THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 29, Issue of July 18, pp. 26466 –26473, 2003 Printed in U.S.A.
Phosphoinositide 3-Kinase Activation Regulates Cell Division Time by Coordinated Control of Cell Mass and Cell Cycle Progression Rate* Received for publication, January 21, 2003, and in revised form, April 2, 2003 Published, JBC Papers in Press, April 21, 2003, DOI 10.1074/jbc.M300663200
Beatriz Alvarez, Elia Garrido, Jose A. Garcia-Sanz, and Ana C. Carrera‡ From the Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas, Universidad Auto´noma de Madrid, Cantoblanco, Madrid E-28049, Spain
Cells must increase their mass in coordination with cell cycle progression to ensure that their size and macromolecular composition remain constant for any given proliferation rate. To this end, growth factors activate early signaling cascades that simultaneously promote cell mass increase and induce cell cycle entry. Nonetheless, the mechanism that controls the concerted regulation of cell growth and cell cycle entry in mammals remains unknown. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B pathway regulates cell cycle entry by inactivating forkhead transcription factors and promoting cyclin D synthesis. PI3K/protein kinase Bderived signals also affect activation of p70 S6 kinase and the mammalian target of rapamycin, enzymes involved in cell growth control. We previously showed that enhancement of PI3K activation accelerates cell cycle entry, whereas reduction of PI3K activation retarded this process. Here we examined whether expression of different PI3K mutants affects cell growth during cell division. We show that diminishing or enhancing the magnitude of PI3K activation in a transient manner reduces or increases, respectively, the protein synthesis rate. Alteration of cell growth and cell cycle entry by PI3K forms appears to be concerted, because it results in lengthening or shortening of cell division time without altering cell size. In support of a central role for PI3K in growth control, expression of a deregulated, constitutive active PI3K mutant affects p70 S6 kinase and mammalian target of rapamycin activities and increases cell size. Together, the results show that transient PI3K activation regulates cell growth and cell cycle in a coordinated manner, which in turn controls cell division time.
Cell division is the process by which a cell duplicates its DNA content and cell mass to produce two daughter cells. In mammals, cell division is essential during development and in the adult for tissue regeneration. Most cell division is a symmetrical process that gives rise to two virtually identical daughter * This work was supported by Grant QLRT-2001-02171 from the European Union, Grant 08.3/0030 from the Community of Madrid, and Grant SAF2001-2278 from the Spanish Direccio´n General de Ciencia y Desarrollo Tecnolo´gico. The Department of Immunology and Oncology was founded and is supported by the Spanish Council for Scientific Research and by the Pharmacia Corporation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: Dept. of Immunology and Oncology, Centro Nacional de Biotecnologı´a/CSIC, Carretera de Colmenar Km 15, Cantoblanco, Madrid E-28049, Spain. Tel.: 34-91-5854849; Fax: 34-91-372-0493; E-mail:
[email protected].
cells (1–5). To initiate symmetrical cell division, mitogens trigger a number of early signals that culminate in the activation of G1 cyclin/CDKs (required for the G1-S transition) and induce an increase in cell mass. This increase is required to ensure that macromolecular composition and cell size are conserved in daughter cells (1–5). Signaling pathways that control the cell mass increase, referred to as cell growth, have recently been elucidated and include proteins such as phosphoinositide 3-kinase (PI3K),1 p70 S6 kinase (p70 S6K), the mammalian target of rapamycin (mTOR), and the tuberous sclerosis complex (TSC1/2) (4, 5). These proteins regulate synthesis of ribosomal components and activation of the translational machinery (4, 5). It is nonetheless unknown how the two independent processes of cell cycle entry and cell growth are coordinated during cell division, particularly in mammals. Studies in Saccharomyces cerevisiae show that whereas blockage of cell division by inactivation of most cell division cycle (cdc) genes allows cell growth to continue, inhibition of cell growth impairs cell cycle progression (6). The cell cycle thus appears to be linked to cell growth. Similar results were obtained in Drosophila (7–10). PI3K is an enzyme that transfers phosphate to the 3-position of the inositol ring of membrane phosphoinositides. The PI3Ks are divided into three subclasses based on their primary structure and substrate specificity, but only class I enzymes generate phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate products in vivo. Basal levels of these lipids are very low in quiescent cells but increase rapidly and transiently following growth factor receptor stimulation, regulating a variety of cell responses including survival and division (11–14). The 3-polyphosphoinositides recruit pleckstrin homology domain-containing proteins such as phosphoinositide dependent kinase-1 and protein kinase B (PKB), which mediate PI3K signal propagation (15–18). Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit (11–14, 19 –23). Activation of this enzyme following growth factor receptor stimulation controls cell cycle entry by regulating cyclin D synthesis and inactivation of FOX0 forkhead transcription factors, events required for G1-to-S transition (24 –27). Nonetheless, subsequent inactivation of PI3K is also important for completion of the cell cycle, because expression of constitutive active PI3K mutants inhibits forkhead activity in G2, which is required for mitotic progression (27). In addition to controlling these events, PI3K activation governs
1 The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; mTOR, mammalian target of rapamycin; p70 S6K, p70 S6 kinase; TSC, tuberous sclerosis complex; 4EBP1, initiation factor 4E-binding protein 1; DMEM, Dulbecco’s modified Eagle’s medium; GFP, green fluorescence protein; CS, calf serum; TOP, terminal oligopyrimidine tract.
