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• W1999310504 abstract "Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-ε. In transfection studies, the isolated variable domain of PKC-ε selectively blocked exogenous activation of the megakaryocyte-specific αIIb promoter. Constitutively active mutants of PKC-ε, but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the αIIb promoter. The functional cooperation between GATA-1 and PKC-ε displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-ε specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters. Multiple studies have shown that intracellular signal transduction by the protein kinase C (PKC) family participates in the initiation of megakaryocyte differentiation. In this study, multiple approaches addressed the functional contributions by specific PKC isozymes to megakaryocytic lineage commitment of two independent cell lines, K562 and human erythroleukemia (HEL). Pharmacologic profiles of induction and inhibition of megakaryocytic differentiation in both cell lines suggested a role for the calcium-independent novel PKCs, in particular PKC-ε. In transfection studies, the isolated variable domain of PKC-ε selectively blocked exogenous activation of the megakaryocyte-specific αIIb promoter. Constitutively active mutants of PKC-ε, but not of other PKC isozymes, cooperated with the transcription factor GATA-1 in the activation of the αIIb promoter. The functional cooperation between GATA-1 and PKC-ε displayed dependence on cellular milieu, as well as on the promoter context of GATA binding sites. In aggregate, the data suggest that PKC-ε specifically participates in megakaryocytic lineage commitment through functional cooperation with GATA-1 in the activation of megakaryocytic promoters. protein kinase C(s) classical or conventional PKC(s) novel PKC(s) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase extracellular signal-regulated kinase ingenol 3,20-dibenzoate human embryonic kidney constitutively active hemagglutinin N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium salts catalytic domains human erythroleukemia A role for protein kinase C (PKC)1 signaling in megakaryocytic differentiation has been established by numerous experiments over the past two decades. In early studies, the PKC agonist phorbol diester selectively enhanced megakaryocyte colony formation by primary mouse bone marrow cells (1Long M.W. Smolen J.E. Szczepanski P. Boxer L.A. J. Clin. Invest. 1984; 74: 1686-1692Crossref PubMed Scopus (35) Google Scholar). More recent studies using primary human progenitors confirmed the promegakaryocytic effects of phorbol ester and showed such effects to be inhibitable by the PKC antagonists GF-109203X and Ro-31–8220 (2Lumelsky N.L. Schwartz B.S. Biochim. Biophys. Acta. 1997; 1358: 79-92Crossref PubMed Scopus (15) Google Scholar). In numerous cell line models of megakaryocytic differentiation, PKC activation induced an array of features including the following: cell cycle arrest, secretion of megakaryocytic cytokines, up-regulation of megakaryocytic surface antigens, cellular enlargement, polyploidization, development of proplatelet processes, and appearance of demarcation membranes (3Alitalo R. Leuk. Res. 1990; 14: 501-514Crossref PubMed Scopus (126) Google Scholar,4Long M.W. Heffner C.H. Williams J.L. Peters C. Prochownik E.V. J. Clin. Invest. 