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• W2066016982 abstract "Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Cζ (PKCζ) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCζ and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCζ in steady-state megakaryopoiesis. Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Cζ (PKCζ) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCζ and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCζ in steady-state megakaryopoiesis. thrombopoietin phosphoinositide 3-kinase extracellular signal-regulated kinase mitogen-activated protein kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase megakaryocyte 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide Src homology 2 domain-containing protein tyrosine phosphatase insulin-like receptor substrate protein kinase C interleukin dominant negative polyacrylamide gel electrophoresis bisindolylmaleimide I glutathioneS-transferase myelin basic protein Binding of TPO1 to its receptor, the product of the proto-oncogene c-mpl, activates a wide variety of signaling molecules and pathways. As for other cytokine systems, it is becoming clear that the response to TPO is characterized by networks of multiple branching and converging signaling pathways, which display extensive cross-talk. As such, blockade of one signaling pathway can be compensated by alternate pathways. This may partially explain relatively mild hematopoietic phenotypes of mice in which supposedly critical signaling pathways are disrupted by homologous recombination (1Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, 2Kirito K. Osawa M. Shimizu R. Oda A. Nakajima K. Morita H. Yamamoto M. Ozawa K. Komatsu N. Blood. 2000; 96 Suppl. 1 (abstr.): 538Google Scholar, 3Luoh S.M. Stefanich E. Solar G. Steinmetz H. Lipari T. Pestina T.I. Jackson C.W. de Sauvage F.J. Mol. Cell. Biol. 2000; 20: 507-515Crossref PubMed Scopus (42) Google Scholar). We demonstrated previously that the ERK/MAPK pathway is activated in response to TPO in both a BaF3 cell line engineered to express full-length Mpl (BaF3/Mpl) and in primary MKs, and plays an important role in MK endomitosis (4Rojnuckarin P. Drachman J.G. Kaushansky K. Blood. 1999; 94: 1273-1282Crossref PubMed Google Scholar). Consistent with our results, MKs from mice engineered to express only a truncated Mpl receptor missing 60 residues from the COOH terminus of the cytoplasmic domain (T61 or Δ60 mice) display a reduced capacity to activate ERK and have significantly decreased endomitotic capability after TPO administration in vivo (3Luoh S.M. Stefanich E. Solar G. Steinmetz H. Lipari T. Pestina T.I. Jackson C.W. de Sauvage F.J. Mol. Cell. Biol. 2000; 20: 507-515Crossref PubMed Scopus (42) Google Scholar). The classic pathway of ERK activation is via growth factor-induced Shc phosphorylation followed by its association with Grb2 (5Rozakis-Adcock M. McGlade J. Mbamalu G. Pelicci G. Daly R. Li W. Batzer A. Thomas S. Brugge J. Pelicci P.G. Schlessinger J. Pawson T. Nature. 1992; 360: 689-692Crossref PubMed Scopus (827) Google Scholar), which then activates Sos, a nucleotide exchange factor for Ras (6Buday L. Downward J. Cell. 1993; 73: 611-620Abstract Full Text PDF PubMed Scopus (933) Google Scholar). Consequently, ERK can be activated by Ras-GTP through Raf and MEK phosphorylation. Several groups have reported that Shc is strongly activated in response to TPO (7Drachman J.G. Griffin J.D. Kaushansky K. J. Biol. Chem. 1995; 270: 4979-4982Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Hence, Shc-dependent activation of Ras is likely to be an important mechanism of TPO-induced ERK activation. However, we also demonstrated that TPO stimulation of BaF3 expressing a truncated form of Mpl missing 52 residues from the COOH terminus of the cytoplasmic domain (BaF3/T69) could activate ERK without Shc phosphorylation (4Rojnuckarin P. Drachman J.G. Kaushansky K. Blood. 