Phosphoinositide 3-Kinases p110α and p110β Regulate Cell Cycle Entry, Exhibiting Distinct Activation Kinetics in G1 Phase

ABSTRACT Phosphoinositide 3-kinase (PI3K) is an early signaling molecule that regulates cell growth and cell cycle entry. PI3K is activated immediately after growth factor receptor stimulation (at the G0/G1 transition) and again in late G1. The two ubiquitous PI3K isoforms (p110α and p110β) are essential during embryonic development and are thought to control cell division. Nonetheless, it is presently unknown at which point each is activated during the cell cycle and whether or not they both control S-phase entry. We found that p110α was activated first in G0/G1, followed by a minor p110β activity peak. In late G1, p110α activation preceded that of p110β, which showed the maximum activity at this time. p110β activation required Ras activity, whereas p110α was first activated by tyrosine kinases and then further induced by active Ras. Interference with p110α and -β activity diminished the activation of downstream effectors with different kinetics, with a selective action of p110α in blocking early G1 events. We show that inhibition of either p110α or p110β reduced cell cycle entry. These results reveal that PI3Kα and -β present distinct activation requirements and kinetics in G1 phase, with a selective action of PI3Kα at the G0/G1 phase transition. Nevertheless, PI3Kα and -β both regulate S-phase entry.

The exposure of quiescent cells to growth factors (GF) activates a number of early signaling pathways that trigger cell cycle entry. Class I phosphoinositide 3-kinase (PI3K) represents one of the GF-stimulated pathways that regulate G 0 /G 1 and G 1 /S transitions. There are four class I PI3K enzymes, composed of a regulatory subunit and a conserved p110 catalytic subunit that triggers phosphatidylinositol (3,4)-biphosphate and phosphatidylinositol (3,4,5)-triphosphate (PIP 3 ) production. Class I PI3K enzymes are further classified as the GF receptor-controlled class I A enzymes and the G proteincoupled receptor-regulated p110␥ (class I B PI3K) (12,42). Three genes encode class I A catalytic subunits (p110␣, p110␤, and p110␦) (12,14,42). Class I A enzymes are activated by tyrosine kinases (TyrK) and Ras and regulate cell growth and DNA synthesis (5,14,17). Of the three class I A catalytic subunits, p110␦ is expressed mainly in hematopoietic cells and regulates the immune response (30), whereas p110␣ and -␤ are ubiquitous and they might control cell division. Mice deficient in p110␣ or -␤ isoforms are embryonic lethal, suggesting that at least in development, these two isoforms have nonredundant functions (3,4).
PI3K activity increases within minutes after GF receptor (GFR) stimulation (first peak) and again in advanced G 1 phase (second peak) (18,19,24). PI3K has been implicated in the induction of cell growth and regulation of Cdk activity. Pharmacological inhibition of PI3K at the time of GFR stimulation blocks cell division (2). In addition, enhanced PIP 3 production after GFR binding accelerates cell cycle entry, whereas PIP 3 reduction diminishes this process (1). PI3K regulates cell mass increase by activating p70S6 kinase (p70S6K) and mTOR (9,10,23,34,35). The upregulation of PI3K activity also enhances Cdk2 activation (21). The mechanisms by which PI3K controls Cdk activity include the induction of cyclin D synthesis and inhibition of cyclin D degradation, an effect mediated by protein kinase B (PKB)-induced glycogen synthase kinase 3␤ inactivation (31,33,41). PI3K also regulates cell cycle entry through PKB-mediated FoxO transcription factor (TF) phosphorylation, which reduces FoxO TF-controlled cyclin G2 and p27 INK expression (25,27). Finally, the late G 1 PI3K activity stabilizes c-Myc, an event required for correct cyclin A expression and Cdk2 activation (24).
