From the Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Cantoblanco, Madrid E-28049, Spain
Received for publication, January 21, 2003 , and in revised form, April 2, 2003.
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ABSTRACT |
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INTRODUCTION |
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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 (1114). 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 (1518). Class IA PI3K is a heterodimer composed of a p85 regulatory and a p110 catalytic subunit (1114, 1923). 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 (2427). 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 cell growth by regulating activation of p70 S6K and mTOR (2832).
mTOR is a large (289 kDa), evolutionarily conserved Ser/Thr kinase that is inhibited by the drug rapamycin (3335). 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 (3739). mTOR activity is sensitive to nutrient (amino acid) and energy (ATP) levels and is also regulated by mitogens (4042). A number of recent studies show that mTOR action on p70 S6K and 4EBP1 is negatively controlled by the TSC1/2 tumor suppressor complex (3032, 43). PI3K/PKB controls mTOR function by regulating TSC2 phosphorylation (3032). 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 (3032).
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,
3739).
mTOR activity is required for the phosphorylation of p70 S6K in several
residues, including Thr389
(3739).
PI3K/PKB regulates TSC2 phosphorylation and, in turn, mTOR activation
(3032).
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 (710). In mammals, deregulation of p70S6K, mTOR, or the PI3K/PKB pathway was shown to affect cell size (35, 5153). 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 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.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and TransfectionNIH 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 µg/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
µM etoposide (Sigma), which yielded 4050% cells in
G2. For M phase arrest, cells were incubated (20 h) with 0.1
µg/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 BlottingCells 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 LabelingCells 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 µCi; 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 µM 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 µg of total protein were resolved in SDS-PAGE and autoradiographed.
Cell Size DeterminationsTo examine cell size after transient transfection and sorting, cells were seeded in 60 mm dishes (2.5 x 105 cells/plate), transfected the following day at 80% confluence using 0.5 µg pEGFP C1 (Clontech) plus 2 µg 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, Schärfe System).
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RESULTS |
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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.
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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). G0-arrested
p65PI3K-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
p110caax-expressing cells
(Fig. 2). Thus, compared with
controls, p85
- or p65PI3K-expressing cells showed increased
and decreased protein synthesis rates, particularly in G1.
p110caax-expressing cells nonetheless exhibited
high rates of biosynthesis in all cell cycle phases.
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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
p65PI3K-expressing 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.
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PI3K Deregulation Alters Cell Division TimeWe 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
t
(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
t
without significantly altering cell size. In
contrast, cells expressing p110caax, which
exhibit sustained PI3K activity, show a t
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).
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p110caax Inhibits Down-regulation of Pathways Controlling
Cell GrowthTo 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 lines (not shown).
p110caax expression thus induces prolonged p70
S6K activation.
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In addition to regulating p70 S6K activation, the PI3K/PKB pathway also regulates mTOR by controlling TSC2 phosphorylation (3032, 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 (3032). 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.
Enhanced Activity of p70 S6K and mTOR Mediates p110caax Cell
Size IncreaseTo 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).
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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
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.
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DISCUSSION |
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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 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 (3032), 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, 710, 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, 3032), 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, post-mitotic 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, 2832, 50). Regulation of cell cycle entry down-stream of PI3K requires Rac, Cdc42, and PKB activation, which affects cyclin/CDK activities or stability (4, 2427, 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 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 (3032) 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 t 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.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Immunology and Oncology,
Centro Nacional de Biotecnología/CSIC, Carretera de Colmenar Km 15,
Cantoblanco, Madrid E-28049, Spain. Tel.: 34-91-585-4849; Fax: 34-91-372-0493;
E-mail:
acarrera{at}cnb.uam.es.
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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ACKNOWLEDGMENTS |
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REFERENCES |
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