1999; 94: 1273-1282Crossref PubMed Google Scholar) and support cell growth. Consistent with these results, platelets from T61 mice also retain some ability to activate ERK, independent of Shc (3Luoh S.M. Stefanich E. Solar G. Steinmetz H. Lipari T. Pestina T.I. Jackson C.W. de Sauvage F.J. Mol. Cell. Biol. 2000; 20: 507-515Crossref PubMed Scopus (42) Google Scholar). Therefore, Shc is not absolutely essential for ERK activation. Because the signals emanating from the full-length receptor are very diverse and redundant, studies of signaling from these truncated receptors allowed us to investigate only the minimally required set of signals for resting-state megakaryopoiesis. Furthermore, the pathways from the truncated Mpl to MAPK may be novel, as they are not mediated by the conventional Shc/Grb2/Sos/Ras pathway. Therefore, potentially new mechanisms of ERK activation have been explored in this study, including phosphoinositide 3-kinase (PI3K) and isoforms of protein kinase C (PKC). Two types of PI3K have been shown to play important roles in cytokine-mediated signal transduction. Class IA PI3Ks, comprising p85 adapter and p110 catalytic subunits, are activated by cytokines and growth factors, whereas class IB PI3K (PI3Kγ), comprising p101 adapter and p110 catalytic subunits, is activated by heterotrimeric G protein-coupled receptors (reviewed in Ref. 8Vanhaesebroeck B. Waterfield M.D. Exp. Cell. Res. 1999; 253: 239-254Crossref PubMed Scopus (763) Google Scholar). A constitutively active form of class IA PI3K has been demonstrated to activate MAPK by stimulating Ras (9Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar). Interference with the PI3K pathway, either by using pharmacological inhibitors (10Marra F. Pinzani M. DeFranco R. Laffi G. Gentilini P. FEBS Lett. 1995; 376: 141-145Crossref PubMed Scopus (80) Google Scholar, 11Duckworth B.C. Cantley L.C. J. Biol. Chem. 1997; 272: 27665-27670Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 12King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar, 13Grammer T.C. Blenis J. Oncogene. 1997; 14: 1635-1642Crossref PubMed Scopus (177) Google Scholar, 14Sarbassov D.D. Peterson C.A. Mol. Endocrinol. 1998; 12: 1870-1878Crossref PubMed Scopus (58) Google Scholar) or expression of a dominant negative protein (9Hu Q. Klippel A. Muslin A.J. Fantl W.J. Williams L.T. Science. 1995; 268: 100-102Crossref PubMed Scopus (517) Google Scholar, 12King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (387) Google Scholar) also blocks ERK activation, suggesting that PI3K is necessary for ERK activation in these systems. We have shown previously that TPO-induced PI3K activation is dependent on the recruitment of the active enzyme into signaling complexes containing Gab/IRS docking proteins (15Miyakawa Y. Rojnuckarin P. Habib T. Kaushansky K. J. Biol. Chem. 2001; 276: 2494-2502Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Consistent with our results, a docking protein, Gab1, has been implicated in PI3K and thus ERK activation in gp130 receptor signaling (16Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (251) Google Scholar). However, in other systems, constitutively active forms of this class of PI3K are insufficient to activate ERKs (17Klippel A. Reinhard C. Kavanaugh W.M. Apell G. Escobedo M.A. Williams L.T. Mol. Cell. Biol. 1996; 16: 4117-4127Crossref PubMed Scopus (417) Google Scholar, 18Frevert E.U. Kahn B.B. Mol. Cell. Biol. 1997; 17: 190-198Crossref PubMed Scopus (156) Google Scholar). In one report, wortmannin-sensitive ERK activation was mediated by the class IB PI3K, PI3Kγ (19Lopez-Ilasaca M. Crespo P. Pellici P.G. Gutkind J.S. Wetzker R. Science. 