APC/C(Cdh1)-mediated degradation of the F-box protein NIPA is regulated by its association with Skp1

Abstract

NIPA (Nuclear Interaction Partner of Alk kinase) is an F-box like protein that targets nuclear Cyclin B1 for degradation. Integrity and therefore activity of the SCF(NIPA) E3 ligase is regulated by cell-cycle-dependent phosphorylation of NIPA, restricting substrate ubiquitination to interphase. Here we show that phosphorylated NIPA is degraded in late mitosis in an APC/C(Cdh1)-dependent manner. Binding of the unphosphorylated form of NIPA to Skp1 interferes with binding to the APC/C-adaptor protein Cdh1 and therefore protects unphosphorylated NIPA from degradation in interphase. Our data thus define a novel mode of regulating APC/C-mediated ubiquitination.

Figure 1. Phosphorylated NIPA is degraded during mitotic exit.

(A) NIH3T3 cells stably overexpressing Flag-NIPA were arrested in prometaphase by a thymidine-nocodazole block and subsequently released into fresh medium either containing cycloheximide (CHX) or without supplements. Cells were collected at the indicated timepoints. The degree of synchronization was confirmed by analysis of the expression of cyclin B1 and FACS analysis (data not shown). (B) HeLa cells were arrested in prometaphase, released into fresh growth medium, and harvested for Western blot at the indicated timepoints. (C) HEK293T cells were transfected with (+) or without (-) HA-Ubiquitin and Flag-NIPA as indicated. Following treatment with MG132, extracts were prepared, denatured and subjected to Flag immunoprecipitation. Ub_n: polyubiquitinated forms. (D) NIH3T3 cells retrovirally infected with a Flag-NIPA construct were treated with cycloheximide (CHX) and either DMSO or the proteasome inhibitor MG132. Cells were harvested for Western blot at the indicated timepoints.

Figure 2. NIPA is degraded in an APC/C^Cdh1-dependent manner.

(A) Autoradiogram of ³⁵S-labelled NIPA after incubation in HeLa cell extract prepared from G1 cells. Where indicated, the APC/C was depleted from the extract using a Cdc27 antibody. The Cdc27 Western blot shows efficient removal of Cdc27 from extracts. (B) Flag-NIPA and either HA-Cdh1 or HA-Cdc20 were expressed in HEK293T cells, and MG132 was added 6 h before the cells were collected. Cell extracts were immunoprecipitated (IP) with an antibody against Flag. (C) Flag-NIPA was expressed in HEK293T cells, and MG132 was added 6 h before the cells were collected. Cell extracts were immunoprecipitated (IP) with an antibody against Flag. (D) Autoradiogram of ³⁵S-labelled NIPA after in vitro ubiquitination by APC/C immunoprecipitates derived from HeLa cells. Cdh1 was supplemented, where indicated. Ub_n: polyubiquitinated forms. (E) and (F) Flag-NIPA was transiently expressed in HEK293T cells and immunoprecipitated using an agarose-bound anti-Flag antibody. Immunoprecipitates were used in APC in vitro ubiquitination reactions. Cdh1 and E1 ubiquitin-activating enzyme were supplemented as indicated. (G) HeLa cells were transfected with either control (firefly Luciferase) siRNA or NIPA siRNA, synchronized in prometaphase and subsequently released for the indicated times. (H) NIH3T3 cells stably overexpressing Flag-NIPA were transfected with empty vector or Cdh1, synchronized in prometaphase and then released for the indicated periods of time.

Figure 3. Degradation motifs in NIPA.

(A) Schematic presentation of the NIPA protein, indicating the position of the D-box motifs. C: carboxyterminus; C3HC: Zinc-Finger motif; N: aminoterminus; NLS: nuclear localization signal; S: serine residue. (B) Alignment of NIPA D-box-like motifs in different species. C, Immunopurified APC/C supplemented with recombinant Cdh1 ubiquitinates wildtype NIPA in vitro but not the Dbox1+2 mutant.

Figure 4. Binding to Skp1 protects NIPA from APC/C^Cdh1-mediated degradation.

(A) HEK293T cells transfected with Flag-NIPA wt or the F-Box mutant were treated with cycloheximide (CHX) for the indicated times and analyzed for total NIPA levels by immunoblotting. (B) HEK293T cells were transfected with HA-Ubiquitin and different amounts of Flag-NIPA wt or F-Box mutant to balance their expression. Following treatment with MG132, extracts were prepared, denatured and subjected to Flag immunoprecipitation. (C) Autoradiogram of an in vitro degradation assay using HeLa cell extract prepared from G1 cells. ³⁵S-labelled Flag-NIPA was incubated with GST-Skp1 or GST and subsequently immunoprecipitated with agarose-bound anti-Flag antibody before being used in degradation assays. (D) Flag-NIPA was transiently expressed in HEK293T cells and immunoprecipitated using an agarose-bound anti-Flag antibody. Immunoprecipitates were used in APC in vitro ubiquitination reactions. Cdh1 and Skp1 were supplemented, where indicated. (E) HA-Cdh1 was co-expressed with Flag-NIPA and Skp1 in HEK293T cells as indicated. Cell extracts were immunoprecipitated (IP) with an antibody against Flag and analysed by immunoblotting.