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This paper is available on line at http://www.jbc.org
PI3K Controls Cell Division Time cell growth by regulating activation of p70 S6K and mTOR (28 –32). mTOR is a large (289 kDa), evolutionarily conserved Ser/Thr kinase that is inhibited by the drug rapamycin (33–35). mTOR, also termed FRAP (FKBP-12 rapamycin-associated protein), phosphorylates and inactivates the eukaryotic initiation factor 4E-binding protein 1 (4EBP1), an inhibitor of the translation initiation complex (36, 37). This complex regulates 5⬘ cap translation, which accounts for the majority of cellular translation (36). In addition, mTOR regulates p70 S6K activation (37–39). mTOR activity is sensitive to nutrient (amino acid) and energy (ATP) levels and is also regulated by mitogens (40 – 42). A number of recent studies show that mTOR action on p70 S6K and 4EBP1 is negatively controlled by the TSC1/2 tumor suppressor complex (30 –32, 43). PI3K/PKB controls mTOR function by regulating TSC2 phosphorylation (30 –32). In fact, the PI3K effector PKB was shown to phosphorylate TSC2; this phosphorylation destabilizes TSC2 and disrupts its association with TSC1, restoring mTOR-regulated phosphorylation of 4EBP1 and p70 S6K (30 –32). p70 S6K is a Ser/Thr kinase that phosphorylates the 40 S ribosomal protein S6 (44). S6 phosphorylation facilitates recruitment of a specific mRNA subset containing a polypyrimidine tract at the 5⬘ transcriptional start site (5⬘ TOP) to translating polysomes. 5⬘ TOP transcripts include those encoding ribosomal proteins (S3, S6, S14, and S24) and translation elongation factors (eEF1A and eEF2) (44, 45). In addition, p70 S6K regulates eEF2 activity (46). p70 S6K is activated by the ordered phosphorylation of residues in the C-terminal pseudosubstrate region, followed by phosphorylation of Thr389 and Thr229 (44, 45). p70 S6K triggering requires activation of both mTOR and PI3K (28, 29, 37–39). mTOR activity is required for the phosphorylation of p70 S6K in several residues, including Thr389 (37–39). PI3K/PKB regulates TSC2 phosphorylation and, in turn, mTOR activation (30 –32). In addition, PI3K controls p70 S6K activation via mTOR-independent mechanisms, as supported by the observation that PI3K activity is still required for activation of rapamycin-resistant p70 S6K mutants (47). The PI3K effectors phosphoinositide-dependent kinase-1 and protein kinase C were shown to stabilize Thr389 phosphorylation, and phosphoinositide-dependent kinase-1 phosphorylates Thr229 (48, 49). Finally, in addition to controlling mTOR and p70 S6K, PI3K also regulates cell growth by controlling translation of 5⬘ TOP mRNAs via a p70 S6K-independent mechanism (50). In addition to the biochemical studies mentioned, genetic experiments in Drosophila support PI3K/PKB, p70 S6K, and mTOR involvement in cell growth control (7–10). In mammals, deregulation of p70S6K, mTOR, or the PI3K/PKB pathway was shown to affect cell size (35, 51–53). Nonetheless, despite the well demonstrated function of PI3K/PKB, p70 S6K, and mTOR in cell growth control, little is known of how cell growth induction is linked to cell cycle progression. Based on the capacity of PI3K to regulate pathways that control cell growth and cell cycle entry, we hypothesized that PI3K activation may contribute to the concerted regulation of these processes during cell division in mammals. We previously described the consequences on cell cycle progression of interfering with physiological PI3K regulation in NIH 3T3 cells by expressing different p85/p110 PI3K forms (27). These studies indicated that enhancement of PI3K activation in a transient manner accelerates cell cycle progression, whereas reduction of PI3K activation decreases this process (27). Here we show that expression of p65PI3K, a mutant that enhances transient PI3K activation, augmented the protein synthesis rate of cycling cells. This increase was concerted with
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the cell cycle progression rate, because p65PI3K expression shortened division time without altering cell size. Accordingly, expression of the recombinant p85␣ regulatory subunit, which reduces the magnitude of transient PI3K activation, increased cell division time without altering cell size. These observations illustrate the concerted regulation of cell growth and cell cycle progression rates by PI3K, thereby controlling cell division time. The key role of PI3K in growth control is supported by the observation that expression of a deregulated, constitutive active PI3K form altered p70 S6K and mTOR activation kinetics, giving rise to larger cells. EXPERIMENTAL PROCEDURES