1997; 275: 394-397Crossref PubMed Scopus (629) Google Scholar), and the effect was dependent on its protein kinase, not lipid kinase activity (20Bondeva T. Pirola L. Bulgarelli-Leva G. Rubio I. Wetzker R. Wymann M.P. Science. 1998; 282: 293-296Crossref PubMed Scopus (302) Google Scholar). Furthermore, PI3K-induced ERK activation has been shown to depend on both cell type and signal intensity; ERK activation depends on PI3K only at low signaling intensities (11Duckworth B.C. Cantley L.C. J. Biol. Chem. 1997; 272: 27665-27670Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Therefore, several questions remain to be explained: how truncated Mpl receptors activate ERK, whether PI3K is activated by the truncated receptor in response to TPO, how it is activated, and whether it plays a role in ERK activation. PKC is an expanding family of serine-threonine kinases, comprising numerous isoforms, which display varied patterns of tissue distribution and different physiological functions. Pharmacological agents modifying PKC that function in an isoform-specific manner have been generated, potentially providing clinically useful strategies to provide desirable therapeutic effects while minimizing adverse reactions. PKC was reported to be activated by TPO in UT7/Mpl cells (21Kunitama M. Shimizu R. Yamada M. Kato T. Miyazaki H. Okada K. Miura Y. Komatsu N. Biochem. Biophys. Res. Commun. 1997; 231: 290-294Crossref PubMed Scopus (21) Google Scholar). However, the specific isoform utilization of PKCs and its contribution to ERK activation has not been reported in the TPO system. Several isoforms of PKCs have been shown to activate ERK (22Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Crossref PubMed Scopus (684) Google Scholar). Interestingly, PDK1, a kinase dependent on PI3K, can activate atypical isoforms of PKCs (23Le Good J.A. Ziegler W.H. Parekh D.B. Alessi D.R. Cohen P. Parker P.J. Science. 1998; 281: 2042-2045Crossref PubMed Scopus (973) Google Scholar, 24Chou M.M. Hou W. Johnson J. Graham L.K. Lee M.H. Chen C.S. Newton A.C. Schaffhausen B.S. Toker A. Curr. Biol. 1998; 8: 1069-1077Abstract Full Text Full Text PDF PubMed Google Scholar, 25Sajan M.P. Standaert M.L. Bandyopadhyay G. Quon M.J. Burke Jr., T.R. Farese R.V. J. Biol. Chem. 1999; 274: 30495-30500Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Standaert M.L. Bandyopadhyay G. Perez L. Price D. Galloway L. Poklepovic A. Sajan M.P. Cenni V. Sirri A. Moscat J. Toker A. Farese R.V. J. Biol. Chem. 1999; 274: 25308-25316Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). An atypical PKC isoform, PKCζ, has been implicated in Ras-independent ERK activation, either by direct phosphorylation of Raf1 (27van Dijk M.C. Hilkmann H. van Blitterswijk W.J. Biochem. J. 1997; 325: 303-307Crossref PubMed Scopus (63) Google Scholar) or of MEK (22Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Crossref PubMed Scopus (684) Google Scholar), serving as a linker between PI3K and ERK pathway. BaF3/Mpl cells were maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) with 10% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 2 mmol/literl-glutamine, 100 units/liter penicillin, 100 mg/ml streptomycin, and 0.25 mg/ml amphotericin B (BioWhittaker, Walkersville, MD) supplemented with murine interleukin-3 (IL-3), obtained from the conditioned medium of engineered baby hamster kidney (BHK) cells. BaF3 cells expressing various truncation mutants of Mpl (28Drachman J.G. Kaushansky K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2350-2355Crossref PubMed Scopus (137) Google Scholar) and primary MK isolation from BDF1 mice by unit gravity sedimentation (4Rojnuckarin P. Drachman J.G. Kaushansky K. Blood. 