYTHROID PROGENITOR CELLS Meredith Mason-Garcial,
C IN
Cheryl L. Weill2, and Barbara S. Beckman1
1Department of Pharmacology, Tulane University School of Medicine, and 2Departments of Anatomy and Neurology, Louisiana State University School of Medicine, New Orleans, Louisiana 70112 Received
February
27,
1990
SUMMARY: The glycoprotein hormone erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells by a signal transduction system which is not well understood. It has recently been reported that prolactin, a mitogen and trophic hormone for liver, will activate a nuclear protein kinase C in hepatocytes. As similarities exist in the actions of Ep and prolactin in their target cells, we tested the hypothesis that Ep could activate protein kinase C in nuclei isolated from erythroid progenitor cells. In a pure population of such nuclei, Ep induced a rapid, time- and dose-dependent increase in phosphorylation of endogenous nuclear substrate which could be blocked by inhibitors of protein kinase C or by antibody to Ep. Other known activators of protein kinase C were also effective in this system. These findings show that Ep may exert its effects by a novel signalling pathway, the activation of a nuclear protein kinase C. 01990 Academic Press, Inc.
Erythropoietin (Ep) is a glycoprotein hormone (MW=34,000) which acts at a specific cell surface receptor to induce both proliferation and differentiation in its target erythroid progenitor cell, the functionally characterized colony forming unit-erythroid (CFU-E) (1). Recently, the Ep receptor was cloned (2) and found to consist of a 507 amino acid polypeptide with a single predicted membrane-spanning domain and without homology to any known protein. The ligand-receptor complex formed by the binding of Ep to this receptor undergoes rapid endocytosis (3), which suggests that the mechanism of action of Ep may be mediated in part by this event . The signal transduction system which is activated by Ep remains largely uncharacterized, despite many years of research devoted to its elucidation (for a review, see 1). It is clear that an Ep receptor-mediated activation of adenylate cyclase or guanylate cyclase does not occur, although cyclic AMP and cyclic GMP may play Abbreviations: Ep, erythropoietin; PKC, protein kinase C; H-7, 1-(5isoquinolynylsuIfonyl)-2-methylpiperazine; PMA, phorbol myristate acetate; PDBu, phorbol dibutyrate; OAG, oleoyl acetyl glycerol; EGF, epidermal growth factor; FGF, fibroblast growth factor; NGF, nerve growth factor; GM-CSF, granulocyte-macrophage colony stimulating factor; TCA, trichloroacetic acid; EGTA, ethylene glycol-bis(f3aminoethyl ether) N,N,N’,N’-tetraacetic acid. ooo6-291w90 $1.50 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
490
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
modulatory roles farther along the signalling pathway (4,5). Likewise, there is no evidence of a tyrosine kinase-like motif in the amino acid sequence of the receptor (2). The role of calcium in the action of Ep is less clear. While the presence of extracellular calcium seems to be an absolute requirement for Ep-induced proliferation and differentiation (6) the positive changes induced by Ep in intracellular calcium have been reported to occur in a time course (3-5 min or longer) indicative of cell membrane channel opening mediated by a diffusible second messenger (7) rather than release from intracellular stores by inositol trisphosphate (IP3). One of the earliest intracellular events reported to occur in response to Ep is a rapid increase in phosphorylation of a number of cellular proteins (8). A role for the calciumand phospholipid-dependent protein kinase (protein kinase C; PKC) in the actions of Ep is suggested by the significant inhibition of Ep-induced colony formation by the PKC inhibitors, I-(5isoquinolynylsulfonyl)-2-methylpiperazine
(H-7) and
staurosporine, and the stimulation of colony formation in the absence of Ep by the PKC activator, phorbol myristate acetate (PMA)(S). Protein kinase C activation has also been implicated in the regulation of erythroid colony formation in normal murine cells (10) and in human hematopoietic progenitor cells (11). Alterations in cellular response to PKC have been reported in hematologic malignancies as well (12). Russell and her colleagues have recently reported a PKC which is activated by prolactin in isolated nuclei of hepatocytes (13). Prolactin is a liver mitogen which, like Ep, undergoes receptor-mediated endocytosis; it is also a growth factor which is not associated with any of the classical signal transduction pathways. We show here that Ep activates a protein kinase C in nuclei isolated from its target cells in murine fetal liver in a manner similar to that of prolactin’s action in hepatocyte nuclei, suggesting that this may represent a new intracellular signalling system for growth factors. MATERIALS
AND METHODS
All chemicals used were of reagent grade and were purchased from Sigma (St. Louis, MO). PMA, 4-alpha PMA, PDBu, OAG, insulin, EGF, and sphingosine were also from Sigma. H-7 was purchased from Molecular Probes, Inc. (Eugene, OR). Staurosporine was from Kamiya Biomedical Co. (Thousand Oaks, CA). Gamma-32PATP (#NEG-002X) was obtained from DuPont/NEN or from ICN (#36017)., Basic FGF and NGF were purchased from Bioscience Products for Science (Indianapolis, IN). GM-CSF was from AmGen (Thousand Oaks, CA) and recombinant human Ep was a gift of the McDonnell-Douglas Corp. (St. Louis, MO). Polyclonal rabbit anti-Ep was produced in our laboratory using highly purified Ep as immunogen; the neutralizing capacity of this antiserum is 4000U/ml. PreDaration of nuclei: The livers of fetal CD-l strain mice were harvested at day 12-l 3 of gestation, when these cells represent a highly enriched population of erythroid progenitor cells (CFU-E). Fetal livers were placed in 5 ml ice-cold Buffer A (0.32 M sucrose, 3 mM MgCl2, 0.5 mM EGTA, 1 mM Hepes, pH 6.8, with 0.1% Ttiton X-l 00) on ice and minced with a razor blade to 1 mm pieces. The tissue and buffer were transferred to a Dounce homogenizer and homogenized with 10 strokes of the B pestle followed by 10 strokes of the A pestle. The homogenate was filtered through 491
Vol.