Cell cycle and immunofluorescence analysis. Cells were synchronized in G 0 by serum starvation as reported previously (25). Synchronous cell cycle entry was induced by the addition of serum. Cell cycle distribution was examined by DNA staining using propidium iodide and analyzed by flow cytometry (Beckman-Coulter, Fullerton, CA). U2OS cell cultures were synchronized at the G 1 /S boundary by double-thymidine block (11) or were synchronized in metaphase with colcemid (13). For retrovirus production, Phoenix cells were transfected by using JetPei-NaCl according to the manufacturer's protocols (Qbiogene, Irvine, CA). Retroviral infection and immunofluorescence analysis were performed as described previously (24).
Quantitation of gel bands and statistical analyses. Statistical analyses were performed by using StatView 512ϩ (Calabasas, CA). Gel bands and fluorescence intensities were quantitated with ImageJ software and were normalized according to the fluorescence intensity of the loading control band. Cell cycle profiles were analyzed with multicycle AV for Windows (Phoenix Flow Systems, CA).

RESULTS
p110␣ and -␤ contribute differently to downstream signaling. We investigated specific functions of p110␣ and -␤ PI3K catalytic subunits in G 1 phase by comparing the consequences of interfering with their activation for the induction of different effectors. We examined several PI3K downstream targets, including PKB, FoxO3a, and p70S6K. To synchronize the cells, we arrested immortal nontransformed murine NIH 3T3 cells in G 0 phase by serum deprivation and then released them by low-density replating in serum-containing medium for different time periods, as described previously (25). We confirmed that the PI3K effector PKB is activated at G 0 /G 1 , in late G 1 , and at M-phase entry (Fig. 1A), as reported previously (7,38). We also synchronized human U2OS cells at the G 1 /S boundary or in metaphase ( Fig. 1B and C), as these cells fail to arrest in G 0 FIG. 1. PI3K is activated at S-phase entry in different cell lines. (A) NIH 3T3 cells were arrested in G 0 ; (B and C) U2OS cells were synchronized at the G 1 /S boundary (B) or in metaphase (C) and released for different times. Extracts were examined with WB by using the indicated Ab. Cell cycle distribution was examined in parallel; transits through G 1 , S, or G 2 /M are indicated (arrows). Graphs represent the means Ϯ standard deviations of the p-PKB signals in arbitrary units, normalized in comparison to control PKB levels (n ϭ 3).

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MARQUÉS ET AL. MOL. CELL. BIOL. (7,38). We confirmed PI3K activation at G 1 /S transition and M-phase entry in U2OS cells ( Fig. 1B and C). We confirmed the specificities of the p110 Ab used for this study by transfection of wild-type p110␣ and p110␤ under the control of the simian virus 40 promoter (pSG5 vector) in COS cells, which gives rise to high levels of overexpression of recombinant proteins ( Fig. 2A). Anti-p110␣ Ab selectively detected p110␣ despite the high expression levels of recombinant p110␤; similarly, anti-p110␤ Ab only detected endogenous and recombinant p110␤ ( Fig. 2A). To interfere with p110's cellular activity, we first used the active p110␣* (1) and p110␤* forms, as well as the kinase-inactive myc-K802R-p110␣ and myc-K805R-p110␤ mutants (see Materials and Methods). The interference activities of the mutants were tested by transient transfection of these PI3K forms in asynchronous cultures of NIH 3T3 cells. The expression levels of exogenous p110 were approximately double those of the endogenous proteins (Fig.  2B). Transient transfection of the mutants showed that K802R-p110␣ and K805R-p110␤ reduced and p110␣* and -␤* increased (p110␣* had a greater effect) the p-PKB cellular levels (Fig. 2C). Thus, these mutants interfere with endogenous PI3K pathway activation.
We then examined PKB, FoxO, and p70S6K activities during early G 1 (until 6 h following serum addition) in synchronized populations of stable p110␣* and p110␤* transfectants (see Materials and Methods). In these cells, the exogenous p110 expression levels were similar to the levels of endogenous proteins (1) (data not shown). In synchrony, p110␣*-expressing cell lines showed sustained p-PKB activation (1) and p110␤*-expressing cells showed a minor increase in basal levels of p-PKB and an increase in the p-PKB signal at ϳ4 h ( Fig.  2D). We failed to stably maintain cells expressing inactive mutants; these mutants were transduced by transient transfection (or infection) of NIH 3T3 cells, which yielded expression levels similar to those of endogenous proteins (Fig. 2B). The expression of the K802R-p110␣ mutant, but not of the K805R-p110␤ mutant, reduced p-PKB activation in early G 1 (ϳ1 h) (Fig. 2E), as p110␣ activity is greater at this point (see below).