1999; 94: 1273-1282Crossref PubMed Google Scholar) were performed as described previously. T61 mice were a gift of Dr Frederick de Sauvage (Genentech Inc., San Francisco, CA). T61 MKs were isolated by a procedure similar to that for the wild type MKs, except that 10% fetal bovine serum was added to initial bone marrow cultures because these MKs developed poorly in the presence of TPO alone. At 48 h after culture initiation, nonadherent cells were transferred to a new flask because of excessive growth of adherent macrophages in serum-containing media. An MTT assay for cellular proliferation/survival in response to TPO was performed as reported previously (4Rojnuckarin P. Drachman J.G. Kaushansky K. Blood. 1999; 94: 1273-1282Crossref PubMed Google Scholar). Anti-IRS2, anti-PI3K (p85 subunit), and anti-phosphotyrosine (4G10) antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Dr. Toshio Hirano (Osaka, Japan) generously provided rabbit anti-Gab2 antibodies. Anti-phospho-Akt (Ser473) antibody and anti-phospho-ERK were obtained from New England Biolabs (Beverly, MA). All chemical inhibitors were purchased from Calbiochem (La Jolla, CA). BaF3 cells and MKs were deprived of serum and cytokines for 14 and 6 h, respectively, before stimulation with 14 ng/ml murine TPO for 10 min, washed once with ice-cold phosphate-buffered saline, and lysed in a buffer containing 0.5% Nonidet P-40 as reported previously (15Miyakawa Y. Rojnuckarin P. Habib T. Kaushansky K. J. Biol. Chem. 2001; 276: 2494-2502Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). The protein concentration of lysates was measured by Protein/DC assay (Bio-Rad). Specific proteins were immunoprecipitated from cell lysates by overnight incubation at 4 °C with the indicated antibodies. Protein A/G-conjugated agarose beads (Santa Cruz) were then added and incubated for an additional 2 h at 4 °C. The pelleted beads were then washed three times with lysis buffer, resuspended in gel electrophoresis loading buffer, and heated to 90 °C for 5 min. The immunoprecipitates were subjected to SDS-PAGE and Western blot analysis. Bands were scanned and quantified by the ImageQuant software. The PI3K activity assay was performed as described previously (15Miyakawa Y. Rojnuckarin P. Habib T. Kaushansky K. J. Biol. Chem. 2001; 276: 2494-2502Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). A DN p85 PI3K cDNA in SRα plasmid was a gift from Dr. Wataru Ogawa (Kobe University School of Medicine, Kobe, Japan). A DN SHP2 construct in a eukaryotic expression vector, pCAGGS, was a generous gift from Dr. Hiroshi Maegawa (Shiga University, Kyoto, Japan). To generate cell lines, 100 µg of DN plasmids were co-transfected with 10 µg of pMX-puro (a gift from Dr. Toshio Kitamura) into BaF3/T69 cells by electroporation. A control culture was transfected with pMX-puro plasmid alone. Puromycin at a final concentration of 1 µg/ml was added 24 h after transfection. Expression of the dominant negative protein was determined by Western blot analysis of whole cell lysates. Cells were lysed with MLB buffer (25 mm HEPES pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 10% glycerol, 25 mm NaF, 10 mmMgCl2, 1 mm EDTA, 1 mmNaVO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin), immediately frozen in a dry ice/ethanol bath, and stored at −70 °C until assayed. Lysates were clarified by centrifugation for 5 min, and 20 µl of packed GST-Ras-binding domain beads were added to 1 ml of supernatant. The mixture was rocked for 30 min at 4 °C. Beads were washed three times with MLB before being subjected to Western blot analysis and probed with a pan-Ras antibody (Transduction Laboratory, Lexington, KY). Cells were lysed with 50 mm Tris, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 10 mm NaF, 10 mm sodium pyrophosphate, 500 mm phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/ml pepstatin A, 1 mmNaVO4 and sonicated for 20 s. Five hundred µg of protein was pre-cleared with 1 µg