1990; 85: 1072-1084Crossref PubMed Scopus (149) Google Scholar). The PKC serine/threonine kinase family consists of at least 11 distinct isozymes organized into three subgroups, based on biochemical, pharmacologic, and structural properties (5Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1219) Google Scholar). The classical or conventional PKCs (cPKCs) require diacylglycerol and Ca2+ for activation and consist of the α, βI, βII, and γ isozymes. The novel PKCs (nPKCs) require only diacylglycerol for activation and consist of the δ, ε, θ, η, and μ isozymes. The atypical PKCs lack responsiveness to diacylglycerol and Ca2+ and consist of the λ and ζ isozymes. Striking functional differences exist among PKC isozymes, with divergent functions noted even for factors with high structural homology (6Thompson L.J. Fields A.P. J. Biol. Chem. 1996; 271: 15045-15053Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 7Walker S.D. Murray N.R. Burns D.J. Fields A.P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9156-9160Crossref PubMed Scopus (40) Google Scholar, 8Gubina E. Rinaudo M.S. Szallasi Z. Blumberg P.M. Mufson R.A. Blood. 1998; 91: 823-829Crossref PubMed Google Scholar) PKC signaling may influence megakaryocytic differentiation through several isozymes. In K562 cells, PKC-mediated, sustained activation of the Raf-MEK-ERK signaling pathway is necessary for initiation of megakaryocytic differentiation (9Racke F.R. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 10Whalen A.M. Galasinski S.C. Shapiro P.S. Stines Nahreini T. Ahn N.G. Mol. Cell. Biol. 1997; 17: 1947-1958Crossref PubMed Scopus (203) Google Scholar). Multiple PKC isozymes, in particular α, βI, η, and δ, possess the capacity to activate the Raf-MEK-ERK pathway (11Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Crossref PubMed Scopus (684) Google Scholar, 12Ueda Y. Hirai S.-I. Osada S.-I. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (512) Google Scholar). However, activation of the Raf-MEK-ERK pathway appears not to be required for megakaryocytic differentiation of primary progenitor cells, suggesting that PKC signaling plays an additional role in megakaryopoiesis independent of ERK activation (13Fichelson S. Freyssinier J.-M. Picard F. Fontenay-Roupie M. Guesnu M. Cherai M. Gisselbrecht S. Porteu F. Blood. 1999; 94: 1601-1613Crossref PubMed Google Scholar). To examine the contribution of specific PKC isozymes to megakaryocyte differentiation, we initially employed isozyme-selective pharmacologic agents in two independent cell line models of megakaryocytic differentiation, K562 and HEL. GF-109203X, an inhibitor of cPKCs and nPKCs but not of atypical PKCs, potently blocked megakaryocytic induction in both cell lines. By contrast, Gö 6976, an inhibitor only of cPKCs (14Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar), failed to block megakaryocytic differentiation, suggesting a specific requirement for nPKC signaling. For both cell lines, the PKC-ε-selective agonist, ingenol 3,20-dibenzoate (IDB) (15Asada A. Zhao Y. Kondo S. Iwata M. J. Biol. Chem. 1998; 273: 28392-28398Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar, 17Weller S.G. Klein I.K. Penington R.C. Karnes W.E. Gastroenterology. 1999; 117: 848-857Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) induced megakaryocytic differentiation, as well as selective nuclear translocation of PKC-ε. In transfection assays, the isolated variable domain of PKC-ε, but not that of PKC-α, completely blocked exogenous activation of the megakaryocytic αIIb promoter. Constitutively active mutants of PKC-ε activated the αIIb promoter 3–6-fold. We also addressed whether PKC-ε signaling influenced the function of GATA-1, a transcription factor known to play a critical role in megakaryopoiesis and in activation of the αIIb promoter (18Martin F. Prandini M.-H. Thevenon D. Marguerie G. Uzan G. J. Biol. Chem. 1993; 268: 21606-21612Abstract Full Text PDF PubMed Google Scholar, 19Lemarchandel V. Ghysdael J. Mignotte V. Rahuel C. Romeo P.