We also examined p70S6K activation. In asynchronous cultures, the transient expression of p110-interfering forms decreased and p110-activating mutations enhanced (p110␣* had a greater effect than p110␤*) p-p70S6K cellular levels ( Fig.  2F). In synchronized populations, however, p110␣* enhanced p70S6K activation even in G 0 , whereas p110␤* increased p-p70S6K levels most notably at ϳ4 h after the addition of serum (Fig. 2G). This suggested a selective action of p110␣ at the first PI3K activity peak; accordingly, the expression of the K802R-p110␣ mutant selectively inhibited the initial p-p70S6K peak (ϳ1 h), whereas the K805R-p110␤ mutant moderately reduced late p-p70S6K levels (Fig. 2H). The K805R-p110␤ mutant did not reduce p70S6K activation at 1 h, probably because p110␤ exhibits a notably lower activity than p110␣ in early G 1 (see below). Quantitation of the gel bands in several assays confirmed the selective effect of the K802R-p110␣ mutant on the early p-PKB and p-p70S6K activity peaks following the addition of GF ( Fig. 2E and H). The reduction of p110␣ and -␤ levels with shRNA yielded consistent results (not shown). These results indicate that both p110␣ and p110␤ modified PKB and p70S6K activation but that only p110␣ regulated their early G 1 (ϳ1 h) activity peaks.
VOL. 28,2008 PI3K␣ AND PI3K␤ CONTROL CELL CYCLE ENTRY 2805 longer incubation periods than p110␣ shRNA (minimum 96 h versus 48 h for p110␣ shRNA), probably due to the greater stability of the p110␤ protein (unpublished data). In control cells, the p-FoxO3a signal peaked at 1 to 1.5 h and was reduced by 2 h after GF addition (Fig. 3B). p110␣ shRNA greatly decreased p-FoxO3a levels at 1 to 1.5 h, whereas p110␤ shRNA had only a moderate effect on FoxO3a phosphorylation (Fig. 3B). Similar results were obtained by using a differ-ent set of shRNA (see Materials and Methods) or the K802R-p110␣ or K805R-p110␤ mutant (Fig. 3C). Control cells showed two peaks of increased p-FoxO3a content in cells in G 1 phase, an early G 1 peak and another peak coincident with the PI3K activity peak in late G 1 (Fig. 3C). Whereas the K802R-p110␣ mutant significantly reduced p-FoxO3a levels throughout G 1 , the K805R-p110␤ mutant moderately diminished the duration of the early G 1 peak and slightly postponed late G 1 FoxO3a phosphorylation. Accordingly, stable p110␣*-expressing cell lines exhibited sustained and high p-FoxO3a levels (1) (Fig.  3D), whereas p110␤* only moderately and transiently increased FoxO3a phosphorylation ( Fig. 3D and data not shown). The more-prominent action of p110␣ in FoxO TF control in early G 1 was confirmed by examining cyclin D (see below). Thus, p110␣ plays a dominant role in FoxO phosphorylation in early G 1 . The parallel examination of cell cycle profiles in these assays showed that both the K802R-p110␣ and the K805R-p110␤ mutant reduced cell cycle entry (Fig. 3E); the levels of inhibition varied in different assays (see below) but were of similar magnitudes for interference with p110␣ or p110␤. Accordingly, p110␣*-expressing cells entered cell cycle earlier (1, 21) (Fig. 3E) and p110␤*-expressing cells entered S phase even more efficiently than p110␣*-expressing cells (Fig. 3E). p110␣ and -␤ control cyclin E and A levels, but only p110␣ regulates cyclin D. Early signaling pathways promote cell growth and the expression of G 1 cyclins (14). We subsequently examined the consequences for G 1 cyclin expression of interfering with p110␣ and -␤ activity. Comparison of synchronous stable p110␣*-and p110␤*-expressing cells showed that enhanced activation of p110␣, but not -␤, increased cyclin D3 levels ( Fig. 4A and B). In contrast, both p110␣*-and p110␤*expressing cells upregulated cyclin E levels even before the addition of serum, and p110␣*, but not -␤*, prolonged cyclin E expression ( Fig. 4A and B). Neither p110␣* nor -␤* expression was sufficient to induce cyclin A expression in G 0 , but cyclin A appeared earlier in these cells than in controls, and its expression was greater and more prolonged in p110␣*-expressing cells ( Fig. 4A and B). In p110␣*-expressing cells, the higher cyclin D3 levels (Fig. 4A) correlated with their greater p-FoxO3a content (Fig. 3) (1), as p-FoxO3a controls cyclin D synthesis (36).
