A comparison between Skp2 and FOXO1 for their cytoplasmic localization by Akt1

The F-box protein Skp2 functions as the substrate recruitment subunit of the SCFSkp2 E3 ubiquitin ligase, whose best-known target is p27Kip1 in the nucleus. Two recent articles reported that kinase Akt1 could phosphorylate Skp2 at Ser72 1, 2. One consequence of this phosphorylation is cytoplasmic retention of Skp2, which raised a number of questions. Since p27 is a nuclear protein, how does cytoplasmic retention of Skp2 affect the regulation of its best-established ubiquitination substrate? Does cytoplasmic Skp2 have a distinct set of substrates from nuclear Skp2? Before addressing these questions, we compared the abilities of Akt1 to induce cytoplasmic retention of Skp2 and FOXO1, another nuclear-to-cytoplasm translocation target of Akt1 3, 4, to determine whether and to what degree Skp2 differs from FOXO1 in the extent of this regulation by Akt1. Consistent with previous studies including those reported by these two articles 1, 2, flag-Skp2 was nuclear-localized when ectopically expressed in 293T cells (Figure 1A). Deletion of the N-terminal 100 residues, which include a canonical nuclear localization signal (NLS), resulted in cytoplasmic retention of the Skp2 (Figure 1H). In the same experiment, GFP-FOXO1 was also primarily nuclear (Figure 1J). When a constitutively active Akt1 (HA-myrAkt1) was coexpressed, its membrane association was visible (Figure 1F) and it induced cytoplasmic localization of GFP-FOXO1 in about 80% of the GFP-FOXO1-expressing cells (Figure 1L). In the same experiment, coexpression of the same HA-myrAkt1 induced cytoplasmic localization of flag-Skp2 in less than 1% of the flag-Skp2-expressing cells with the remaining cells showing nuclear Skp2 (Figure 1E). These findings are in contrast to those reported by Lin et al and Gao et al 1, 2 showing cytoplasmic localization of Skp2 in about 40% of the transfected cells by coexpression of myrAkt1. Although certain differences in experimental conditions might exist to explain the discrepancy, our results suggest that, compared to FOXO1, Skp2 is not robustly regulated by Akt1 for its cytoplasmic localization and activation of Skp2 function in the cytoplasm might not be the major purpose of regulation of Skp2 by Akt1. Another functional consequence of Skp2 phosphorylation at Ser72 by Akt1 was to promote the formation of SCFSkp2 complex (Lin et al)1. Using an Skp2S72A mutant and coexpression of HA-myrAkt1, we also failed to detect robust effects of Akt1 on the assembly of SCFSkp2 (Supplemental Figure 1). Based on these findings, we suggest the need for further studies to determine the regulation of Skp2 by Akt1. Figure 1 Comparison between Skp2 and FOXO1 for their cytoplasmic localization by a constitutively active Akt1. 293T cells were transfected with flag-tagged wild type Skp2 or the ΔN-Skp2, or GFP-tagged FOXO1 (as indicated on top), alone or together with ...