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Phosphatidylinositol 3-Kinase Activity Regulates α-Thrombin-stimulated G1 Progression by Its Effect on Cyclin D1 Expression and Cyclin-dependent Kinase 4 Activity*

Open AccessPublished:June 16, 2000DOI:https://doi.org/10.1074/jbc.M909194199
      In this study, we present evidence that PI 3-kinase is required for α-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signal-regulated kinase (ERK)) pathway controls transit through G1 phase of the cell cycle by regulating the induction of cyclin D1 mRNA levels and cyclin dependent kinase 4 (CDK4)-cyclin D1 activity. In IIC9 cells, PI 3-kinase activation also is an important controller of the expression of cyclin D1 protein and CDK4-cyclin D1 activity. Pretreatment of IIC9 cells with the selective PI 3-kinase inhibitor, LY294002 blocks the α-thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect α-thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-negative Ras significantly decreased both α-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the α-thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase activities, both of which are required for expression of cyclin D1 protein and progression through G1.
      CDK
      cyclin-dependent kinase
      ERK
      extracellular signal-related kinase
      PI and PtdIns
      phosphatidylinositol
      Rb
      retinoblastoma
      HA
      hemagglutinin
      PBS
      phosphate-buffered saline
      Progression through the mammalian cell cycle requires the mitogen-stimulated induction of cyclin D1. In the presence of growth factor, cyclin D1 accumulates in the G1 phase of the cell cycle and assembles with its catalytic partner, CDK41 or CDK6 (
      • Matsushime H.
      • Ewen M.E.
      • Strom D.K.
      • Kato J.-Y.
      • Hanks S.K.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Matsushime H.
      • Quelle D.E.
      • Shurtleff S.A.
      • Shibuya M.
      • Sherr C.J.
      • Kato J.
      ,
      • Sherr C.J.
      ,
      • Bates S.
      • Bonetta L.
      • MacAllan D.
      • Parry D.
      • Holder A.
      • Dickson C.
      • Peters G.
      ). The cyclin D1-CDK4 or CDK6 complex controls transit through the G1/S phase transition by phosphorylating and inactivating the growth suppressor, retinoblastoma protein (Rb) (
      • Kato J.
      • Matsushime H.
      • Hiebert S.W.
      • Ewen M.E.
      • Sherr C.J.
      ,
      • Ewen M.E.
      • Sluss H.K.
      • Sherr C.J.
      • Matsushime H.
      • Kato J.
      • Livingston D.M.
      ,
      • Dowdy S.F.
      • Hinds P.W.
      • Louie K.
      • Reed S.I.
      • Arnold A.
      • Weinberg R.A.
      ,
      • Resnitzky D.M.
      • Gossen M.
      • Bujard H.
      • Reed S.I.
      ,
      • Weinberg R.A.
      ). In early G1, cyclin D1 levels increase and remain elevated. However, withdrawal of mitogen results in the rapid decline of cyclin D1 and growth arrest in G1 (
      • Sherr C.J.
      ,
      • Matsushime H.
      • Roussel M.F.
      • Ashmun R.A.
      • Sherr C.J.
      ). The importance of cyclin D1 as a regulator of transit through the G1 phase is emphasized by its ability to accelerate passage through the G1 phase of the cell cycle when it is overexpressed (
      • Resnitzky D.M.
      • Gossen M.
      • Bujard H.
      • Reed S.I.
      ,
      • Quelle D.E.
      • Ashmun R.A.
      • Shurtleff S.A.
      • Kato J.
      • Bar-Sagi D.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Liu J.
      • Chao J.
      • Jiang M.
      • Ng S.
      • Yen J.J.
      • Yang-Yen H.
      ). In addition, inhibition of cyclin D1 using antisense cDNA or microinjection of cyclin D1-specific antibodies results in withdrawal from the cell cycle and G1 growth arrest (
      • Quelle D.E.
      • Ashmun R.A.
      • Shurtleff S.A.
      • Kato J.
      • Bar-Sagi D.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Baldin V.
      • Lukas J.
      • Marcote M.J.
      • Pagano M.
      • Draetta G.
      ).
      It is well established that the Ras/ERK pathway is an important regulator of mitogen-stimulated expression of cyclin D1 (
      • Albanese C.
      • Johnson J.
      • Watanabe G.
      • Eklund N.
      • Vu D.
      • Arnold A.
      • Pestell R.G.
      ,
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Müller R.