We performed a complementary analysis and examined the effects of interfering with p110␣ and -␤ expression on G 1 cyclin expression. We examined the effect of reducing p110␣ and -␤ expression levels by shRNA in murine NIH 3T3 cells (not shown) and human U2OS cells synchronized at the G 1 /S border (Fig. 5). hp110␣ shRNA selectively reduced p110␣ expression, and p110␤ shRNA acted only on p110␤ (Fig. 5A). Nonetheless, p110␣, but not -␤, shRNA reduced cyclin D3 expression ( Fig. 5B and C). In contrast, both shRNA (for p110␣ or -␤) delayed the expression of cyclins E and A ( Fig. 5B and C). Thus, p110␣ and p110␤ regulate the expression of cyclins E and A, but only p110␣ controls cyclin D levels.
p110␣ and -␤ control late G 1 c-Myc levels and RB phosphorylation. Late G 1 PI3K activation stabilizes c-Myc (24); we attempted to determine which of the two ubiquitous isoforms regulated c-Myc levels in late G 1 . Stable p110␣*-and p110␤*expressing NIH 3T3 cell lines, as well as NIH 3T3 cells infected with retroviruses expressing the K802R-p110␣ or K805R-p110␤ mutant, were synchronized as described above. The control cells exhibited two peaks of increased c-Myc levels ( Fig. 6A and B), as reported previously (24). In p110␣*-expressing cells, the c-Myc levels were higher and peaked earlier but the cells still showed the two peaks of c-Myc expression ( Fig. 6A and B). p110␤* expression also moderately enhanced c-Myc stability, but only in late G 1 (Fig. 6A and B). The effect of p110␣* at increasing c-Myc levels is consistent with its ac-tion on FoxO TF, since FoxO TF represses c-Myc expression (8); it also concurs with the higher cyclin A levels observed in these cells, as c-Myc regulates cyclin A expression (26). Nonetheless, both p110␣* and p110␤* prolonged c-Myc stability in late G 1 . Interference with either p110␣ or -␤ postponed or reduced, respectively, the c-Myc expression levels in late G 1 (Fig. 6C and D), suggesting that both isoforms control c-Myc levels in advanced G 1 , although they do so differently. We also examined the consequences of interfering with p110␣ and -␤ activities for the phosphorylation of RB, a major Cdk2/cyclin substrate (37). In synchronized NIH 3T3 control cells, RB was hyperphosphorylated at ϳ12 h after GF addition (Fig. 7A). Both p110␣* and -␤* expression affected RB phosphorylation, which was observed at low levels even in quiescent cells; in late G 1 , p110␣* and -␤* also increased and accelerated (p110␤* more so) the appearance of hyperphosphorylated RB (Fig. 7A). Accordingly, interference with p110␣ or -␤ activity by the expression of the K802R-p110␣ or K805R-p110␤ mutant delayed RB phosphorylation (the K805R-p110␤ mutant had a greater effect) (Fig. 7B), suggesting that both p110␣ and -␤ activities regulate RB phosphorylation.
