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The Roles of Phosphatidylinositol 3-Kinase and Protein Kinase Cζ for Thrombopoietin-induced Mitogen-activated Protein Kinase Activation in Primary Murine Megakaryocytes*

Open AccessPublished:November 02, 2001DOI:https://doi.org/10.1074/jbc.M106508200
      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.
      TPO
      thrombopoietin
      PI3K
      phosphoinositide 3-kinase
      ERK
      extracellular signal-regulated kinase
      MAPK
      mitogen-activated protein kinase
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      MK
      megakaryocyte
      MTT
      3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide
      SHP
      Src homology 2 domain-containing protein tyrosine phosphatase
      IRS
      insulin-like receptor substrate
      PKC
      protein kinase C
      IL
      interleukin
      DN
      dominant negative
      PAGE
      polyacrylamide gel electrophoresis
      BIM
      bisindolylmaleimide I
      GST
      glutathioneS-transferase
      MBP
      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 (
      • Teglund S.
      • McKay C.
      • Schuetz E.
      • van Deursen J.M.
      • Stravopodis D.
      • Wang D.
      • Brown M.
      • Bodner S.
      • Grosveld G.
      • Ihle J.N.
      ,
      • Kirito K.
      • Osawa M.
      • Shimizu R.
      • Oda A.
      • Nakajima K.
      • Morita H.
      • Yamamoto M.
      • Ozawa K.
      • Komatsu N.
      ,
      • Luoh S.M.
      • Stefanich E.
      • Solar G.
      • Steinmetz H.
      • Lipari T.
      • Pestina T.I.
      • Jackson C.W.
      • de Sauvage F.J.
      ). 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 (
      • Rojnuckarin P.
      • Drachman J.G.
      • Kaushansky K.
      ). 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 (
      • Luoh S.M.
      • Stefanich E.
      • Solar G.
      • Steinmetz H.
      • Lipari T.
      • Pestina T.I.
      • Jackson C.W.
      • de Sauvage F.J.
      ). The classic pathway of ERK activation is via growth factor-induced Shc phosphorylation followed by its association with Grb2 (
      • Rozakis-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.
      ), which then activates Sos, a nucleotide exchange factor for Ras (
      • Buday L.
      • Downward J.
      ). 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 (
      • Drachman J.G.
      • Griffin J.D.
      • Kaushansky K.
      ). 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 (
      • Rojnuckarin P.
      • Drachman J.G.
      • Kaushansky K.
      ) and support cell growth. Consistent with these results, platelets from T61 mice also retain some ability to activate ERK, independent of Shc (
      • Luoh S.M.
      • Stefanich E.
      • Solar G.
      • Steinmetz H.
      • Lipari T.
      • Pestina T.I.
      • Jackson C.W.
      • de Sauvage F.J.
      ). 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.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). A constitutively active form of class IA PI3K has been demonstrated to activate MAPK by stimulating Ras (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ). Interference with the PI3K pathway, either by using pharmacological inhibitors (
      • Marra F.
      • Pinzani M.
      • DeFranco R.
      • Laffi G.
      • Gentilini P.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ,
      • Grammer T.C.
      • Blenis J.
      ,
      • Sarbassov D.D.
      • Peterson C.A.
      ) or expression of a dominant negative protein (
      • Hu Q.
      • Klippel A.
      • Muslin A.J.
      • Fantl W.J.
      • Williams L.T.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ) 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 (
      • Miyakawa Y.
      • Rojnuckarin P.
      • Habib T.
      • Kaushansky K.
      ). Consistent with our results, a docking protein, Gab1, has been implicated in PI3K and thus ERK activation in gp130 receptor signaling (
      • Takahashi-Tezuka M.
      • Yoshida Y.
      • Fukada T.
      • Ohtani T.
      • Yamanaka Y.
      • Nishida K.
      • Nakajima K.
      • Hibi M.
      • Hirano T.
