Skp2 inhibits FOXO1 in tumor suppression through ubiquitin-mediated degradation

Abstract

Forkhead transcription factors FOXO1 (FKHR), FOXO3a (FKHRL1), and FOXO4 (AFX) play a pivotal role in tumor suppression by inducing growth arrest and apoptosis. Loss of function of these factors due to phosphorylation and proteasomal degradation has been implicated in cell transformation and malignancy. However, the ubiquitin ligase necessary for the ubiquitination of the FOXO factors and the relevance of this regulation to tumorigenesis have not been characterized. Here we demonstrate that Skp2, an oncogenic subunit of the Skp1/Cul1/F-box protein ubiquitin complex, interacts with, ubiquitinates, and promotes the degradation of FOXO1. This effect of Skp2 requires Akt-specific phosphorylation of FOXO1 at Ser-256. Moreover, expression of Skp2 inhibits transactivation of FOXO1 and abolishes the inhibitory effect of FOXO1 on cell proliferation and survival. Furthermore, expression of the FOXO1 protein is lost in a mouse lymphoma model, where Skp2 is overexpressed. These data suggest that the Skp2-promoted proteolysis of FOXO1 plays a key role in tumorigenesis.

Fig. 1.

Skp2 induces ubiquitination and degradation of FOXO1. (A) Stable clones of LNCaP cells expressing Skp2 (Skp2-C1, Skp2-C8, and Skp2-C12) or empty vector (EV-C5) were established, and cell extracts were prepared for Western blot analysis. (B) A FLAG-tagged FOXO1 expression vector was cotransfected transiently with Skp2 or an empty vector (pcDNA3.1) into LNCaP cells, and whole cell lysates were prepared for Western bot analysis. (C) HepG2 cells were transfected with a pool of nonspecific (n.s.) siRNAs or siRNAs for Skp2, and cell extracts were prepared at the indicated times for Western blot analysis. (D) LNCaP cells were transfected with the indicated plasmids including HA-tagged ubiquitin, and cell extracts were either immunoprecipitated with an anti-FLAG antibody and immunoblotted with an anti-HA antibody or immunoblotted directly with antibodies for the FLAG tag or Skp2. Samples were analyzed by Western blots.

Fig. 4.

Effect of Skp2 on transactivation of FOXO1. (A) NIH 3T3 cells were transfected with luciferase reporter 3xIRS-Luc and the wild-type FOXO1. At 36 h after transfection, luciferase activity, levels of FOXO1 and Skp2 proteins as well as the status of phosphorylated FOXO1 were analyzed. Activity of luciferase was determined by normalizing the measured light units of firefly luciferase with the measured Renilla luciferase activity. *, P < 0.05 comparing the effect of Skp2 and Akt with that of the empty vector pcDNA3.1. (B) NIH 3T3 cells were transfected with luciferase reporter 3xIRS-Luc and FOXO1-S256A. Luciferase analysis and immunoblotting were performed as in A. **, P < 0.01 comparing the effect of Akt and Akt plus Skp2 with that of the empty vector pcDNA3.1. (C) NIH 3T3 cells were transfected with luciferase reporter 3xIRS-Luc and FOXO1-AAA. Luciferase analysis and immunoblotting were performed as in A.(D) NIH 3T3 cells were transfected with luciferase reporter 3xIRS-Luc and FOXO1. At 24 h after transfection, cells were treated with 15μM of MG132 or DMSO for 12 h. Luciferase analysis and immunoblotting were performed as in A. **, P < 0.01 comparing the effect of MG132 with the vehicle.

Fig. 2.

Skp2 interacts with FOXO1 in vivo and in vitro. LNCaP cells were transfected with the indicated plasmids, and protein lysates were immunoprecipitated (IP) with anti-FLAG antibody (M2) (A) or an anti-HA antibody (12CA5) (B). A mouse IgG was used as a negative control. Immunoprecipitates were analyzed by immunoblotted (IB) with anti-FLAG and anti-HA antibodies. (C) Protein lysates from NIH 3T3 cells were immunoprecipitated with an anti-Skp2 antibody and immunoblotted with antibodies for FOXO1 and Skp2. (D) Lysates of NIH 3T3 cells transfected with FLAG-FOXO1 were subjected to GST pull-down by GST-Skp2 purified from bacteria.

Fig. 3.

Interaction and ubiquitination of FOXO1 by Skp2 depend on phosphorylation of FOXO1 at Ser-256. (A) FOXO1 plasmids were cotransfected with Skp2 into LNCaP cells, and cell extracts were prepared for immunoprecipitation with an anti-FLAG antibody and immunoblotted with antibodies for FLAG, Skp2, or phospho-Ser-256 FOXO1 (FOXO1-p). (B) LNCaP cells were transfected with the indicated FOXO1, Skp2, and HA-tagged ubiquitin plasmids, and cell extracts were immunoprecipitated with the anti-FLAG antibody and immunoblotted with the anti-HA antibody. (C) Bacterially expressed GST-Skp2 was incubated in vitro with beads coupled to the FOXO1 phosphopeptide GKSPRRRAApSMDNNSKFAKS (FOXO1-p), which contains a phosphoserine at position 256. A corresponding nonphosphopeptide (FOXO1) was included as a control. (D) In vitro ubiquitination of FOXO1. Anti-FLAG immunoprecipitates immobilized on beads from LNCaP cells transfected with either wild-type (WT) or mutant (S256A) FLAG-FOXO1 were incubated with Skp2 proteins immunoprecipitated from Skp2-stable LNCaP cell lines (see Fig. 1) in a reaction system that contained 20 μM MG132 and rabbit reticulocyte lysate, which was precleared with anti-Skp2 antibody. Reactions were terminated by washing the pellets three times in RIPA buffer, and samples were analyzed by SDS/PAGE and immunoblotting.

Fig. 6.

The FOXO1 proteins are lost in lymphomas in CBP knockout mice. Representative immunoblot analyses of Skp2, FOXO1, phosphorylated Akt at Ser-473 (Akt-p), and Akt in normal mouse thymocytes (n = 4), CBP^–/– lymphomas (n = 8) (A), or CBP^–/–p27^KIP1+/– lymphomas (n = 3) (B). Erk2 was used as a loading control. (C) Jurkat cells were transfected with luciferase reporter 3xIRS-Luc and Skp2 siRNAs. At 36 h after transfection, luciferase activity, levels of FOXO1 and Skp2 proteins were analyzed. *, P < 0.05. (D) A diagram depicts the role of Skp2 in tumorigenesis via promoting degradation of FOXO1. Dashed lines indicate the tumor suppression functions of FOXO1 in the absence of interference of Skp2.

Fig. 5.

Skp2 antagonizes the tumor suppression function of FOXO1. (A and B) LNCaP cells were cotransfected with pEGFP vector along with control vector pcDNA3.1, wild-type FOXO1, or two mutants FOXO1-S256A and FOXO1-AAA in the presence or absence of Skp2. At 48 h after transfection, transfected viable cells were photographed under both UV and transmitted light (A) and quantified by using trypan blue (B). (C and D) 786-O cells were cotransfected with pEGFP vector along with control vector pcDNA3.1, wild-type FOXO1, or Skp2. At 48 h after transfection, cells were fixed briefly with formaldehyde and subjected to two-color FACS analysis for cell cycle distribution (C) and lysed for immunoblotting with FOXO1 and Skp2 antibodies (D).