cDNA Constructs, Antibodies, and Materials—pcDNA3-TSC1 and pcDNA3-TSC2 cDNA and anti-TSC2 antibodies were kindly provided by Mark Nellist (54). Prk5-Myc-D3Ep70S6K was kindly donated by George Thomas (55). Anti-p70 S6K antibodies were from Santa Cruz Biotechnology, and anti-Thr(P)389 and anti-Thr(P)421/Ser(P)424 p70 S6K antibodies were from New England Biolabs. Horseradish peroxidaseconjugated antibodies were from Dako, and the enhanced chemiluminiscence developing kit was from Amersham Biosciences. Rapamycin was from Calbiochem. Cell Culture and Transfection—NIH 3T3 cells were cultured (37 °C, 10% CO2) in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker) with 10% calf serum (Invitrogen). Stable NIH 3T3 cell lines expressing p110caax, p65PI3K and p85␣ have been described (56, 57). Stable cell lines expressing D3Ep70S6K were obtained by transfection of cells with Prk5-Myc-D3Ep70S6K cDNA combined with p-Pur cDNA (Clontech); clones were selected in medium containing 2 g/ml puromycin (Sigma). Transient transfection was performed using LipofectAMINE Plus (Invitrogen) according to manufacturer’s instructions. Cell cycle arrest was as described (27). Briefly, for G0 phase arrest, cells were incubated without serum for 20 h. For G2 phase arrest, cells were incubated (20 h) with 5 M etoposide (Sigma), which yielded 40 –50% cells in G2. For M phase arrest, cells were incubated (20 h) with 0.1 g/ml colcemid (Invitrogen), yielding ⬃70% cells in M phase. For G1 samples, cells were arrested in G0 for 19 h and incubated with serum for 1 h. Extract Preparation and Western Blotting—Cells were lysed in 50 mM HEPES pH 8, 150 mM NaCl and 1% Triton X-100 containing phosphatase and protease inhibitors (27, 58). For p70S6K immunoblotting, cells were lysed in 10 mM Hepes pH 7.8, 20 mM  glycerol phosphate, 15 mM KCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 0.2% Nonidet P-40 containing phosphatase and protease inhibitors (58). Protein concentration was estimated by the BCA assay (Pierce) and equal protein amounts were resolved in SDS-PAGE. Gels were transferred to nitrocellulose and probed with the indicated antibodies. Cell Labeling—Cells were washed in methionine/cysteine-free RPMI (BioWhittaker) and incubated in this medium supplemented with 10% dialyzed fetal calf serum for 2 h prior addition of 35S Met/Cys (20 Ci; Amersham Biosciences) for the times indicated. For 35S Met/Cys labeling of cells in G0 and G2, cells were incubated 16 h in serum-free medium or in medium containing 10% serum and 5 M etoposide, respectively, then labeled as above. For G1 labeling, cells were incubated as for G0 conditions, labeled, and then incubated with 10% dialyzed calf serum for 1 h. The cells were collected and lysed in Triton X-100 lysis buffer (50 mM HEPES pH 8, 150 mM NaCl and 1% Triton X-100 containing phosphatase and protease inhibitors, 58). Protein concentration was estimated and 20 g of total protein were resolved in SDS-PAGE and autoradiographed. Cell Size Determinations—To examine cell size after transient transfection and sorting, cells were seeded in 60 mm dishes (2.5 ⫻ 105 cells/plate), transfected the following day at 80% confluence using 0.5 g pEGFP C1 (Clontech) plus 2 g of plasmids encoding p110caax or p70S6K (58), and incubated overnight. The cells were replated in 10-mm dishes, incubated alone or in the presence of rapamycin (20 nM, 72 h), harvested, and sorted for GFP expression. Forward scatter profiles were analyzed by live cell flow cytometry using a Becton Dickinson fluorescence-activated cell sorter. To determine cell size in stable transfected cell lines, the cells were maintained in exponential growth, alone or in the presence of rapamycin (20 nM, 4 days). The cell diameters and volumes were determined using a particle size counter (CASY, Scha¨ rfe System).
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PI3K Controls Cell Division Time
FIG. 1. PI3K enhances growth in NIH 3T3 cells. Exponentially growing stable cell clones expressing p65PI3K, p85␣, or p110caax were labeled with 20 Ci of [35S]methionine. A, cells were collected after 30 min of labeling, and 20 g of total lysate was analyzed by SDS-PAGE and autoradiography. B, total cpm at each time point were quantified using phosphorimaging; the graph shows the mean cpm incorporation of five different experiments at 30 min of labeling. Ctr, control.
FIG. 2. Sustained PI3K activation enhances growth throughout the cell cycle. Stable cell clones expressing p65PI3K, p85␣, or p110caax were arrested in G0, G1, or G2 and were labeled with 20 Ci of [35S]methionine. A, cells were collected after 30 min, and 20 g of total lysate was analyzed by SDS-PAGE and autoradiography. B, total cpm at each time point were quantified using phosphorimaging; the graph shows the mean cpm incorporation of two experiments. Ctr, control; FS, forward scatter.
