Regulating the stability and localization of CDK inhibitor p27(Kip1) via CSN6-COP1 axis

Abstract

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27(Kip1) stability, and that COP1 is a negative regulator of p27(Kip1). Ectopic expression of CSN6 can decrease the expression of p27(Kip1), while CSN6 knockdown leads to p27(Kip1) stabilization. Mechanistic studies show that CSN6 interacts with p27(Kip1) and facilitates ubiquitin-mediated degradation of p27(Kip1). CSN6-mediated p27 degradation depends on the nuclear export of p27(Kip1), which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.

Keywords: COP1; COP9 signalosome; CSN6; p27; ubiquitination.

Figure 1.

CSN6 interacts with p27 and regulates its stability. (A) Indicated expression vectors were transfected into 293T cells. Lysates were analyzed by IP with Flag and IB with anti-HA. CSN6 interacts with endogenous p27. Lysates of HCT116 cells were prepared and equal amounts of cell lysates were analyzed by immunoprecipitation (IP) with either control mouse IgG or CSN6 and analyzed by immunoblotting (IB) with anti-p27. (B) CSN6 reduced the steady-state expression of p27 in a dose-dependent manner. 293T cells were co-transfected with the indicated expression vectors. Equal amounts of protein from cell lysates were analyzed by immunoblotting with the indicated antibodies. Knockdown of CSN6 upregulates the expression of p27 protein level. Lysates of HCT116 cells infected with either CSN6-shRNA or control shRNA were analyzed by IB with the indicated antibodies. (C) CSN6-mediated destabilization of p27 is proteasome-dependent. 293T cells co-transfected with the either Myc-CSN6 or vector control was treated with or without proteasome inhibitor MG132 before collecting lysates. Lysates were immunoblotted with indicated antibodies. (D) CSN6 increases the turnover of p27. 293T cells co-transfected with the indicated expression vectors were treated with cycloheximide (CHX) (100 µg/ml) for the indicated times. Equal amounts of protein from cell lysates were immunoblotted with indicated antibodies.

Figure 2.

CSN6 increases p27 poly-ubiquitination. (A) 293T cells were transfected with indicated expressing plasmids. MG132 was added 6 h before they were harvested. The cell lysates was then immunoprecipitated with anti-Flag and immunoblotted with anti-HA antibody. Equal amount of whole cell lysates were immunoblotted with anti-myc or Actin. (B) HCT116 cells were transfected with CSN6 or knocked down with CSN6 shRNA. The cell lysates from indicated cells were immunoprecipitated with anti-p27 and immunoblotted with anti-ubiquitin antibody. Equal amounts of cell lysates were analyzed by IB with the indicated antibodies.

Figure 3.

COP1-mediated nuclear export of p27 is involved in CSN6-mediated p27 ubiquitination (A) COP1 is downregulated following the CSN6 knockdown. 293T cells were co-transfected with the indicated expression vectors. Lysates were immunoblotted with the indicated antibodies. (B) CSN6-mediated p27 downregulation is diminished by the leptomycin B. 293T cells were co-transfected with increasing myc-CSN6 expression vectors. Cells were treated with or without leptomycin B (20 ng/ml) for 6 hours before lysates were collected. Lysates were immunoblotted with the indicated antibodies. (C) Leptomycin B rescued COP1-mediated p27 downregulation. 293T cells were co-transfected with the indicated expression vectors. Cells were treated with or without leptomycin B (20 ng/ml) for 6 hours before lysates were collected. Lysates were immunoblotted with the indicated antibodies. (D) Mutation in the COP1 nuclear export signal (NES) sequence impaired the ability of COP1 to downregulate p27 expression. 293T cells were co-transfected with the indicated expression vectors. Lysates were immunoblotted with the indicated antibodies.

Figure 4.

COP1 induces p27 nuclear export. (A) Leptomycin B increased p27 nuclear accumulation in the presence of COP1. 293T cells co-transfected with either wild-type (wt) or NES mutant GFP-COP1 and RFP-p27, cultured with or without leptomycin B (LMB), were stained with DAPI. (B) Percentages of nuclear p27 signals among GFP-positive cells in A are shown.

Figure 5.

COP1 interacts with p27 to mediate nuclear export. (A) The Flag-p27 (VPAA) mutant nuclear staining is not diminished in the presence of COP1. 293T cells were co-transfected with either wild-type (wt) or VPAA mutant RFP-p27 and GFP-COP1 and stained with DAPI. (B) Percentages of nuclear p27 signals among GFP-COP1-positive cells in A are shown.

Figure 6.

COP1 overexpression leads to upregulation of p27-mediated suppressed genes. (A, B) COP1 regulated the genes that are suppressed through p27 mediation. COP1-overexpression or COP1 knockdown on gene expression of p27 target genes are shown. mRNA levels of the indicated p27 target genes were determined by quantitative reverse transcriptase PCR. (C) COP1 antagonized the suppressive impact of p27 on the expression of several targeted genes.

Figure 7.

COP1-p27 link correlates with poor survival. (A) COP1 overexpression in cancer led to increased expression of genes suppressed through p27 mediation. Relative expression of COP1 and genes suppressed through p27 mediation (COQ5, CDC123, GEMIN5, AURKA, KIF11, ING4, HEARTR1, and MED18) were analyzed from a data set of multiple myeloma samples. Data are presented as a heat map. (B) High expression of COP1 was associated with poor overall survival. Lysates of indicated multiple myeloma cells (RPMI8226) or ovarian cancer cells (2008) infected with either COP1-shRNA or control shRNA were analyzed by IB with the indicated antibodies. Kaplan-Meier overall survival curves for 414 patients with multiple myeloma (left) or ovarian cancer (right), classified by COP1 expression, are shown. (C) Protein levels of p27 were regulated according to COP1 expression in xenograft mouse models. HCT116 cells with CSN6 overexpression or knockdown were subcutaneously injected into the nude mice. Tumor samples were collected and lysates of indicated tumor samples were analyzed by IB with the indicated antibodies.