Phosphatidylinositol 3-Kinase Activity Regulates a -Thrombin-stimulated G 1 Progression by Its Effect on Cyclin D1 Expression and Cyclin-dependent Kinase 4 Activity*

In this study, we present evidence that PI 3-kinase is required for a -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 G 1 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 a -thrombin-stimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect a -thrombin-induced cyclin D1 steady state message levels, indicating that PI 3-kinase acts independent of the ERK pathway. Interestingly, expression of a dominant-nega-tive Ras significantly decreased both a -thrombin-stimu-lated ERK and PI 3-kinase activities. These data clearly demonstrate that the a -thrombin-induced Ras activation coordinately regulates ERK and PI 3-kinase wortmannin. g/ml pepstatin). sonicated Molecular PhosphorImager TM representative of two independent

In this study, we present evidence that PI 3-kinase is required for ␣-thrombin-stimulated DNA synthesis in Chinese hamster embryonic fibroblasts (IIC9 cells). Previous results from our laboratory demonstrate that the mitogen-activated protein kinase (extracellular signalregulated kinase (ERK)) pathway controls transit through G 1 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 ␣-thrombinstimulated increase in cyclin D1 protein and CDK4 activity. However, LY294002 does not affect ␣-thrombininduced 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 ␣-thrombin-stimulated ERK and PI 3-kinase activities. These data clearly demonstrate that the ␣-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 G 1 .
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 G 1 phase of the cell cycle and assembles with its catalytic partner, CDK4 1 or CDK6 (1)(2)(3)(4). The cyclin D1-CDK4 or CDK6 complex controls transit through the G 1 /S phase transition by phosphorylating and inactivating the growth suppressor, retinoblastoma protein (Rb) (5)(6)(7)(8)(9). In early G 1 , cyclin D1 levels increase and remain elevated. However, withdrawal of mitogen results in the rapid decline of cyclin D1 and growth arrest in G 1 (3,10). The importance of cyclin D1 as a regulator of transit through the G 1 phase is emphasized by its ability to accelerate passage through the G 1 phase of the cell cycle when it is overexpressed (8,11,12). In addition, inhibition of cyclin D1 using antisense cDNA or microinjection of cyclin D1-specific antibodies results in withdrawal from the cell cycle and G 1 growth arrest (11,13).
It is well established that the Ras/ERK pathway is an important regulator of mitogen-stimulated expression of cyclin D1 (14 -17). Inhibition of the Ras/ERK pathway blocks mitogeninduced up-regulation of cyclin D1 in several cell types (14,15,18,19), including Chinese hamster fibroblasts (16,17), 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 (16).
In addition to the Ras/mitogen-activated protein kinase pathway, recent data suggest a role for the phosphatidylinositol (PI) 3-kinase in cell growth (20 -24). 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)P 2 to generate PtdIns(3)P, PtdIns(3,4)P 2 , and PtdIns(3,4,5)P 3 , respectively. PI 3-kinase lipid products have been implicated as second messengers in several cellular processes including cell survival, mitogenesis, protein trafficking, and metabolism (25)(26)(27). Activation of PI 3-kinase activity has been shown to be required for DNA synthesis in response to several mitogens (22)(23)(24). In addition, the intracellular levels of PI 3-kinase lipid products are elevated in response to mitogen stimulation or oncogenic transformation (25, 28 -30). 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 (31)(32)(33)(34)(35)(36). 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.
␣-Thrombin is a potent mitogen in IIC9 cells. The addition of ␣-thrombin to growth-arrested IIC9 cells stimulates an increase in endogenous ERK1 activity, and this activity is required for growth. 2 In this study, we show that PI 3-kinase is required for ␣-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.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-IIC9 cells, a subclone of Chinese hamster embryo fibroblasts, were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/liter glucose and 2 mM L-glutamine (BioWhittaker, Walkersville, MD) supplemented with 5% (v/v) fetal calf serum. Subconfluent IIC9 cells (80%) were growth-arrested by washing once with ␣-minimal essential medium (Life Technologies, Inc.), containing 2 mM L-glutamine (BioWhittaker) followed by a 48-h incubation in the same media. Human ␣-thrombin isolated from plasma (Sigma) was used at 1 unit/ml in all experiments. PD98059 (New England Biolabs, Beverly, MA) was used at 15 M. Wortmannin (Calbiochem) was used at 100 nM. LY294002 (Calbiochem) was used at 10 M. Calphostin C (Calbiochem) was used at 10 M.