of rabbit IgG and 20 µl of protein A agarose beads and immunoprecipitated overnight with 3 µg of anti-PKCζ. Forty µl of Protein A-agarose beads were added and incubated for another 2 h. Beads were then washed two times with the high salt buffer (500 mm Tris, pH 7.4, 500 mm NaCl, 1% Nonidet P-40), three times with lysis buffer, and two times with kinase buffer (25 mm HEPES, pH 7.4, 25 mm MgCl2, 20 mmβ-glycerophosphate, 20 mmp-nitrophenol phosphate, 20 mm NaVO4, 2 mmdithiothreitol). One-tenth of the last wash was collected for Western blot analysis of PKCζ to assess protein levels in the immune complexes. The kinase buffer was then added with 0.02 mmATP, 5 µCi of [γ-32P]ATP (Amersham Pharmacia Biotech), and either 5 µg of recombinant MEK1 (Santa Cruz, CA) or 16 µg of myelin basic protein (MBP) in a total volume of 25 µl. The kinase reaction was performed at 25 °C for 1 h, stopped by adding 20 µl of Western loading buffer and boiling for 5 min, and subjected to SDS-PAGE. The gel was then dried and exposed to film, and the intensity of bands was quantitated by PhosphorImaging. In searching for pathways responsible for Shc-independent ERK activation, the PI3K pathway was explored. As shown in Fig. 1A, TPO stimulation of BaF3/T69 clearly induced ERK phosphorylation, but to a lower extent than that seen in BaF3/Mpl cells. Using phosphorylation of Akt as a marker of PI3K activity, BaF3/T69 cells were found to retain the ability to activate PI3K in response to TPO, although weakly compared with that seen in BaF3/Mpl. Our group has shown that PI3K is essential for cellular proliferation in BaF3/Mpl and primary MK (31Geddis A. Fox N.E. Kaushansky K. J. Biol. Chem. 2001; 276: 34473-34479Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). As TPO-induced Akt activation is much weaker in BaF3/T69 cells than in cells with full-length Mpl, its physiological relevance needed to be determined. BaF3/T69 cell proliferation in response to various concentrations of TPO was assessed by an MTT assay in the presence or absence of a specific PI3K inhibitor, Ly 294002. This inhibitor significantly decreased TPO-induced BaF3/T69 survival/proliferation in a dose-dependent manner (Fig. 1B). At 16 µm final concentration, there were almost no viable cells remaining in culture. Therefore, despite only modest activation, PI3K plays an important role in proliferation of BaF3/T69 cells. Our previous studies have shown that the major mechanism of TPO-induced PI3K activation was via recruiting active enzyme into Gab- or IRS-containing signaling complexes (15Miyakawa Y. Rojnuckarin P. Habib T. Kaushansky K. J. Biol. Chem. 2001; 276: 2494-2502Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). To expand upon our Western blot analyses, PI3K activity associated with Gab2-and IRS2-complexes was determined, comparing BaF3/Mpl and BaF3/T69. Phosphoinositol was used as a substrate. After TPO stimulation, cell lysates were immunoprecipitated with either Gab2 or IRS2, and in vitro PI3K activity was assessed by measuring [γ-32P]ATP incorporation into phosphoinositol separated by thin layer chromatography (Fig.2A). Each reaction was conducted with an equal amount of immunoprecipitated docking protein, as determined by parallel Western blot experiments (Fig. 2A,lower panel). PI3K activity was clearly detectable after TPO stimulation in Gab2 and IRS2 signaling complexes derived from both BaF3/Mpl and BaF3/T69 cells. In BaF3/Mpl, the Gab2-associated PI3K activity was more prominent than the IRS2-associated activity. In contrast, the TPO-induced Gab2-associated PI3K activity was significantly lower, and the IRS2-containing complex became more prominent in BaF3/T69 cells. To further explore the mechanisms of PI3K activation in BaF3/T69 cells, we studied the two PI3K-associated signaling complexes in response