The PI3K-Akt signaling pathway controls many cellular functions, such as cell survival, cell cycle progression, cell proliferation, and glucose metabolism. A large number of cancers were reported to aberrantly activate this pathway, resulting in cell proliferation. Although proliferation is strictly regulated by the cell cycle, the aberrant activation of the PI3K-Akt pathway breaks this regulation in proliferating cancer cells. For example, the PI3K-Akt signaling pathway was reported to promote G1
progression through activation of the cyclin D1/Cdk4 complex (4
). Cyclin D1 phosphorylation at Thr286
by glycogen synthase kinase 3 (GSK-3) is associated with the translocation of cyclin D1 from the nucleus to the cytoplasm (4
). Rapid degradation induced by GSK-3 is inhibited by activation of the PI3K-Akt pathway because Akt directly phosphorylates and inactivates GSK-3 (4
). By contrast, Akt is known to inactivate the Cdk inhibitors p21Waf1/Cip1
). Akt phosphorylates p21Waf1/Cip1
, resulting in cytoplasmic translocation and suppression of the functions (4
). These results indicate that the PI3K-Akt signaling pathway promotes cell cycle progression in G1
phase by activating positive regulators and inactivating negative ones. However, only G1
progression by the PI3K-Akt signaling pathway is not enough to account for cell proliferation because the cell cycle is strictly regulated at each checkpoint.
Several previous studies suggested that some anticancer drugs inhibit the kinase activity of Akt (15
). The 90-kDa heat shock protein Hsp90 is reported to control the PDK1-Akt signaling pathway because it binds to PDK1 through its kinase domain and protects against the proteasome-dependent degradation of PDK1 (15
). 17-AAG, which is a derivative of geldanamycin, was reported as an Hsp90 inhibitor that protects against Hsp90 binding to PDK1, resulting in inactivation of the PDK1-Akt signaling pathway (3
). UCN-01 (7-hydroxystaurosporine), a drug now in clinical trials and with a unique fingerprint pattern, is also known to be a direct inhibitor of PDK1 because UCN-01 binds to PDK1 in an ATP-binding motif and inhibits its kinase activity, resulting in turnoff of survival signaling and apoptosis induction (36
). Furthermore, cisplatin, etoposide, adriamycin, and camptothecin were reported to down-regulate the PI3K-Akt pathway in some cell lines (30
Although it is clear that some anticancer drugs induce S or G2
/M arrest (1
), the relationship between the arrest and down-regulation of Akt kinase has not been clarified. Thus, we first elucidated the relationship between anticancer drug-induced G2
/M arrest and Akt inactivation in 293T and HeLa cells. We found that VP-16, cDDP, and 17-AAG could induce S or G2
/M arrest accompanying down-regulation of Akt (Fig. ). Simultaneously, inhibitory phosphorylation of Cdc2 (Tyr15
) was observed and Cdc2 kinase activities were decreased under these conditions, and this phosphorylation depended on WEE1Hu activation (Fig. ). Moreover, the PI3K inhibitor LY294002 also increased the population in the G2
/M phase and the WEE1Hu-dependent inhibitory phosphorylation of Cdc2 (Fig. ). These findings indicate the possibility that Akt negatively regulates WEE1Hu in a phosphorylation-dependent manner.
Because WEE1Hu has some putative Akt consensus phosphorylation sequences, we examined the phosphorylation of WEE1Hu by Akt. Actually, Akt directly bound to and phosphorylated WEE1Hu at the Ser642
residue in the COOH terminus during the S to G2
phase (Fig. and ). Although Thr161
on Cdc2, which is known to be essential for Cdc2 kinase activity (7
), has homology to a putative Akt consensus sequence, Akt could not phosphorylate Cdc2 at the site in vitro and in vivo (data not shown). Moreover, Cdc25C, which also has a putative Akt consensus sequence, could not be phosphorylated by Akt in vitro (data not shown). These results indicate that Akt regulates the cell cycle at the G2
/M transition, mainly by phosphorylating WEE1Hu.