Transient Transfection-The cDNA encoding pcDNA3 (Invitrogen) or a HA-tagged dominant-negative Ras mutant, HA-RasN17 (a kind gift from Gary L. Johnson, University of Colorado) was transfected into subconfluent (60 -80%) IIC9 cells using LipofectAMINE TM (Life Technologies) following the manufacturer's protocol. 2 g of each plasmid was mixed with 10 l of LipofectAMINE TM per 1 ml of medium. Six hours post-transfection, an equal volume Dulbecco's modified Eagle's medium supplemented with 0.2% (v/v) fetal calf serum was added to the transfection mix, and the cells were incubated overnight. The following day, cells were growth-arrested by washing once with ␣-minimal essential medium followed by a 48-h incubation in the same medium prior to agonist stimulation. Transient transfection using LipofectAMINE TM resulted in 80 -90% expression efficiency as visualized by ␤-galactosidase staining.
Western Blot Analysis-Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin after preincubation in the absence or presence of 100 nM wortmannin, 10 M LY294002, or 15 M PD98059 for 30 min. At the indicated times, cells were washed twice in cold PBS and harvested by scraping into 150 l of cold lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (v/v) Tween 20, 10% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin). The lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation at 14,000 ϫ g at 4°C for 5 min. Protein concentrations of the supernatants were determined using Coomassie ® Plus (Pierce) as recommended by the manufacturer. Protein lysates (10 -25 g) were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Boston, MA). Membranes were probed with polyclonal antibodies to cyclin D1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA), Akt (Santa Cruz Biotechnology), phopsho-Akt (New England Biolabs, Beverly, MA), or phospho-ERK1/2 (Santa Cruz Biotechnology). Immunoreactive bands were visualized by enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech) as recommended by the manufacturer.
Northern Blot Analysis-Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin after preincubation in the absence or presence of 100 nM wortmannin, 10 M LY294002, or 15 M PD98059 for 30 min. At the indicated times, total RNA was isolated using TRIZOL reagent (Life Technologies, Inc.) according to the manufacturer's protocol. RNA (20 g) was electrophoresed in a 2% (w/v) agarose-formaldehyde gel. After electrophoresis, formaldehyde was removed from the gel washing gels in 0.5% ammonium acetate. RNA was then transferred to a Hybond N ϩ nylon membrane (Amersham Pharmacia Biotech) using the Turboblotter TM system (Schleicher & Schuell) and cross-linked onto the membrane using an ultraviolet cross-linker (Amersham Pharmacia Biotech) as recommended by the manufacturer. Randomly labeled [␣-32 P]dCTP cDNA probes were made using the Random Primed DNA Labeling Kit (Roche Molecular Biochemicals). Membranes were preincubated with rapid hybridization buffer (Amersham Pharmacia Biotech) for 1 h at 65°C and then probed simultaneously with cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase probes for 2 h at 65°C. After hybridization, the membrane was washed twice with 5ϫ SSPE (20 mM EDTA, 1 M NaCl, 50 mM NaH 2 PO 4 -H 2 O), 0.1% (w/v) SDS at room temperature and once with 1ϫ SSPE, 0.1% SDS at 65°C. The membrane was developed using a PhosphorImager TM (Molecular Dynamics).
Immune Complex Kinase Assay for ERK1-Growth IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin after preincubation in the absence or presence of 100 nM wortmannin, 10 M LY294002, or 15 M PD98059 for 30 min. At the indicated times, cells were washed twice in cold PBS and harvested by scraping into 100 l of cold ERK lysis buffer and assayed as described previously (16).