      ). However, in other systems, constitutively active forms of this class of PI3K are insufficient to activate ERKs (
      • Klippel A.
      • Reinhard C.
      • Kavanaugh W.M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Frevert E.U.
      • Kahn B.B.
      ). In one report, wortmannin-sensitive ERK activation was mediated by the class IB PI3K, PI3Kγ (
      • Lopez-Ilasaca M.
      • Crespo P.
      • Pellici P.G.
      • Gutkind J.S.
      • Wetzker R.
      ), and the effect was dependent on its protein kinase, not lipid kinase activity (
      • Bondeva T.
      • Pirola L.
      • Bulgarelli-Leva G.
      • Rubio I.
      • Wetzker R.
      • Wymann M.P.
      ). 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 (
      • Duckworth B.C.
      • Cantley L.C.
      ). 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 (
      • Kunitama M.
      • Shimizu R.
      • Yamada M.
      • Kato T.
      • Miyazaki H.
      • Okada K.
      • Miura Y.
      • Komatsu N.
      ). 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 (
      • Schonwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ). Interestingly, PDK1, a kinase dependent on PI3K, can activate atypical isoforms of PKCs (
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ,
      • Chou M.M.
      • Hou W.
      • Johnson J.
      • Graham L.K.
      • Lee M.H.
      • Chen C.S.
      • Newton A.C.
      • Schaffhausen B.S.
      • Toker A.
      ,
      • Sajan M.P.
      • Standaert M.L.
      • Bandyopadhyay G.
      • Quon M.J.
      • Burke Jr., T.R.
      • Farese R.V.
      ,
      • Standaert 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.
      ). An atypical PKC isoform, PKCζ, has been implicated in Ras-independent ERK activation, either by direct phosphorylation of Raf1 (
      • van Dijk M.C.
      • Hilkmann H.
      • van Blitterswijk W.J.
      ) or of MEK (
      • Schonwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ), serving as a linker between PI3K and ERK pathway.

      DISCUSSION

      The TPO receptor Mpl generates a wide variety of signals that impact upon cellular survival, proliferation, and differentiation. Based on the capacity of a truncated TPO receptor to support cellular growth in vitro and to maintain base-line platelet production in vivo, yet fail to stimulate a full repertoire of signals, it appears that several TPO-induced signaling pathways are superfluous, at least for steady-state thrombopoiesis. Moreover, several essential Mpl-induced signaling molecules can be activated by more than one pathway. For example, ERK is activated by both Shc-dependent and Shc-independent pathways (
      • Luoh S.M.
      • Stefanich E.
      • Solar G.
      • Steinmetz H.
      • Lipari T.
      • Pestina T.I.
      • Jackson C.W.
      • de Sauvage F.J.
      ,
      • Rojnuckarin P.
      • Drachman J.G.
      • Kaushansky K.
      ). In addition, our data have shown previously that PI3K can be activated by either Gab2 or IRS2 complex formation (
      • Miyakawa Y.
      • Rojnuckarin P.
      • Habib T.
      • Kaushansky K.
      ). Recent reports have illustrated the effects of disrupting one (or a few) Mpl signaling pathways in whole animals (
      • Kirito K.
      • Osawa M.
      • Shimizu R.
      • Oda A.
      • Nakajima K.
      • Morita H.
      • Yamamoto M.
      • Ozawa K.
      • Komatsu N.
      ,
      • Luoh S.M.
      • Stefanich E.
      • Solar G.
      • Steinmetz H.
      • Lipari T.
      • Pestina T.I.
      • Jackson C.W.
      • de Sauvage F.J.
      ). Interestingly, these mice display normal base-line platelet counts, but respond to platelet demand poorly, displaying delayed recovery following the administration of myelosuppressive agents. Therefore, these apparent signaling redundancies are beneficial, serving as means to augment signal transduction intensity when necessary. Using cells expressing truncated Mpl receptors as models to dissect ERK activation from Shc/Ras, our study helps to define such additional pathways of ERK activation other than the well characterized Ras-dependent mechanism.
