FBXO11 promotes the Neddylation of p53 and inhibits its transcriptional activity

Abstract

The p53 tumor suppressor is regulated by post-translational modification, including ubiquitination, phosphorylation and acetylation. It has previously been shown that the ubiquitin ligase Mdm2 also promotes the conjugation of Nedd8, a ubiquitin-like protein, to p53, inhibiting its transcriptional activity. We report the identification of FBXO11, a member of the F-box protein family and a component of the Skp1.Cullin1.F-box (SCF) complex, as a new p53-interacting protein. We show that FBXO11 promotes the neddylation of p53 both in vitro and in vivo. In addition to the C-terminal lysine residues, FBXO11 can also promote Nedd8 conjugation to Lys-320 and Lys-321, and neddylation of p53 leads to suppression of p53 function. This is consistent with recent studies showing that a lysine to arginine mutation at Lys-320 significantly enhances p53 function, although Lys-320 was originally identified as an acetylation site involving PCAF-mediated activation of p53. Our study provides an example of an F-box protein acting as an adaptor protein that can mediate the neddylation of a non-cullin substrate.

FIGURE 1. Identification of FBXO11 in the p53 nuclear complex

A, structure of the FLAG-HA-p53(R175H) construct used in the purification of p53-interacting proteins. TAD, transactivation domain; DBD, DNA binding domain. B, silver staining of the SDS-polyacrylamide gel containing protein standard (lane 1), control eluate from M2/HA IP of H1299 cell nuclear extract (lane 2), and p53 nuclear complex obtained by M2/HA immunoprecipitation of H1299/FLAG-HA-p53(R175H) stable cell line nuclear extract (lane 3). Cells used for purification were incubated for 6 h with proteasome inhibitor (25 μM MG132 and 25 μM MG101). Specific p53-interacting protein bands were analyzed by mass spectrometry, and FBXO11 peptide sequences are presented.

FIGURE 2. FBXO11 is a component of the SCF complex

A, structure of FBXO11. B, FLAG-FBXO11 transfected into HEK 293 cells co-immunoprecipitates endogenous Cullin1, Skp1, and Roc1 (lane 2), as detected by Western blot, whereas FLAG-FBXO11(del-F-box) does not (lane 3).

FIGURE 3. FBXO11 interacts with p53 in vitro and in vivo

A, GST (lane 2) and GST-p53 (lane 3) were used in a GST-pulldown assay with in vitro translated ³⁵S-labeled FBXO11. B, HCT116 cellular extract was used for immunoprecipitation using normal rabbit IgG (lane 2) or anti-FBXO11 antibody (lane 3), and endogenous p53 protein was detected in the eluate.

FIGURE 4. FBXO11 does not promote the in vivo ubiquitination or degradation of p53

A, H1299 cells were transfected with His-Ub, p53, Mdm2, and FBXO11 plasmid as indicated. Ubiquitinated proteins were purified using nickel-agarose beads and run on an SDS gel. Ubiquitinated p53 was detected by Western blot using p53-specific antibody. B, FBXO11 overexpression has no effect on p53 stability. H1299 cells were transfected with p53 (lanes 3–7), FBXO11 (lanes 2, 4 –7), and GFP (lanes 1–7) expression plasmids, and the level of expressed protein was detected by Western blot.

FIGURE 5. FBXO11 promotes the neddylation of p53 in vivo and in vitro

A, upper panel, scheme of p53 lysine to arginine mutants used in this experiment. TAD, transactivation domain; DBD, DNA binding domain; NLS, nuclear localization signal; RD, C-terminal regulatory domain. Lower panel, H1299 cells were transfected with His-Nedd8, p53, and FBXO11 constructs as indicated. Neddylated proteins were purified using nickel-agarose beads and run on an SDS gel. Neddylated p53 was detected by Western blot using p53-specific antibody. p53 with lysine to arginine mutations on the six C-terminal lysines (6KR) has decreased neddylation by FBXO11 (lane 4), while p53 with two additional mutations (Lys-320 and Lys-321, 8KR) is not neddylated by FBXO11 (lane 6). B, SCF^FBXO11 complex purified from a FLAG-FBXO11-expressing H1299 stable line promotes the neddylation of p53 in an in vitro neddylation reaction.

FIGURE 6. Fusion of Nedd8 to the C terminus of p53 inhibits its activity

A, schematic representation of the wild-type p53 and p53-Nedd8 constructs used in this experiment. B, activation of a p21-luciferase reporter by wild-type p53 and p53-Nedd8. The luciferase reporter construct was transfected into mouse embryonic fibroblast (p53^−/−, Mdm2^−/−) cells along with the indicated amounts of p53 plasmid, and luciferase activity was measured. C, sub-cellular localization of wild-type p53 and p53-Nedd8 protein. H1299 cells were transfected with expression plasmids for both constructs, and the expressed proteins were detected by immunofluorescence staining. Cells were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) to visualize nuclei.

FIGURE 7. FBXO11 inhibits p53 transcriptional activity

siRNA knockdown of FBXO11 using three different oligos (upper panel, lanes 2, 3, and 4) in U2OS results in an increase in p21 levels compared with control GFP siRNA (lane 1), whereas p53 levels are unaffected. Knockdown of FBXO11 in p53-null H1299 cells (lower panel, lanes 5 and 6) has no effect on p21 levels.