-H. Mol. Cell. Biol. 1993; 13: 668-676Crossref PubMed Scopus (179) Google Scholar, 20Shivdasani R.A. Fujiwara Y. McDevitt M.A. Orkin S.H. EMBO J. 1997; 16: 3965-3973Crossref PubMed Scopus (587) Google Scholar). Indeed, GATA-1 and constitutively active PKC-ε showed synergistic activation of the αIIb promoter. Notably, the ability to synergize with GATA-1 distinguished PKC-ε from other PKC isozymes, depended on cellular milieu, and depended on the context of GATA binding sites within the promoter. K562 and HEL, obtained from the ATCC, were grown in RPMI 1640 with 10% fetal bovine serum at 37 °C, 5% CO2. C3H10T1/2, obtained from ATCC, was grown in Dulbecco's modified Eagle's medium with 10% neonatal calf serum at 37 °C, 5% CO2. HEK-293T, provided by Dr. Kevin Lynch (Department of Pharmacology, University of Virginia School of Medicine), was grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C, 5% CO2. All experiments using K562 and HEL employed mid-log phase cells at a density of 0.5–1.0 × 106 cells/ml. Conditioned media was obtained as described previously by 72 h of treatment of either K562 or HEL cells with 25 nm12-O-tetradecanoylphorbol-13-ester (Sigma) followed by harvesting and dialysis of supernatant (9Racke F.R. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). For megakaryocytic induction, cells were resuspended in conditioned media and incubated at 37 °C, 5% CO2 for 1–3 days, as indicated. The compounds GF-109203X, Gö 6976, and IDB were purchased from LC Laboratories. The PKC inhibitors GF-109203X and Gö 6976 were added, at indicated concentrations, to conditioned media at the initiation of megakaryocytic induction. Staining of cells for surface CD41 employed the fluorescein isothiocyanate-conjugated antibody PLT1-FITC (Coulter) at 25 μg/ml. Staining of cells for surface glycophorin A employed the phycoerythrin-conjugated antibody GA-R2-PE (Pharmingen) at 10 μg/ml. Appropriate fluorochrome-conjugated, isotype-matched antibody controls were used at concentrations identical to the corresponding experimental antibodies. Flow cytometric analysis was performed on a FACScan system utilizing Lysys II software (Becton Dickinson). Treated cells were cytospun onto glass slides and fixed for 2 min in ice-cold methanol followed by 2 min in ice-cold acetone. After blocking for 30 min at room temperature with 1% normal goat serum in phosphate-buffered saline, primary antibodies in 0.1% normal goat serum/phosphate-buffered saline were applied for 1 h at room temperature. Murine monoclonal antibodies to PKC-ε (Santa Cruz Biotechnology, Santa Cruz, CA) and to PKC-α (Transduction Laboratories, Lexington, KY) were used at 200 ng/ml and at 1.25 μg/ml, respectively. Control murine antibody (NOR 3.2;BIOSOURCE International) was used at 1 μg/ml. Secondary antibody, consisting of phycoerythrin-conjugated goat anti-mouse (Tago, Inc.) diluted 1:100 in 0.1% normal goat serum/phosphate-buffered saline, was applied for 30 min at room temperature. For nuclear visualization, 4,6-diamidino-2-phenylindole was included in the coverslip mounting medium. Cells were visualized by confocal laser scanning fluorescence microscopy on a Zeiss LSM 410 (Jena, Germany) using Zeiss LSM analysis software. The αIIb-luciferase reporter constructs were made by polymerase chain reaction amplification of bases −598 to +32, bases −98 to +32, or bases −348 to +32 from a human αIIb promoter fragment kindly provided by Dr. Samuel Santoro (21Fong A.M. Santoro S.A. J. Biol. Chem. 1994; 269: 18441-18447Abstract Full Text PDF PubMed Google Scholar). The polymerase chain reaction products were co-digested with XhoI plus HindIII and ligated into the corresponding sites of pGL3-Basic (Promega). The β-galactosidase expression vector consisted of pCMVβ (CLONTECH). The GATA-1 expression vector employed the EF-1-α-neo expression plasmid and has been previously described (22Visvader J.E. Crossley M. Hill J. Orkin S.H. Adams J.M. Mol. Cell. Biol. 1995; 15: 634-641Crossref PubMed Google Scholar). Mammalian expression of a full-length constitutively active (CA) mutant of PKC-ε employed SRD-ε-K155A/R156A/A159E (AE3), kindly provided by