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Molecular and Cellular Biology, December 2004, p. 10868-10881, Vol. 24, No. 24
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.24.10868-10881.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Cooperative Regulation of the Cell Division Cycle by the Protein Kinases RAF and AKT

Amer M. Mirza,^1, Stephan Gysin,¹ Nisar Malek,^2, Kei-ichi Nakayama,³ James M. Roberts,² and Martin McMahon^1*

Cancer Research Institute and Department of Cellular and Molecular Pharmacology, UCSF Comprehensive Cancer Center, San Francisco, California,¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington,² Department of Molecular and Cellular Biology, Kyushu University, Fukuoka, Japan³

Received 24 March 2004/ Returned for modification 20 May 2004/ Accepted 9 September 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
The RAS-activated RAFMEKextracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)PDK1AKT signaling pathways are believed to cooperate to promote the proliferation of normal cells and the aberrant proliferation of cancer cells. To explore the mechanisms that underlie such cooperation, we have derived cells harboring conditionally active, steroid hormone-regulated forms of RAF and AKT. These cells permit the assessment of the biological and biochemical effects of activation of these protein kinases either alone or in combination with one another. Under conditions where activation of neither RAF nor AKT alone promoted S-phase progression, coactivation of both kinases elicited a robust proliferative response. Moreover, under conditions where high-level activation of RAF induced G₁ cell cycle arrest, activation of AKT bypassed the arrest and promoted S-phase progression. At the level of the cell cycle machinery, RAF and AKT cooperated to induce cyclin D1 and repress p27^Kip1 expression. Repression of p27^Kip1 was accompanied by a dramatic reduction in KIP1 mRNA and was observed in primary mouse embryo fibroblasts derived from mice either lacking SKP2 or expressing a T187A mutated form of p27^Kip1. Consistent with these observations, pharmacological inhibition of MEK or PI3'-kinase inhibited the effects of activated RAS on the expression of p27^Kip1 in NIH 3T3 fibroblasts and in a panel of bona fide human pancreatic cancer cell lines. Furthermore, we demonstrated that AKT activation led to sustained activation of cyclin/cdk2 complexes that occurred concomitantly with the removal of RAF-induced p21^Cip1 from cyclin E/cdk2 complexes. Cumulatively, these data strongly suggest that the RAFMEKERK and PI3'KPDKAKT signaling pathways can cooperate to promote G₀G₁S-phase cell cycle progression in both normal and cancer cells.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Coordinate regulation of intracellular signaling pathways is central to the ability of mitogens and oncogenes to promote cell cycle progression (24). Two pathways thought to play an important role in committing quiescent cells into S phase are the RAS-activated RAFMEKextracellular signal-regulated kinase (ERK) and phosphatidylinositol 3'-kinase (PI3'-kinase)PDK1AKT pathways (35, 36). These pathways are reported to influence the expression, activity, or subcellular localization of key components of the cell cycle machinery such as cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) leading to the appropriate activation of E2F transcription factors (50, 51). In addition to sensing the activation of specific signaling pathways, cells are also able to integrate the extent and timing of signal pathway activation and convert that information into an appropriate biological response (32, 48, 64, 66). For example, depending on the level of expression or activation, activated RAS can promote either cellular immortalization, oncogenic transformation, or cell cycle arrest in the same cell type (20, 48, 64, 66). Under these circumstances, RAS (or RAF)-induced cell cycle arrest is mediated by induced expression of CKIs of the INK4 or CIP/KIP family (29, 37). Interestingly, activation of "parallel" signaling pathways can modify the ability of RAS to regulate the G₀G₁S-phase cell cycle transition. For example, RAS-induced cell cycle arrest in Swiss 3T3 cells is prevented by coactivation of Rho signaling pathways (9, 43). Under these circumstances activated RhoA is reported to prevent the expression of the CKI p21^Cip1, a major mediator of RAS- or RAF-induced cell cycle arrest in mouse fibroblasts (43).

Previous work has suggested that the RAS effectors RAF and PI3'-kinase can cooperate to promote both mitogenesis and oncogenic transformation of mammalian cells (19, 25). Here we use NIH 3T3 cells harboring conditionally active forms of RAF and AKT (3T3-RA cells) to explore the biochemical mechanism(s) that underlies the ability of these RAS effectors to cooperate (39). We observed that activated RAF and AKT cooperated to promote S-phase progression in 3T3-RA cells. Moreover, AKT was able to bypass RAF-induced G₁ cell cycle arrest mediated by CKI-dependent inhibition of cyclin E/cdk2 (64). Coactivation of AKT and RAF had at least three effects on the cell division cycle machinery: (i) cooperation to induce cyclin D1 expression, (ii) cooperation to repress KIP1 mRNA and p27^Kip1 expression, and (iii) promotion by AKT of the removal of RAF-induced p21^Cip1 from cdk2 complexes, leading to sustained cyclin E/cdk2 activity. Finally, using pharmacological inhibitors of RAFMEKERK or PI3'-kinasePDK1AKT signaling, we demonstrate that these pathways play a role in the regulation of the cell cycle machinery downstream of both the platelet-derived growth factor (PDGF) receptor and activated forms of RAS in mouse fibroblasts and in bona fide human cancer cells. These data suggest that RAS-activated signaling pathways cooperate with one another in a variety of biochemical ways to promote cell cycle progression in response to both mitogenic stimulation and oncogenic transformation.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of retrovirus expression vectors and derivation of cell lines. The construction of retrovirus vectors for the expression of M⁺Akt:ER* and EGFPRaf-1:AR in mammalian cells has previously been described (27, 39). NIH 3T3 cells constitutively expressing K-RAS4B (G12V) were derived by retrovirus infection with virus derived from the pLXSN:KRAS^G12V vector. NIH 3T3:iRAS cells, in which the expression of activated HRAS^G12V is induced by the addition of 5 mM IPTG (isopropyl-ß-D-thiogalactopyranoside), have been described previously (16, 34). Primary cultures of mouse embryo fibroblasts (MEFs) from either normal mice or mice expressing the KIP1^T187A allele or nullizygous for SKP2 were isolated and cultured by standard techniques (31, 42).

