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First published online March 19, 2008
doi: 10.1242/10.1242/jcs.009530

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Akt-mediated phosphorylation of CDK2 regulates its dual role in cell cycle progression and apoptosis

Subbareddy Maddika^1,2,*, Sudharsana Rao Ande¹, Emilia Wiechec^1,3,4, Lise Lotte Hansen⁴, Sebastian Wesselborg^5, and Marek Los^1,2,,,¶

¹ Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba, Winnipeg, MB, R3E 0V9, Canada
² Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, MB, R3E 0V9, Canada
³ Department of Human Genetics, University of Aarhus, DK-8000 C Aarhus, Denmark
⁴ Department of Experimental and Clinical Radiobiology, Oncology Center, Maria Sklodowka-Curie Memorial Institute, Wybrzeze Armii Krajowej 15, PL-44100 Gliwice, Poland
⁵ Department of Internal Medicine I, University of Tübingen, Tübingen, Germany

View larger version (30K): [in this window] [in a new window]   Fig. 1. Akt phosphorylates CDK2 both in vitro and in vivo. (A) A non-radioactive in vitro kinase assay was performed with GST-CDK2 alone, using a recombinant active Akt or inactive Akt, and the phosphorylation of CDK2 was detected by immunoblotting with an anti-phospho substrate Akt antibody (upper panel). The levels of GST-CDK2 were detected by immunoblotting with CDK2 antibodies. Constitutively active Akt (Akt-CA) or catalytically inactive Akt (Akt-K179M) immunoprecipitated from 293T cells were used in a similar kinase assay using GST-CDK2 as a substrate, and the phosphorylation of CDK2 was detected as above (lower panel). (B) CDK2 wild-type (WT) proteins or mutated ones at T39, T160 or both were used in a kinase assay with a recombinant active Akt (Rec. Akt) or immunoprecipitated active Akt, and the CDK2 phosphorylation was detected by immunoblotting as in A. The expression of GST-CDK2 wild-type and mutant proteins was monitored by immunoblotting with CDK2 antibody. (C) The CDK2 phosphorylation in vivo was monitored by transfecting 293T cells with either pSR-CDK2 wild-type vector or the mutant proteins all tagged with HA epitope, immunoprecipitated using anti-HA antibodies after 48 hours and blotted with anti-phospho-Akt substrate antibody. The expression of CDK2 wild-type and mutant proteins was detected by anti-HA antibodies. (D) Murine 3T3 fibroblasts were transiently transfected with a wild-type CDK2 or CDK2-T39A mutant and serum-starved for 30 hours, then incubated with 20 ng/ml EGF, in combination with EGF and wortmannin (5 nM) or 10% fetal calf serum. The phosphorylation of CDK2 was detected as in A. Total CDK2 expression, phosphorylated Akt and total Akt were detected by the respective antibodies. (E) 3T3 fibroblasts were serum-starved for 30 hours, then treated exactly as in D. The phosphorylation of endogenous CDK2 was detected by immunoprecipitating with CDK2 antibodies and then immunoblotting with phospho-Akt substrate-specific antibody. Total CDK2 expression was detected by anti-CDK2 antibody. (F) Murine 3T3 fibroblasts were transfected with HA-tagged CDK2 wild-type plasmid and then serum-starved for 30 hours. Then they were incubated with a combination of EGF (20 ng/ml) and specific Akt inhibitor (210 nM, Calbiochem) or EGF alone or in combination with EGF and rapamycin. The phosphorylation of CDK2 was detected by immunoprecipitating with CDK2 antibodies and then immunoblotting with phospho-AKT substrate-specific antibody. Total CDK2 expression was detected by CDK2 antibodies. (G) 3T3 fibroblasts were transfected with HA-tagged CDK2 wild-type plasmid and then serum-starved for 30 hours. The phosphorylation of CDK2 was detected by immunoprecipitating with CDK2 antibodies and then immunoblotting with phospho-Akt substrate-specific antibody after stimulating the cells with EGF at different indicated time points. For detection of phosphorylated GSK3, cell lysates at indicated time points were immunoblotted with phospho-GSK3 antibody (cell signaling). Total GSK3 and CDK2 were detected using respective antibodies.