Transforming activity of Fbxo7 is mediated specifically through regulation of cyclin D/cdk6

Abstract

D cyclins (D1, D2 and D3) and their catalytic subunits (cyclin-dependent kinases cdk4 and cdk6) have a facilitating, but nonessential, role in cell cycle entry. Tissue-specific functions for D-type cyclins and cdks have been reported; however, the biochemical properties of these kinases are indistinguishable. We report that an F box protein, Fbxo7, interacted with cellular and viral D cyclins and distinguished among the cdks that bind D-type cyclins, specifically binding cdk6, in vitro and in vivo. Fbxo7 specifically regulated D cyclin/cdk6 complexes: Fbxo7 knockdown decreased cdk6 association with cyclin and its overexpression increased D cyclin/cdk6 activity and E2F activity. Fbxo7 interacted with p27, but its enhancement of cyclin D/cdk6 activity was p21/p27 independent. Fbxo7 overexpression transformed murine fibroblasts, rendering them tumorigenic in athymic nude mice. Transformed phenotypes were dependent on cdk6, as knockdown of cdk6 reversed them. Fbxo7 was highly expressed in epithelial tumors, but not in normal tissues, suggesting that it may have a proto-oncogenic role in human cancers.

Figure 1

(A) Schematic diagram of Fbxo7, with F box motif (black), proline-rich domain (medium gray) and Ubl motif (light gray). Sequences contained within pGAD-Fbxo7 are indicated. (B) pGAD-Fbxo7 interacted specifically with pGBD-HVS cyclin. Yeast were grown on media selecting for the plasmids (SC-ura-leu) and on media selecting for activation of reporter genes (SC-ura-leu-his-ade). (C) Western blot of T7-Fbxo7(129–398) detected in anti-flag immunoprecipitates of D cyclins, but not cyclins E and A (bottom panels). Western blot of cdk2 is shown as a control for equivalent immunoprecipitation of these cyclins. (D) Fbxo7 co-immunoprecipitated with D cyclins, but not cyclins E or A. Western blots of cyclins D2 and D3 (middle), and cdk2 (bottom) shown as controls for cyclin immunoprecipitation. (E) Fbxo7 was detected in immunoprecipitates of cdk6, but not cdk4 or cdk2. Western blots for cdk subunits are shown below.

Figure 2

(A) Coomassie staining of an SDS–PAGE gel showing GST fusion proteins to cyclin and cdk subunits. (B) GST-cdk6, but not GST, GST-cyclin D1/D3 or GST-cdk4, bound in vitro-translated Fbxo7 protein. (C) Western blots for Skp1 and T7 of anti-flag immunoprecipitates of lysates of U2OS cells cotransfected with flag-Fbxo7 and T7-Rbx1. (D) Western blots for cell cycle proteins of lysates from cells transfected with control (C) or Fbxo7 (F) dsRNA. (E) Western blots of anti-cdk6 or isotype control (iso) immunoprecipitates for cdk6 and cyclin D1 from cells with endogenous or reduced Fbxo7. (F) Timeline of de novo association experiments conducted in panel G and Figure 3E. (G) Western blots for cdks and flag of anti-flag immunoprecipitates from cells with endogenous or reduced Fbxo7. Western blots of total lysates for Fbxo7 are shown. ^* indicates a nonspecific band used as an internal loading control. (H) FACS analysis of BrdU incorporation by control (C) or Fbxo7 (F) dsRNA-treated cells stained with FITC-conjugated anti-BrdU antibodies (FL1-H) and propidium iodide (FL3-H).

Figure 3

(A) Western blots for T7-Fbxo7, p21 and p27 in anti-p21 and p27 immunoprecipitates from lysates of T7-Fbxo7-transfected cells. (B) GST-cdk6 and GST-p27(N-term) interacted with in vitro-transcribed and -translated Fbxo7. (C) Western blot for cdk6 in binding assays using GST-cyclin D1 and GST-cyclin D3 and in vitro-translated proteins, cdk6, p27 and Fbxo7. Western blot of 10% input is shown in the left panel. (D) A representative result of an in vitro kinase assay of anti-cdk immunoprecipitates from p21^−/−p27^−/−cells, with or without Fbxo7, using pRb as a substrate. The average percent increase from three experiments is noted. (E) Western blots for cdk6 and flag of anti-flag immunoprecipitates from cells with endogenous or reduced Fbxo7 and of total lysates for Fbxo7. Experiment was performed as in Figure 2G. (F) Western blots of nuclear or cytoplasmic fractions from cells with endogenous or overexpressed Fbxo7. (G) Images of confocal fluorescence microscopy of untreated or leptomycin B (LMB)-treated cells expressing dsRED-Fbxo7.

Figure 4

(A) Western analysis for T7-Fbxo7 and cdks in lysates from 3T3/vec or 3T3/Fbxo7 cells. (B) A representative result of an in vitro kinase assay of anti-cdk immunoprecipitates from 3T3/vec or 3T3/Fbxo7 cells using pRb as substrate. The average percent increase from three trials is noted. (C) Heat maps of E2F-regulated genes from GEM analysis of 3T3/vec and 3T3/Fbxo7 cells. Arrays were performed in triplicate. Red shows increased and blue decreased relative expression. (D) Western blots of flag and cdk subunits in anti-flag cyclin immunoprecipitates from 3T3/vec and 3T3/Fbxo7 cells. (E) FACS analysis on serum-starved 3T3/vec and 3T3Fbxo7 cells stained with FITC-conjugated antibodies to annexin V (FL1-H) and propidium iodide (FL3-H).

Figure 5

(A) Images of colony growth in soft agar assays (top) and graph of colony number formed by 3T3/vec and 3T3/Fbxo7 cells (bottom). (B) Tumor formation assay with 3T3/vec and 3T3/Fbxo7 cells. (C) Images of tumors from 3T3/Fbxo7 cells stained for histology with hematoxylin and eosin (H&E), and immunohistochemistry for Fbxo7. (D) Images from confocal fluorescence microscopy of cells stained with rhodamine-conjugated phalloidin. 3T3/Fbxo7 cells had disorganized F-actin and fewer stress fibers (indicated by white arrows). (E) Images of ECM invasion assays where invasive cells stain violet.

Figure 6

(A) Western blot for Fbxo7(129–522) expression in NIH3T3 cells. (B) Images from ECM invasion assays with 3T3/Fbxo7Δ129 cells. (C) Graph of colony number formed by 3T3/vec and 3T3/Fbxo7Δ129 cells in soft agar assay. (D) Western blot for Fbxo7ΔFbox expression in NIH3T3 cells. (E) Images from ECM invasion assays with 3T3/Fbxo7ΔFbox cells. (F) Graph of colony number for 3T3/Fbxo7ΔFbox cells in soft agar assay. (G) Western blot for the T7 from total lysate and anti-cdk6 immunoprecipitates from lysates of cells transfected with T7-Fbxo7 or T7-Fbxo7(ΔFbox). (H) Western analysis for cdk6 in cell lysates as indicated. (I) Images from ECM invasion assays with indicated cell lines. (J) Graph of number of colonies in soft agar assay with cell lines as indicated.

Figure 7

(A) Images of immunohistochemistry for Fbxo7 on normal lung tissue and biopsies from lung cancers. Brown color indicates Fbxo7 staining, and hematoxylin counterstaining for nuclei is blue. Scoring of subcellular localization of Fbxo7 is summarized. (B) Images of immunohistochemistry for Fbxo7 in normal colon and in malignancies of the colon. Staining and analysis are as above.