Retrovirus stocks were obtained by Lipofectamine (Gibco-BRL)-mediated transfection of the appropriate vectors into Bosc23 or Phenix-E cells as described previously (39, 44). Cells were serially infected with M⁺Akt:ER*- and EGFPRaf-1:AR-encoding retroviruses to generate cells expressing both conditional protein kinases. Target cells were infected and then cultured in medium containing 2 to 10 µg of puromycin/ml or 12 to 25 µg of blasticidin/ml (Sigma), depending on the vector, to select for virus-infected cells. Standard virus stocks gave rise to 10⁶ drug-resistant colonies of virus/ml. Following selection cells were pooled, expanded, and tested for the expression of M⁺Akt:ER* or EGFPRaf-1:AR proteins by Western blotting and, where appropriate, by FACScan at 490 nm. Under these conditions 95% of the pooled blasticidin-resistant cells expressed the EGFPRaf-1:AR fusion proteins.

Cell culture and cell treatments. All cells were cultured in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 25 mM HEPES, glutamine, penicillin, and streptomycin (Gibco-BRL) at 37°C in a humidified atmosphere containing 5% (vol/vol) CO₂. Methyltrienolone (R1881; New England Nuclear) and 4-hydroxytamoxifen (4-HT; Sigma) were prepared as 1 mM stocks in ethanol, stored at –20°C, and diluted immediately prior to use. Cells were routinely treated with 100 nM R1881 to activate EGFPRaf-1:AR or with 100 nM 4-HT to activate M⁺Akt:ER*, unless otherwise indicated. Serum or PDGF stimulation of cells was achieved by the addition of either 20% (vol/vol) fetal calf serum or 10 ng of PDGF (R&D Systems)/ml to cells that had been rendered quiescent. Confluent monolayers of cells were rendered quiescent by culture in DMEM containing glutamine, penicillin, streptomycin (Gibco-BRL), 25 mM HEPES (pH 7.4), and 2.5 mg of linoleic acid/liter and 500 mg of bovine serum albumin (BSA)/liter (linoleic acid-BSA complex; Becton Dickinson) for the times indicated (3, 39). Human pancreatic cancer cell lines were cultured in DMEM containing 10% (vol/vol) fetal calf serum. Stock solutions of U0126, CI-1040, or LY294002 were prepared in dimethyl sulfoxide, and cells were treated with these agents as described elsewhere in the text.

Preparation of cell extracts and analysis by Western blotting. Triton X-100-soluble cell lysates were prepared using Gold Lysis buffer (1% [vol/vol] Triton X-100, 20 mM Tris [pH 8.0], 137 mM NaCl, 15% [vol/vol] glycerol, and 5 mM EDTA) plus protease inhibitors (1 µM phenylmethylsulfonyl fluoride and 10 µM pepstatin) and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM tetrasodium pyrophosphate, 100 µM ß-glycerophosphate, and 1 mM sodium orthovanadate) as previously described (47). Protein concentrations were measured using the bicinchoninic acid protein assay kit (Pierce). Aliquots of cell lysates were electrophoresed through polyacrylamide gels and Western blotted onto Immobilon P polyvinylidene difluoride membranes (Millipore). Western blots were probed with the appropriate dilutions of primary antibodies for at least 1 h at room temperature. Most antibodies used were obtained commercially. Anti-estrogen receptor (hbER), anti-p44 ERK1/mitogen-activated protein kinase, and anti-PCNA antibodies were from Santa Cruz Biotechnology; anti-AKT (PKB) was from Upstate Biotechnology Inc.; and the antiphospho-S473-AKT antibody was either from New England Biolabs or generously supplied by David Stokoe (UCSF Cancer Center). Antibodies to p27^Kip1, p21^Cip1, cyclin E, and cdk4 were obtained from Transduction Laboratories. Anti-pan-RAS antibody was from Oncogene Science. Anti-cyclin D1 rabbit polyclonal antiserum was a generous gift from D. Parry and E. Lees (DNAX Research Institute). Antigen-antibody complexes were detected using the appropriate secondary antibody or protein A coupled to horseradish peroxidase and visualized using the enhanced chemiluminescence detection system (ECL; Amersham).

Immune complex kinase assays. cdk2 immune complex kinase assays were performed as previously described (39). Briefly, cells were lysed in NP-40 lysis buffer (50 mM HEPES [pH 7.5], 0.1% [vol/vol] NP-40, and 250 mM NaCl). Lysates were precleared with protein A-Sepharose (Sigma) and immunoprecipitated with a rabbit anti-cdk2 polyclonal antibody (Upstate Biotechnology Inc.). Immunoprecipitates were washed three times in NP-40 lysis buffer and once in cdk2 kinase reaction buffer (50 mM Tris [pH 7.4], 10 mM MgCl₂, 1 mM dithiothreitol). The reaction was carried out at 30°C for 30 min in 40 µl of kinase reaction buffer containing 2.5 µg of histone H1, 10 µCi of [-³²P]ATP, and 0.01 mM ATP. The reactions were quenched by addition of 5x sample buffer containing ß-mercaptoethanol followed by boiling for 5 min. Kinase reaction mixtures were electrophoresed and Western blotted, with the phosphorylation of substrates being quantitated using a Molecular Dynamics Storm Phosphoimager, and subsequently exposed to X-ray film (Kodak). Following quantitation of ³²P-histone H1, the Western blots were probed with the appropriate antibody to detect the presence of specific proteins in each immunoprecipitate.

DNA synthesis and cell cycle analysis. DNA synthesis was assessed by the incorporation of bromodeoxyuridine (BrdU) into cellular DNA as described previously (14, 39). Briefly, cells were rendered quiescent by culture in DMEM-BSA-linoleic acid for 5 days with medium being changed every 24 h. Cells were then treated as described elsewhere in the text. BrdU was added to the cells to a final concentration of 50 µM, and the incubation continued for an additional 18 h. Ethanol-fixed cells were serially stained with an anti-BrdU antibody and propidium iodide (Becton Dickinson). Following staining, the cells were washed and analyzed using a Becton Dickinson FACScan instrument.