RESULTS
PI3K Deregulation Alters Cell Growth—We previously examined the consequences on cell cycle progression of interfering with physiological PI3K activation kinetics by expressing different PI3K forms (27). These studies indicated that enhanced PI3K activation accelerates cell cycle entry, whereas decreased PI3K activation reduces this transition, supporting the role of PI3K in cell cycle entry. PI3K activation must nonetheless be transient to allow completion of the cell cycle, because expression of a constitutive active PI3K or PKB mutant deregulated forkhead transcription factor activity throughout the cell cycle, impairing mitotic progression (27). Here we analyze whether PI3K regulates cell growth in exponentially dividing mammalian cells. We examined growth in NIH 3T3 cell lines expressing p110caax, a constitutively active p110 catalytic subunit mutant (57), or p65PI3K, a mutant of the PI3K p85␣ regulatory subunit that binds to p110 and enhances its activation by growth factors (56). We also studied cell lines expressing recombinant p85␣ at levels double those of the endogenous protein; this modification reduces the magnitude of endogenous p110 activation (56, 57). The cells were maintained in exponential growth and labeled for short periods with [35S]Met/Cys to compare protein synthesis rates. A 30-min labeling period was adequate to obtain sufficient labeling without saturation; Fig. 1 illustrates a representative experiment and quantification of several assays. Whereas p85␣ expression reduced [35S]Met/Cys incorporation, the two activating PI3K mutations, p110caax and p65PI3K, increased the protein synthesis rate.
FIG. 3. Sustained PI3K activation increases cell size. p65PI3K, p85␣, or p110caax clones were cultured in DMEM with 10% calf serum (CS) and then harvested for analysis by live cell flow cytometry. Overlaid forward scatter profiles are shown.
We also compared protein synthesis rates in these cell lines that had been arrested in different phases of the cell cycle. As reported, protein synthesis in control cells was maximal in G1 (1, 3) and was moderate in G0- and G2-arrested cells (Fig. 2). Protein synthesis was also low in M phase arrested cells (not shown). G 0 -arrested p65 PI3K -expressing cells showed a higher rate of protein synthesis than control or p85 ␣ expressing cells. This may be due to the modest basal activation of PI3K seen in p65PI3K cells (56). Nonetheless, p110caax-cells exhibited a remarkably higher level of protein synthesis than control cells (Fig. 2). In G1, both p65PI3K and p110caax cells showed a higher protein synthesis rate than controls, whereas [35S]Met/Cys incorporation was lower in p85␣-expressing cells (Fig. 2). Finally, in all cell lines, protein synthesis was lower in G2 than in G1 and had a distinct protein labeling pattern but remained higher in p110caaxexpressing cells (Fig. 2). Thus, compared with controls, p85␣or p65PI3K-expressing cells showed increased and decreased protein synthesis rates, particularly in G 1 . p110 caax -
PI3K Controls Cell Division Time
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TABLE I Size determination of NIH 3T3 cells stably transfected with p65P13K, p85␣, or p110caax cDNA NIH 3T3 cell lines, stably transfected as indicated, were cultured in DMEM with 10% CS and harvested, and cell diameter and volume were measured using a particle size counter. The means of 10 determinations are shown. Cell type
Mean diameter ⫾ S.D.
⌬ diametera
Diameter p valueb
Mean volume ⫾ S.D.
⌬ volumea
m
Control p65P13K p85␣ p110caax
17.13 ⫾ 0.84 17.70 ⫾ 1.17 17.90 ⫾ 1.28 19.34 ⫾ 0.93
m
fl
fl
0.57 0.77 2.21
0.19 0.15 ⬍ 0.001
2936 ⫾ 509 3289 ⫾ 694 3398 ⫾ 593 4124 ⫾ 461
353 462 1188
Volume p valueb
0.18 0.26 ⬍0.001
a ⌬ diameter or volume was calculated by subtracting the mean diameter or volume of the control cell line from the mean diameter or volume of each individual cell line. b Student’s t test. p values were obtained by comparing the raw values for diameter or volume of each stable cell line with those for control cells.