Thymidine Incorporation-Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin for 17 h after preincubation in the absence or presence of 100 nM wortmannin or 10 M LY294002 for 30 min. Following the 17-h incubation, 1 Ci/ml [ 3 H]thymidine (NEN Life Science Products) was added to the cells for an additional 3-h incubation. 3 H-Labeled cells were washed twice with cold PBS, and the DNA was precipitated by incubating the cells in 5% (v/v) trichloroacetic acid for 30 min at 4°C. The trichloroacetic acidprecipitated DNA was washed twice with cold 5% trichloroacetic acid and solubilized with 500 l of 2% (w/v) sodium bicarbonate, 0.1 N NaOH. The solution was neutralized with 100 l of 5% trichloroacetic acid, and the trichloroacetic acid-precipitated [ 3 H]DNA was quantified by scintillation counting.
Ras Activation Assay-Growth-arrested IIC9 cells were labeled for 4 h with [ 32 P]P i at 0.2 mCi/100-mm dish in phosphate-free Dulbecco's modified Eagle's medium (BioWhittaker). Cells were then washed twice with phosphate-free medium and once with a saline buffer (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). Cells were incubated in the presence or absence of 1 unit/ml ␣-thrombin for 5 min following preincubation in the absence or presence of 100 nM wortmannin or 10 M LY294002 for 30 min. After stimulation, cells were washed two times with PBS and harvested by scraping into 500 l of IP buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl 2 , 150 mM NaCl, 1% Triton X-100, 2 mM p-nitrophenylphosphate, 10 g/ml pepstatin, 10 g/ml aprotinin, and 10 g/ml leupeptin). Homogenates were incubated for 10 min on ice and then centrifuged at 750 ϫ g for 5 min. The supernatants were treated with 100 l of bovine serum albumin-coated charcoal for 5 min at 4°C and then centrifuged at 750 ϫ g to remove charcoal. Ras immune complexes were immunoprecipitated by incubation with a monoclonal p21 ras antibody (Oncogene) for 3 h at 4°C, followed by an incubation with protein G plus agarose (Oncogene) overnight at 4°C. Ras complexes were washed twice with IP buffer and three times with wash buffer (Tris-HCl, pH 7.5, 20 mM MgCl 2 , and 150 mM NaCl). Final pellets were drained and bound nucleotides were eluted in 20 l of elution buffer (20 mM Tris-HCl, pH 7.5, 20 mM EDTA, 2% SDS, 0.5 mM GDP, and 0.5 mM GTP). Eluants were heated at 65°C for 5 min and centrifuged. The supernatants were spotted onto a polyethyleneimine-cellulose thin layer plate (Merck) and developed with 0.75 M KH 2 PO 4 , pH 3.4. GDP and GTP 32 P-labeled fractions were quantified using a Phos-phorImager TM (Molecular Dynamics).
Phosphatidylinositol 3-Kinase Assay-Growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin for 10 min after preincubation in the absence or presence of wortmannin. After stimulation, cells were washed twice with PBS and harvested by scraping into in 400 l of cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl 2 , 1% (v/v) Triton X-100, 10% (v/v) glycerol, 1 mM EGTA, 100 mM sodium vanadate, 50 mM ␤-glycerophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g/ml pepstatin). The lysates were sonicated briefly, and the insoluble material was pelleted by centrifugation at 14,000 ϫ g at 4°C for 5 min. p85 immune complexes were immunoprecipitated from lysates containing 300 -400 g of protein by incubation with polyclonal p85 antibody (Upstate Biotechnology, Inc., Lake Placid, NY) at 4°C for 3 h, followed by an incubation with protein A-agarose (Sigma) at 4°C overnight. The p85 immune complexes were pelleted by centrifugation and washed three times with lysis buffer, three times with TNE (100 mM Tris-HCl, pH 7.4, 5 M LiCl, and 100 mM sodium vanadate), and twice with 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, and 100 mM sodium vanadate. After the last wash, 50 l of TNE, 10 l of phosphatidylinositol (suspended by sonication in 10 mM Tris-HCl, pH 7.4, and 1 mM EGTA at 2 g/ml), and 10 l of 100 mM MgCl 2 was added to the beads. The reaction was started by the addition of 5 l of reaction buffer (0.88 mM ATP, 10 Ci of [␥-32 P]ATP, and 20 mM MgCl 2 ). Samples were incubated for 10 min at 37°C, and the reactions were stopped by the addition of 20 l of 6 N HCl. Lipids were extracted by adding 160 l of CHCl 3 /MeOH (1:1) to the samples and vortexing briefly. Labeled lipids were resolved by spotting on a silicon TLC plate (J. T. Baker Inc.) and developed with CHCl 3 /MeOH/4 N NH 4 OH (9:7:2). Phosphatidylinositol-3-phosphate production was quantitated using a PhosphorImager TM (Molecular Dynamics). Phosphatidylinositol 4-phosphate isolated from bovine brain (Avanti Polar Lipids, Inc., Alabaster, AL) was included as a standard for TLC resolution of the lipids and visualized by iodine vapor.