Quantitative reverse transcription-PCR and RNase protection assay. Cells were pretreated with PD98059 (100 µM) or LY294002 (30 µM) or both for 10 min and then restimulated with 10 ng of PDGF/ml for 18 h. Total RNA was isolated using the RNeasy minikit (Qiagen); total cDNA was generated using 500 ng of total RNA in a final volume of 100 µl with murine leukemia virus reverse transcriptase; and random hexamers were incubated at 25°C for 10 min, then at 48°C for 40 min, and finally at 95°C for 5 min. Expression of mouse KIP1 mRNA was analyzed using the 5'-nuclease assay (Real-Time TaqMan reverse transcription-PCR) with the ABI PRISM 7700 instrument (ABI). PCR was conducted in triplicates with 50-µl reaction volumes of 1x PCR buffer A (ABI), 5.5 mM MgCl₂, 0.9 mM (each) primer, 200 mM deoxynucleoside triphosphates, 200 nM probe, and 0.025 U of Taq Gold (ABI)/ml. The PCR cycling conditions were 95°C for 15 min and 40 cycles of 95°C for 30 s and 60°C for 1 min. Sequences of the PCR primers and TaqMan probe (Integrated DNA Technologies) were as follows: mouse KIP1 forward, 5'-GGTTAGCGGAGCAGTGTCCA-3'; mouse KIP1 reverse, 5'-GGGAACCGTCTGAAACATTTTC-3'; mouse KIP1 TaqMan probe, FAM 5'-CCTGCTGCAGAAGATTCTTCTTCGCAAAAC-3' BHQ1; mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5'-TGCACCACCAACTGCTTAG-3'; mouse GAPDH reverse, 5'-GGATGCAGGGATGATGTTC-3'; mouse GAPDH TaqMan probe, FAM 5'-CAGAAGACTGTGGATGGCCCCTC-3' TAMRA.

RNase protection assays for the simultaneous detection of KIP1 and GAPDH mRNAs were carried out as described previously (33, 34). Plasmids containing the coding sequences of mouse KIP1 (Emma Lees, DNAX Research Institute) or GAPDH were digested and transcribed in vitro with T7 RNA polymerase to generate RNase protection probes by using the MAXIscript in vitro transcription kit (Ambion). Probes were gel purified, and RNase protection assays were performed using an RNase protection assay kit (Ambion). The protected fragments were resolved using denaturing polyacrylamide gel electrophoresis on a 6% (vol/vol) gel containing urea. KIP1 and GAPDH mRNA expression was quantitated using a Molecular Dynamics Storm Phosphoimager, and then the fragments were exposed to X-ray film (Kodak).

Confocal immunofluorescence microscopy. The effects of the coactivation of RAF with AKT on the subcellular localization of p21^Cip1 were assessed by confocal immunofluorescence microscopy with the use of a Zeiss LSM510 Meta microscope. NIH 3T3 cells expressing both EGFPRaf-1:AR and M⁺Akt:ER* were plated on coverslips, rendered quiescent, and then treated with R1881 or 4-HT to activate EGFPRaf-1:AR and/or M⁺Akt:ER* as appropriate. Cells were fixed using ice-cold methanol at 4°C for 15 min. p21^Cip1 was detected by staining with a mouse anti-p21^Cip1 monoclonal antibody (Santa Cruz Biotechnology) for 1 h at 4°C. Following three washes in cold phosphate-buffered saline plus 1% (wt/vol) BSA, fixed cells were incubated with a Texas Red-conjugated goat anti-mouse antiserum (Becton Dickinson) for 1 h at 4°C. Coverslips were washed extensively with cold phosphate-buffered saline plus 1% (wt/vol) BSA prior to being mounted. Analysis of stained cells was conducted using a Zeiss LSM510 Meta microscope.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Activation of RAF and AKT cooperates to induce cell cycle reentry. To explore the ability of RAF and AKT to cooperate in regulating the cell division cycle, we derived NIH 3T3 cells (3T3-RA cells) expressing both a conditionally active form of AKT (M⁺Akt:ER*) and a conditionally active form of RAF-1 (EGFPRaf-1:AR) as described previously (39). 3T3-RA cells were serum deprived and then treated with the estrogen analog 4-HT to activate Akt:ER*, the androgen analog R1881 to activate EGFPRaf-1:AR, or the coaddition of both hormones to elicit activation of both protein kinases. Cell cycle progression was measured by either propidium iodide staining (Fig. 1) or BrdU (data not shown) labeling. Under conditions where activation of neither RAF nor AKT alone was sufficient to induce DNA synthesis, coactivation of both kinases elicited a strong cooperative response. By varying the concentration of R1881 added to 3T3-RA cells, we observed that AKT was also able to cooperate to promote cell cycle progression under conditions where RAF was activated at either a low level or a high level where RAF induces a late G₁ cell cycle arrest (48, 64). Moreover, AKT activation could be delayed for up to 24 h after high-level RAF activation and still promote cell cycle progression (data not shown). These data indicate that RAF and AKT are able to cooperate to promote NIH 3T3 cell cycle progression and, like activated RhoA, AKT can overcome RAF-induced cell cycle arrest (43, 64).

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FIG. 1. RAF and AKT cooperate to promote cell cycle progression. NIH 3T3 cells engineered to express conditionally active EGFPRaf-1:AR and M+Akt:ER* (3T3-RA) were serum deprived for 36 h (Control) prior to the addition of 4-HT to activate AKT, R1881 to activate RAF, or a combination of 4-HT and R1881 to activate both RAF and AKT. Cell cycle progression was assessed by staining fixed cells with propidium iodide to estimate the percentage of cells in G0/G1 (2N DNA content), G2/M (4N DNA content), and S phase (2 to 4N DNA content) (A). The percentage of cells in each phase of the cell cycle was quantitated using CellQuest software and plotted (B).