Distinct activation kinetics of p110␣ and -␤ during G 1 phase. The distinct contributions of p110␣ and -␤ to early G 1 events suggested that p110␣ and -␤ might present different activation kinetics. To determine the PI3K isoform(s) activated in early and late G 1 , we examined NIH 3T3 cells that permit the synchronization of the cultures in G 0 phase (25). To isolate p110␣ and -␤, we could not use p85 Ab as it brings down both catalytic subunits, nor we could use anti-p110␣ and -␤ Ab, since most of them reduce PI3K activity (unpublished observations). Thus, to evaluate specific isoform activation through G 1 phase, we cotransfected NIH 3T3 cells simultaneously with cDNA encoding wild-type p110␣ and -␤ fused to two different tags. Recombinant Myc-tagged p110␣ and His-tagged p110␤ were expressed at slightly above basal levels (Fig. 8A). p110␣ and p110␤ were efficiently immunoprecipitated by using Myctagged or His-tagged Ab, as determined by WB using the specific p110␣ or p110␤ Ab, respectively (Fig. 8B). Moreover, p110␣-p85 and p110␤-p85 complexes were at similar levels, as estimated by comparison of the amounts of p85 present in p110␣ and p110␤ immunoprecipitates (Fig. 8B, bottom). We immunopurified p110␣ and -␤ with the corresponding anti-tag Ab and tested their enzyme activities in vitro. After the addition of serum, p110␣ activated early, at 5 to 10 min following serum addition; this activity increased at 1 h and then diminished to basal levels, increasing again at ϳ7 h (Fig. 8C). p110␤ exhibited modest activity peaks at 1 and 4 h and a maximum activity at ϳ8 h after the addition of serum (Fig. 8C). In NIH 3T3 cells, part of p110␤, but not p110␣, localizes in the nuclei (our unpublished results); nuclear PI3K activity peaked at ϳ8 h after the addition of serum, confirming maximum endogenous p110␤ activity in late G 1 (not shown). We checked that similar amounts of p85 were associated with either p110␣ or p110␤ at different time points (Fig. 8D). Therefore, most PI3K activity in early G 1 corresponds to that of p110␣; p110␤ exhibits another minor peak by 4 h. In late G 1 , both p110␣ and -␤ are activated and p110␤ exhibits its maximum activity. p110␣ and -␤ have different activation requirements. The different activation kinetics of p110␣ and -␤ suggested that they exhibit distinct activation requirements. Since TyrK and Ras regulate class I A PI3K activation (17), we tested whether the p110␣ and -␤ activities in G 1 phase were affected by treatment with inhibitors of TyrK (herbamycin) and Ras (lovastatin). We first checked the selective action of these inhibitors in reducing p-Tyr or active Ras levels (24 and data not shown).
Herbamycin treatment, but not treatment with lovastatin, reduced p110␣ activity at 7 min. Both herbamycin and lovastatin inhibited p110␣ activation at 1 and 7 h (Fig. 8C). This suggests that the first increase in the activity of p110␣ is TyrK dependent, but TyrK and Ras contribute to p110␣ activation at 1 and 7 h. In contrast, the modest p110␤ activity at 1 h was sensitive to lovastatin, but not to herbamycin, although both inhibitors blocked later p110␤ activation peaks (at 4 and 8 h) (Fig. 8C and F). The results of these assays illustrate the distinct activation requirements for p110␣ and -␤ activities. The maximum p110␣ (at 1 h) and p110␤ (at 8 h) activities, nonetheless, were herbamycin and lovastatin sensitive, suggesting that TyrK and Ras activation contribute to optimal p110␣ and p110␤ activities.
To confirm the distinct activation requirements of p110␣ and p110␤, we examined whether the response of purified p85-p110␤ complex to activated TyrK and Ras is similar to that of p85-p110␣ (17). We used Tyr-phosphorylated platelet-derived growth factor receptor (PDGF-R) peptide and purified active Ras in vitro; this analysis confirmed that the Tyr-phosphorylated peptide activates p110␣, that active Ras alone exerts a moderate activation effect, and that Ras synergizes with p-Tyr phosphopeptides to enhance p110␣ activity ( Fig. 9B and C) (17). In contrast, although p110␤ activity also increased with the phosphopeptides and with active Ras and together they induced a greater activation effect ( Fig. 9B and C), there was a consistent difference between p110␣ and p110␤ activation. Whereas p110␣ responds better to Tyr phosphopeptides than to V12-Ras alone, Ras consistently induced a greater activating effect than phosphopeptides on p110␤ ( Fig. 9B and  C). These assays confirmed the TyrK activation requirement for p110␣ induction (17) and demonstrated the greater intrinsic Ras dependence for p110␤ activation.