      • Pouysségur J.
      ,
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ,
      • Weber J.D.
      • Cheng J.
      • Raben D.M.
      • Gardner A.
      • Baldassare J.J.
      ). Inhibition of the Ras/ERK pathway blocks mitogen-induced up-regulation of cyclin D1 in several cell types (
      • Albanese C.
      • Johnson J.
      • Watanabe G.
      • Eklund N.
      • Vu D.
      • Arnold A.
      • Pestell R.G.
      ,
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Müller R.
      • Pouysségur J.
      ,
      • Takuwa N.
      • Fukui Y.
      • Takuwa Y.
      ,
      • Gille H.
      • Downward J.
      ), including Chinese hamster fibroblasts (
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ,
      • Weber J.D.
      • Cheng J.
      • Raben D.M.
      • Gardner A.
      • Baldassare J.J.
      ), demonstrating the requirement of this pathway in the integration of extracellular signals responsible for cyclin D1 expression. We have shown previously that in IIC9 cells, platelet-derived growth factor-induced cyclin D1 accumulation is dependent on the sustained activation of ERK (
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ).
      In addition to the Ras/mitogen-activated protein kinase pathway, recent data suggest a role for the phosphatidylinositol (PI) 3-kinase in cell growth (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Coughlin S.R.
      • Escobedo J.A.
      • Willimas L.T.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Hu S.-W.
      • Shi X.-Y.
      • Lin R.Z.
      • Hoffman B.B.
      ). The PI 3-kinases comprise a family of lipid kinases that phosphorylate the 3-position of the inositol ring of phosphatidylinositol (PtdIns), PtdIns(4)P, and PtdIns(4,5)P2 to generate PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3, respectively. PI 3-kinase lipid products have been implicated as second messengers in several cellular processes including cell survival, mitogenesis, protein trafficking, and metabolism (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ,
      • Leevers S.J.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      ). Activation of PI 3-kinase activity has been shown to be required for DNA synthesis in response to several mitogens (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Hu S.-W.
      • Shi X.-Y.
      • Lin R.Z.
      • Hoffman B.B.
      ). In addition, the intracellular levels of PI 3-kinase lipid products are elevated in response to mitogen stimulation or oncogenic transformation (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Toker A.
      • Cantley L.C.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      ,
      • Rameh L.E.
      • Cantley L.C.
      ). The role of PI 3-kinase in growth probably involves the serine/threonine kinase, Akt (PKB), a downstream effector of PI 3-kinase, thought to be important for cell proliferation and antiapoptotic responses (
      • Franke T.F.
      • Yang S.-I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.T.
      • Coffer P.J.
      ,
      • Kennedy S.G.
      • Wagner A.J.
      • Conzen S.D.
      • Jordán J.
      • Bellacosa A.
      • Tsichlis P.N.
      • Hay N.
      ,
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Coffer P.J.
      • Jin J.
      • Woodgett J.R.
      ,
      • Downward J.
      ). Although the importance of the PI 3-kinase pathway in cell growth is well established, its role in the regulation of growth in not understood.
      α-Thrombin is a potent mitogen in IIC9 cells. The addition of α-thrombin to growth-arrested IIC9 cells stimulates an increase in endogenous ERK1 activity, and this activity is required for growth.
      A. J. Gardner and J. J. Baldassare, unpublished results.
      2A. J. Gardner and J. J. Baldassare, unpublished results.
      In this study, we show that PI 3-kinase is required for α-thrombin-stimulated growth in IIC9 cells. We provide evidence that PI 3-kinase is required for cyclin D1 accumulation independent of the ERK pathway. Furthermore, these pathways are regulated at the level of Ras. Our data indicate that both the PI 3-kinase and ERK pathways coordinately regulate cyclin D1 expression to promote cell cycle progression. This is the first study to examine the role of PI 3-kinase stimulated by a G-protein-coupled receptor in the regulation of cyclin D1.