168,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
nylon cloth, diluted with Buffer A and distilled water such that the final sucrose concentration was 0.28 M, and layered over Buffer A. After a 10 min centrifugation at 700 rcf, the pellet was harvested, suspended in Buffer B ( 2.1 M sucrose, 1 mM MgCl2, 0.5 mM EGTA, 1 mM Hepes, pH 6.8, with 0.1% Triton X-100 ), and centrifuged at 50,000 rcf at 4O C for 1 hr in a swinging bucket rotor. The nuclear pellet was washed twice by resuspending it in Buffer C (0.25 M sucrose, 1 mM MgCl2, 1 mM Hepes, pH 6.8 ) and centrifuging at 700 rcf for 10 min. The washed nuclei were resuspended in incubation buffer (0.25 M sucrose, 4 mM MgCI2, 20 mM NaF, 1 mM dithiothreitol, 40 mM Tris, pH 8.0) and used immediately in the nPKC assay. All buffer solutions contained leupeptin at 50 pg/ml.
Electron m’c OSCQP~LLFor electron microscopic
analysis, an aliquot of the nuclei was pelleted in ‘a\ .5 ml microcentrifuge tube, the pellet incubated overnight in a 100mM Na phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde, fixed in 2% osmium tetroxide, dehydrated in acetone, and embedded in LX1 12 epoxy resin. Sectioned samples (1000 A) were stained with lead citrate followed by alcoholic uranyl acetate before being examined with a Siemens model 101 transmission electron microscope.
Nuclear motein kinase C assa-v- Nuclear PKC activity was determined using an in vitro assay in which the phosphbrylation of endogenous substrate is measured (13). At room temperature, purified nuclei (106/l 00 pl) were preincubated for 15 min with 50 pl of ATP (50 PM), 50 PI of CaC12 (17.5 mM) and 25 p.1of either incubation buffer or H-7 (60 PM). Following a 1 min incubation at 30’ C, 25 pl of incubation buffer containing agonist and 0.6 uCi gamma- 32P ATP was added to each tube. Final concentration of ATP was 10 PM, of CaCl2, 3.5 mM, and of H-7, 6 PM. At the end of a further incubation period (0.5, 1 .O, 2.0 or 3.0 min; see individual experiments), the reaction was terminated by adding 3 ml of ice-cold TCA (10%) to each tube. The tubes were placed on ice for 30 min, after which time the TCA preciptitates were collected onto a GF/C filter with a Brandel cell harvester. The filters were dried and counted in a liquid scintillation counter. PKC activity was determined to be the difference in the amount (pmol) of PO4 incorporated into endogenous substrate in the absence and presence of H-7. Each determination was performed in triplicate (k H-7) in 3-6 different assays. Statistical analyses were done using Student’s t-test for unpaired data.