Phosphoinositide Hydrolysis Assay-Subconfluent IIC9 cells were incubated for 24 h in myoinositol-free basal medium and then an additional 24 h with 1 Ci of [ 3 H]myoinositol (NEN Life Science Products) in the same medium. LiCl was added to the cells 1 min prior to incubation in the absence or presence of ␣-thrombin for 15 min. Cells were washed with cold PBS, and total inositol phosphates were extracted in 600 l of cold 4% HClO 4 (v/v) for 30 min at 4°C. To each supernatant, 50 l of phenol red, 60 mM Hepes was added, and supernatants were neutralized with Na 4 OH and then centrifuged. The supernatants were applied to a 0.5-ml column of Dowex AG1 ϫ 8 (200 -400 mesh size), formate form (Bio-Rad). Columns were washed three times with 3 ml of water and three times with 3 ml of 0.05 M ammonium formate, 0.005 M Borax. [ 3 H]Inositol phosphates were eluted with 3 ml of formic acid, 1.8 M ammonium formate, and 1 ml was quantified by scintillation counting.

PI 3-Kinase Is
Required for ␣-Thrombin-induced DNA Synthesis-Recently, much attention has focused on the role of PI 3-kinase in the regulation of cell growth. PI 3-kinase activity has been shown to be required for DNA synthesis in response to several mitogens (22)(23)(24). To determine whether PI 3-kinase is essential for ␣-thrombin-stimulated growth in IIC9 cells, we investigated the effect of a selective PI 3-kinase inhibitor, LY294002, on ␣-thrombin-induced DNA synthesis (Fig. 1). Pre-treatment of growth-arrested IIC9 cells with LY294002 inhibits ␣-thrombin-induced DNA synthesis as measured by [ 3 H]thymidine incorporation in a dose-dependent manner (Fig.  1). Maximal inhibition of ␣-thrombin-induced DNA synthesis occurs at a concentration of 10 M LY294002 (Fig. 1). Wortmannin, a less selective inhibitor of PI-3-kinase activity, also blocks ␣-thrombin-induced DNA synthesis in a dose-dependent manner (data not shown).
We next examined whether ␣-thrombin-induces the activation of endogenous PI 3-kinase activity. ␣-Thrombin stimulates a 2-fold increase in PI 3-kinase activity over levels found in growth-arrested IIC9 cells (Fig. 2A). Although LY294002 is a potent selective inhibitor of PI 3-kinase activity, its effects are reversible (37) and subject to error when assayed in vitro by immunocomplex assays. To overcome this problem, we investigated whether LY294002 inhibits ␣-thrombin-induced Akt phosphorylation. This phosphorylation can be quantified by immunoblot analysis with a specific anti-phospho-Akt antibody. We reasoned that because the phosphorylation of Akt is dependent on PI 3-kinase activation (31,32,34), changes in its phosphorylation reflect changes in PI 3-kinase activity. ␣-Thrombin induces a rapid increase in Akt phosphorylation as detected by immunoblot analysis (Fig. 2B). Increased activity is detectable within 10 min and remains elevated for at least 60 min. Preincubation of IIC9 cells with 10 M LY294002 blocks Akt phosphorylation (Fig. 2C). Importantly, treatment with 10 M LY294002 decreased Akt phosphorylation to values seen in growth-arrested IIC9 cells, in agreement with its effects on DNA synthesis. To confirm our results, we also determined the effect of wortmannin, a nonreversible but less selective inhibitor of PI 3-kinase activity on both PI 3-kinase ( Fig. 2A) and Akt phosphorylation (data not shown). When IIC9 cells are incubated with 100 nM wortmannin 30 min prior to stimulation with ␣-thrombin, PI 3-kinase activity ( Fig. 2A) and Akt phosphorylation (data not shown) are below levels found in growtharrested IIC9 cells.