to TPO, Gab2/SHP2/PI3K and IRS2/PI3K. Gab2 and SHP2 phosphorylation in BaF3 cells expressing full-length Mpl and various COOH-terminally truncated forms of Mpl (designated by T followed by the number of remaining cytoplasmic residues) is shown in a diagram (Fig.2B). A truncation of Mpl that eliminates the two terminal tyrosine residues of Mpl (T98) significantly reduced Gab2 phosphorylation and SHP2 association (compare Mpl to T98; Fig.2C, upper panel). Formation of the complex was further reduced in cells expressing shorter receptors, but as long as a receptor supported JAK2 phosphorylation (T98 through T53; Ref. 28Drachman J.G. Kaushansky K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2350-2355Crossref PubMed Scopus (137) Google Scholar) a modicum of Gab2 and SHP2 phosphorylation was seen. A different result was obtained with the IRS2 complex. TPO-induced p85 PI3K association with IRS2 was not decreased in BaF3/T69, compared with that of BaF3/Mpl cells (data not shown). Consistent with the PI3K activity data, IRS2-containing complex formation was not found to be dependent on the terminal half of Mpl. Specific PI3K inhibitors (Ly 294002 and wortmannin) were used to determine the roles of PI3K in ERK activation. In both BaF3/Mpl cells and BaF3/T69 cells, either Ly 294002 or wortmannin inhibited ERK phosphorylation in response to TPO (Fig.3). Compared to cells without inhibitors, TPO-induced ERK1 and ERK2 phosphorylation in BaF3/Mpl cells was reduced by wortmannin at 100 nm final concentration to 62 and 32%, respectively (n = 2). As BaF3/T69 supported less ERK activation, wortmannin decreased ERK1 and ERK2 phosphorylation to 17 to 12% of control, respectively (n = 3). These data suggest that PI3K is critical for ERK activation by this truncated receptor. To assess the role of PI3K by a different means, a dominant negative construct of p85 PI3K was used. The dominant negative p85 contained a small deletion, rendering it unable to bind to the catalytic subunit of PI3K, p110 (32Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda H. Stephens L. Jackson T.R. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (418) Google Scholar). However, its SH2 domains are intact, allowing it to compete with the wild type protein for binding to Gab and IRS docking proteins. Similar to the results with the chemical inhibitor, DN p85 significantly inhibited both Akt and ERK activation in BaF3/T69 cells. In two separate experiments, Akt and ERK phosphorylation were decreased by dominant negative expression to 57 and 55% (pAkt) and 47 and 25% (pERK2) of that seen in control cells not expressing the DN p85 construct. However, TPO-induced JAK2 phosphorylation was unchanged by the presence of DNp85, suggesting that the inhibition was specific (data not shown). SHP2 has been implicated in ERK activation in insulin and other growth factor systems (33Milarski K.L. Saltiel A.R. J. Biol. Chem. 1994; 269: 21239-21243Abstract Full Text PDF PubMed Google Scholar, 34Xiao S. Rose D.W. Sasaoka T. Maegawa H. Burke Jr, T.R. Roller P.P. Shoelson S.E. Olefsky J.M. J. Biol. Chem. 1994; 269: 21244-21248Abstract Full Text PDF PubMed Google Scholar, 35Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (350) Google Scholar, 36Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (347) Google Scholar). The pathway from SHP2 to MAPK has been shown to be dependent on Ras (35Noguchi T. Matozaki T. Horita K. Fujioka Y. Kasuga M. Mol. Cell. Biol. 1994; 14: 6674-6682Crossref PubMed Scopus (350) Google Scholar, 36Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (347) Google Scholar). We demonstrated above that SHP2 is phosphorylated and associates with Gab2 in response to TPO in BaF3/T69. In the lysophosphatidic acid and epidermal growth factor systems, binding of SHP2 to Gab1 is required