The data we present here clearly demonstrate that Akt1 and -2 are functionally nonredundant and play opposing roles in the modulation of cell proliferation in nontransformed cells. Potentially nonredundant function was already implied from the in vivo knockout mouse models, where only Akt2 appeared necessary in glucose homeostasis whereas Akt1 knockout principally affected organ growth and overall mouse size (9
). However, opposing actions of Akt1 and -2 in the control of cell proliferation were not previously identified. Although numerous in vitro studies have concentrated on PKB/Akt using transformed cell lines and Akt1, very few reports have analyzed specific functional activities of Akt1 or -2 and none had concluded in differences in the ability to promote cell growth between Akt1 and -2 (reviewed in references 6
). Indeed, a recent study analyzing isoform-specific functions in IGF-R1 overexpressing epithelial cells reported that Akt1 knockout results in an epithelial-to-mesenchymal transition with enhanced ERK activation, a phenotype reversed by cosilencing Akt2 without any significant effect of Akt2 knockout alone (22
). In addition, use of constitutively active membrane-localized forms of Akt in some of these studies likely gave rise to misleading results, since the predominant endogenous localization of Akt2 is clearly not membrane bound but nuclear. We also show for the first time that Akt1 and -2 differentially modulate the cell cycle inhibitor p21 and identify a specific domain of interaction between Akt2 and p21.
The small cyclin-dependent kinase inhibitor p21 is one of a family of cdk modulators which play an active role in regulating cell cycle transitions through interactions with cdk2, -4, and -6 (46
) and is an active inhibitor of cdk2/cyclin E and A. The inhibitory activity of p21 is reduced by its phosphorylation, which inhibits interactions with cdk2, the cyclins, or PCNA (2
). Previous reports have detailed the phosphorylation of p21 by PKB/Akt, without specifying the isoform (31
). Phosphorylation of p21-Thr145 both promotes the cytoplasmic delocalization of p21 and prevents interaction with PCNA (33
). We have shown here that the nuclear import/export process of p21 is modulated differentially by Akt1 and Akt2: in cells in which Akt1 is silenced by siRNA, p21 is localized in the nucleus. Considering its small size, p21 would normally be free to diffuse in and out of the nucleus, suggesting that in the absence of Akt1, p21 stays in the nuclear compartment in part through association with Akt2, and the different intracellular localizations of Akt1 and -2 would support such a role.Moreover, cells overexpressing Akt2 (which associates specifically with p21 in vivo and in vitro) show increased nuclear localization of p21. Although other proteins bind p21 and may retain it in the nucleus (16
), we believe that the interaction with Akt2 is an important component of this system due to the stability of the complex between p21 and Akt2 and the decreased level of p21 we immunoprecipitated from cells knocked down for Akt2 in comparison to control and cells knocked down for Akt1 (Fig. ). Detailed in vitro analysis of the interaction between Akt2 and p21 showed that Akt2 binds to p21 through one of the two cyclin binding regions of p21 previously mapped to amino acids 150 to 158 (8
). This region is very close to the site phosphorylated by Akt1 (T145), and prior phosphorylation of this site by Akt1 also prevented the interaction with Akt2, whereas it did not abolish cyclin A binding, probably due to a second cyclin A binding domain on p21 (8
). Interestingly, the T145 site could not be phosphorylated by Akt1 if it was already occupied by Akt2, whereas it could be partially phosphorylated if p21 was previously incubated with cyclin A (Fig. ). This would imply that the interaction site for Akt2 with p21 more effectively covers T145 than the cyclin A site, which has been defined as slightly more C terminal of T145 (150 to 158) (8
). This observation that the Akt2-bound form of the p21 complex is more stable than with cyclin A is consistent with the potential role for Akt2 in cell cycle exit, since it would provide a nuclear stabilized form of p21 which still associates with cdk2 (binding through the N terminal of p21 [17
]), thus hindering normal proliferative growth. Figure shows a schematic interpretation of our data concerning Akt1/2 in the modulation of proliferation via p21 and its role in regulating cell cycle progression through nuclear accumulation.
An important physiological implication of our observations of opposing roles for Akt1 and Akt2 in proliferation concerns the therapeutic development of anticancer agents targeting Akt/PKB (20
). Deregulation or overexpression of Akt is frequently associated with malignant transformation (35
). There has been abundant evidence that Akt/PKB is involved in tumorigenesis, and inactivation of Akt/PKB has been proposed as an effective means of simultaneously inhibiting cancer cell proliferation and resistance to apoptosis (37
). Among the targeted effects of Akt activation is the cytoplasmic relocalization and inactivation of p21 (26
). This cytoplasmic relocalization of the cdk inhibitor p21 has been implicated in both proliferative and anti-apoptotic effects (4
; see the review by Blagosklonny [5
]). It is therefore highly relevant to find p21 as a key effector of the differential roles of Akt1 and -2 in cell proliferation. Our data show that Akt1 levels are higher in human tumors or transformed cells than in their normal counterpart, while p21 levels show the opposite. This implies that isoform-specific inhibitors of Akt1 should be developed for use in cancer therapy. In support of such isoform targeting, the angiogenic factor vascular endothelial growth factor is known to activate Akt/PKB, and a recent finding from knockout mice suggests that Akt1/Akt1 is the primary effector of vascular endothelial growth factor in postnatal angiogenesis (1
). In this respect some specific inhibitors of Akt isoforms have recently been reported (14
), and it will be interesting to determine if they also bring about the differential effects on cell proliferation which we report here.
Another important question raised by our observations concerns the physiological relevance of these differences in Akt isoform activity in normal cells and tissues. To date, the large majority of Akt studies have concentrated on Akt1 and involved overexpression in transformed cells. As we have previously observed by microinjection of isoform-specific antibodies (41
) and confirmed here, in siAkt-injected cells, inhibition of Akt2 leads to increased myoblast proliferation and inhibition of their differentiation (Fig. ). These data are complementary to our previous observations that in postmitotic cells which no longer divide (41
), Akt2 is more highly expressed than Akt1. When analyzed at the protein level, we have observed that Akt1 is the principle isoform detected by Western blotting in proliferating cells and tissues, whereas in postmitotic cells and tissue (muscle and pancreatic islets), Akt2 becomes the more-abundant isoform (A. Fernandez and N.J.C. Lamb, unpublished observations). These differences in protein levels between mitotically active and postmitotic differentiated cells and tissue strongly support the role of Akt2 in cell cycle exit, an essential initial event in differentiation.
Taken together, we show here for the first time contrasting functions of the two Akt/PKB isoforms in cell cycle progression and identify a biochemical basis for a differential interaction of Akt1 and -2 with the cyclin kinase inhibitor p21Cip1.