The deubiquitylase USP7 is a novel cyclin F-interacting protein and regulates cyclin F protein stability

Abstract

Cyclin F, unlike canonical and transcriptional cyclins, does not bind or activate any cyclin-dependent kinases. Instead, it harbors an F-box motif and primarily functions as the substrate recognition subunit of the Skp1-Cul1-F-box E3 ubiquitin ligase complex, SCF^{Cyclin F}. By targeting specific proteins for ubiquitin-mediated proteasomal degradation, cyclin F plays a critical role in the regulation of centrosomal duplication, DNA replication and repair, and maintenance of genomic stability. Cyclin F abundance and activity are tightly regulated throughout the cell cycle. However, the molecular mechanisms regulating cyclin F are scantily understood. Here, we identify the deubiquitylase USP7 as a novel cyclin F-interacting protein. We observe that USP7 stabilizes cyclin F protein and that this function is independent of the deubiquitylase activity of USP7. Additionally, our data suggest that USP7 is also involved in the regulation of cyclin F mRNA. Pharmacological inhibition of the deubiquitylase activity of USP7 resulted in downregulation of cyclin F mRNA.

Keywords: USP7; atypical cyclins; cell cycle; cyclin F; genomic integrity.

Figure 1

Cyclin F interacts with USP7. (A) HEK-293T cells transfected with Flag-HA-Cyclin F were lysed and immunoprecipitated with anti-HA antibody or non-specific mouse immunoglobulin (IgG) as loading control. Immunocomplexes were immunoblotted as indicated. (B) Schematic representation of cyclin F WT and cyclin F^1-270 truncated mutant, highlighting the F-box, cyclin-box and PEST regions. (C) HEK-293T cells transfected with Flag-HA-Cyclin F WT or Flag-HA-Cyclin F^1-270 were immunoprecipitated and immunoblotted as indicated. (D) Endogenous USP7 was immunoprecipitated from HEK-293T cell extracts, with anti-USP7 antibody or an unrelated, anti-p-TAK1 antibody as a loading control (denoted as control). Immunocomplexes were immunoblotted as indicated. Asterisk denotes non-specific band.

Figure 2

SCF^{Cyclin F} does not regulate USP7 protein levels. (A) HCT116 cells treated with either DMSO or MLN4924 (3 μM) for 5 h were lysed, and immunoblotted as indicated. β-Actin was the loading control. Asterisk denotes non-specific band. (B) HEK-293T cells were transfected with an empty vector or Flag-HA-Cyclin F for 48 h, lysed, and immunoblotted as indicated. (C) HeLa cells treated with DMSO, etoposide (10 μM) or MG132 (10 μM) for 4 h were lysed, and immunoblotted as indicated. Asterisk denotes non-specific band.

Figure 3

USP7 regulates cyclin F protein levels. (A) HeLa cells treated with DMSO, MLN4924 (10 μM), or P22077 (25 μM) for the indicated hours were lysed, and immunoblotted as indicated. β-Actin was the loading control. Asterisks denote non-specific bands. Fold changes were calculated with densitometric values for cyclin F and cyclin A2 blots using β-Actin as loading control. (B) HCT116 cells were treated with DMSO or P22077 (25 μM) for 5 h. Where indicated, cells were treated with MG132 (10 μM) for 4 h prior to harvest. Cells were then lysed and immunoblotted as indicated. Asterisks denote non-specific bands. Fold changes were calculated with densitometric values for cyclin F and cyclin A2 blots using β-Actin as loading control. (C) HCT116 cells were treated with DMSO or cycloheximide (CHX; 50 μg/mL) for 75 min. Where indicated, bafilomycin A1 (BafA1; 100 nM) and/or MG132 (12.5 μM) were added along with CHX. Cells were lysed, and immunoblotted as indicated. Asterisks denote non-specific bands.

Figure 4

USP7 regulates cyclin F mRNA levels. (A) HeLa cells treated with DMSO or P22077 (25 μM) for the indicated hours were collected and total RNA was extracted. cDNAs prepared from each sample were amplified using primer pairs specific to each gene as indicated. The PCR products were analyzed by agarose gel electrophoresis and stained using ethidium bromide. RPL35A was the loading control. Fold change in cyclin F:RPL35A ratios were determined using densitometry values of the amplicons. The values represent an average from two independent experiments. (B) HCT116 cells treated with DMSO or P22077 (25 μM) for 4 h were analyzed as in (A).

Figure 5

USP7 regulates cyclin F protein stability. (A) HCT116 cells were co-transfected with Flag-HA-Cyclin F WT and Flag-USP7 WT or empty vector. Twenty-four hours post transfection, cells were treated with DMSO or cycloheximide (CHX; 50 μg/mL) for the indicated minutes, lysed, and immunoblotted as indicated. α-Tubulin was the loading control. (B) HeLa cells were co-transfected with Flag-HA-Cyclin F WT and Flag-USP7 WT or empty vector. Forty-eight hours post transfection, cells were treated with CHX (50 μg/mL) for 75 min. Where indicated, P22077 (25 μM) was added to cells 2 h prior to the addition of CHX. Cells were then lysed, and immunoblotted as indicated. (C) HeLa cells were co-transfected with Flag-HA-Cyclin F WT and Flag-USP7 WT, Flag-USP7 CS, or empty vector. Forty-eight hours post transfection, cells were treated with CHX (50 μg/mL) for 75 min, lysed, and immunoblotted as indicated.

Figure 6

Interaction between USP7 and cyclin F under DNA-damaging condition. HeLa cells were treated with DMSO (control), etoposide (10 μM), or MG132 (10 μM) for 4 h. Endogenous USP7 was immunoprecipitated (IP) with anti-USP7 antibody or an unrelated, anti-p-TAK1 antibody (mock IP) as a loading control. Immunocomplexes were immunoblotted as indicated; β-actin was the loading control. Asterisk denotes non-specific band.