expressing cells nonetheless exhibited high rates of biosynthesis in all cell cycle phases. We postulated that PI3K may control cell growth and cell cycle progression rates in a concerted manner, giving rise to cells that are normal in size but that divide more rapidly or more slowly, depending on the intensity of PI3K activation. We measured the size of the stable cell lines expressing the different PI3K forms by flow cytometry. Both p85␣- and p65PI3Kexpressing cells showed a size similar to that of NIH 3T3 control cells (Fig. 3). Nonetheless, cells expressing the constitutive active p110caax mutant were larger than controls (Fig. 3). We also analyzed cell diameter and volume using a particle size counter and found that only p110caax cells showed a statistically significant volume and diameter difference compared with controls (Table I). We conclude that alteration of the magnitude of PI3K activation in a transient manner does not modify cell size; in contrast, constitutive activation of PI3K increases cell size. PI3K Deregulation Alters Cell Division Time—We next examined the cell division time. Stable cell lines were seeded at similar density, and the division rate was calculated by cell counting at different time points after the initiation of culture. p65PI3K protein expression reduced doubling time, whereas increased expression of the p85␣ form increased t1⁄2 (Fig. 4). Cells expressing mutants that alter the magnitude but not the transient kinetics of PI3K activation are thus able to alter cell cycle progression in concert with cell growth, inducing variations in t1⁄2 without significantly altering cell size. In contrast, cells expressing p110caax, which exhibit sustained PI3K activity, show a t1⁄2 similar to that of wild type cells (Fig. 4), as well as a larger size (Fig. 3); this suggests that cell growth and cell cycle progression are not coordinated in these cells. Similar results were obtained (not shown) using CTLL2 cells expressing different PI3K forms (56, 58). p110caax Inhibits Down-regulation of Pathways Controlling Cell Growth—To analyze the mechanisms by which PI3K deregulation affects cell growth, we examined the activation kinetics of the PI3K effector p70 S6K in p110caax cells. p70 S6K activation is a multistep process that begins with phosphorylation of pseudosubstrate residues (Ser411, Ser418, Thr421, and Ser424), followed by phosphorylation of Thr389 (44, 45). Once Thr389 is phosphorylated, the enzyme is susceptible to Thr229 phosphorylation by phosphoinositide-dependent kinase-1 kinase (48, 49, 55). Thr389 phosphorylation is considered a limiting step and was analyzed as an indicator of p70 S6K activation in p110caax and control cells at distinct cell cycle phases. Phosphorylation of Thr389 was increased in G1 after serum addition and was low in G2 and M phase arrested control cells (Fig. 5A). In contrast, p110caax-expressing cells retained a low level of p70 S6K Thr389 phosphorylation in G0, and Thr389 remained phosphorylated in G2 and M. Similar results were obtained when we examined Thr421/Ser424 phosphorylation (Fig. 5A). Thr389 phosphorylation was transient in the p65PI3K and p85␣ stable cell
FIG. 4. Effect of interfering PI3K mutants on population doubling time. p65PI3K, p85␣, or p110caax clones of NIH 3T3 fibroblasts were seeded at a similar densities in DMEM with 10% CS, and cell numbers were counted at 24-h intervals. The figure represents the mean of five assays.
lines (not shown). p110caax expression thus induces prolonged p70 S6K activation. In addition to regulating p70 S6K activation, the PI3K/PKB pathway also regulates mTOR by controlling TSC2 phosphorylation (30 –32, 43, 59). mTOR in turn regulates 4EBP1 and p70 S6K phosphorylation (36, 37, 39). Thus, although p70 S6K was deregulated in p110caax cells (Fig. 5A), this effect may reflect an mTOR defect or a direct effect of PI3K on p70 S6K activity, because PI3K also controls p70 S6K by mTOR-independent mechanisms (47). To analyze whether mTOR activity is altered in p110caax cells, we thus examined 4EBP1 phosphorylation. We found a more slowly migrating, hyperphosphorylated 4EBP1 species in control cells following serum stimulation (G1) (Fig. 5A). This species was nonetheless already found in serum-starved p110caax cells and was more clearly detectable in p110caax cells than in controls in all cell cycle phases (Fig. 5A). This suggests that p110caax expression affects mTOR activation. These results show that deregulation of PI3K affects mTOR and p70 S6K activity. To analyze whether p70 S6K deregulation was exclusively a consequence of defective mTOR inactivation, we overexpressed TSC1 and TSC2, which inhibit mTOR (30 –32). Transfection of the exogenous TSC1/2 complex in p110caax cells reduced 4EBP1 mobility as well as p70 S6K activation levels in G1 (Fig. 5B) but did not correct the prolonged activation kinetics of p70 S6K in G2/M (Fig. 5B). This supports mTOR deregulation as a contributory mechanism to cell mass increase in p110caax cells. In addition, even when mTOR is inhibited by TSC1/2 expression, p110caax induces prolonged p70 S6K activation, reflecting a direct PI3K effect on p70 S6K activation that may also contribute to increasing the size of p110caax cells.
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PI3K Controls Cell Division Time TABLE II Volume of NIH 3T3 cells stably transfected with p110 caax or D3Ep70 S6K cDNA NIH 3T3 cell lines, stably transfected as indicated, were cultured in DMEM/10% CS alone or in the presence of rapamycin (20 nM) and harvested, and cell diameter and fluid volume were measured using a particle size counter. The mean of 10 determinations is shown. ⫺Rapamycin Cell type
Mean volume ⫾ S.D.
Control p110caax S6K D3E
2936 ⫾ 509 4124 ⫾ 461 3910 ⫾ 527
⫹Rapamycin Mean volume ⫾ S.D.