PI 3-Kinase Is Required for ␣-Thrombin-induced CDK4 Activity and Accumulation of Cyclin D1-Previous data from our laboratory (16) and others (15) have shown that inhibition of mitogen-induced signaling pathways affects CDK4 activity and progression through the G 1 phase of the cell cycle. We therefore decided to determine whether inhibition of PI 3-kinase activity affects cyclin D1-CDK4 activation by quantifying the effects of LY294002 on ␣-thrombin-stimulated CDK4 activity. Stimulation with ␣-thrombin produces a 4-fold induction of CDK4 activity that is blocked by preincubation of IIC9 cells with 10 M LY294002 (Fig. 3). Interestingly, this concentration of LY294002 also reduces DNA synthesis and Akt phosphorylation to growth-arrested levels ( Figs. 1 and 2C).
The effect of PI 3-kinase on CDK4 activity is similar to that seen with inhibitors of the ERK pathway in IIC9 cells (16). Previously, we (16) found that the ERK pathway controls platelet-derived growth factor-induced CDK4 activity by regulating expression of cyclin D1. We next determined the effect of inhibition of ERK activation on ␣-thrombin-induced cyclin D1 expression. Pretreatment of IIC9 cells with 15 M PD98059, a selective MEK inhibitor, prior to treatment with ␣-thrombin blocks the ␣-thrombin-stimulated increase in cyclin D1 expression (Fig. 4B) and CDK4 activity (Fig. 3). These data suggested to us that PI 3-kinase also may regulate CDK4 activity by regulating cyclin D1 expression. Moreover, others have suggested that PI 3-kinase regulates mitogen-stimulated cyclin D1 expression (19, 38 -40). Western blot analysis shows that in IIC9 cells, ␣-thrombin stimulates a 3-5-fold induction in cyclin D1 by 4 h, and the increase in cyclin D1 protein is sustained for at least 10 h (Fig. 4A). Indeed, pretreatment of IIC9 cells with 10 M LY294002 (Fig. 4B) or 100 nM wortmannin (data not shown), completely blocks the ␣-thrombin-induced up-regulation of cyclin D1, suggesting that PI 3-kinase regulates CDK4 activity through its effect on cyclin D1 expression.
PI 3-Kinase Does Not Affect Cyclin D1 Steady State Message Levels-If PI 3-kinase prevents expression of cyclin D1 identi-cal to ERK, we expect both pathways to block cyclin D1 protein in a similar manner. Previous data from our laboratory (16) and others (14,15,19) demonstrate that ERK controls cyclin D1 mRNA expression. ␣-Thrombin stimulates a 1.9-fold induction in cyclin D1 mRNA levels by 4 h (Fig. 5) and reaches 2.7-fold by 8 h poststimulation. Preincubation with LY294002 (Fig. 5) or wortmannin (data not shown) does not affect the ␣-thrombin-induced increase in cyclin D1 mRNA 6 h poststimulation, indicating that PI 3-kinase is not regulating cyclin FIG. 2. ␣-Thrombin-stimulated PI 3-kinase activity is sensitive to inhibition by wortmannin and LY294002. A, growth-arrested IIC9 cells were preincubated in the absence or presence of wortmannin at 1, 10, 50, and 100 nM, 30 min prior to incubation in the absence or presence of 1 unit/ml ␣-thrombin for 10 min. Cells were harvested by scraping into cold PI 3-kinase lysis buffer (see "Experimental Procedures"). PI 3-kinase complexes were immunoprecipitated from lysates containing equal protein using an anti-p85 polyclonal antibody and assayed for their ability to phosphorylate PI in vitro as described under "Experimental Procedures." Total lipids were extracted and resolved by TLC. [ 32 P]PI3P was quantitated using a Molecular Dynamics Phospho-rImager TM . B, growth-arrested IIC9 cells were incubated in the absence or presence of 1 unit/ml ␣-thrombin for 0, 10, 20, 30, 45, or 60 min. Cells lysates were prepared in 1ϫ Laemmli buffer, separated by SDS-polyacrylamide gel electrophoresis (9.75%), and immunoblotted with an anti-phospho-Akt or anti-Akt polyclonal antibody. C, growth-arrested IIC9 cells were incubated in the absence or presence of 10 M LY294002 30 min prior to incubation in the absence or presence or 1 unit/ml ␣-thrombin for 30 min. Cell lysates were prepared, and immunoblots were performed as described above. Data are representative of at least two independent experiments. D1 steady state message levels. In contrast, PD98059 completely blocks the ␣-thrombin-induced increase in cyclin D1 mRNA.