To dissect the biochemical mechanism(s) underlying the ability of these protein kinases to promote the cell division cycle, we used Western blotting to assess the effects of RAF and AKT on the expression of a number of proteins known to be targets of RAF or AKT signaling. Of the various immediate-early targets of RAF signaling that we analyzed (e.g., Fos, Jun, c-Myc, and HB-EGF), none showed evidence of cooperative regulation in response to coactivation of AKT and RAF (data not shown) (11, 33, 34). However, we observed cooperative effects of RAF and AKT on the expression of two key cell cycle regulators, cyclin D1 and p27^Kip1. Activation of AKT or RAF alone led to a two- or threefold induction of cyclin D1 expression, respectively (Fig. 2A). However, coactivation of both signaling pathways led to a sixfold induction of cyclin D1. These observations are consistent with previous reports of cooperative regulation of cyclin D1 by RAS effector pathways and with the reported effects of the ERK mitogen-activated protein kinase pathway on cyclin D1 gene transcription and of AKT on cyclin D1 protein stabilization (1, 2, 13, 19).

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FIG. 2. RAF and AKT cooperate to regulate the expression of cyclin D1 and p27Kip1. Serum-deprived 3T3-RA cells were treated with 4-HT, R1881, or both hormones to activate RAF and/or AKT appropriately. At different times (6 to 24 h) after hormone addition cell extracts were prepared with the expression of cyclin D1 and p27Kip1 being assessed by Western blotting (A). The abundance of KIP1 mRNA was assessed using a simultaneous RNase protection assay with the use of GAPDH mRNA as an internal loading control (B) as described previously (34). The ratio of KIP1 to GAPDH mRNA was quantitated and is represented as a bar graph (C).

Activation of AKT alone had only a modest effect on p27^Kip1 expression, consistent with previous observations that suggest that AKT activation reduces p27^Kip1 expression by 50% (38). By contrast, activation of RAF alone led to significant repression of p27^Kip1 expression, but this was not clearly manifest until 12 to 24 h after RAF activation. Strikingly, coactivation of RAF and AKT led to a rapid and profound repression of p27^Kip1 expression that was clearly evident at 6 h and continued up to 24 h (Fig. 2A). It is noteworthy that the effects of RAF activation alone on cyclin D1 and p27^Kip1 expression, especially at later time points, are likely enhanced due to activation of PI3'-kinase signaling through the release of autocrine growth factors such as HB-EGF (34, 58).

The expression of p27^Kip1 is reported to be under both transcriptional and posttranscriptional control (31, 38, 42, 54, 61). To investigate the mechanism(s) underlying the regulated expression of p27^Kip1 by RAF and AKT, we assessed the effects of these protein kinases on KIP1 mRNA expression in 3T3-RA cells (Fig. 2B and C). Consistent with previous observations, activation of AKT alone led to a 50% reduction of KIP1 mRNA that was evident after 24 h (38). Activation of RAF alone led to a 65% reduction of KIP1 mRNA that was best evident after 24 h. Strikingly, coactivation of RAF and AKT led to a 70% reduction of KIP1 mRNA at 6 h and a 90% reduction at 24 h. These data suggest that RAF and AKT can cooperate to increase both the rate and the extent of repression of KIP1 mRNA consistent with their effects on p27^Kip1 expression in these cells. However, since p27^Kip1 expression is also controlled by regulated proteolysis, we suspect that the cooperative effects of RAF and AKT are likely to be a composite of effects of these pathways on KIP1 mRNA expression and p27^Kip1 proteolysis.

PI3'-kinase and MEK signaling pathways are required for repression of p27^Kip1 by growth factors and activated RAS. Although activation of RAF and AKT is clearly sufficient to repress p27^Kip1 expression, these experiments do not address whether the PI3'-kinasePDK1AKT and the RAFMEKERK pathways are required for the repression of p27^Kip1 in response to mitogenic stimulation or oncogenic transformation. To address this, we used pharmacological inhibitors of the signaling proteins PI3'-kinase (LY294002), mTor (rapamycin), and MEK (PD98059, U0126, or CI-1040) either alone or in combination with one another. PDGF stimulation of quiescent NIH 3T3 cells leads to activation of both the PI3'-kinasePDK1AKT and the RAFMEKERK signaling pathways and repression of p27^Kip1 expression (Fig. 3A). Perhaps surprisingly, addition of rapamycin had no effect on p27^Kip1 repression by PDGF. By contrast, blockade of either the RAFMEKERK or the PI3'-kinasePDK1AKT pathway alone inhibited the effects of PDGF on p27^Kip1 expression by 50%. However, blockade of both pathways in concert entirely inhibited the effects of PDGF on p27^Kip1 expression. These data suggest that the RAFMEKERK and the PI3'-kinasePDK1AKT pathways play an important role in the repression of p27^Kip1 that occurs in response to mitogenic stimulation. These data also suggest that the mTorp70^S6K pathway does not play a role in the regulation of p27^Kip1 expression by PDGF in these cells.

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FIG. 3. PDGF repression of p27Kip1 requires MEK and PI3'-kinase. Serum-deprived 3T3-RA cells were restimulated with 10 ng of recombinant PDGF/ml for 24 h in the absence or presence of either a pharmacological inhibitor of MEK (PD98059 or U0126), PI3'-kinase (LY294002), or mTor (rapamycin) or a combination of inhibitors as indicated. The expression of p27Kip1 and the activation status of ERK1/2 and AKT were assessed by Western blotting with appropriate antisera (A). Total AKT was used as a loading control in these experiments. The expression of KIP1 mRNA was assessed by real-time PCR (TaqMan) with GAPDH mRNA as an internal control (B).

To determine if the RAFMEKERK and PI3'-kinasePDK1AKT signaling pathways had an effect on KIP1 mRNA levels in these cells, we performed quantitative real-time PCR analysis (TaqMan) on mRNA derived from quiescent NIH 3T3 cells stimulated with PDGF in the absence or presence of LY294002 or PD98059 (Fig. 3B). Consistent with observations described above, PDGF repressed KIP1 mRNA by 60%. Inhibition of either the PI3'-kinasePDK1AKT or the RAFMEKERK pathway alone partially inhibited the effects of PDGF on KIP1 mRNA expression. However, blockade of both pathways eliminated the effects of PDGF on KIP1 mRNA. These data are in accord with the effects of conditional RAF and AKT on KIP1 mRNA (Fig. 2) and suggest that PDGF signaling through both the PI3'-kinasePDK1AKT and RAFMEKERK pathways is required for the regulated expression of both KIP1 mRNA and p27^Kip1 in these cells.

