Akt phosphorylates CDK2 both in vitro and in vivo
To determine whether Akt phosphorylates CDK2 in vitro, we used a recombinant bacteria-expressed GST-CDK2 protein as a substrate in the kinase assay. Recombinant active Akt, but not its inactive form, is able to phosphorylate CDK2 (). Further, the measurement of stoichiometry of CDK2 phosphorylation using γ-32P ATP revealed a high level of phosphorylation in the presence of active Akt but not catalytically inactive Akt (supplementary material Fig. S1A). These results were also confirmed by an in vitro kinase assay using a constitutively active form of Akt (Akt-CA), or a catalytically inactive Akt (Akt-K179M) immunoprecipitated from 293T cells (, lower panel). A preliminary analysis of CDK2 protein for the putative Akt phosphorylation sites revealed two partial Akt consensus sites at threonine 39 (T39) and T160 residues. To determine if these residues are the possible Akt phosphorylation sites, the mutants replaced with a non-phosphorylatable alanine instead of the T39, T160 or both residues were used in the kinase assay. Both, active recombinant Akt or immunoprecipitated Akt phosphorylated CDK2-T160A mutant to the same level as the wild-type protein. No phosphorylation is seen with T39A and T39A/T160A mutant ( and supplementary material Fig. S1B), indicating that the T39 residue is the only Akt target on CDK2.
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 (more ...)
To determine whether Akt phosphorylates CDK2 in vivo, within cultured cells, we constructed HA-tagged CDK2 wild type, T39A and T160A vectors and transiently transfected them into 293T cells. In the presence of serum, the phosphorylation of CDK2 revealed that both CDK2 and CDK2 T160A mutants were phosphorylated, but T39A and T39A/T160A mutants were not phosphorylated in vivo (). This was checked using anti-phospho-Akt substrate antibody after immunoprecipitation of CDK2. Further, to determine if Akt is the kinase phosphorylating CDK2 at the T39 site in vivo, either CDK2 wild type or CDK2 T39A vector was transiently expressed in transformed mouse 3T3 fibroblasts. The serum starvation of cells for 30 hours, which inactivated Akt, completely abolished the phosphorylation of CDK2. The activation of Akt, stimulated by either EGF or by reintroduction of 10% serum to the cells restored the CDK2 phosphorylation, which is inhibited in the presence of wortmannin, a selective PI3K/Akt pathway inhibitor ( and supplementary material Fig. S1B). As expected, no Akt-mediated phosphorylation was observed with the CDK2 T39A mutant. This indicates that CDK2 phosphorylation at T39 residue occurs in vivo and is mediated by the PI3K/Akt pathway. In addition, we also tested for the Akt-mediated T39 phosphorylation of endogenous CDK2. Addition of EGF or serum enhances the T39 phosphorylation of endogenous CDK2, which is inhibited by wortmannin (). To further support the role of Akt as a direct kinase phosphorylating CDK2 at T39 residue, we used an Akt-specific inhibitor (Barnett et al., 2005
) in the presence of EGF. Pretreatment of cells with an Akt-specific inhibitor abolished the EGF-induced CDK2 phosphorylation (), but no effect was seen with pretreatment with rapamycin, an inhibitor of S6 kinase (Akt downstream molecule). Further, we performed a timecourse for T39 phosphorylation of CDK2 and GSK-3β, a known substrate for Akt. The phosphorylation of GSK-3 was spontaneous and was seen as early as 15 minutes after EGF stimulation, whereas CDK2 phosphorylation occurred around 6 hours after EGF stimulation (), indicating that CDK2 phosphorylation by Akt is a late event and differs from other early Akt substrate phosphorylations.