⌬ volumea
fl
2639 ⫾ 222 3253 ⫾ 356 3046 ⫾ 273
297 871 864
a ⌬ volume was calculated by subtracting the mean volume of each rapamycin-treated cell line from the mean volume of the same untreated cell line.
or in the presence of rapamycin, and GFP-positive and -negative cells were isolated by cell sorting (transfection efficiency, ⬃60%). p110caax-transfected cells were larger than control cells (Fig. 6). Nonetheless, control and p110caax cells were similar in size when incubated with rapamycin (Fig. 6). The cells were also transfected with cDNA encoding p70 S6K, which gave rise to larger cells; this phenotype was also attenuated by rapamycin addition (Fig. 6). Similar results were obtained using the constitutive active p70 S6K mutant D3E p70 S6K (not shown). As the size of p110caax cells decreases upon inhibition of p70 S6K and mTOR, these results indicate that PI3K increases cell growth by affecting mTOR and p70 S6K regulation. Our observations support the hypothesis that transient variations in the magnitude of PI3K activation modify growth and cell cycle progression rates in concert. In contrast, sustained PI3K activation deregulates cell growth machinery throughout the cell cycle, uncoupling the protein synthesis rate from cell cycle progression rates, giving rise to larger cells. FIG. 5. Constitutive PI3K activation impairs p70 S6K downregulation. A, lysates (50 g) from control and p110caax-expressing cells arrested at the indicated cell cycle phases were resolved by SDSPAGE. The gels were analyzed in Western blot using anti-phosphoThr389-p70 S6K, anti-phosphoThr421/Ser424-p70 S6K, anti-p70 S6K, and anti-4EBP1 antibodies. B, NIH 3T3 cells cultured in DMEM with 10% CS were transiently transfected with a vector encoding p110caax alone or in combination with vectors encoding TSC1 and TSC2. At 24 h post-transfection, the cells were arrested in cell cycle phases as indicated, and the lysates (50 g) were examined in Western blot using anti-phosphoThr389-p70 S6K, anti-p70 S6K, anti-TSC1, anti-TSC2, and anti-4EBP1 antibodies.
Enhanced Activity of p70 S6K and mTOR Mediates p110caax Cell Size Increase—To examine whether increased p110caax cell size was a consequence of enhanced p70 S6K and mTOR activation, we inhibited these enzymes using rapamycin (33). Control and p110caax stable transfectants were cultured alone or with rapamycin, and their volumes were measured in a particle size counter (Table II). Rapamycin decreased the volume of control cells moderately (⬃10%) and that of p110caax cells more intensely (⬎20%) (Table II). We also measured the volume of stable transfectants of the p70 S6K mutant D3E p70 S6K, an activating mutation with acidic substitutions in the pseudosubstrate region residues (Ser411, Ser418, Thr421, and Ser424) but whose activity requires Thr389 and Thr229 phosphorylation, remaining sensitive to rapamycin (55). As for p110caax cells, the D3E p70 S6K-expressing cells were larger, and their size decreased by ⬃20% following incubation with rapamycin (Table II). In an alternative approach, the cells were transiently transfected with a vector encoding GFP and either a control vector or cDNA encoding p110caax. The cells were then incubated alone
DISCUSSION
The observations presented show that alteration of endogenous PI3K activation by expression of PI3K-interfering forms (p65PI3K, p85␣, or p110caax) affects the rate of cell growth in dividing mammalian cells. Moreover, expression of p65PI3K or p85␣, which induces transient enhancement or reduction in the magnitude of PI3K activation, alters cell growth without significantly modifying cell size (Fig. 3). This shows that transient changes in the intensity of PI3K activation modify cell cycle progression in concert with cell growth rates. In fact, the regulated increase in PI3K activation induced by p65PI3K accelerated cell division (decreased t1⁄2), whereas the p85␣-triggered reduction in the magnitude of PI3K activation delayed cell division (Fig. 4). These observations suggest that cells sense the magnitude of PI3K activation and establish a cell cycle progression rate that is proportional to the cell growth rate, ensuring that daughter cells maintain appropriate cell size. The critical role of PI3K in the control of cell growth during cell division is supported by the observation that expression of a deregulated, constitutive active PI3K mutant impairs coordination of these two processes, inducing a cell size increase. This is the first description that links transient PI3K activation to the concerted regulation of cell growth and cell cycle progression rates during cell division in mammals. Expression of the constitutive active PI3K mutant p110caax induces enlargement in cell size (Fig. 3). This mutant increases the protein synthesis rate (Fig. 1) and accelerates cell cycle entry but retards G2/M progression and cell cycle exit (27). A partial explanation for the lack of balance between cell growth and cell cycle progression rates in p110caax cells may thus be the delayed transition through G2/M. We nonetheless show
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FIG. 6. Incubation with rapamycin reduces p110caax cell size. NIH 3T3 cells cultured in DMEM with 10% CS were transiently transfected with a PSG5 empty vector, a vector encoding p110caax, or a vector encoding p70S6K, all in combination with a vector encoding GFP (4:1). At 24 h post-transfection, the cells were plated alone or with rapamycin (20 nM) and incubated for 72 h. The cells were harvested and sorted for GFP expression and then analyzed by live cell flow cytometry. Overlaid forward scatter profiles of GFP(⫹) and GFP(⫺) cells are shown. Ctr, control.