Mutationally activated RAS is implicated in the aberrant proliferation of many human cancer cells (35). Since both PI3'-kinase and RAF are reported to be key effectors of RAS signaling, we determined the requirement for these pathways in the ability of oncogenic RAS to repress expression of p27^Kip1. Serum deprivation of parental NIH 3T3 cells leads to elevated p27^Kip1 expression and cell cycle arrest (Fig. 4A). By contrast, serum deprivation of NIH 3T3 cells expressing activated K-RAS^G12V had no effect on the already low level of p27^Kip1 expression. Treatment of serum-deprived K-RAS^G12V-transformed NIH 3T3 cells with either PD98059 or LY294002 alone had only a modest effect on p27^Kip1. However the combination of both inhibitors led to a marked elevation of p27^Kip1 expression to the level observed in serum-deprived parental NIH 3T3 cells. To confirm these results, we utilized NIH 3T3 cells harboring an IPTG-inducible H-RAS^G12V allele (3T3:iRAS cells) (15, 16, 34). Serum-deprived 3T3:iRAS cells displayed elevated p27^Kip1 that was reduced by subsequent induction of H-RAS^G12V expression by addition of IPTG (Fig. 4B). Pharmacological inhibition of the PI3'-kinasePDK1AKT pathway, the RAFMEKERK signaling pathway, or both pathways together prevented the H-RAS^G12V-mediated reduction of p27^Kip1 expression. Cumulatively, these data suggest that the level of p27^Kip1 expression in RAS-transformed cells is under the dual control of the PI3'-kinasePDK1AKT and RAFMEKERK pathways.

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FIG. 4. Repression of p27Kip1 by activated RAS is MEK and PI3'-kinase dependent. (A) NIH 3T3 cells expressing activated K-RASG12V were generated by retroviral infection with pLXSN:KRASG12V. Asynchronously (A) proliferating parental and K-RASG12V-expressing NIH 3T3 cells were serum deprived (Q) in the absence or presence of pharmacological inhibitors of MEK (PD98059) or PI3'-kinase (LY294002) as indicated. The expression of p27Kip1 and ERK1/2 was assessed by Western blotting. (B) NIH 3T3 cells harboring an IPTG-inducible allele of activated H-RASG12V (3T3:iRAS cells) (16, 34) were either untreated or treated with 5 mM IPTG to induce the expression of H-RASG12V in the absence or presence of pharmacological inhibitors of MEK (PD98059) or PI3'-kinase (LY294002). The expression of p27Kip1, H-RASG12V, and ERK1/2 was assessed by Western blotting. (C) Hs766T pancreas cancer cells were either untreated or treated with LY294002 (LY), U0126 (U), or CI-1040 (CI) at the indicated concentrations for 24 h. Cell extracts were prepared and assayed by Western blotting for pERK1/2, pAKT, pan-ERK1/2, and p27Kip1 as described in Materials and Methods.

To address whether the regulation of p27^Kip1 expression observed in RAS-transformed NIH 3T3 cells is relevant to its regulation in bona fide human cancer cell lines, we utilized a panel of pancreatic cancer cell lines (57). We chose to analyze pancreatic cancer cells since this tumor type displays the highest frequency of somatic mutations of RAS genes of all human malignancies (5, 22). Hs766T cells express activated KRAS and display elevated levels of pERK1/2 and pAKT (Fig. 4C). Treatment of Hs766T cells with the PI3'-kinase inhibitor LY294002 led to a selective decrease in pAKT with little or no effect on pERK1/2. By contrast, treatment of the cells with the MEK inhibitor U0126 or CI-1040 led to a selective decrease in pERK1/2 with little or no effect on pAKT. Strikingly, both the PI3'-kinase and the MEK inhibitors led to accumulation of p27^Kip1 expression. These data are consistent with the observation that both MEK and PI3'-kinase inhibitors elicited a G₀/G₁ cell cycle arrest in Hs766T cells (data not shown).

Further consistent with these observations, treatment of a panel of pancreatic cancer cells (eight cell lines) (57) with a MEK inhibitor (U0126 or CI-1040) led to a consistent G₀/G₁ cell cycle arrest accompanied by induced expression of p27^Kip1 in all eight cell lines tested (S. Gysin and M. McMahon, submitted for publication). For the most part, treatment of cells with MEK inhibitors led to a cytostatic rather than a cytotoxic effect, although cell death was often observed after prolonged treatment (6 days). Treatment of pancreatic cancer cells with LY294002 led in some cases to a cytostatic effect and in others to a more rapid cytotoxic effect. Consistent with the observations in Fig. 4C, inhibition of PI3'-kinase by LY294002 led to elevated p27^Kip1 expression in five of eight pancreas cancer cell lines tested. These data are fully consistent with the hypothesis that the RAS-activated RAFMEKERK and PI3'-kinasePDKAKT pathways play a role in the regulation of p27^Kip1 expression in bona fide human cancer cell lines.