Akt-mediated CDK2 phosphorylation regulates its cellular localization
To test the functional significance of CDK2 phosphorylation by Akt, we checked the CDK2 kinase activity by using histone H1 as its substrate. The co-transfection of Akt CA, a constitutively active form of Akt along with CDK2 wild type, significantly enhanced the CDK2 kinase activity (). However, the expression of dominant-negative Akt restored the kinase activity to normal levels, similar to transfection with an empty vector. The expression of active Akt has no effect on total CDK2 protein levels. Interestingly, the kinase activity assessment with the wild-type CDK2 along with the non-phosphorylatable T39A mutant and T39E phosphomimetic mutants revealed that both the mutants were able to phosphorylate histone H1 as effectively as the wild-type protein (). This indicates that although Akt enhances the CDK2 kinase activity, the T39 phosphorylation has no significant role during basal CDK2 kinase activity. The increase in CDK2 activity during the presence of active Akt might be an indirect effect, as Akt is also known to target other regulators of CDK2 such as p27kip1
(Liang and Slingerland, 2003
). As expected, the other CDK2 mutants lacking the phosphorylation site T160 have no kinase activity. Furthermore, to determine the effect of T39 phosphorylation on CDK2, we analyzed the association of cyclin A and cyclin E with CDK2 under different conditions. The expression of active Akt significantly enhanced the binding of cyclin A to CDK2, which was reduced to normal levels with the expression of dominant-negative Akt (). By contrast, the activation of Akt had no effect on the binding of cyclin E to CDK2. Interestingly, although the CDK2 T39E phosphomimetic mutant showed slightly enhanced cyclin A binding compared with wild-type CDK2 (), the T39A mutation on CDK2 again had no effect on the binding of either cyclin A or cyclin E, thus indicating that T39 phosphorylation might not be required for cyclin A or cyclin E binding.
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 (more ...)
We next tested whether the activation of Akt and CDK2 T39 phosphorylation affects the intracellular localization of CDK2. As shown in , the wild-type CDK2 was predominantly localized in the cytoplasm in the presence of Akt-CA, but was relocated to the nucleus when dominant-negative Akt was co-expressed. The mutation of CDK2 T39 to a non-phosphorylatable alanine abolished the cytoplasmic localization even in the presence of Akt-CA. CDK2 T39A was predominantly nuclear in the presence of either Akt-CA or Akt-DN. The CDK2 T39E mutant, which mimics the phosphorylation status, was predominantly localized in the cytoplasm irrespective of the activation status of the Akt pathway. As the expression of different forms of Akt might perturb the cell cycle distribution and thus might affect the localization of CDK2 mutants, we further confirmed that the CDK2 localization is dependent on Akt-mediated T39 phosphorylation by checking the localization of T39A mutant in cells synchronized in different phases of the cell cycle. The CDK2 T39A mutant is nuclear irrespective of the cell cycle phase (supplementary material Fig. S2). Thus, these results indicate that the T39 site is important for the regulation of CDK2 intracellular localization and the phosphorylation of this residue by Akt results in CDK2 cytoplasmic localization. The phosphorylation-dependent cytoplasmic localization of CDK2 is similar to the effect of Akt-mediated phosphorylation of its substrates Foxo3a and Bad. The Akt-dependent phosphorylation of Foxo3a and Bad leads to their cytoplasmic export from the nucleus via 14-3-3 interactions. Thus, we tested the possibility of phosphorylation-dependent interaction of CDK2 with 14-3-3 by transfecting the cells with either wild-type CDK2 or T39 mutants. Both wild-type CDK2 and T39E phosphomimetic mutants, but not the T39A mutant, interact with 14-3-3 (), suggesting that Akt-dependent phosphorylation of CDK2 leads to its nuclear exclusion via 14-3-3 binding.
Akt-regulated nucleo-cytoplasmic CDK2-relocation is required for cell cycle progression
CDK2 is a nuclear protein and is required for cell cycle progression at the G1-S phase, but although Akt is known to regulate the cell cycle at various stages, it is primarily a cytoplasmic protein. We thus tested whether Akt has differential activation status and further differential localization during various phases of the cell cycle, so that it can act on distinct substrates either in the cytosol or in the nucleus. The murine 3T3 fibroblasts were arrested in different phases of the cell cycle as described in the Materials and Methods section, and tested for the activation of Akt by immunoblotting with anti-phospho-Akt antibodies (, upper panel). Akt, which was inactive in G0 phase, rapidly became activated in G1 phase. Akt activity was downregulated in S-phase arrested cells, but peaked again in cells arrested in G2 phase of the cell cycle. The M-phase arrested cells had a lower Akt activity. The arrest in different phases of the cell cycle had no effect on the total Akt levels. Furthermore, we tested the Akt-mediated phosphorylation of CDK2 at T39 by immunoprecipitating CDK2 and blotting with anti-phospho-Akt substrate antibody at different phases of the cell cycle. CDK2 was heavily phosphorylated at T39 residue in G2-phase arrested cells, whereas weaker phosphorylation was seen during S phase of the cell cycle (, lower panel). No phosphorylation was seen during other phases.