Since p110␣ activation is greater than that of p110␤ in early FIG. 7. p110␣ and -␤ control RB phosphorylation. (A and B) Stable NIH 3T3 cells expressing p110␣* or p110␤* or infected with viruses encoding the K802R-p110␣ (p110␣) or K805R-p110␤ (p110␤) mutant were treated as described in the Fig. 6 legend; RB expression levels were analyzed by WB. Graphs represent the mean percentages Ϯ standard deviations (SD) of the signals for p-RB (pRB) compared to the maximum p-RB signal in wild-type cells at 15 h (100%) (n ϭ 3). Quantitation was as described in the Fig. 6 legend. (*), P Ͻ 0.05. VOL. 28,2008 PI3K␣ AND PI3K␤ CONTROL CELL CYCLE ENTRY G 1 (at 7 min to 1 h), we compared the binding of p110␣ and -␤ to PDGF-R at early time points. Both isoforms associated with the PDGF-R at 7 min after the addition of serum (not shown), arguing against a selective binding of p110␣ as the cause for its selective activation at this point. To gain insight into the mechanisms of p110␣ and -␤ activation in early G 1 , we considered the greater Ras dependence of p110␤ in vitro and postulated that the activation of p110␤ in vivo might also rely more on active Ras than that of p110␣ does. To determine the relative Ras dependence for p110␣ and -␤, we examined the sensitivities of p110␣ and -␤ to interference with Ras activation induced by the coexpression of N17-Ras with Myc-tagged versions of p110␣ and -␤. Whereas the first p110␣ activity peak at 7 min decreased only partially in the presence of N17-Ras (approximately one-third), p110␤ activation, which was lower than that of p110␣, occurred later and was drastically reduced (more than 90%) following the expression of N17-Ras ( Fig. 9D and E). These observations show that both in vitro and in vivo, the activation of p110␤ is more Ras dependent than that of p110␣. Considering that Ras activation is moderate at 1 h and maximal in late G 1 (24), the greater Ras dependence of p110␤ explains its activation kinetics in G 1 phase. Interference with p110␣ or -␤ expression/activity results in cell cycle entry defects. During the course of the experiments using synchronized populations, we noticed that cells expressing p110␣* and -␤* showed an earlier S-phase entry (Fig. 3E,  4, 6A, and 7A). Accordingly, the expression of K802R-p110␣ and K805R-p110␤ mutants (Fig. 3E, 6B, and 7B) or the reduction of p110␣ and -␤ levels by shRNA in U2OS cells (Fig.  5B) induced a delayed G 1 /S transition. We also interfered with p110␣ or -␤ expression in NIH 3T3 cells by using p110␣ or -␤ shRNA, as described above. p110␣ shRNA selectively reduced p110␣ expression and p110␤ shRNA diminished only p110␤ levels (Fig. 10A). Despite partial reductions in p110␣ and -␤ expression, both shRNA delayed S-phase entry (Fig. 10A).
We also examined cell cycle entry by the incorporation of bromodeoxyuridine (BrdU). Interference with endogenous p110␣ and -␤ kinase activity in COS cells by the transfection of the inactive K802R-p110␣ or K805R-p110␤ mutants reduced BrdU incorporation (Fig. 10B). We also analyzed primary cells. Homozygous deletion of p110␣ or -␤ causes embryonic lethality (3,4). We thus examined MEF from p110␣ and -␤ heterozygous mice. Since G 0 arrest by serum deprivation or growth to confluence is inefficient in MEF, we analyzed Sphase entry by measuring BrdU incorporation in exponentially growing cultures. Both heterozygous deletions reduced the fraction of BrdU-positive cells compared to the BrdU-positive fraction of wild-type fibroblasts (Fig. 10C). These results demonstrate that both p110␣ and -␤ control cell cycle entry.