Dr. S. Ohno (23Hirai S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar). Expression of a full-length CA mutant of PKC-δ employed SRD-δR144/145A (DRA), also kindly provided by Dr. S. Ohno (23Hirai S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar). Expression of a full-length CA mutant of PKC-α employed pRc-CMV-PKC-α A25E, kindly provided by Dr. Gottfried Baier (24Uberall F. Giselbrecht S. Hellbert K. Fresser F. Bauer B. Gschwendt M. Grunicke H.H. Baier G. J. Biol. Chem. 1997; 272: 4072-4078Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Expression of a full-length CA mutant of PKC-θ employed pRc/CMV-PKC-θ R145I/R146W, kindly provided by Dr. J. Anthony Ware (25Tang S. Morgan K.G. Parker C. Ware J.A. J. Biol. Chem. 1997; 272: 28704-28711Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Mammalian expression constructs for HA-epitope-tagged, isolated PKC catalytic and regulatory domains have been previously described (26Soh J.-W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (249) Google Scholar). In brief, fragments encoding the catalytic domains of α (amino acids 326–672) and ε (amino acids 395–737) were ligated into the pHANE vector. The fragment encoding the catalytic domain of δ (amino acids 334–674) was ligated into the pHACE vector. Fragments encoding the variable domains of α (amino acids 2–325) and ε (amino acids 2–394) were ligated into the pHANE vector. Transfection of K562 and C3H10T1/2 cells employed the liposomal reagent DOTAP (Roche Molecular Biochemicals) using ∼6 μg of DNA in 200 μl of HBS (20 mm HEPES, 150 mm NaCl, pH 7.4) combined with 30 μl of DOTAP in 200 μl HBS. The DNA/DOTAP mixture was incubated for 10 min at room temperature and was then added dropwise to 1. 6 × 106 K562 cells in 2 ml of RPMI 1640 with 5% fetal bovine serum. For C3H10T1/2 cell transfections, the DNA/DOTAP mixture was added to ∼60% confluent cells in 6-well plates. Transfection of HEK-293T cells was performed as described for C3H10T1/2 cells except that 4 μg of DNA in 50 μl of HBS was combined with 20 μl of DOTAP in 100 μl of HBS. After overnight incubation, cells were changed to fresh complete medium. Cells were subsequently incubated 24 h prior to harvesting for luciferase and β-galactosidase assays. Luciferase assays were performed using the commercial luciferase assay system (Promega), and β-galactosidase assays were performed using the O-nitrophenyl β-d-galactopyranoside (Sigma) colorimetric substrate. All transfections were performed at least in triplicate, and all luciferase values were normalized according to β-galactosidase readings. K562, C3H10T1/2, and HEK-293T cells were transfected and harvested as for the luciferase and β-galactosidase assays, except that whole cell lysates were prepared by resuspending cells in 1× SDS polyacrylamide gel electrophoresis loading buffer. Samples were resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Equivalent lane loading was confirmed by Ponceau staining of membranes. Probing of membranes was carried out as described previously (27Goldfarb A.N. Goueli S. Mickelson D. Greenberg J.M. Blood. 1992; 80: 2858-2866Crossref PubMed Google Scholar). For detection of HA-tagged PKC regulatory and catalytic domains, the primary antibody consisted of 12CA5, a murine monoclonal directed to the HA epitope tag, employed as ascites fluid diluted 1:2000. The secondary antibody consisted of peroxidase-conjugated goat anti-mouse (Sigma) used at a dilution of 1:3000. For detection of GATA-1, the primary antibody consisted of the rat monoclonal N6 (Santa Cruz Biotechnology) at a final concentration of 0.4 μg/ml. The secondary antibody consisted of peroxidase-conjugated goat anti-rat (Sigma) used at a dilution of 1:5000. Signal detection employed enhanced chemiluminescence. Previous work in our laboratory indicated that sustained activation of the Raf-MEK-ERK pathway in the K562 hematopoietic cell line resulted in production of autocrine factors promoting megakaryocytic maturation (9Racke F.R. