The deubiquitinating enzyme BAP1 regulates cell growth via interaction with HCF-1

Abstract

The deubiquitinating enzyme BRCA1-associated protein 1 (BAP1) possesses growth inhibitory activity and functions as a tumor suppressor. In this study we report that BAP1 also plays positive roles in cell proliferation. BAP1 depletion by RNAi inhibits cell proliferation as does overexpression of a dominant negative mutant of BAP1. Mass spectrometry analyses of copurified proteins revealed that BAP1 is associated with factors involved in chromatin modulation and transcriptional regulation. We show that the interaction with host cell factor-1 (HCF-1), a cell-cycle regulator composed of HCF-1N and HCF-1C, is critical for the BAP1-mediated growth regulation. We found that HCF-1N is modified with Lys-48-linked polyubiquitin chains on its Kelch domain. The HCF-1 binding motif of BAP1 is required for interaction with HCF-1N and mediates deubiquitination of HCF-1N by BAP1. The importance of the BAP1-HCF-1 interaction is underscored by the fact that growth suppression by the dominant negative BAP1 mutant is entirely dependent on the HCF-1 binding motif. These results suggest that BAP1 regulates cell proliferation by deubiquitinating HCF-1.

FIGURE 1.

Interference of BAP1 functions results in growth retardation. A, shown is the effect of BAP1 depletion on cell proliferation. MCF10A cells were infected with retroviruses expressing the indicated shRNAs, and BAP1 protein levels were assessed by Western blotting (left). β-Actin is shown as a loading control. Proliferation of MCF10A cells expressing the indicated shRNAs were measured by counting cells after a 10-day culture. The ratio of cell count relative to that in shControl population is expressed as the mean ± S. D. (n = 3). B, shown is the effect of BAP1 overexpression on cell proliferation. MCF10A cells were infected with retroviruses expressing BAP1 or BAP1 C91S. BAP1 levels were examined by immunoblotting (left). β-Actin is shown as a loading control. Proliferation of MCF10A cells expressing the indicated proteins were measured by counting cells after a 10-day culture. Ratio of cell count relative to vector control is expressed as the mean ± S. D. (n = 3).

FIGURE 2.

Purification of BAP1-associated proteins. A, shown is purification of BAP1 interactors. 293T cells were infected with retroviruses expressing 3× FLAG/His₈-tagged BAP1 and BAP1-associated proteins were purified by tandem affinity purification. Purified proteins were examined by silver staining. Mock purification from cells infected with empty viruses is shown as a control. B, shown is cell fractionation. Cellular proteins were fractionated into cytosolic- (S2), nucleoplasmic- (S3), and chromatin-enriched (P3) fractions as described under “Experimental Procedures.” WCE, whole cell extracts. Where indicated, P3 fractions were treated with micrococcal nuclease (+MNase) to release chromatin-bound proteins into S3 fractions. ERK and ORC2 are shown as markers of cytosolic and chromatin-bound proteins, respectively.

FIGURE 3.

Mapping the BAP1-interacting domain in HCF-1. A, shown is the association of endogenous BAP1 and HCF-1. BAP1 and HCF-1 proteins were immunoprecipitated (IP) with specific antibodies, and the indicated proteins were examined by Western blotting. B, shown is a schematic representation of the HCF-1 domain structure. C, shown is in vivo association of BAP1 with HCF-1N. 293T cells were transfected with expression plasmids for the indicated proteins. HCF-1N and HCF-1C proteins were immunoprecipitated by anti-FLAG antibodies. Coprecipitation of BAP1 proteins was examined by anti-HA Western blotting (WB). A schematic representation of the HCF-1 domain structure is shown (top). D, shown is mapping the BAP1 binding domain of HCF-1N. Cells were transfected with plasmids expressing 4HA-BAP1 and various truncation mutants of HCF-1N tagged with FLAG-2NLS (nuclear localization signal). Association of BAP1 with each fragment was assessed by anti-FLAG immunoprecipitation followed by anti-HA Western blotting (bottom). An asterisk indicates Ig heavy chains. Note that FLAG-HCF 1–450 migrates just below the IgH chain. A schematic representation of the HCF-1N domain structure is shown (top). FL, full-length.