DISCUSSION
The activation of PI3K is essential for cell division. We examined which one of the two ubiquitous PI3K isoforms (p110␣ and -␤) regulates cell cycle entry. We describe results showing that p110␣ activated before p110␤ at the G 0 /G 1 transition exerts a selective action in inducing G 1 entry events. In fact, the first activity peak of p110␣ had already occurred at 5 to 10 min following the addition of GF and it required TyrK activation; p110␣ further increased its activity at ϳ1 h in a TyrK-and Ras-dependent manner and activated again in advanced G 1 . In contrast, p110␤ displayed low activity in early G 1 , with a moderate increase at ϳ1 h; Ras induction was essential for p110␤ activation. p110␤ displayed another low activity peak in mid-G 1 and maximal activation in late G 1 . p110␣ and -␤ activate in a sequential manner in late G 1 . This concurs with their distinct sensitivities to TyrK and Ras since, also in late G 1 , the activation of TyrK precedes that of Ras, which is maximal at this point (24). In agreement with the greater activation of p110␣ in early G 1 , this isoform regulated early G 1 events (such as cyclin D levels and FoxO phosphorylation) more than p110␤ did. Nonetheless, interference with either p110␣ or p110␤ reduced S-phase entry, showing that both isoforms control the G 1 3S transition. p110␣ and -␤ regulated the expression of c-Myc and cyclins E and A, RB phosphorylation, and, in turn, S-phase entry.
The critical role of p110␣ and -␤ in the control of cell division was taken into account during the preparation of the cell lines for this study. We used stable cell lines expressing low levels of p110␣* and p110␤*, since the transient overexpression of high levels of p110␣* impairs progression through the G 2 /M phases (1). p110␣*-expressing cells entered cell cycle faster than controls, and p110␤*-expressing cells divided at a lower half-life than both p110␣*-expressing cells and controls. For the analysis of the consequences of reducing the p110␣ and p110␤ activities, we had to use transient transfection or infection, as cell lines of kinase-inactive mutants or shRNA were unstable, showing that p110␣ and p110␤ activities control cell survival and/or division.
An open question regarding the select functions of class I A PI3K isoforms is how the specificities of the different isoforms are acquired, as p110 catalytic subunits produce the same lipid products and all class I A p110s associate with p85 molecules, which bring p110 to activated receptors (42,12). p110␦'s specificity seems related to its tissue-specific expression pattern (30). However, in the case of p110␣ and -␤, they are ubiquitous and still they exhibit distinct functions in development (3,4) and cell division ( Fig. 2 and 3). The observations presented here illustrate mechanisms for the p110␣ and -␤ functional  3). Double-ended arrows indicate the two values being compared. (D) NIH 3T3 cells were transfected with empty vector or cotransfected with cDNAs encoding p85 and Myc-p110␣ or Myc-p110␤. p85-p110 cDNAs were transfected alone or with a vector encoding N17-Ras. After 36 h, cells were synchronized in G 0 and released by serum addition for different times. p110␣ or p110␤ was immunopurified, and their activities assayed in vitro. We examined the amount of p85 in the p85-p110 complexes by WB (middle panels). The different samples expressed similar N17-Ras levels (bottom), as determined by WB. ϩ, present; Ϫ, absent; IP, immunoprecipitate. (E) Graphs compare the mean percentages Ϯ standard deviations (SD) of p110␣ and -␤ activities of three different assays as described for panel C to the activity of p110␣ or p110␤ at 1 h (100%), normalized in comparison with the p85 loading control. (*), P Ͻ 0.05. V-Ras/ VRas, V12-Ras; N-Ras/ 17 N-Ras, N17-Ras. VOL. 28,2008 PI3K␣ AND PI3K␤ CONTROL CELL CYCLE ENTRY 2811 specificities that are delimited by the different activation requirements determining when these isoforms are activated. The phenotype of mice expressing a Ras-resistant p110␣ mutant supports the observation that, physiologically, p110␣ activity is partially independent of Ras. These mice present a number of defects, including reduced cell proliferation