      We first investigated PI3K as an upstream activator of ERKs, because it has been shown previously to activate MAPKs in other signaling systems (
      • Marra F.
      • Pinzani M.
      • DeFranco R.
      • Laffi G.
      • Gentilini P.
      ,
      • Duckworth B.C.
      • Cantley L.C.
      ,
      • King W.G.
      • Mattaliano M.D.
      • Chan T.O.
      • Tsichlis P.N.
      • Brugge J.S.
      ,
      • Grammer T.C.
      • Blenis J.
      ,
      • Sarbassov D.D.
      • Peterson C.A.
      ). Furthermore, by itself, PI3K is critical for cellular proliferation and survival (
      • Geddis A.
      • Fox N.E.
      • Kaushansky K.
      ) and may thus provide essential signals for the growth of cells containing truncated receptors. Akt, a downstream effector of PI3K, is phosphorylated in response to TPO in BaF3/T69 cells, suggesting that the PI3K pathway can be activated by the truncated receptor. Consistent with this result, p85 associated with Gab2- and IRS2-containing signaling complexes and was activated in response to TPO. Similar to the results in BaF3 cells with the full-length receptor, PI3K activation was required for BaF3/T69 cell proliferation and survival as demonstrated by the use of the PI3K inhibitor Ly 294002 at concentrations just sufficient for Akt blockade, suggesting that this modest PI3K activation is physiologically important. Furthermore, we also demonstrate that class IA PI3K contributes TPO-induced ERK activation in BaF3/T69 cells using both pharmacological inhibitors and a dominant negative p85 construct.
      To investigate the site within the Mpl receptor from which PI3K activation originates, BaF3 cells expressing various COOH-terminal truncation mutants of Mpl were tested for PI3K-containing complexes after TPO stimulation. Our group demonstrated previously that mutations of two terminal tyrosine residues, Tyr117 and Tyr112, of Mpl reduced, but did not abolish, TPO-induced BaF3 cell proliferation (
      • Drachman J.G.
      • Kaushansky K.
      ). In this study, deletion of these terminal two tyrosine residues (the T98 construct) significantly decreased Gab2 phosphorylation and its association with SHP2 in BaF3 cells. A novel function of Shc was found to link Gab2 to the phosphotyrosine on the IL-2β receptor via Grb2 (
      • Gu H.
      • Maeda H.
      • Moon J.J.
      • Lord J.D.
      • Yoakim M.
      • Nelson B.H.
      • Neel B.G.
      ). Consistent with these data, a recent report showed that the Shc docking site on Mpl, Tyr112, serves a similar function in promoting Gab phosphorylation by JAK2 (
      • Bouscary D.
      • Lecoq-Lafon C.
      • Chretien S.
      • Zompi S.
      • Fichelson S.
      • Muller O.
      • Porteu F.
      • Dusanter-Fourt I.
      • Gisselbrecht S.
      • Mayeox P.
      • Lacombe C.
      ). However, in our hands, Gab2/SHP2/p85PI3K complex formation was still present in BaF3/T69, albeit reduced compared with BaF3/Mpl cells. This finding suggests that the Gab2/SHP2/PI3K complex contributes to PI3K activation in TPO-stimulated BaF3/Mpl cells, as TPO-induced Akt phosphorylation is significantly diminished in BaF3/T69 cells. In contrast to the Gab2-based PI3K complex, the IRS2/PI3K complex formed in BaF3/T69 was of similar intensity to that seen in BaF3/Mpl cells and the PI3K activity associated with this complex was also similar. Therefore, phosphorylation of IRS2 and Gab2 does not absolutely require phosphotyrosine docking sites or other sites within the distal 52 residues of c-Mpl.
      PI3K activation of ERKs can either be Ras-dependent or Ras-independent. Recent studies have shown that PI3K positively enhances Gab-containing complex formation, SHP2 recruitment (
      • Rodrigues G.A.