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). To identify signaling pathways triggered by such autocrine factors, we analyzed the effects of pharmacologic inhibitors on megakaryocytic induction in two independent cell lines, K562 and HEL. The induction stimulus consisted of conditioned media from 12-O-tetradecanoylphorbol-13-ester-treated HEL cells, which show identical activity to that previously reported for K562 cells (9Racke F.R. Lewandowska K. Goueli S. Goldfarb A.N. J. Biol. Chem. 1997; 272: 23366-23370Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The only compound in our screening that potently and specifically blocked megakaryocytic induction by conditioned media was GF-109203X. GF-109203X inhibits both cPKC and nPKC isozymes, as well as pp90rsk2 (14Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar, 28Swanson K.D. Taylor L.K. Haung L. Burlingame A.L. Landreth G.E. J. Biol. Chem. 1999; 274: 3385-3395Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Therefore, parallel experiments were carried out using the compound Gö 6976, known to inhibit cPKCs and pp90rsk2 but not nPKCs (14Martiny-Baron G. Kazanietz M.G. Mischak H. Blumberg P.M. Kochs G. Hug H. Marme D. Schachtele C. J. Biol. Chem. 1993; 268: 9194-9197Abstract Full Text PDF PubMed Google Scholar, 28Swanson K.D. Taylor L.K. Haung L. Burlingame A.L. Landreth G.E. J. Biol. Chem. 1999; 274: 3385-3395Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). As shown in the flow cytometric profiles in Fig. 1, conditioned media alone induced up-regulation of the megakaryocyte surface antigen CD41 and down-regulation of the erythroid surface antigen glycophorin A. Whereas GF-109203X completely blocked both responses, Gö 6976 at similar doses showed no inhibition of either response. Analysis of cellular morphology supported the flow cytometric results in Fig. 1. In particular, HEL cells exposed to conditioned media undergo spreading and enlargement as part of their megakaryocytic differentiation. As shown in Fig. 2, the morphologic changes induced in HEL cells by conditioned media were abrogated by GF-109203X. By contrast, Gö 6976 strikingly enhanced the cellular spreading and enlargement induced by conditioned media. These data confirm that biologically active doses of Gö 6976 acted to enhance rather than inhibit features of megakaryocytic differentiation. In an alternative approach, the isozyme-selective PKC agonist IDB was applied directly to K562 and HEL cells in standard growth media. Multiple previous studies have indicated that IDB is a selective activator of nPKCs, particularly PKC-ε (15Asada A. Zhao Y. Kondo S. Iwata M. J. Biol. Chem. 1998; 273: 28392-28398Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16Ohoka Y. Kuwata T. Asada A. Zhao Y. Mukai M. Iwata M. J. Immunol. 1997; 158: 5707-5716PubMed Google Scholar, 17Weller S.G. Klein I.K. Penington R.C. Karnes W.E. Gastroenterology. 1999; 117: 848-857Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). As shown in Fig.3, IDB caused CD41 up-regulation and glycophorin A down-regulation in K562 and HEL cells. In addition, HEL cells treated with IDB manifested the standard morphologic changes seen with megakaryocytic induction (not shown). Immunofluorescent staining (Fig. 4) showed that treatment of K562 cells with IDB induces rapid nuclear translocation of PKC-ε but no change in the subcellular localization of PKC-α. Rapid nuclear translocation of PKC-ε was also observed in HEL cells treated with IDB (data not shown). Thus, both agonists and antagonists implicate nPKC, in particular PKC-ε, in the induction of megakaryocytic differentiation.Figure 4Immunofluorescent subcellular localization of PKC-ε and PKC-α in K562 