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, (more ...)
In addition to the differential Akt activation status, the localization of Akt in cells arrested in different phases of the cell cycle revealed Akt either in the cytosol or nucleus, depending on the phase of the cell cycle. Akt was mainly cytoplasmic during G0 and G1 phases, whereas during G2 phase it was exclusively seen in the nucleus. During S phase, Akt was dispersed both in the cytoplasm and the nucleus (, upper panel). As Akt regulates the cellular localization of CDK2, in a separate experiment we also tested the localization of CDK2 in cells arrested in different phases of the cell cycle. CDK2 was mainly nuclear in G1 phase, whereas it was exclusively cytoplasmic during G2 phase of the cell cycle. During G0 and S phases, CDK2 was found both in the nucleus and the cytoplasm (, lower panel). This correlates with the Akt nuclear localization at S and G2 phases of the cell cycle. These results indicate that activated Akt may translocate to the nucleus during the S phase, phosphorylate CDK2 and promote cytoplasmic CDK2 localization during late S and G2 phases of the cell cycle. To provide further conclusive evidence on the role of phosphorylation on the cellular localization of CDK2, we performed kinetics studies of Akt activation and CDK2 phosphorylation as the cells progressed through the cell cycle. Murine 3T3 fibroblasts arrested in G0 phase by 30-hour serum starvation, were released from G0, and Akt and CDK2 phosphorylation was assessed at different time points as the cells progressed through the cell cycle. Akt was activated immediately after G0 release as the cells entered G1, but was only activated transiently. As the cells progressed from G1 to S phase, the Akt was deactivated. Once S phase was initiated, as the cells prepared for S-to-G2 progression, a second peak of Akt activation was seen around 16–18 hours after G0 release in these cells (). By contrast, CDK2 activation was not seen during G1 or early S phases but only during late S and G2 phases, as it was evident only after 16–18 hours of G0 release in correlation with a second peak of Akt activation (, lower panel). As the cells went from G2 to M phase, both Akt and CDK2 phosphorylation was decreased (data not shown). The level of total Akt or total CDK2 remained constant for the duration of the experiment. CDK2 was mainly nuclear during G1 and early S phases, but upon Akt-mediated phosphorylation translocated to the cytoplasm during last S phase and G2 phase around 16–18 hours after G0 release () and re-localized to the nucleus during late phases of the cell cycle. To test further whether CDK2 alone or an active cyclin-Abound CDK2 complex is exported to the cytoplasm during G2 phase, we isolated the nuclear and cytoplasmic fractions from the cells synchronized in either early S phase or G2 phase and assessed for the localization of CDK2, cyclin A and their activity. As shown in , the nuclear S-phase-active cyclin-A bound CDK2 was exported to the cytoplasm during G2 phase in an active cyclin-Abound complex.
To further determine the effect of CDK2 phosphorylation on the progression of the cell cycle, we assessed both the G1-S and G2-M population under different conditions. The overexpression of a non-phosphorylatable mutant of CDK2, CDK2-T39A, or inactivation of Akt by overexpression of Akt-DN, resulted in an increase in G1-S cell population and a significant decrease of cells in G2-M phase of the cell cycle. However, the transfection of Akt-CA or wild-type CDK2 increased the G2-M population (). This indicates that Akt activation and CDK2 phosphorylation are important for S to G2-M progression. To determine the role of CDK2 T39 phosphorylation on the progression through the S- and G2 phases of the cell cycle, we examined the progression of S to G2 phase in the presence of both phosphorylation active and inactive CDK2 mutants. The 3T3 fibroblasts arrested in S phase were released and the percentage of cells entering G2 was assessed. After 3 hours of S-phase release, the presence of wild-type CDK2 and the CDK2-T160A mutant had no significant effect on the percentage of cells entering G2 phase compared to controls, whereas the CDK2-T39A transfection resulted in the reduction of cells entering from S to G2 (). Although the CDK2-T39A/T160A has a non-phosphorylatable T39 site, it has no effect on the percentage of G2 cells, as it is inactive as CDK2 kinase. Thus, these results indicate that Akt-mediated transient phosphorylation of CDK2 at the T39 site and the resulting CDK2 cytoplasmic localization occurs in late S and G2 phases and is absolutely necessary for the cells to progress from S to G2-M phases.