      • Falasca M.
      • Zhang Z.
      • Ong S.H.
      • Schlessinger J.
      ,
      • Yart A.
      • Laffargue M.
      • Mayeux P.
      • Chretien S.
      • Peres C.
      • Tonks N.
      • Roche S.
      • Payrastre B.
      • Chap H.
      • Raynal P.
      ), and thus Ras-dependent ERK activation (
      • Milarski K.L.
      • Saltiel A.R.
      ,
      • Xiao S.
      • Rose D.W.
      • Sasaoka T.
      • Maegawa H.
      • Burke Jr, T.R.
      • Roller P.P.
      • Shoelson S.E.
      • Olefsky J.M.
      ,
      • Noguchi T.
      • Matozaki T.
      • Horita K.
      • Fujioka Y.
      • Kasuga M.
      ,
      • Bennett A.M.
      • Tang T.L.
      • Sugimoto S.
      • Walsh C.T.
      • Neel B.G.
      ). To elucidate the unconventional mechanisms of ERK activation originating from the membrane proximal region of Mpl, the roles of Ras and SHP2 were explored. In contrast to the result using cells with the full-length receptor, there was no Ras activation by TPO in BaF3/T69 cells, although ERK was clearly activated. Furthermore, SHP2 disruption by a dominant negative form had no effect on ERK activation. These findings led us to search for a Ras-independent, SHP2-independent mechanism of ERK activation in BaF3/T69 cells.
      Various isoforms of PKCs have been shown to activate ERK by directly activating Raf and MEK (
      • Schonwasser D.C.
      • Marais R.M.
      • Marshall C.J.
      • Parker P.J.
      ). The role of PKC isoforms were explored initially using pharmacological inhibitors. ERK activation in BaF3/T69 cells was inhibited by Ro 31-8220, a specific inhibitor of atypical isoform of PKCs, but was not affected by an inhibitor of conventional and novel isoforms of PKC, low dose BIM. This finding implicated a role for atypical isoforms of PKCs in ERK activation. The cell-permeable PKCζ pseudosubstrate peptide significantly inhibited TPO-induced ERK activation in BaF3/T69 cells, consistent with the results of chemical inhibitors. Therefore, kinase assays were performed using PKCζ immunoprecipitates in the presence or absence of TPO. Using either MEK1 or MBP as a substrate TPO significantly enhanced cellular PKCζ activity. Therefore, PKCζ may activate ERK, at least partly, by direct phosphorylation of the MAPK kinase, MEK, in cells. However, the phosphorylation of the MAPK kinase kinase, Raf1, by PKCζ has also been reported (
      • van Dijk M.C.
      • Hilkmann H.
      • van Blitterswijk W.J.
      ) and may contribute to this Ras-independent ERK activation. As PKCζ has previously been shown to be a target of the PI3K-dependent kinase, PDK1 (
      • Le Good J.A.
      • Ziegler W.H.
      • Parekh D.B.
      • Alessi D.R.
      • Cohen P.
      • Parker P.J.
      ,
      • Sajan M.P.
      • Standaert M.L.
      • Bandyopadhyay G.
      • Quon M.J.
      • Burke Jr., T.R.
      • Farese R.V.
      ,
      • Romanelli A.
      • Martin K.A.
      • Toker A.
      • Blenis J.
      ), we hypothesized that the membrane proximal region of Mpl activates ERK through IRS2/PI3K and PKCζ. Pretreatment of cell with Ly 294002, a PI3K inhibitor, decreased PKCζ activation in four separate experiments. However, the standard deviation in these experiments was too great to statistically establish that PI3K is upstream of PKCζ. It has been reported that PDK1 phosphorylates PKCζ at Thr410 on its activation loop, resulting in its activation (
      • Sajan M.P.
      • Standaert M.L.
      • Bandyopadhyay G.
      • Quon M.J.
      • Burke Jr., T.R.
      • Farese R.V.
      ,
      • Standaert 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.