PI 3-Kinase Is Not Required for ␣-Thrombin-induced ERK1 Activation-Several recent reports indicate a role for PI 3-kinase in the regulation of ERK in response to activation by G-protein-coupled receptors. In COS-7 cells, treatment with wortmannin or LY294002 or expression of a dominant-negative mutant of PI 3-kinase blocks ERK activation by lysophosphatidic acid (41). Similar results are seen with carbachol in COS-7 cells expressing the m2 muscarinic receptor (42). Our data, however, indicate differential regulation of cyclin D1 expression by ERK and PI 3-kinase, suggesting that these pathways are parallel and not dependent on each other. To confirm that PI 3-kinase is not required for ␣-thrombin-stimulated ERK activity in IIC9 cells, we examined the effect of LY294002 and wortmannin on endogenous ERK1 activity. ␣-Thrombin stimulates a biphasic activation of ERK, with a rapid initial phase within the first few minutes of activation and a late sustained phase lasting at least 4 h (Fig. 6A). Pretreatment of IIC9 cells with 10 M LY294002 (Fig. 6A) or 100 nM wortmannin (data not shown) does not affect early or sustained ERK1 activity, although these concentrations completely block PI 3-kinase activation ( Fig. 2C and data not shown) as determined by the phosphorylation of Akt. In addition, pretreatment with PD98059 does not affect Akt phosphorylation (data not shown). These data clearly demonstrate that PI 3-kinase does not regulate the ERK pathway and differentially contribute to both cyclin D1 up-regulation and cell cycle progression. As expected, pretreatment of IIC9 cells with PD98059 (Fig. 6A) blocks ␣-thrombin-stimulated ERK1 activity. In agreement with these data, pretreatment of IIC9 cells with LY294002 (Fig. 6B) or wortmannin (data not shown) had no effect on ␣-thrombinstimulated Ras activity, which is required for ␣-thrombin-stimulated ERK1 activation in IIC9 cells (see below).
Ras Coordinately Regulates PI 3-Kinase and ERK1 Activities-Previous data from our laboratory (16) clearly demonstrate that activation of Ras results in cyclin D1 expression and transformation of IIC9 cells. We reasoned that coordinate regulation of the ERK and PI 3-kinase pathway could account for the ability of Ras to transform IIC9 cells. To determine whether PI 3-kinase and ERK are coordinately controlled by Ras, we quantified the effect of expression of HA-tagged dominant neg-ative Ras (HA-RasN17) on endogenous Ras, ERK, and PI 3-kinase activities (Fig. 7). Because of variation in the transfection efficiency between experiments, the endogenous activities were quantified from the same lysates. In IIC9 cells, ␣-thrombin induces a 4-fold increase (GTP/(GTP ϩ GDP) increased from 9 to 35%) in GTP loading of Ras (Fig. 7A). Expression of HA-FIG. 5. ␣-Thrombin-stimulated PI 3-kinase does not regulate cyclin D1 steady-state message levels. Growth-arrested IIC9 cells were preincubated in the absence or presence of 15 M PD98059 or 10 M LY294002, 30 min prior to incubation in the absence or presence of 1 unit/ml ␣-thrombin for 0, 4, 6, and 8 h. At the indicated times, total RNA was isolated with TRIZOL reagent (Life Technologies, Inc.) according to the manufacturer's protocol. RNA (15 g) was separated on a 2% agarose/formaldehyde gel and transferred to Hybond Nϩ nylon membrane. The membrane was probed simultaneously with randomly [␥-32 P]dCTP-labeled cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase and washed as described under "Experimental Procedures." Cyclin D1 and glyceraldehyde-3-phosphate dehydrogenase transcripts were visualized using a Molecular Dynamics PhosphorImager TM . Data are representative of two independent experiments.