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Akt-mediated CDK2 phosphorylation regulates its cellular localization but is dispensable for basal CDK2 kinase activity. (A) 293T cells were transiently transfected with HA-tagged CDK2, co-transfected with Akt-CA or dominant-negative Akt (Akt-DN), or with vectors coding for CDK2-mutants. CDK2 was immunoprecipitated using anti-HA antibody and the kinase activity was assessed using histone H1 as substrate. The phosphorylated histone H1 was detected by immunoblotting with phospho-H1 antibody. The expression of wild-type CDK2 and mutant proteins was detected by anti-HA antibody. (B) The wild-type CDK2 or CDK2-T39A coding vectors were transiently transfected with either vector alone, constitutively-active Akt (Akt-CA) or Akt-DN, and CDK2 was immunoprecipitated using anti-HA antibody. The presence of cyclin A and cyclin E co-immunoprecipitated with CDK2 was detected by immunoblotting with their respective antibodies. (C) HA-tagged wild-type CDK2, CDK2 T39E or CDK2 T39A mutants were trasfected into 293T cells. The association of cyclin A and cyclin E with CDK2 was detected by western blotting after immunoprecipitating CDK2 with anti-HA antibodies. (D) Wild-type CDK2, CDK2-T39A or CDK2-T39E and Akt-CA or Akt-DN vectors were co-transfected into 293T cells. After 24 hours, the localization of CDK2 was detected by confocal microscopy after immunostaining with anti-CDK2 antibodies followed by Cy3-conjugated secondary antibody. DAPI was used to counterstain nuclei. Corresponding micrographs with nuclear and CDK2 staining were overlaid to visualize changes in CDK2 cellular localization. (E) HA-tagged wild-type CDK2 or T39 mutants were co-transfected into 293T cells with either Akt-CA or Akt-DN vectors and the association of 14-3-3 with CDK2 was determined by immunoblotting with 14-3-3 antibodies after immunoprecipitating with either HA antibody or IgG control antibody. FOXO3A interaction with 14-3-3 was used as a positive control. The input controls for CDK2, 14-3-3 and FOXO3A were shown by immunoblotting the total cell lysates with the respective antibodies.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Akt-regulated CDK2 relocalization is required for cell cycle progression. (A) Murine 3T3 fibroblasts were arrested in G0, G1, S, G2 and M phases of the cell cycle, as described in the Materials and Methods section, and the phosphorylated Akt, total Akt, phosphorylated CDK2 and total CDK2 were detected by immunoblotting with their respective antibodies. The CDK2 kinase activity is shown at different phases of the cell cycle using histone H1 as substrate. (B) The 3T3 fibroblasts were arrested in different phases of the cell cycle and the localization of Akt was detected by using confocal microscopy after immunostaining with Akt antibody followed by Cy3-conjugated secondary antibody (upper panel). In parallel samples, the localization of CDK2 was detected by using CDK2 antibody followed by Cy-3 conjugated secondary antibody (lower panel). (C) 3T3 fibroblasts were serum-starved for 36 hours and then serum was added to allow the cells to progress sequentially through different phases of the cell cycle. At the indicated time points, cells were sampled, the phosphorylated Akt was detected by immunoblotting and the phosphorylated CDK2 was detected by immunoprecipitation of CDK2 followed by immunoblotting with anti-phospho-substrate-Akt antibody. The stages of the cell cycle were confirmed by `FITC-BrdU Flow Kit' staining, followed by flow cytometry at the indicated time points; each cell cycle phase is denoted below the lower immunoblotting panel. (D) The cells grown on coverslips were synchronically put through the cell cycle as described above; they were then collected at indicated time points, and the localization of CDK2 was detected by confocal microscopy preceded by immunostaining with anti-CDK2 antibodies followed by a Cy-3 conjugated secondary antibody. DAPI is used to counterstain the nucleus. (E) The nuclear (N) and cytoplasmic (C) fractions from 3T3 fibroblasts synchronized in either S phase or in G2 phase were isolated and the CDK2 kinase activity measured using histone H1 as substrate after immunoprecipitating with CDK2 antibodies. The presence of CDK2 and cyclin A were also detected in both the fractions using their respective antibodies. (F) 3T3 fibroblasts were transfected with Akt-CA, Akt-DN, wild-type CDK2, or with CDK2-T39A-expressing vectors. Twenty-four hours later the percentage of G1-S and G2-M population was gated and analyzed by flow cytometry following propidium iodide staining. (G) The cells were transfected with wild-type CDK2-, CDK2-T39A-, CDK2-T160A- or CDK2-T39A/T160A-expressing vectors, then arrested in S phase using 2 µg/ml aphidicolin for 20 hours and released from S phase for 3 hours. The percentage of cells moving from S to G2 phase was assessed by BrdU labeling, as described in the Materials and Methods section, followed by flow cytometric analysis.