      ). We investigated Thr410 phosphorylation of PKCζ using a phosphothreonine-specific antibody (a gift from Dr. Alex Toker). However, we did not identify an increase in Thr410phosphorylation after TPO stimulation in either BaF3 cells or primary MKs (data not shown). Consistent with our data, insulin is also unable to induce PKCζ Thr410 phosphorylation, but PKCζ activity is enhanced by insulin-induced autophosphorylation of the enzyme (
      • Standaert 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.
      ). Therefore, it remains possible that PKCζ is activated by PI3K in response to TPO.
      Finally, we explored ERK activation in wild type MKs and MKs expressing a truncated Mpl receptor, T61. We demonstrated that TPO-induced ERK activation was inhibited by Ro 31-8220, but not by BIM, again suggesting an important role for one or more atypical isoforms of PKC. Furthermore, ERK, PI3K, and SHP2 were activated by TPO in T61 MKs, but Shc was not phosphorylated, suggesting that Shc-independent ERK activation also exists in primary cells. Interestingly, base-line ERK activation in T61 MKs was higher than that of wild type MKs (Fig.8A). This is possibly caused by a deletion of a negative regulatory region of Mpl.
      In primary MKs, a Tyr-phosphorylated protein that runs with a mass of ∼100 kDa, termed pp100, can be co-immunoprecipitated with p85 PI3K (
      • Miyakawa Y.
      • Rojnuckarin P.
      • Habib T.
      • Kaushansky K.
      ). We postulated that pp100 is a Gab-related adapter protein in MKs. We believe that PI3K can be activated in T61 MKs because of 1) the clear data in BaF3/T69 showing TPO-induced PI3K activation, 2) PI3K association with pp100 and SHP2 after TPO stimulation in T61 MKs, and 3) PI3K-dependent ERK activation in these MKs. However, we cannot demonstrate TPO-induced Akt activation in these cells. Consistent with our data, a previous report could not find TPO-induced Akt phosphorylation in T61 platelets (
      • Luoh S.M.
      • Stefanich E.
      • Solar G.
      • Steinmetz H.
      • Lipari T.
      • Pestina T.I.
      • Jackson C.W.
      • de Sauvage F.J.
      ). It is possible that Akt activation is under the limit of detection because of the small amount of protein as Akt phosphorylation detectable in BaF3/T69 cells required a large amount of protein, unobtainable from primary cells. Alternatively, PI3K activation in these MKs is not sufficient to support Akt phosphorylation, but is still able to activate ERKs via another (PKCζ) pathway. Blockade of PI3K reduced ERK activation only in T61 MKs, not in wild type MKs. This finding suggests that the Shc-dependent ERK activation pathway is predominant in normal MKs and the significance of the PI3K-dependent pathway is revealed only after the major pathway is disrupted. As we found evidence of a pp100/SHP2/PI3K complex in T61 MKs, our results suggest that PI3K is activated in these cells by a mechanism similar to that in MKs expressing the full-length receptor.
      In conclusion, we have shown that ERK, SHP2, PI3K, and PKCζ are activated in response to TPO in cells engineered to express a truncated Mpl receptor, BaF3/T69, and T61 MKs. Both PI3K and PKCζ contribute to ERK activation. PI3K may be an upstream activator of PKCζ, which in turn phosphorylates MEK (or possibly Raf1), resulting in ERK phosphorylation. The mechanism of PI3K activation by the proximal region of the Mpl receptor is probably via recruitment into TPO-induced signaling complexes similar to those found in cells bearing the Mpl receptor. This novel Shc-independent TPO-induced ERK activation may play an important role in megakaryopoiesis.

      Acknowledgments

      We thank Drs. Frederick de Sauvage, Toshio Hirano, Donald Foster, Wararu Ogawa, Hiroshi Maegawa, Toshio Kitamura, Jonathan G. Drachman, and Alex Toker for their gifts of mice, cell lines, and reagents.

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