RESULTS
Puritv of nuclear preparation: the preparation
evaluated
Nuclei were isolated from the liver cells and the purity of with electron microscopy as described. A representative
population of nuclei is shown contamination.
in Figure 1; nuclei were judged to be free of membrane
Activation of protein kinase C in isolated nuclei: When nuclear PKC activity was assayed
as described,
PKC activity, as shown
erythropoietin
(0.2 U/ml) induced a time-dependent
increase
in
in Figure 2. The amount of PKC activity was significantly
higher (p
RAPID
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 490-497
ACTIVATION BY ERYTHROPOIETIN OF PROTEIN KINASE NUCLEI OF ERYTHROID PROGENITOR CELLS Meredith Mason-Garcial,
C IN
Cheryl L. Weill2, and Barbara S. Beckman1
1Department of Pharmacology, Tulane University School of Medicine, and 2Departments of Anatomy and Neurology, Louisiana State University School of Medicine, New Orleans, Louisiana 70112 Received
February
27,
1990
SUMMARY: The glycoprotein hormone erythropoietin (Ep) regulates the proliferation and differentiation of erythroid progenitor cells by a signal transduction system which is not well understood. It has recently been reported that prolactin, a mitogen and trophic hormone for liver, will activate a nuclear protein kinase C in hepatocytes. As similarities exist in the actions of Ep and prolactin in their target cells, we tested the hypothesis that Ep could activate protein kinase C in nuclei isolated from erythroid progenitor cells. In a pure population of such nuclei, Ep induced a rapid, time- and dose-dependent increase in phosphorylation of endogenous nuclear substrate which could be blocked by inhibitors of protein kinase C or by antibody to Ep. Other known activators of protein kinase C were also effective in this system. These findings show that Ep may exert its effects by a novel signalling pathway, the activation of a nuclear protein kinase C. 01990 Academic Press, Inc.
Erythropoietin (Ep) is a glycoprotein hormone (MW=34,000) which acts at a specific cell surface receptor to induce both proliferation and differentiation in its target erythroid progenitor cell, the functionally characterized colony forming unit-erythroid (CFU-E) (1). Recently, the Ep receptor was cloned (2) and found to consist of a 507 amino acid polypeptide with a single predicted membrane-spanning domain and without homology to any known protein. The ligand-receptor complex formed by the binding of Ep to this receptor undergoes rapid endocytosis (3), which suggests that the mechanism of action of Ep may be mediated in part by this event . The signal transduction system which is activated by Ep remains largely uncharacterized, despite many years of research devoted to its elucidation (for a review, see 1). It is clear that an Ep receptor-mediated activation of adenylate cyclase or guanylate cyclase does not occur, although cyclic AMP and cyclic GMP may play Abbreviations: Ep, erythropoietin; PKC, protein kinase C; H-7, 1-(5isoquinolynylsuIfonyl)-2-methylpiperazine; PMA, phorbol myristate acetate; PDBu, phorbol dibutyrate; OAG, oleoyl acetyl glycerol; EGF, epidermal growth factor; FGF, fibroblast growth factor; NGF, nerve growth factor; GM-CSF, granulocyte-macrophage colony stimulating factor; TCA, trichloroacetic acid; EGTA, ethylene glycol-bis(f3aminoethyl ether) N,N,N’,N’-tetraacetic acid. ooo6-291w90 $1.50 Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.
490
Vol.
166,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
modulatory roles farther along the signalling pathway (4,5). Likewise, there is no evidence of a tyrosine kinase-like motif in the amino acid sequence of the receptor (2). The role of calcium in the action of Ep is less clear. While the presence of extracellular calcium seems to be an absolute requirement for Ep-induced proliferation and differentiation (6) the positive changes induced by Ep in intracellular calcium have been reported to occur in a time course (3-5 min or longer) indicative of cell membrane channel opening mediated by a diffusible second messenger (7) rather than release from intracellular stores by inositol trisphosphate (IP3). One of the earliest intracellular events reported to occur in response to Ep is a rapid increase in phosphorylation of a number of cellular proteins (8). A role for the calciumand phospholipid-dependent protein kinase (protein kinase C; PKC) in the actions of Ep is suggested by the significant inhibition of Ep-induced colony formation by the PKC inhibitors, I-(5isoquinolynylsulfonyl)-2-methylpiperazine
(H-7) and
staurosporine, and the stimulation of colony formation in the absence of Ep by the PKC activator, phorbol myristate acetate (PMA)(S). Protein kinase C activation has also been implicated in the regulation of erythroid colony formation in normal murine cells (10) and in human hematopoietic progenitor cells (11). Alterations in cellular response to PKC have been reported in hematologic malignancies as well (12). Russell and her colleagues have recently reported a PKC which is activated by prolactin in isolated nuclei of hepatocytes (13). Prolactin is a liver mitogen which, like Ep, undergoes receptor-mediated endocytosis; it is also a growth factor which is not associated with any of the classical signal transduction pathways. We show here that Ep activates a protein kinase C in nuclei isolated from its target cells in murine fetal liver in a manner similar to that of prolactin’s action in hepatocyte nuclei, suggesting that this may represent a new intracellular signalling system for growth factors. MATERIALS
AND METHODS
All chemicals used were of reagent grade and were purchased from Sigma (St. Louis, MO). PMA, 4-alpha PMA, PDBu, OAG, insulin, EGF, and sphingosine were also from Sigma. H-7 was purchased from Molecular Probes, Inc. (Eugene, OR). Staurosporine was from Kamiya Biomedical Co. (Thousand Oaks, CA). Gamma-32PATP (#NEG-002X) was obtained from DuPont/NEN or from ICN (#36017)., Basic FGF and NGF were purchased from Bioscience Products for Science (Indianapolis, IN). GM-CSF was from AmGen (Thousand Oaks, CA) and recombinant human Ep was a gift of the McDonnell-Douglas Corp. (St. Louis, MO). Polyclonal rabbit anti-Ep was produced in our laboratory using highly purified Ep as immunogen; the neutralizing capacity of this antiserum is 4000U/ml. PreDaration of nuclei: The livers of fetal CD-l strain mice were harvested at day 12-l 3 of gestation, when these cells represent a highly enriched population of erythroid progenitor cells (CFU-E). Fetal livers were placed in 5 ml ice-cold Buffer A (0.32 M sucrose, 3 mM MgCl2, 0.5 mM EGTA, 1 mM Hepes, pH 6.8, with 0.1% Ttiton X-l 00) on ice and minced with a razor blade to 1 mm pieces. The tissue and buffer were transferred to a Dounce homogenizer and homogenized with 10 strokes of the B pestle followed by 10 strokes of the A pestle. The homogenate was filtered through 491
Vol.