cells, untreated and treated with IDB. Cells were treated for 10 min at 37 °C with or without 100 nm IDB. Following stimulation, cells were fixed and stained as described under “Materials and Methods.” The images represent the following:Left column, blue staining indicates 4,6-diamidino-2-phenylindole staining of nuclei. Middle column, anti-PKC immunoreactivity indicated by red staining. Right column, composite image superimposing left and middle columns demonstrating nuclear localization of PKC-ε following IDB treatment. This experiment was performed on three separate occasions, each yielding similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The amino-terminal regulatory sequences of PKCs, when expressed as isolated fragments, function as dominant-negative PKC inhibitors (29Hundle B. McMahon T. Dadgar J. Chen C.H. Mochly-Rosen D. Messing R.O. J. Biol. Chem. 1997; 272: 15028-15035Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 30Parissenti A.M. Kirwan A.F. Kim S.A. Colantonio C.M. Schimmer B.P. J. Biol. Chem. 1998; 273: 8940-8945Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Accordingly, we examined whether transfection of isolated regulatory domains from either PKC-α or PKC-ε could interfere with exogenous activation of the megakaryocyte-specific αIIb-598 promoter (31Era T. Takagi T. Takahashi T. Bories J.-C. Nakano T. Blood. 2000; 95: 870-878Crossref PubMed Google Scholar). In cells transfected with control vector, 24 h of conditioned media caused an ∼14-fold up-regulation of the αIIb-598-luciferase reporter activity (Fig. 5 A). Expression in cells of the isolated α regulatory fragment (α-Reg) minimally inhibited conditioned media activation of αIIb-598-luciferase. In striking contrast, expression in cells of the isolated ε regulatory fragment (ε-Reg) almost completely eliminated responsiveness to the conditioned media stimulus. Immunoblotting showed similar expression levels of the HA-epitope-tagged α and ε regulatory domains in HEK-293T transfectants (Fig. 5 B). We next tested whether constitutively active PKC mutants could activate the αIIb megakaryocytic promoter. Fig. 6 A demonstrates the similar results obtained with two different types of constitutively active mutants, inhibitory domain point mutants (CA mutants in left graph), and isolated PKC catalytic domains (CAT mutants in right graph). The inhibitory domain point mutants consist of full-length PKCs with point mutations in the autoinhibitory regulatory domains (23Hirai S. Izumi Y. Higa K. Kaibuchi K. Mizuno K. Osada S. Suzuki K. Ohno S. EMBO J. 1994; 13: 2331-2340Crossref PubMed Scopus (110) Google Scholar, 24Uberall F. Giselbrecht S. Hellbert K. Fresser F. Bauer B. Gschwendt M. Grunicke H.H. Baier G. J. Biol. Chem. 1997; 272: 4072-4078Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 25Tang S. Morgan K.G. Parker C. Ware J.A. J. Biol. Chem. 1997; 272: 28704-28711Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The isolated PKC CAT completely lack regulatory domains, which have also been implicated in Ca2+and lipid binding, interaction with RACKs, and kinase-independent signaling (26Soh J.-W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (249) Google Scholar). Using the megakaryocyte-specific αIIb-598 reporter, we tested the effects of PKC isozymes alone and in conjunction with GATA-1, a known positive regulator of the αIIb promoter (18Martin F. Prandini M.-H. Thevenon D. Marguerie G. Uzan G. J. Biol. Chem. 1993; 268: 21606-21612Abstract Full Text PDF PubMed Google Scholar, 19Lemarchandel V. Ghysdael J. Mignotte V. Rahuel C. Romeo P.