and diminished Ras-dependent tumor formation (15); however, they exhibit a milder phenotype than p110␣-deficient mice (3). This shows that despite the fact that K227A p110␣ is not activated by Ras, it still exerts some of the p110␣ actions in vivo (15). Interestingly, the expression of wild-type p110␤, -␦, and -␥ is sufficient to induce chicken embryo fibroblast focus formation; in contrast, p110␣ requires an activating mutation to trigger transformation (20). The crystal structure analysis of the inter-Src homology 2 domain of p85 in complex with the N-terminal part of p110␣ suggests that activation by Tyr kinases releases p110␣ from the inhibition exerted by p85; it is possible that the p85-mediated p110 structural constraint is stronger in the case of p110␣ (16,29). The H1047R mutant activates p110␣; following the additional K227E mutation, this mutant no longer binds Ras but contributes to cell transformation. In contrast, wild-type p110␤ loses its transforming activity when Ras binding is impaired (20). It is possible that in the absence of Ras binding, p110␤ simply exhibits low enzymatic activity, since we show that purified p110␤ shows a higher Ras dependence for activation than p110␣ (Fig. 9). In this regard, although late G 1 p110␤ activation was greatly inhibited by the addition of herbamycin at 7 h (Fig. 8C), this treatment reduced late G 1 Ras activity (not shown). Future studies will attempt to determine which residues in the p110 Ras-binding site determine the greater Ras dependence of p110␤.
Whereas the results of our studies support the existence of activation specificities for p110␣ and -␤, downstream of p110␣ and -␤ we find that they are both capable of regulating the same substrates. In fact, both the mutants interfering with p110␣ and those interfering with p110␤ affected PKB and p70S6K activities, illustrating that these p110 isoforms have the potential of regulating the same effectors. The distinct kinetics of p110␣ activation in early G 1 phase explains the specific function for p110␣ at this point. In fact, in synchronized cells, p110␣ selectively controlled the first activation wave of PKB and p70S6K and, in turn, FoxO3a phosphorylation and the expression of its effector, cyclin D. Since p110␤'s activity was low in early G 1 , interference with its kinase activity affected PKB and p70S6K activities at this point only slightly, although it modulated their activities at later time points (Fig. 2). In contrast, in late G 1 , both p110␣ and p110␤ exhibited remarkable increases in activity and regulated c-Myc and cyclin E and A levels, as well as RB phosphorylation. Therefore, both the p110␣ and -␤ isoforms controlled cell cycle regulators at the G 1 /S boundary, offering a mechanism for the involvement of these isoforms in the control of cell cycle entry.
The expression of p110␣ shRNA inhibits carcinoma cell growth (28), supporting the role of p110␣ in cell division. Selective interference with p110␣ inhibited the early activation of the cell growth regulator p70S6K (Fig. 2). Since cell cycle entry cannot occur without cell growth (35), p110␣ mutations in human cancer might facilitate G 0 exit by upregulating protein synthesis and inhibiting FoxO TF-controlled cell cycle inhibitors. Later in the cell cycle, p110␣ and -␤ contribute to enhancing c-Myc stability and Cdk2 activation (24). p110␣ is thus a potential target for cancer treatment; nonetheless, the inhibition of p110␣ interferes with glucose metabolism (22). Alternatively, interference with p110␤ might also be considered a promising approach, since although no activating mutations in p110␤ in human cancer have been described, the overexpression of the wild-type p110␤ does promote cell transformation (20). In fact, shRNA for p110␤ show an antiproliferative effect in tumor cell lines (6,32) and interference with p110␤ blocks S-phase entry (Fig. 10). Altogether, we report that p110␣ and -␤ are activated with distinct kinetics during G 1 phase, as they respond differently to the activation of TyrK and Ras. p110␣ primarily controls early G 1 events, such as FoxO TF inactivation and cyclin D expression, whereas both p110␣ and -␤ regulate later G 1 events and G 0 /G 1 transition.