Phosphorylation of threonine 187 is not required for suppression of p27^Kip1 following activation of AKT and RAF. Phosphorylation of p27^Kip1 on threonine 187 (T187) by active cdk2 leads to its recognition by the F-box protein SCF^Skp2, thereby promoting p27^Kip1 ubiquitination and destruction by the 26S proteasome (45). However, it appears that this mechanism for p27^Kip1 regulation may operate largely in S phase. Knock-in of an AG mutation in exon 2 of the mouse KIP1 gene encodes a T187A form of p27^Kip1. The expression of p27^Kip1T187A is elevated in serum-deprived MEFs and is dramatically reduced following subsequent serum stimulation. However, compared to normal p27^Kip1, p27^Kip1T187A is aberrantly reexpressed during S phase, thereby delaying cell cycle progression (31). These data suggest that there are likely two mechanisms for the repression of p27^Kip1 during the G₀G₁S-phase cell cycle transitions. Repression of p27^Kip1 during the G₀G₁ transition is independent of T187 phosphorylation, whereas maintenance of low levels of p27^Kip1 during S phase is dependent on T187. Although the mechanism(s) that leads to repression of p27^Kip1 early in G₁ is poorly understood, it is nonetheless reported to be dependent on SCF^Skp2 and therefore likely to be dependent on regulated proteolysis of p27^Kip1 (21, 31, 42, 51). These observations prompted us to test whether the effects of RAF and AKT on p27^Kip1 expression might be mediated by T187 phosphorylation or by SCF^Skp2. To address these questions, we assessed the effects of RAF and AKT activation on p27^Kip1 expression by using primary MEFs derived from mice expressing either normal (+/+) or the T187A form of p27^Kip1 (31). These cells were engineered to express conditionally active RAF and AKT as described in Materials and Methods. Activation of RAF or AKT alone in either normal or KIP1^T187A MEFs had only a modest effect on the expression of p27^Kip1 (Fig. 5A). By contrast, coactivation of RAF and AKT led to a significant reduction in p27^Kip1 that was not diminished by the T187A mutation. These data indicate that phosphorylation of T187 is not required for the reduced expression of p27^Kip1 following activation of RAF and AKT. Importantly, in these cells as well as in control MEFs, treatment of cells with PDGF led to decreased KIP1 mRNA expression. These data indicate that KIP1 mRNA remains subject to repression by mitogens in MEFs expressing p27^Kip1T187A (Fig. 5B).

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FIG. 5. RAF and AKT repress the expression of KIP1T187A. (A) Primary MEFs isolated from normal (KIP1+/+) and KIP1T187A mice engineered to express conditionally active RAF and AKT were serum deprived for 36 h prior to the addition of 4-HT to activate AKT, R1881 to activate RAF, or a combination of 4-HT and R1881 to activate both RAF and AKT. The expression of p27Kip1, PCNA, and ERK1/2 was assessed by Western blotting. (B) Primary MEFs isolated from normal (KIP1+/+) and KIP1T187A mice were serum deprived for 36 h prior to the addition of PDGF, at which time the expression of KIP1 mRNA was assessed by real-time PCR (TaqMan) with GAPDH mRNA as an internal control (B). The relative abundance of KIP1 mRNA is expressed as a ratio to the level of GAPDH mRNA.

To test a possible requirement for the F-box protein SCF^Skp2 in the repression of p27^Kip1 in response to mitogenic stimulation, we used primary MEFs from SKP2^–/– mice (42). It has previously been reported that repression of p27^Kip1 following mitogen stimulation is entirely SCF^Skp2 dependent (21, 31, 42). Unfortunately SKP2^–/– cells grow very poorly in culture, making it impossible to generate derivatives expressing conditionally active RAF and/or AKT. Consequently we used PDGF stimulation to assess the regulation of p27^Kip1 expression in the G₀G₁ transition. In initial experiments we noted a striking effect of cell density on the repression of p27^Kip1 expression by PDGF in SKP2^–/– cells. SKP2 null cells at different degrees of confluency (80 to 100%) were serum deprived and then restimulated with PDGF. When 90 to 100% confluent SKP2^–/– cells were stimulated with PDGF, we observed no decrease in p27^Kip1 expression, consistent with previous reports that SKP2 is required for loss of p27^Kip1 expression (Fig. 6A). However, when subconfluent cells (80%) were stimulated with PDGF or serum, we observed a clear reduction in p27^Kip1 expression (Fig. 6A and B). However, the extent of p27^Kip1 repression was always less profound in SKP2^–/– cells than in normal MEFs. These data suggest that cell density influences the requirement for SKP2 in the regulation of p27^Kip1 expression in response to mitogen stimulation and may explain previous observations on the requirement for SKP2 in the repression of p27^Kip1. However, it is possible that cell density also influences the ability of mitogen stimulation to target the transcriptional machinery that regulates KIP1 mRNA expression.

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FIG. 6. Density-dependent repression of p27Kip1 in SKP2 null cells. (A) Normal (+/+) and SKP2 null (–/–) cells at different degrees of confluency (80 to 100%) were serum deprived prior to restimulation by 5 ng of PDGF/ml. The expression of p27Kip1 and ERK1/2 was assessed by Western blotting. (B) Normal (+) and SKP2 null (–) cells at 80% confluency were serum deprived prior to restimulation with 5 ng of PDGF/ml or 20% (vol/vol) fetal calf serum (FCS). The expression of p27Kip1 and ERK1/2 was assessed by Western blotting. (C) Serum-deprived SKP2 null cells were either untreated or treated with PDGF in the absence or presence of pharmacological inhibitors of MEK (PD98059) or PI3'-kinase (LY294002) as indicated. The expression of p27Kip1 and ERK1/2 at 16 or 24 h after PDGF addition was assessed by Western blotting. In parallel, the percentage of cells in S phase was assessed by labeling with BrdU and then costaining to detect BrdU-positive cells (y axis), and DNA content was determined with propidium iodide (x axis).

To assess the requirement for the RAFMEKERK and PI3'-kinasePDK1AKT pathways in the repression of p27^Kip1 in SKP2^–/– cells, we assessed the effects of pharmacological inhibitors of these pathways on p27^Kip1 expression. PDGF stimulation of subconfluent SKP2^–/– cells led to a clear reduction of p27^Kip1 expression (Fig. 6C). Pharmacological blockade of either the RAFMEKERK or the PI3'-kinasePDK1AKT pathway led to a striking inhibition of the effects of PDGF on p27^Kip1 expression. Importantly, the reduction of p27^Kip1 in response to PDGF occurred prior to the onset of DNA synthesis, arguing that the repression of p27^Kip1 was not simply a consequence of progression through S phase. These data are further consistent with the hypothesis that there are at least two independent mechanisms for the repression of p27^Kip1, one of which is active in G₀G₁ and the other of which is active in G₁SG₂ phase. Although the repression of p27^Kip1 that occurs in S phase is most likely dependent on phosphorylation-induced ubiquitination leading to proteolysis, the repression that occurs in the G₀G₁ transition appears to be independent of T187 phosphorylation and SCF^Skp2.