      DISCUSSION

      Progression from the G1 to S phase of the cell cycle requires activation of CDK4, and CDK4 activation is controlled, in part, by complex formation with its catalytic partner, cyclin D1 (
      • Matsushime H.
      • Ewen M.E.
      • Strom D.K.
      • Kato J.-Y.
      • Hanks S.K.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Matsushime H.
      • Quelle D.E.
      • Shurtleff S.A.
      • Shibuya M.
      • Sherr C.J.
      • Kato J.
      ,
      • Sherr C.J.
      ,
      • Bates S.
      • Bonetta L.
      • MacAllan D.
      • Parry D.
      • Holder A.
      • Dickson C.
      • Peters G.
      ). Cyclin D1 levels are low in serum-arrested cells and increase in the G1 phase of the cell cycle. Several laboratories have demonstrated that the Ras/ERK pathway is an essential regulator of mitogen-stimulated expression of cyclin D1 and its assembly with its catalytic partner, CDK4 or -6 (
      • Albanese C.
      • Johnson J.
      • Watanabe G.
      • Eklund N.
      • Vu D.
      • Arnold A.
      • Pestell R.G.
      ,
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Müller R.
      • Pouysségur J.
      ,
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ,
      • Weber J.D.
      • Cheng J.
      • Raben D.M.
      • Gardner A.
      • Baldassare J.J.
      ). Although the importance of PI 3-kinase activity as a controller of cell growth is recognized (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Coughlin S.R.
      • Escobedo J.A.
      • Willimas L.T.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ), little is known of how PI 3-kinase affects growth. Recent data with several cell types suggest that PI 3-kinase contributes to the up-regulation of cyclin D1 (
      • Takuwa N.
      • Fukui Y.
      • Takuwa Y.
      ,
      • Gille H.
      • Downward J.
      ,
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Muise-Helmericks R.C.
      • Grimes H.L.
      • Bellacosa A.
      • Malstrom S.E.
      • Tsichlis P.N.
      • Rosen N.
      ,
      • Treinies I.
      • Paterson H.F.
      • Hooper S.
      • Wilson R.
      • Marshall C.J.
      ). Here we examine the effect of PI 3-kinase in α-thrombin-stimulated cyclin D1 expression and CDK4 activity. Our results clearly demonstrate that PI 3-kinase activity is required for the up-regulation of cyclin D1 protein expression. Treatment of IIC9 cells with LY294002, a selective inhibitor of PI 3-kinase, results in the significant reduction in α-thrombin-induced cyclin D1 protein expression (Fig. 4 B), CDK4 activity (Fig.3), and DNA synthesis (Fig. 1). In agreement with previous data from our laboratory (
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ) and others (
      • Lavoie J.N.
      • L'Allemain G.
      • Brunet A.
      • Müller R.
      • Pouysségur J.
      ,
      • Gille H.
      • Downward J.
      ), the inhibition of ERK activation also markedly suppresses mitogen-induced cyclin D1 expression. As previously reported for platelet-derived growth factor-induced cyclin D1 in IIC9 cells (
      • Weber J.D.
      • Raben D.M.
      • Phillips P.J.
      • Baldassare J.J.
      ), inhibition of ERK affects cyclin D1 mRNA expression stimulated by α-thrombin in IIC9 cells. In contrast, no detectable change in cyclin D1 mRNA is observed when PI 3-kinase is inhibited to levels found in growth-arrested IIC9 cells (Fig. 5). These data indicate that ERK and PI 3-kinase activities regulate α-thrombin-induced cyclin D1 protein expression by different mechanisms.