168,
No.
BIOCHEMICAL
2, 1990
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
nylon cloth, diluted with Buffer A and distilled water such that the final sucrose concentration was 0.28 M, and layered over Buffer A. After a 10 min centrifugation at 700 rcf, the pellet was harvested, suspended in Buffer B ( 2.1 M sucrose, 1 mM MgCl2, 0.5 mM EGTA, 1 mM Hepes, pH 6.8, with 0.1% Triton X-100 ), and centrifuged at 50,000 rcf at 4O C for 1 hr in a swinging bucket rotor. The nuclear pellet was washed twice by resuspending it in Buffer C (0.25 M sucrose, 1 mM MgCl2, 1 mM Hepes, pH 6.8 ) and centrifuging at 700 rcf for 10 min. The washed nuclei were resuspended in incubation buffer (0.25 M sucrose, 4 mM MgCI2, 20 mM NaF, 1 mM dithiothreitol, 40 mM Tris, pH 8.0) and used immediately in the nPKC assay. All buffer solutions contained leupeptin at 50 pg/ml.
Electron m’c OSCQP~LLFor electron microscopic
analysis, an aliquot of the nuclei was pelleted in ‘a\ .5 ml microcentrifuge tube, the pellet incubated overnight in a 100mM Na phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde, fixed in 2% osmium tetroxide, dehydrated in acetone, and embedded in LX1 12 epoxy resin. Sectioned samples (1000 A) were stained with lead citrate followed by alcoholic uranyl acetate before being examined with a Siemens model 101 transmission electron microscope.
Nuclear motein kinase C assa-v- Nuclear PKC activity was determined using an in vitro assay in which the phosphbrylation of endogenous substrate is measured (13). At room temperature, purified nuclei (106/l 00 pl) were preincubated for 15 min with 50 pl of ATP (50 PM), 50 PI of CaC12 (17.5 mM) and 25 p.1of either incubation buffer or H-7 (60 PM). Following a 1 min incubation at 30’ C, 25 pl of incubation buffer containing agonist and 0.6 uCi gamma- 32P ATP was added to each tube. Final concentration of ATP was 10 PM, of CaCl2, 3.5 mM, and of H-7, 6 PM. At the end of a further incubation period (0.5, 1 .O, 2.0 or 3.0 min; see individual experiments), the reaction was terminated by adding 3 ml of ice-cold TCA (10%) to each tube. The tubes were placed on ice for 30 min, after which time the TCA preciptitates were collected onto a GF/C filter with a Brandel cell harvester. The filters were dried and counted in a liquid scintillation counter. PKC activity was determined to be the difference in the amount (pmol) of PO4 incorporated into endogenous substrate in the absence and presence of H-7. Each determination was performed in triplicate (k H-7) in 3-6 different assays. Statistical analyses were done using Student’s t-test for unpaired data.
RESULTS
Puritv of nuclear preparation: the preparation
evaluated
Nuclei were isolated from the liver cells and the purity of with electron microscopy as described. A representative
population of nuclei is shown contamination.
in Figure 1; nuclei were judged to be free of membrane
Activation of protein kinase C in isolated nuclei: When nuclear PKC activity was assayed
as described,
PKC activity, as shown
erythropoietin
(0.2 U/ml) induced a time-dependent
increase
in
in Figure 2. The amount of PKC activity was significantly
higher (p