AKT activation promotes relocalization of Raf-induced p21^Cip1 out of cdk2 complexes. RAS/RAF-induced cell cycle arrest is dependent on the inhibition of cyclin/cdk2 complexes that is in turn mediated by CKIs such as p21^Cip1 (30, 48, 64). Although repression of p27^Kip1 by AKT and RAF would tend to promote cdk2 activity, we sought to determine if AKT activation had any effect on RAF-induced p21^Cip1, the key mediator of RAF-induced NIH 3T3 cell cycle arrest (30, 48, 64). Activation of AKT alone had little or no effect on total p21^Cip1 expression measured in whole-cell lysates (Fig. 7A, lanes 1 to 4), whereas activation of RAF alone led to robust induction of p21^Cip1 as reported previously (Fig. 7A, lanes 5 to7, and 7B). Coactivation of AKT had no effect on the overall level of RAF-induced p21^Cip1 expression and in some experiments appeared to modestly augment its expression (lanes 8 to 10 and data not shown). These data indicate that the mechanism(s) by which AKT overcomes RAF-induced cell cycle arrest is dissimilar to the effects of constitutive RhoA, which acts by inhibiting RAS-induced expression of p21^Cip1 (43). When measured in the same cell extracts, coactivation of RAF and AKT led to sustained cdk2 activity (lanes 8 to 10), whereas activation of RAF or AKT alone elicited, at best, only transient activation of cdk2 (lanes 2 to 7). Consistent with these data, RAF-induced p21^Cip1 was readily detected in complex with cdk2 (lanes 5 to 7). However, when AKT was coactivated with RAF, we detected little or no p21^Cip1 in complex with cdk2, consistent with sustained cdk2 activity. These data indicate that AKT activation elicits sustained cdk2 activation, not by inhibiting p21^Cip1 expression but by promoting the removal of p21^Cip1 from cdk2 complexes. Although p21^Cip1 is known to bind to cyclin D/cdk4 complexes, immunoprecipitation of p21^Cip1 from cells expressing active RAF and AKT did not show elevated cyclin D1 or cdk4 compared to that for RAF alone (Fig. 7B). Hence, the AKT-dependent removal of RAF-induced p21^Cip1 from cdk2 complexes was not simply a consequence of increased association with cyclin D1 or cdk4. Furthermore, immunofluorescence analysis of cells expressing active RAF or RAF and AKT suggested that AKT promoted nuclear export of RAF-induced p21^Cip1 into a cytoplasmic compartment as reported previously (Fig. 7C) (65).

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FIG. 7. AKT-mediated removal of p21Cip1 from cdk2 complexes. (A) Serum-deprived 3T3-RA cells were treated with 4-HT, R1881, or both hormones to activate RAF and/or AKT as appropriate. At different times (6 to 24 h) after hormone addition cell extracts were prepared. The expression of p21Cip1 in whole-cell extracts was assessed by Western blotting. cdk2 was immunoprecipitated from the same cell extracts, and its activity was assessed using [-32P]ATP and histone H1 as a substrate. The abundance of cyclin E, p21Cip1, and cdk2 in each immunoprecipitate was assessed by Western blotting as indicated. (B) Serum-deprived 3T3-RA cells were treated with 4-HT, R1881, or both hormones to activate RAF and/or AKT as indicated. Twenty-four hours after hormone addition cell extracts were prepared with the expression of cyclin D1, cdk4, p21Cip1, cdk2, p27Kip1, and ERK1/2 being assessed by Western blotting of whole-cell lysates (WCL). p21Cip1 was immunoprecipitated from the same cell extracts with the abundance of cyclin D1, cdk4, cdk2, and p21Cip1 in each immunoprecipitate being assessed by Western blotting as indicated. (C) Serum-deprived 3T3-RA cells were treated with R1881, 4-HT, or both hormones to activate RAF and/or AKT as indicated. The expression and localization of p21Cip1 (red) were assessed by indirect immunofluorescence. Cytoplasmic expression of EGFPRaf-1:ER (green) was assessed by virtue of the enhanced green fluorescent protein (EGFP) tag on the protein (64).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Inappropriate activation of RAS and/or its downstream effector RAF or PI3'-kinase is a feature common to a wide variety of human cancers (5, 12, 49). Here we have explored the link between activated RAF and AKT and the regulation of G₀G₁S-phase cell cycle progression. The ability of RAF and AKT to cooperatively promote cell cycle progression correlated with cooperative effects on the expression of cyclin D1 and p27^Kip1 and the cytoplasmic relocalization of p21^Cip1 resulting in sustained activation of cdk2. Under normal circumstances p27^Kip1 expression is elevated in G₀ and reduction in p27^Kip1 is a requirement for entry of cells into DNA synthesis (8). The importance of p27^Kip1 in cell cycle control is demonstrated by the fact that KIP1 nullizygous mice show multiorgan hyperplasia and gigantism and are tumor prone (18, 26, 41). Even more striking, KIP1 heterozygous mice display a haploinsufficient tumor-prone phenotype, indicating that even a 50% reduction of p27^Kip1 expression has serious consequences for cancer susceptibility in the mouse (17). In humans, reduced expression of p27^Kip1 is directly correlated with a poor prognosis in a variety of human malignancies (17, 40, 45, 53, 60).