that constitutive activation of PI3K also interferes with correct down-regulation of cell growth-promoting pathways. Accordingly, incubation with PI3K inhibitors reduces cell growth (35) and impairs cell cycle entry (24, 27, 35). We examined mTOR and p70 S6K and found that p110caax expression extended p70 S6K activation kinetics to the G2/M phases and induced hyperphosphorylation of the mTOR effector 4EBP1 in G0. PI3K activation must thus be transient to allow correct control of cell cycle progression (27) and cell growth throughout the cell cycle. The reduction in p110caax cell size following rapamycin inhibition of mTOR and p70 S6K activity suggests that these PI3K effectors control growth in dividing cells. PI3K may regulate cell growth by additional mechanisms. This possibility is supported by the behavior of NIH 3T3 p65PI3K-expressing cells, in which p70 S6K is transiently triggered and down-regulated, but whose activation levels are lower than in controls (not shown). This concurs with our previous observations showing that p85, but not p65PI3K, forms a complex with p70 S6K and mTOR that is required for p70 S6K activation (58). Stable NIH 3T3 p65PI3K cells express similar levels of p65PI3K and of endogenous p85 (56), which accounts for the moderate p70 S6K activation observed in these cells. Because p65PI3K cells have a higher protein synthesis rate and reduced p70 S6K activation; p70 S6K does not appear to be the main effector mediating enhanced cell growth in these cells. Activation of the p70 S6K 2 isoform (51, 60), TSC inactivation (30 –32), or an as yet undescribed mechanism may cooperate with p70 S6K to enhance cell growth in response to PI3K activation. In addition, it was recently reported that PI3K enhances 5⬘ TOP mRNA translation independently of p70 S6K (50). We found that p65PI3K-expressing cells have a higher proportion of rpL32 mRNA (5⬘ TOP) (50) in heavy polysomes than control cells (not shown), suggesting that 5⬘ TOP translation is enhanced in these cells. The observations presented suggest that PI3K has an essential role in the concerted regulation of cell growth and cell cycle progression. Previous observations in yeast illustrated that inhibition of cell growth blocks cell cycle entry, whereas inhibition of cell cycle progression allows growth to continue (6). This shows that cell cycle entry is linked to the cell growth process. As to the signaling pathways that control cell growth in yeast, no class I PI3K homologues have been found in this organism; TOR function in control of cell growth is nonetheless conserved from yeast to mammals (61). In the fruit fly Drosophila melanogaster, disruption of cell cycle regulatory genes (dE2F and cdc2) results in cell cycle arrest at a larger cell size (62, 63). This shows that growth without division can also be observed in this organism, but division requires growth. With regard to the pathways that control cell growth and cell division, mutations in Inr, dp110,
dIRS (Chico), dPTEN, and dRas affect cell growth and cell cycle simultaneously, whereas mutations in dTOR, d4EBP, and dS6K affect only cell size (reviewed in Refs. 4, 7–10, and 64). In addition, deletion of the negative regulator TSC1 (which participates in negative control of TOR) affects cell size (4, 5, 65). PI3K regulates the TSC complex and TOR (7, 30 –32), suggesting that one signaling branch downstream of PI3K regulates cell growth, and the other controls cell cycle progression. dAKT appears to regulate only cell size, suggesting that AKT lies in the growth branch of the PI3K pathway in flies (66). Another difference compared with mammals is that dS6K appears to lie in a pathway different from that of dPI3K (67), although the dPI3K pathway still controls cell growth and cell division. Most of the mutations mentioned above were described in the Drosophila wing imaginal disc, in which cell growth and cell cycle increase in parallel. The study of these processes in Drosophila has the additional difficulty that organ size is subject to internal regulatory mechanisms (reviewed in Refs. 4, 5, 7, and 64). Moreover, division is not coupled to growth in some organs; for example, in the pupal stage, postmitotic cells in the eye grow without undergoing division (4). This explains the observation that flies carrying a dp110 mutation exhibit a cell growth and cell division phenotype in the wing but only a growth phenotype in the eye (8). In mammals, inhibition of cell growth also blocks cell cycle entry (3), although growth continues following inhibition of cell cycle entry (35). This also shows that growth in mammals can be separated from the cell cycle but that the cycle is linked to growth. Cell growth in mammals requires PI3K and TOR activities; in fact, expression of the p16 cell cycle inhibitor blocks the cycle in G1, but the resulting cells are larger (35). This cell size increase is partially blocked by TOR inhibition and even more clearly by PI3K inhibitors, illustrating the relevance of PI3K and TOR in cell growth control (35). Nonetheless, only PI3K, but not TOR, appears to mediate the concerted