FIG. 6. PI 3-kinase acts independently of the mitogen-activated protein kinase pathway. A, growth-arrested IIC9 cells were preincubated in the absence (ⅷ) or presence of 10 M LY294002 (OE) or 15 M PD98059 (f) prior to incubation in the absence or presence of 1 unit/ml ␣-thrombin for 5, 30, 120, 240, and 360 min. Cells were harvested by scraping into cold ERK lysis buffer (see "Experimental Procedures"). ERK1 complexes were immunoprecipitated from lysates containing equal protein and assayed for their ability to phosphorylate myelin basic protein in vitro as described under "Experimental Procedures." ERK1 activity was quantitated using a Molecular Dynamics PhosphorImager TM . Data are representative of three independent experiments. B, 32 P-labeled IIC9 cells were growth-arrested and preincubated in the absence or presence of 10 M LY294002 30 min prior to incubation in the absence or presence of 1 unit/ml ␣-thrombin for 5 min. Cells were harvested by scraping into cold lysis buffer (see "Experimental Procedures"). Ras immune complexes were immunoprecipitated with a monoclonal Ras antibody and analyzed for 32 P-labeled guanine nucleotides by TLC as described under "Experimental Procedures." Resolved nucleotides were quantitated using a Molecular Dynamics PhosphorImager TM , and Ras activity is reported as the ratio of GTP/ (GTP ϩ GDP). The results are the mean Ϯ S.D. of two independent experiments. FIG. 7. PI 3-kinase and ERK activities are mediated by Ras. IIC9 cells were transiently transfected with HA-RasN17 using LipofectAMI-NE TM as described under "Experimental Procedures." Transfected IIC9 cells were 32 P-labeled and growth-arrested. In those samples treated with calphostin C, cells were preincubated with 10 M calphostin C for 1 h prior to incubation with 1 unit/ml ␣-thrombin for 5 min. A, HA-RasN17 was removed by immunoprecipitation with anti-HA antibody for 1 h. Immediately after treatment with antibodies directed against HA, endogenous Ras immune complexes were immunoprecipitated by treatment with a monoclonal Ras antibody and analyzed for 32 P-labeled guanine nucleotides by TLC as described under "Experimental Procedures." Resolved nucleotides were quantitated using a Molecular Dynamics PhosphorImager TM , and Ras activity is reported as the ratio of GTP/(GTP ϩ GDP). B, after removal of unreacted 32 P by treatment with albumin-coated charcoal, the lysates containing equal protein were prepared in 1ϫ Laemmli buffer, separated by SDS-polyacrylamide gel electrophoresis (9.75%), and immunoblotted with an anti-phospho-Akt antibody. C, ERK1 complexes were immunoprecipitated from lysates containing equal protein and assayed for their ability to phosphorylate myelin basic protein in vitro as described under "Experimental Procedures." ERK1 activity was quantitated using a Molecular Dynamics PhosphorImager TM . Data are representative of three independent experiments. RasN17 (Fig. 7A) blocks the increase of GTP loading of Ras by approximately 75% (GTP/(GTP ϩ GDP) increases from 9 to 14%). Transient expression of HA-RasN17 inhibits PI 3-kinase activity by approximately 70% as measured by Akt phosphorylation (Fig. 7B). Taken together, these data strongly indicate that ␣-thrombin-induced PI 3-kinase activity is mediated solely through Ras.