Based on a wealth of previous research, the best evidence suggests that the regulation of p27^Kip1 expression by oncogenes and growth factors occurs at multiple levels. For example, transcription of the KIP1 gene in quiescent cells is promoted by members of the FOXO family of transcription factors (38). Activated AKT phosphorylates FOXO3a, leading to translocation of the protein into the cytoplasm, thereby repressing KIP1 gene transcription. In addition, there is strong evidence for regulation of p27^Kip1 by phosphorylation-induced ubiquitination followed by proteasome-mediated destruction. Under certain circumstances this is mediated by phosphorylation of p27^Kip1 on T187 followed by SCF^Skp2-mediated ubiquitination. However, analysis of knock-in mice expressing KIP1^T187A suggests that this latter mechanism may be of critical importance only during S phase and is dispensable for the repression of p27^Kip1 that occurs early in G₀/G₁ (31). Moreover, there is some evidence for regulated proteolysis of p27^Kip1 that is both T187 and SCF^Skp2 independent (21). Finally, evidence suggests that direct phosphorylation of p27^Kip1 by AKT may regulate the subcellular localization of the protein (4, 10, 23, 28, 52). Data presented here suggest that one mode of p27^Kip1 regulation by RAF and AKT is likely at the level of mRNA expression. Although the modest effects of AKT on KIP1 mRNA may be mediated through FOXO transcription factors, the mechanism by which RAF cooperates with AKT is not known. However, it is possible either that RAF may directly regulate a transcription factor involved in KIP1 mRNA expression or that RAF may regulate KIP1 mRNA stability or nucleuscytoplasm mRNA transport. These possibilities are under investigation.

Consistent with an important role for the RAFMEKERK and the PI3'-kinasePDK1AKT pathways in regulating KIP1 mRNA expression in a pathological state, treatment of a panel of human pancreatic cancer cell lines with U0126, CI-1040, or LY294002 leads to a cessation of proliferation, G₀/G₁ cell cycle arrest accompanied by increased KIP1 mRNA and p27^Kip1. Importantly, under these circumstances, inhibition of p27^Kip1 expression can bypass the inhibitory effects of MEK inhibition on the cell division cycle (Gysin and McMahon, submitted). Consequently, we propose that one of the important effects of RAS signaling on cell cycle progression is the repression of p27^Kip1 expression in G₀/G₁, leading to initial activation of CDK2. The initial effects of RAS signaling on p27^Kip1 expression would then be sufficient to promote activation of cyclin/cdk2, leading to further repression of p27^Kip1 throughout S phase by proteolysis in a pT187/SCF^Skp2-dependent manner.

Evidence for signal pathway cooperation has been described in a number of different situations. For example, in colon cancer cells, evidence suggests that the Wnt pathway cooperates with activated RAS to regulate the expression of cyclin D1 to promote G₀/G₁S-phase progression (55, 56). Moreover, in model systems in tissue culture, Rho signaling pathways can influence the ability of RAS or RAF to elicit cell cycle arrest by preventing the RAS/RAF-induced expression of p21^Cip1 (9, 43). Consistent with the notion that RAF and PI3'-kinase pathways can cooperate in oncogenic transformation, we show here that RAF and AKT cooperate to promote the reentry of cells into the cell cycle. In addition, AKT is able to bypass RAF-induced cell cycle arrest. In this case the effects of AKT are not to silence the expression of p21^Cip1 but to promote the removal of the protein out of the cyclin/cdk2 complex. Hence, the cooperative effects of RAF and AKT on cyclin D1, p27^Kip1, and p21^Cip1 together most likely explain the ability of these signaling pathways to cooperate to effectively promote cell cycle progression. These data are consistent with and expand upon the work of others who demonstrated that multiple RAS effector pathways contribute to G₁ cell cycle progression and oncogenic transformation (19, 46). They are also consistent with reports demonstrating the ability of the E7 oncoprotein of human papillomavirus type 16 to bypass the antiproliferative effects of RAF through activation of AKT and alterations in subcellular localization of p21^Cip1 (63). Under what conditions might cooperation between RAF and AKT signaling be directly relevant to cell cycle progression and/or oncogenic transformation? Human melanomas expressing activated NRAS rarely display mutations of BRAF or loss of PTEN expression. By contrast, melanomas expressing mutationally activated BRAF frequently display loss of PTEN expression (59). One explanation for these data is that mutationally activated NRAS can effectively engage both the RAFMEKERK and the PI3'-kinasePDK1AKT signaling pathways. However, in the 70% of human melanomas that express activated BRAF there remains a driving force for subsequent loss of PTEN expression to promote activation of the PI3'-kinasePDK1AKT pathway leading to oncogenic transformation. Conditional systems for the expression of activated RAS, RAF, and AKT, such as those described here, will allow this hypothesis to be tested directly both in primary melanocytes and in appropriate mouse models of melanoma (6, 7, 62). Moreover, since mutational activation of RAS, BRAF, and PI3'-kinase signaling has been reported in a wide variety of human cancers, these observations may have direct implications for the mechanisms underlying cell cycle dysregulation in a variety of human malignancies.

 
We thank all of the members of the McMahon lab for advice, constructive criticism, and continued support, especially David Dankort and Steen Hansen. We are most grateful to Emma Lees and David Parry for the provision of materials and reagents and for critical review of the manuscript. We also thank members of the UCSF Cancer Center for the provision of reagents and materials and for ongoing advice and suggestions, especially Frank McCormick, Osamu Tetsu, David Stokoe, and Christian Brandts.

M.M. gratefully acknowledges Schering-Plough Corporation for initial funding to support this research. This work was also supported by funds from the UCSF Cancer Center and grants from the Lustgarten Foundation for Pancreas Cancer Research and the Black Foundation to M.M. A.M.M. was supported in part by funds from an NIH Institutional Training Grant (T32 CA 09270). S.G. was a recipient of a postdoctoral fellowship from the Swiss National Science Foundation and the Novartis Foundation.

 
* Corresponding author. Mailing address: Cancer Research Institute and Department of Cellular and Molecular Pharmacology, UCSF Comprehensive Cancer Center, 2420 Sutter St., Box 0128, San Francisco, CA 94143-0128. Phone: (415) 502-5829. Fax: (415) 502-3179. E-mail: mcmahon{at}cc.ucsf.edu.

Present address: Cytokinetics, Inc., South San Francisco, CA 94080.

Present address: Department of Gastroenterology and Institute for Molecular Biology, Hannover Medical School, 30156 Hannover, Germany.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, December 2004, p. 10868-10881, Vol. 24, No. 24
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.24.10868-10881.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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