-H. Mol. Cell. Biol. 1993; 13: 668-676Crossref PubMed Scopus (179) Google Scholar). As shown in Fig. 6 A, constitutively active PKC-ε mutants alone modestly activated the αIIb promoter (3–6-fold) but demonstrated clear functional cooperation with GATA-1. In fact, coexpression of GATA-1 with either CA or CAT mutants of ε led to levels of reporter activation analogous to those obtained with conditioned media induction. Functional cooperation with GATA-1 was clearly isozyme-restricted in that constitutively active mutants of α, δ, and θ all failed to augment GATA-1-mediated αIIb activation. In fact, δ caused a 2–3-fold inhibition of GATA-1 activation. Immunoblot analysis demonstrated equivalent levels of GATA-1 expression in all transfectants, indicating that the differential effects of the PKC isozymes were not because of differences in GATA-1 levels (Fig. 6 B). In addition, immunoblot demonstrated analogous expression levels of the HA-epitope-tagged ε, α, and δ catalytic domains in HEK-293T cells (Fig. 6 C). To determine whether functional interaction between PKC-ε and GATA-1 occurred also in non-hematopoietic cells, cotransfections were carried out in C3H10T1/2 fibroblasts rather than in K562 hematopoietic cells. The full-length CA ε mutant, as well as the isolated ε CAT, failed to augment GATA-1-mediated αIIb activation in C3H10T1/2 fibroblasts (Fig.7 A). Immunoblotting demonstrated equivalent expression of GATA-1 in all of the transfectants (Fig. 7 B). Thus, the functional interaction of PKC-ε with GATA-1 clearly depends on the cell type employed for transfection. The αIIb promoter contains multiple GATA binding sites, including a functional site within the promoter-proximal −98 fragment (18Martin F. Prandini M.-H. Thevenon D. Marguerie G. Uzan G. J. Biol. Chem. 1993; 268: 21606-21612Abstract Full Text PDF PubMed Google Scholar, 19Lemarchandel V. Ghysdael J. Mignotte V. Rahuel C. Romeo P.-H. Mol. Cell. Biol. 1993; 13: 668-676Crossref PubMed Scopus (179) Google Scholar). To determine whether specific promoter regions were required for PKC-ε/GATA-1 cooperativity, 5′ truncated reporter constructs, αIIb-98 and αIIb-348, were compared with αIIb-598 for responsiveness to PKC-ε ± GATA-1 in K562 cells. Surprisingly, the αIIb-98 and αIIb-348 reporters showed full activation by GATA-1 alone but no evidence of augmentation by PKC-ε (Fig. 7 C). Thus, the functional GATA binding sites in the αIIb −348 to +32 fragment were insufficient to mediate PKC-ε/GATA-1 cooperativity. Involvement of PKC signaling in hematopoietic lineage commitment decisions has been well documented. In progenitors transformed by the E26 avian leukemia virus, thresholds of PKC activity correlated with cell fate determinations as follows: (a) no kinase activity was associated with undifferentiated cells; (b) low activity was associated with myelomonocytic differentiation; and (c) high activity was associated with eosinophil differentiation (32Rossi F. McNagny K.M. Smith G. Frampton J. Graf T. EMBO J. 1996; 15: 1894-1901Crossref PubMed Scopus (58) Google Scholar). In primary, bipotential granulocyte macrophage colony-forming cells, activation of PKC-α induced commitment to the macrophage lineage (33Pierce A. Heyworth C.M. Nicholls S.E. Spooncer E. Dexter T.M. Lord J.M. Owen-Lynch P.J. Wark G. Whetton A.D. J. Cell Biol. 1998; 140: 1511-1518Crossref PubMed Scopus (42) Google Scholar). Similarly, our data suggest that signaling via PKC-ε may promote megakaryocytic lineage commitment of the bipotential BFU-E/MK progenitor, a cell with capability for either erythroid or megakaryocytic differentiation (34Debili N. Coulombel L. Croisille L. Katz A. Guichard J. Breton-Gorius J. Vainchenker W. Blood. 1996; 88: 1284-1296Crossref PubMed Google Scholar). Previous studies have notably shown that PKC-ε undergoes down-regulation during erythroid differentiation and that inhibition of PKC-ε specifically enhances erythroid differentiation (35Myklebust J.H. Smeland E.B. Josefsen D. Sioud M. Blood. 2000; 95: 510-518Crossref PubMed Google Scholar, 36Bassini A. Zauli G. Migliaccio G. Migliaccio A.R. Pascuccio M. Pierpaoli S. Guidotti L. Capitani S. Vitale M. Blood. 