Selected anticancer drugs constitutively activate the PI3K/Akt pathway
Although Akt and CDK2 are required for cell cycle progression, they have also been assigned an important role in regulating apoptosis, either positively or negatively upon different apoptotic stimuli (Deb-Basu et al., 2006
; Shi et al., 1996
). We thus tested whether the above-described PI3K/Akt-CDK2 pathway has a role in apoptosis induced by selected anticancer drugs. We screened for the activation of the PI3K-Akt pathway in MCF7 cells using several known anticancer drugs and found that methotrexate (, upper panel), docetaxel (, lower panel) and doxorubicin (data not shown) were able to constitutively activate Akt. The activation of Akt by methotrexate occurred very rapidly after around 30 minutes, and the kinase remained constitutively active even upon 12 hours of treatment. Docetaxel-induced Akt activation occurred late, after around 6 hours of treatment, and it remained constitutively active even after 30 hours (data not shown). We further tested the localization of Akt in the presence of methotrexate and docetaxel. Akt is mainly cytoplasmic (, upper panel), but the treatment of MCF7 cells with docetaxel for 6 hours resulted in the nuclear localization of Akt. Also, the treatment of cells with methotrexate for 3 hours induced Akt nuclear localization (). The nuclear localization of Akt is dependent on the upstream activation of PI3K, as the inhibition of PI3K activity by wortmannin abrogated the nuclear localization of Akt even in the presence of the chemotherapeutics. To further demonstrate the importance of nuclear Akt during cell death induced by these drugs, we compared the cells treated with the anticancer drugs alone to those that were transduced with adenovirus encoding nuclear Akt. The transduction of MCF7 cells with nuclear Akt enhanced the cell death induced by both methotrexate and docetaxel, and, interestingly, the cell death was partly inhibited by pretreatment of cells with wortmannin (), indicating that the PI3K/Akt activation and the nuclear Akt are important for cell death induced by these drugs. We also observed a similar trend with another chemotherapeutic drug, doxorubicin (data not shown). In a control experiment we transduced both the 293 and PC-3 cells with a constitutively active Akt, and the cell death was assayed 48 hours after transfection. The overexpression of Akt-CA alone induced up to 10–15% of cell death, compared with 2–5% in the control cells (), preceded by a G2-M cell cycle arrest (, and data not shown).
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 (more ...)
CDK2 is required for methotrexate- and docetaxel-induced cell death
As methotrexate and docetaxel activated Akt and induced nuclear Akt localization constitutively, we tested whether the Akt/CDK2 pathway is involved in the cell death induced by these drugs. The CDK2 phosphorylation detected by phospho-specific antibody after CDK2 immunoprecipitation revealed that CDK2 was phosphorylated at the T39 site upon treatment of methotrexate (, upper panel) for 6 hours and docetaxel (, lower panel) for 9 hours, respectively. The CDK2 phosphorylation induced by methotrexate and docetaxel is dependent on Akt activation, as pretreatment of the cells with wortmannin severely reduced the CDK2 phosphorylation even in the presence of the anticancer drugs. The CDK2 phosphorylation at T39 upon methotrexate and docetaxel treatment results in the constitutive translocation of CDK2 to the cytoplasm (). To determine whether the CDK2 activity in the cytoplasm is required for cell death induced by these drugs, we used two different approaches. First, we inhibited the CDK2 activity using a pharmacologic CDK2 inhibitor, roscovatine, and treated the cells with anticancer drugs. Roscovatine treatment significantly inhibited cell death induced by both methotrexate and docetaxel (). In the second approach, we compared the cell death induced by these anticancer drugs in immortalized CDK2 wild-type fibroblasts and the CDK2-deficient fibroblasts. Both methotrexate and docetaxel were able to induce cell death efficiently in wild-type fibroblasts, whereas the cell death is significantly inhibited in CDK2 knockout cells (). Reconstitution of CDK2-deficient fibroblasts with CDK2 wild-type vector but not T39A mutant restores their sensitivity to cell death induced by methotrexate and docetaxel. These results indicate that Akt-mediated CDK2 phosphorylation at T39 contributes to the cell death induced by methotrexate and docetaxel.
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 (more ...)