View larger version (36K): [in this window] [in a new window]   Fig. 4. Methotrexate and docetaxel activate Akt and induce constitutive nuclear Akt translocation. (A) MCF7 cells were treated with methotrexate (10 µM) (upper panel) or docetaxel (0.05 µM) (lower panel) for the indicated time, then phosphorylated Akt and total Akt were detected by immunoblotting with the respective antibodies. (B) The localization of Akt in the nucleus in either control untreated cells or the drug-treated cells (as indicated) was assessed by immunostaining followed by confocal microscopy. Six hours after docetaxel treatment, or 3 hours after methotrexate treatment, cells were fixed, and the immunostaining was performed with anti-Akt antibodies followed by secondary Cy-3 conjugated antibody. DAPI was used as nuclear counterstain. The superimposed micrographs of Akt- and nuclear staining visualize changes in the cellular localization of Akt. (C) 293T cells were left untreated, treated with methotrexate or docetaxel alone, or in combination with either adenoviral vector coding for nuclear Akt, or with wortmannin. Twenty-four hours later, the cells were stained using the Nicoletti method, and the percentage of cell death was assessed by flow cytometry. (D) 293T and PC-3 cells were transiently transfected with Akt-CA coding vector and the cell death was then assayed by flow cytometry as described above. Comparison was made between the Akt-CA-transfected cells and the non-transfected control cells. The amount of cell death also confirmed using MTT assay in an independent experiment (data not shown).

View larger version (30K): [in this window] [in a new window]   Fig. 5. CDK2 phosphorylation and cytoplasmic distribution is required for methotrexate- and docetaxel-induced cell death. (A) 293T cells were left untreated, treated with methotrexate alone (upper panel), docetaxel alone (lower panel) or given a 15 minute pretreatment with wortmannin. CDK2 was immunoprecipitated with anti-CDK2 antibody, and the phosphorylation of CDK2 was detected by immunoblotting with a phospho-Akt substrate-specific antibody. Total CDK2 was detected by a CDK2-specific antibody. (B) 293T cells were left untreated or treated with methotrexate or docetaxel, and the localization of CDK2 was detected by confocal microscopy after immunostaining with anti-CDK2 antibody followed by a Cy3-conjugated secondary antibody. (C) The cells were left untreated, treated with methotrexate alone or docetaxel alone, or the drugs were added 15 minutes after pretreatment with roscovatine. Twenty-four hours later, the percentage of cell death was assayed by flow cytometry (Nicoletti method). (D) Immortalized CDK2 wild-type (CDK2 WT), CDK2-deficient fibroblasts (CDK2–/–), CDK2 wild-type reconstituted CDK2–/– fibroblasts and CDK2 T39A mutant reconstituted CDK2–/– cells were treated with either methotrexate or docetaxel, and 24 hours later the percentage of cell death was assayed by flow cytometry (propidium iodide staining).

View larger version (12K): [in this window] [in a new window]   Fig. 6. Model for the dual role of the Akt/CDK2 pathway in cell cycle progression and cell death. In the presence of survival signal, PI3K activates Akt and the transiently activated Akt translocates to the nucleus only during late S and G2 phases and shuttles back to the cytoplasm. The transient nuclear Akt phosphorylates CDK2 in the nucleus at late S and G2 phases and leads to the temporary CDK2 relocation to the cytoplasm. When Akt is shuttled back to the cytoplasm, CDK2 rapidly translocates to the nucleus and helps in the cell cycle progression. But in the presence of selected apoptotic signals, PI3K constitutively activates Akt and leads to the constitutive nuclear Akt accumulation, where it phosphorylates CDK2. The constitutively phosphorylated CDK2 is sequestered in the cytoplasm and directed to the different cytoplasmic substrates, which ultimately leads to cell death.