for MAPK activation (37Cunnick J.M. Dorsey J.F. Munoz-Antonia T. Mei L. Wu J. J. Biol. Chem. 2000; 275: 13842-13848Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). A homologous SHP2 and Gab2 association in TPO system may function similarly. Therefore, the roles of SHP2 and Ras for ERK activation were investigated in BaF3/T69 cells. First, Ras activation after TPO stimulation was assessed in BaF3/Mpl and BaF3/T69 cells. Cell lysates were incubated with the Ras-binding domain of Raf1/GST fusion protein coupled to glutathione beads. As Raf1 binds only the active form of Ras, the amount of active Ras in cells was assessed by Western blot analysis probing the amount of Ras bound to Raf1/GST beads in the presence and absence of TPO with a pan-Ras antibody. BaF3/Mpl cells stimulated with TPO activate Shc, leading to Ras activation (Fig. 4A). In contrast, there was no Ras activation in BaF3/T69 cells, although ERKs were activated. Because the known signaling pathway from SHP2 to ERK is mediated by Ras, it is less likely that SHP2 induces ERK activation in BaF3/T69 cells. To more directly investigate the role of SHP2 in ERK activation in BaF3/T69 cells, a dominant negative SHP2 construct was used (38Maegawa H. Hasegawa M. Sugai S. Obata T. Ugi S. Morino K. Egawa K. Fujita T. Sakamoto T. Nishio Y. Kojima H. Haneda M. Yasuda H. Kikkawa R. Kashiwagi A. J. Biol. Chem. 1999; 274: 30236-30243Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). BaF3/T69 cells were stably transfected with a dominant negative SHP2 expression plasmid. Clones that highly expressed DN SHP2 and those that did not express the protein were selected for further study (Fig.4B). Endogenous SHP2 phosphorylation after TPO stimulation was markedly inhibited in DN cells (Fig. 4C,second panel). As shown in the top panel of Fig. 4C, the DN SHP2 had no effect on ERK activation in TPO-stimulated BaF3/T69 cells. Atypical isoforms of PKC, particularly PKCζ, have been implicated in Ras independent ERK activation in other cell systems (30Monick M.M. Carter A.B. Flaherty D.M. Peterson M.W. Hunninghake G.W. J. Immunol. 2000; 165: 4632-4639Crossref PubMed Scopus (111) Google Scholar,39Liao D.F. Monia B. Dean N. Berk B.C. J. Biol. Chem. 1997; 272: 6146-6150Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 40Takeda H. Matozaki T. Takada T. Noguchi T. Yamao T. Tsuda M. Ochi F. Fukunaga K. Inagaki K. Kasuga M. EMBO J. 1999; 18: 386-395Crossref PubMed Scopus (137) Google Scholar). Therefore, we considered PKCζ a potential candidate for mediating Ras-independent ERK activation in BaF3/T69 cells. The function of PKC isoforms was initially evaluated using two pharmacological inhibitors of PKCs, low concentrations of bisindolylmaleimide I (BIM), and Ro 31-8220. At low concentrations BIM is an inhibitor of the conventional and novel isoforms of PKC, displaying only minor effects on atypical isoforms of PKCs. In contrast, Ro 31-8220 is a relatively specific inhibitor of atypical isoforms of the kinase (26Standaert M.L. Bandyopadhyay G. Perez L. Price D. Galloway L. Poklepovic A. Sajan M.P. Cenni V. Sirri A. Moscat J. Toker A. Farese R.V. J. Biol. Chem. 1999; 274: 25308-25316Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). TPO-induced ERK activation in BaF3/T69 cells was not affected by BIM (Fig.5A), although PMA-induced activation of ERK was blocked (indicating that the inhibitor was active), suggesting that conventional and novel isoforms of PKCs are not essential for ERK activation by T69Mpl." @default.
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• W2066016982 title "The Roles of Phosphatidylinositol 3-Kinase and Protein Kinase Cζ for Thrombopoietin-induced Mitogen-activated Protein Kinase Activation in Primary Murine Megakaryocytes" @default.
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