We next determined the effect of expression of HA-RasN17 on ERK activity (Fig. 7C). As shown for other cell types (41), whereas sustained ERK activity (30 min to several hours) is solely dependent on Ras, multiple signaling pathways regulate the initial phase (between 5 and 20 min) of ERK activation in IIC9 cells. Approximately 50% of the initial phase of ␣-thrombin-induced ERK activity is Ras-dependent. In agreement with others (41), the Ras-independent activation is mediated through the G q family by activation of phospholipase C␤1 and protein kinase C. 2 In serum-arrested IIC9 cells, ERK1 activity is approximately 11% of the maximal activity (100%) found in IIC9 cells stimulated with ␣-thrombin for 5 min (Fig. 7C). ERK1 activity in cells transient transfected with HA-RasN17 increases to 53% of the maximal activity when stimulated with ␣-thrombin, indicating that expression of HA-RasN17 inhibits the initial phase by approximately 40 -45% (Fig. 7C). Furthermore, in the presence of the protein kinase C inhibitor, calphostin C, ERK1 activity increases to 23% of maximal level (Fig.  7C). Therefore, treatment with both the calphostin C and HA-RasN17 ERK1 activity blocks ERK1 activation by greater than 80%. Taken together with the data showing that expression of HA-RasN17 blocks Ras GTP-loading by 90%, these data strongly indicate that Ras mediates a significant portion of the initial phase of ␣-thrombin ERK1 activation. Moreover, treatment with calphostin C does not further block Ras activation (GTP/(GTP ϩ GDP) increased from 9 to 15%) in the presence of HA-RasN17 (Fig. 7A), indicating that protein kinase C activity is not involved in Ras activation.
Because expression of RasN17 inhibits growth, we next examined whether the inhibition of the ERK and PI 3-kinase activities are specific or a consequence of the inability of these cells to grow. In IIC9 cells, ␣-thrombin-induced inositol phosphate release is mediated through G q and is independent of Ras. 2 We next examined the effect of expression of HA-RasN17 on PI hydrolysis (Fig. 8). Treatment of IIC9 cells with ␣-throm-bin increases inositol phosphate release approximately 5-fold. Ectopic expression of HA-RasN17 does not affect inositol phosphate release (Fig. 8), indicating that the effect of HA-RasN17 is specific. DISCUSSION Progression from the G 1 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 (1)(2)(3)(4). Cyclin D1 levels are low in serum-arrested cells and increase in the G 1 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 (14 -17). Although the importance of PI 3-kinase activity as a controller of cell growth is recognized (20 -22, 25), 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 (18, 19, 38 -40). Here we examine the effect of PI 3-kinase in ␣-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 ␣-thrombin-induced cyclin D1 protein expression (Fig. 4B), CDK4 activity (Fig. 3), and DNA synthesis (Fig. 1). In agreement with previous data from our laboratory (16) and others (15,19), 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 (16), inhibition of ERK affects cyclin D1 mRNA expression stimulated by ␣-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 ␣-thrombin-induced cyclin D1 protein expression by different mechanisms.
The PI 3-Kinase G 1 Target, Cyclin D1-In IIC9 cells, PI 3-kinase is required for the regulation of cyclin D1 protein expression (Fig. 4B). This data is consistent with several reports indicating a role for PI 3-kinase in mitogen-stimulated cyclin D1 up-regulation (18, 19, 38 -40). However, in IIC9 cells, LY294002 had no effect on ␣-thrombin-induced steady state message levels (Fig. 5), which is inconsistent with results in NIH3T3 cells stimulated with serum (18,19). 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 (38), also working in NIH3T3 cells, found PI 3-kinase to be important in cyclin D1 protein stabilization. They (38) found that glycogen synthase kinase-3␤ phosphorylates and targets cyclin D1 for ubiquitinmediated degradation. PI 3-kinase activates Akt, which phosphorylates and inhibits glycogen synthase kinase-3␤ (38). Furthermore, treatment of ␣-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 ␣-thrombin-stimulated IIC9 cells. 3 Role of Ras-Previous studies in IIC9 cells show that activation of Ras is sufficient for transformation (17,43). Because PI 3-kinase is essential for G 1 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 (19,38,44,45). In agreement with Ras being a 3 P. J. Phillips-Mason and J. J. Baldassare, unpublished results. divergence point for several activities, we (45) 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 ␣-thrombinstimulated 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 (38), who show that PI 3-kinase and ERK act independently to contribute to cyclin D1 accumulation.