regulation of cell growth and cell cycle (Fig. 4). In contrast to the ability of PI3K mutants to regulate cell cycle progression and growth, activation of the mTOR pathway does not trigger cell division (5, 9, 35). PI3K is thus the first signaling pathway reported to link both processes. Two routes would be induced by PI3K, one branch involved in triggering cell cycle entry and the other in promoting cell growth. The branch regulating cell growth includes mTOR and its effectors, among others (5, 28 –32, 50). Regulation of cell cycle entry downstream of PI3K requires Rac, Cdc42, and PKB activation, which affects cyclin/CDK activities or stability (4, 24 –27, 62, 68). Nonetheless, PI3K involvement in coordinating cell growth and cell division was not observed in mice expressing constitutive active forms of PI3K/PKB in the heart (52, 53). Expression of constitutive active PI3K/PKB in post-mitotic
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cells (cardiomyocytes) may mask the contribution of PI3K to triggering cell division (52, 53). In contrast, the phenotype of mice expressing the transient p65PI3K mutation as a transgene in T cells and retina revealed the contribution of the PI3K route in cell division in vivo (69, 70). The mechanism by which PI3K exerts concerted regulation on cell cycle progression and cell growth is incompletely understood. Induction of cell growth and cell cycle entry may simply occur in parallel. Because both cell growth and cell cycle entry are regulated by PI3K, the magnitude of PI3K activation may determine the extent of these processes. It is also possible that translation of a specific cell cycle entry component is sensitive to the availability of the translation machinery. In yeast, G1 cyclin (Cln3) protein expression is highly dependent on the levels of the translation initiation complex, such that Cln3 levels define whether a cell has sufficient translation machinery to enter the cell cycle (71). It has also been shown that overexpression of cyclin D in Drosophila triggers cell growth (4), supporting the possibility that cyclin E rather than cyclin D acts as a growth sensor in this organism. In mammals, PI3K contributes specifically to inducing cyclin D and E synthesis and regulates E2F induction (24, 72). Nonetheless, whether or not translation of mammalian G1 cyclins mRNAs depends on PI3K-controlled translation machinery remains to be determined. The fact that PI3K has a crucial role linking cell growth and cell cycle entry does not imply that this enzyme is in itself sufficient for either of these processes. For instance, 5⬘ cap translation, which accounts for 85% of total translation, requires mTOR activation. Nonetheless, mTOR activity requires not only TSC inactivation by PI3K/PKB (30 –32) but also appropriate ATP and nutrient levels (40, 41). Translation initiation is also regulated by mitogen-activated protein kinase-dependent pathways (73). For cell cycle entry, other signaling cascades in addition to PI3K also modulate cyclin D expression (74, 75). The requirement for signals other than PI3K to induce cell growth or division explains why some receptors that activate PI3K can induce cell growth, whereas others trigger cell division (4, 5, 50, 72). It is thus possible that the pathways that act in conjunction with PI3K to trigger cell growth and cell cycle entry also have a role in coordinating these two processes. Nonetheless, the concerted modification of cell cycle progression and cell growth rates observed after genetic alteration of PI3K points to this early signal as a central player for correct coordination. In conclusion, alteration of t1⁄2 without modification of cell size or cell cycle profiles in p65PI3K and p85␣-expressing cells illustrates the central role of PI3K in the concerted regulation of cell growth and cell cycle progression. The upstream position of PI3K in cell growth- and cell cycle-controlling signaling pathways makes this regulation possible. Coordination of both processes requires PI3K activation to be transient. Acknowledgments—We thank Dr. M. Nellist for pcDNA3-TSC1 and pcDNA3-TSC2 cDNA and for anti-TSC1 and-TSC2 antibodies, Dr. G. Thomas for Prk5-Myc-D3Ep70S6K, Dr. V. Calvo for critical reading of the manuscript, and C. Mark for editorial assistance. REFERENCES 1. 1. Polymenis, M., and Schmidt, E. V. (1999) Curr. Opin. Genet. Dev. 9, 76 – 80 2. Pardee, A. B. (1989) Science 246, 603– 608 3. Zetterberg, A., and Larsson, O. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 5365–5369 4. Tapon, N., Moberg, K. H., and Hariharan, I. K. (2001) Curr. Opin. Cell Biol. 13, 731–737 5. Saucedo, L. J., and Edgar, B. A. (2002) Curr. Opin. Genet. Dev. 12, 565–571 6. Johnston, G. C., Pringle, J. R., and Hartwell, L. H. (1977) Exp. Cell Res. 105, 79 –98 7. Stocker, H., and Hafen, E. (2000) Curr. Opin. Genet. Dev. 10, 529 –535 8. Weinkove, D., Neufeld, T. P., Twardzik, T., Waterfield, M. D., and Leevers, S. J. (1999) Curr. Biol. 9, 1019 –1029 9. Zhang, H., Stallock, J. P., Ng, J. C., Reinhard, C., and Neufeld, T. P. (2000)
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