      The PI 3-Kinase G1 Target, Cyclin D1

      In IIC9 cells, PI 3-kinase is required for the regulation of cyclin D1 protein expression (Fig. 4 B). This data is consistent with several reports indicating a role for PI 3-kinase in mitogen-stimulated cyclin D1 up-regulation (
      • Takuwa N.
      • Fukui Y.
      • Takuwa Y.
      ,
      • Gille H.
      • Downward J.
      ,
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Muise-Helmericks R.C.
      • Grimes H.L.
      • Bellacosa A.
      • Malstrom S.E.
      • Tsichlis P.N.
      • Rosen N.
      ,
      • Treinies I.
      • Paterson H.F.
      • Hooper S.
      • Wilson R.
      • Marshall C.J.
      ). However, in IIC9 cells, LY294002 had no effect on α-thrombin-induced steady state message levels (Fig. 5), which is inconsistent with results in NIH3T3 cells stimulated with serum (
      • Takuwa N.
      • Fukui Y.
      • Takuwa Y.
      ,
      • Gille H.
      • Downward J.
      ). We do not think the effect of PI 3-kinase on cyclin D1 is cell type-specific. In support of this view, Diehl and co-workers (
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ), also working in NIH3T3 cells, found PI 3-kinase to be important in cyclin D1 protein stabilization. They (
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ) found that glycogen synthase kinase-3β phosphorylates and targets cyclin D1 for ubiquitin-mediated degradation. PI 3-kinase activates Akt, which phosphorylates and inhibits glycogen synthase kinase-3β (
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ). Furthermore, treatment of α-thrombin-stimulated IIC9 cells with the ubiquitin-dependent proteosome inhibitor, MG132, blocks the effects of LY294002 and results in levels of cyclin D1 protein seen with α-thrombin-stimulated IIC9 cells.
      P. J. Phillips-Mason and J. J. Baldassare, unpublished results.

      Role of Ras

      Previous studies in IIC9 cells show that activation of Ras is sufficient for transformation (
      • Weber J.D.
      • Cheng J.
      • Raben D.M.
      • Gardner A.
      • Baldassare J.J.
      ,
      • Cheng J.
      • Weber J.D.
      • Baldassare J.J.
      • Raben D.M.
      ). Because PI 3-kinase is essential for G1 transit and Ras activation results in growth, we reasoned that PI 3-kinase must be downstream of Ras. Consistent with this model and the ability of Ras to transform IIC9 cells, expression of RasN17 inhibits both ERK and PI 3-kinase activities (Fig. 7). Several reports have suggested Ras as a key regulator of several divergence signaling pathways (
      • Gille H.
      • Downward J.
      ,
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ,
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ,
      • Weber J.D.
      • Hu W.
      • Jefcoat Jr., S.C.
      • Raben D.M.
      • Baldassare J.J.
      ). In agreement with Ras being a divergence point for several activities, we (
      • Weber J.D.
      • Hu W.
      • Jefcoat Jr., S.C.
      • Raben D.M.
      • Baldassare J.J.
      ) have shown in IIC9 cells that Ras stimulates both ERK and RhoA, which control cyclin D1 up-regulation and p27 kip1 degradation, respectively. Proper transit through the cell cycle depends on the timely synthesis and degradation of cyclin D1. The regulation of cyclin D1 by both synthesis and degradation allows for the rapid changes necessary to secure the timely appearance and disappearance of cyclin D1. We propose that, in α-thrombin-stimulated cells, Ras coordinates the activation of both PI 3-kinase and ERK, leading to the rapid changes in cyclin D1 expression. This model is in agreement with the findings of Diehl and co-workers (
      • Diehl J.A.
      • Cheng M.
      • Roussel M.F.
      • Sherr C.J.
      ), who show that PI 3-kinase and ERK act independently to contribute to cyclin D1 accumulation.

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