The data presented above provide evidence for a novel mechanism by which Akt1-mediated phosphorylation of Skp2 at Ser72 protects Skp2 from Cdh1-mediated destruction through disruption of the interaction between Skp2 and its E3 ligase Cdh1, as well as inducing Skp2 cytoplasmic translocation. The Ser72 phosphorylation site on human Skp2 is not present in the mouse sequence. Similar inter-species differences have been reported for other Akt substrates including p2717
. However, the Ser72 site is conserved in most large mammals (). It is plausible that for larger animals with a longer life span than mice, cell cycle control is more stringent, illustrated by the additional layer of Akt regulation on Skp2 stability.
For most SCF/F-box complexes, the regulation of substrate recognition occurs at the level of the substrate, while the interaction of Cdh1 and Cdc20 with their substrates usually does not require any post-translational modifications40
. Our finding provides another unique mechanism for the selective degradation of Cdh1 downstream targets. This protective mechanism mediated by the Akt pathway is very similar to the Cdk2/cyclin E complex, which protects Cdc6 from Cdh1-mediated destruction41
The Akt pathway functions to promote both cell survival and cell growth by inactivating many of its downstream substrates9
. In the case of p27, p21 and FOXO proteins, Akt phosphorylation triggers the recruitment of 14-3-3 which results in the masking of the NLS and subsequent cytoplasmic translocation37
. We also observed an enhanced interaction of 14-3-3 with Skp2 in cells expressing activated Akt. Moreover, phosphorylation of Skp2 by Akt at Ser72 greatly reduces the interaction between Skp2 and importin. It is possible that both of these mechanisms contribute to the cytoplasmic translocation of Skp2 subsequent to Akt phosphorylation42,43
. Thus our results offer a molecular mechanism for Skp2 cytoplasmic localization, which has been observed in many clinical tumor samples and is correlated with aggressive malignancy and poor diagnosis3,21,22,44
Interestingly, our data point to Akt isoform specificity in the regulation of Skp2 protein stability (). Furthermore, we demonstrated that when overexpressed in 293T cells, human Skp2 specifically interacts with endogenous Akt1, but not Akt2 (), although the precise mechanism by which Akt1 can, whereas Akt2 cannot, signal to Skp2 has yet to be defined.
Collectively, our results provide novel insight into how Akt activity could influence the Skp2/p27 pathway, which is a known hotspot for mutations in human cancer. On one level, our finding provides a mechanism whereby Akt influences cell cycle progression. On another level, we offer a novel mechanism by which Akt affects the degradation order of specific APC/Cdh1 substrates. Ultimately, these data may provide the rationale to develop specific Akt1 inhibitors as efficient anti-cancer drugs.