FIGURE 4.

Mapping the HCF-1-interacting domain in BAP1. A, shown is in vitro association of BAP1 fragments with HCF-1N. GST pulldown assays were performed using GST fusion of the indicated BAP1 fragments and HCF-1N protein produced by in vitro translation. HCF-1N proteins in the pulldown fractions were examined by autoradiography. Precipitated GST fusion proteins are shown by Coomassie staining. B, shown is a schematic representation of BAP1 protein indicating the highly conserved sequence surrounding the HBM. NLS, nuclear localization signal. C, shown is in vivo interaction of HCF-1 with various mutants of BAP1. 293T cells were transfected with expression plasmids for indicated proteins. FLAG-BAP1 proteins were pulled down from cell lysates using anti-FLAG antibodies and probed with indicated antibodies. IP, immunoprecipitate; WB, Western blot; WT, wild type. D, shown is in vivo interaction of BAP1 and the HCF-1 Kelch domain. Wild type or P134S mutant of HCF-1 Kelch domain was coexpressed with BAP1, immunoprecipitated with anti-FLAG antibodies, and associated BAP1 proteins examined by anti-HA Western blotting.

FIGURE 5.

Ubiquitination of HCF-1N in vivo. A, shown is mapping the ubiquitination domain of HCF-1N. Plasmids expressing the indicated proteins were cotransfected to 293T, and ubiquitination (Ub) of HCF-1 proteins was examined by Western blotting (WB) after anti-FLAG immunoprecipitation (IP). B, shown is purification of ubiquitinated FLAG-Kelch from cells. 293T cells were transfected with plasmids expressing HA-ubiquitin and FLAG-Kelch. FLAG-Kelch proteins were purified with anti-FLAG antibodies, separated by SDS-PAGE, and visualized by Coomassie staining. Unmodified Kelch is indicated by an arrow. Triangles indicate bands analyzed by mass spectrometry. C, shown is the amino acid sequence of the HCF-1 Kelch domain. The alignment was adapted from Wilson et al. (52). The ubiquitin acceptors identified in this study are highlighted in black. β-Strands are indicated by gray boxes. D, examination of ubiquitin linkages on the HCF-1 Kelch domain is shown. FLAG-Kelch proteins were expressed in combination with various ubiquitin mutants. Ubiquitination of FLAG-Kelch was analyzed as in A. The unmodified Kelch domain is indicated by arrows. Asterisks indicate Ig heavy chains. WT, wild type.

FIGURE 6.

Deubiquitination of HCF-1N by BAP1. A, shown is in vivo ubiquitination assays. 293T cells were cotransfected with indicated plasmids. FLAG-Kelch was immunoprecipitated (IP) by anti-FLAG antibodies and examined by Western blotting (WB). WT, wild type. Ub, ubiquitin. B, shown is the effect of the ΔHBM mutation on the BAP1-mediated reduction of Kelch ubiquitination. In vivo ubiquitination assays were performed in cells expressing the indicated proteins. C, shown is the effect of the ΔHBM mutation on the dominant negative activity of BAP1 C91S. In vivo ubiquitination assays were performed as in A. D, shown is the effect of the P134S mutation on the Kelch ubiquitination. Cells were transfected with plasmids for the indicated proteins. Ubiquitination of the Kelch domains was examined as in A. Unmodified FLAG-Kelch is indicated by an arrow. Asterisks indicate Ig heavy chains.

FIGURE 7.

Overexpression of the BAP1 C91S mutant suppresses cell growth in an HBM-dependent manner. A, shown is overexpression of various BAP1 mutant proteins. BAP1 mutants were expressed in MCF10A by retrovirus vectors and examined by Western blotting (WB). β-Actin is shown as a loading control. IP, immunoprecipitate. Ub, ubiquitin. B, shown is effect of BAP1 proteins harboring various mutations on cell proliferation. Proliferation of MCF10A cells expressing the indicated proteins were measured by counting cells after a 11-day culture. Values are the means ± S. D. (n = 3).