1999; 93: 1178-1188Crossref PubMed Google Scholar). Mechanisms by which PKC-ε signaling might contribute to the activation of the megakaryocytic αIIb promoter remain unclear. Earlier studies with isolated catalytic domains have shown PKC-ε to activate multiple pathways that converge on the serum-response element of the c-fos promoter, c-Raf-MEK1-ERK, MEK kinase 1-stress-activated protein kinase kinase-c-Jun NH2-terminal kinase, and rhoA (26Soh J.-W. Lee E.H. Prywes R. Weinstein I.B. Mol. Cell. Biol. 1999; 19: 1313-1324Crossref PubMed Scopus (249) Google Scholar). However, those studies showed equivalent activation of the various pathways by the PKC-α catalytic domain. Our results, by contrast, show no activation of the αIIb promoter by the PKC-α catalytic domain. The rapid nuclear translocation of PKC-ε observed with megakaryocytic induction by IDB (Fig. 4) raises the possibility that PKC-ε itself might act directly upon critical nuclear substrates. The functional cooperation of PKC-ε with GATA-1 raises a number of mechanistic possibilities. One scenario is that PKC-ε signaling targets a transcriptional complex containing GATA-1 and enhances GATA-1 function by phosphorylation of one of the members of this complex, such as GATA-1 itself or the cofactor friend of GATA-1. The absence of functional interaction in C3H10T1/2 cells argues against direct phosphorylation of GATA-1 by PKC-ε as a sufficient mechanism. This scenario also fails to account for the dependence of PKC-ε signaling on promoter context, as illustrated in Fig. 7 C. Accordingly another possibility is that PKC-ε signaling targets GATA-1 complexes binding to specific regions of the αIIb promoter. A recent study employing embryonic stem cell hematopoiesis has defined within the human αIIb promoter a 200-base pair critical enhancer region, −398 to −598, that is necessary and sufficient for megakaryocyte-specific transgene expression (31Era T. Takagi T. Takahashi T. Bories J.-C. Nakano T. Blood. 2000; 95: 870-878Crossref PubMed Google Scholar). Interestingly, our data indicate that a similar region (from −348 to −598 of the human αIIb promoter) is required for responsiveness to PKC-ε signaling. Future studies will attempt to correlate PKC-ε response elements within the αIIb promoter with megakaryocyte-specific enhancer function. A major question in the molecular characterization of hematopoietic lineage commitment is how two lineages with highly similar arrays of transcription factors can show non-overlapping, indeed mutually exclusive, patterns of gene expression. Erythroid and megakaryocytic cells share expression of the highly restricted factors GATA-1, GATA-2, Lmo2, NF-E2, friend of GATA-1, and SCL/tal. Most striking among these factors is GATA-1, which dominantly activates erythroid genes only in erythroblasts and dominantly activates megakaryocytic genes only in megakaryocytes. Our current data raise the possibility that isozyme-specific signaling by PKC may modify GATA function in accordance with promoter context. In particular, PKC-ε signaling might specifically augment GATA-1 function in the context of megakaryocytic promoters, thereby redirecting the entire transcriptional program of a cell from erythroid to megakaryocytic. We thank Kristine Lewandowska for excellent technical assistance in the early phases of this project. For generously providing plasmid constructs, thanks go to Drs. Samuel Santoro, Gottfried Baier, J. Anthony Ware, and S. Ohno. For helpful discussions and support, thanks go to Drs. Chi V. Dang, Isa Hussaini, and Julianne J. Sando." @default.
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• W1999310504 title "A Potential Role for Protein Kinase C-ε in Regulating Megakaryocytic Lineage Commitment" @default.
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