With DNA damage (e.g., radiation or UV irradiation), Chk1 is activated and induces G2
/M arrest through phosphorylation of Cdc25C and WEE1Hu. Cdc25C, which is a phosphatase for the phosphorylated Tyr15
of Cdc2 and which activates it, is phosphorylated on Ser216
by Chk1 and the phosphorylated form of Cdc25C binds to 14-3-3 and eliminates the functions through translocation to cytoplasm (25
). Moreover, Chk1 may also phosphorylate WEE1Hu. Although there is no report of this in humans, Lee et al. found that in frog egg extract, Chk1 phosphorylated Xenopus
Wee1 on Ser549
, corresponding to WEE1Hu on Ser642
of WEE1Hu is also conserved in mouse and rat Wee1 homologues. Phosphorylated Ser642
is reported to increase the stability of WEE1Hu in the nucleus by binding to 14-3-3β or -σ (34
). As a result, Cdc2 is continuously phosphorylated at Tyr15
and the cell cycle arrests at the G2
Since Akt also phosphorylated WEE1Hu on Ser642 (Fig. ) and overexpression of wild-type Akt decreased the phosphorylation of Tyr15 of Cdc2 (Fig. ), we questioned how WEE1Hu kinase activity was regulated through phosphorylated Ser642. To answer this question, we looked at whether 14-3-3 proteins bound to WEE1Hu through the phosphorylation of Ser642 by Akt and changed the localization of WEE1Hu. Although exogenous 14-3-3β and -θ could bind to WEE1Hu through phosphorylated Ser642 (Fig. ), only 14-3-3θ could bind to it at the endogenous expression level of 14-3-3 because the endogenous 14-3-3β and -σ expression levels were down-regulated during late S to G2 phase (Fig. and data not shown). 14-3-3θ binding promoted the translocation of WEE1Hu from the nucleus to the cytoplasm (Fig. ) and abolished the WEE1Hu-induced G2/M arrest (Fig. ). Thus, Akt promotes cell cycle progression at the G2/M transition through Akt-dependent WEE1Hu phosphorylation on Ser642 and its binding to 14-3-3θ. On the other hand, the Akt-dependent phosphorylation state of the point-mutated form of Thr257 (T257A) on WEE1Hu was slightly decreased compared with wild-type WEE1Hu, suggesting that Thr257 on WEE1Hu may also be a site phosphorylated by Akt (Fig. ). However, the T257A mutation had no effect on 14-3-3 binding and translocation of WEE1Hu (data not shown). Thus, we need to examine the effects of phosphorylation on Thr257 by Akt in the future.
Although 14-3-3β and -σ were reported to bind to WEE1Hu through phosphorylated Ser642
), we could not observe that they bound to WEE1Hu at endogenous expression levels (Fig. ). These different results could be explained by considering two points. First, many antibodies to 14-3-3 cross-react with other 14-3-3 isotypes. Second, overexpression of 14-3-3 proteins, except 14-3-3θ, was not fully down-regulated through the cell cycle progression, resulting in cell cycle-independent binding to WEE1Hu. We also observed exogenous 14-3-3β binding to WEE1Hu (Fig. ). At endogenous 14-3-3 levels, only 14-3-3θ formed a complex with WEE1Hu. Moreover, the expression levels of the 14-3-3 proteins, except 14-3-3θ, were changed through the cell cycle progression (Fig. ). Actually, the expression levels of 14-3-3β and -σ were down-regulated during late S to G2
phase (Fig. and data not shown). Thus, under physiological conditions, the localization of Ser642
-phosphorylated WEE1Hu may only be regulated by binding to 14-3-3θ.
In summary, we discovered that Akt-mediated WEE1Hu phosphorylation on Ser642
induced WEE1Hu binding to 14-3-3θ and cytoplasmic localization upon growth factor stimulation. Thus, Akt eliminates WEE1Hu functions through translocation to the cytoplasm by 14-3-3θ. Okumura et al. reported that Akt-dependent phosphorylation of Myt1, another member of the Wee1 family, led to meiotic G2
/M transition in starfish oocytes (31
), and now we identify WEE1Hu as a new target of Akt and demonstrate that Akt functions as an M-phase initiator in mammalian cells.