The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2
2003; Springer Nature; Volume: 22; Issue: 13 Linguagem: Inglês
10.1093/emboj/cdg315
ISSN1460-2075
Autores Tópico(s)Protein Degradation and Inhibitors
ResumoArticle1 July 2003free access The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2 Oliver H. Krämer Oliver H. Krämer Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany Search for more papers by this author Ping Zhu Ping Zhu Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany Search for more papers by this author Heather P. Ostendorff Heather P. Ostendorff Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany Search for more papers by this author Martin Golebiewski Martin Golebiewski Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Jens Tiefenbach Jens Tiefenbach Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Marvin A. Peters Marvin A. Peters Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany Search for more papers by this author Boris Brill Boris Brill Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Bernd Groner Bernd Groner Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Ingolf Bach Ingolf Bach Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany Search for more papers by this author Thorsten Heinzel Corresponding Author Thorsten Heinzel Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Martin Göttlicher Corresponding Author Martin Göttlicher Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany Search for more papers by this author Oliver H. Krämer Oliver H. Krämer Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany Search for more papers by this author Ping Zhu Ping Zhu Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany Search for more papers by this author Heather P. Ostendorff Heather P. Ostendorff Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany Search for more papers by this author Martin Golebiewski Martin Golebiewski Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany Search for more papers by this author Jens Tiefenbach Jens Tiefenbach Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Marvin A. Peters Marvin A. Peters Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany Search for more papers by this author Boris Brill Boris Brill Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Bernd Groner Bernd Groner Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Ingolf Bach Ingolf Bach Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany Search for more papers by this author Thorsten Heinzel Corresponding Author Thorsten Heinzel Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany Search for more papers by this author Martin Göttlicher Corresponding Author Martin Göttlicher Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany Search for more papers by this author Author Information Oliver H. Krämer1,4, Ping Zhu2,4, Heather P. Ostendorff3, Martin Golebiewski2, Jens Tiefenbach1, Marvin A. Peters3, Boris Brill1, Bernd Groner1, Ingolf Bach3, Thorsten Heinzel 1 and Martin Göttlicher 2,4 1Georg-Speyer-Haus, Paul-Ehrlich-Straße 42-44, D-60596 Frankfurt, Germany 2Forschungszentrum Karlsruhe, Institute of Toxicology and Genetics, H.-v.-H.-Platz 1, D-76344 Eggenstein, Germany 3Zentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Martinistraße 85, D-20251 Hamburg, Germany 4Present address: GSF National Research Center on Environment and Health, Institute of Toxicology, Ingolstädter Landstraße 1, D-85754 Neuherberg, Germany ‡O.H.Krämer and P.Zhu contributed equally to this work *Corresponding authors. E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2003)22:3411-3420https://doi.org/10.1093/emboj/cdg315 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Histone-modifying enzymes play essential roles in physiological and aberrant gene regulation. Since histone deacetylases (HDACs) are promising targets of cancer therapy, it is important to understand the mechanisms of HDAC regulation. Selective modulators of HDAC isoenzymes could serve as efficient and well-tolerated drugs. We show that HDAC2 undergoes basal turnover by the ubiquitin–proteasome pathway. Valproic acid (VPA), in addition to selectively inhibiting the catalytic activity of class I HDACs, induces proteasomal degradation of HDAC2, in contrast to other inhibitors such as trichostatin A (TSA). Basal and VPA-induced HDAC2 turnover critically depend on the E2 ubiquitin conjugase Ubc8 and the E3 ubiquitin ligase RLIM. Ubc8 gene expression is induced by both VPA and TSA, whereas only TSA simultaneously reduces RLIM protein levels and therefore fails to induce HDAC2 degradation. Thus, poly-ubiquitination and proteasomal degradation provide an isoenzyme-selective mechanism for downregulation of HDAC2. Introduction The recruitment of histone acetyltransferases (HATs) and histone deacetylases (HDACs) is considered as a key element in the dynamic regulation of many genes playing important roles in cellular proliferation and differentiation (Glass and Rosenfeld, 2000; Kouzarides, 2000). Hyperacetylation of the N-terminal tails of histones H3 and H4 correlates with gene activation, whereas deacetylation mediates transcriptional repression (Strahl and Allis, 2000). Consequently, many diseases have been linked to changes in gene expression caused by mutations affecting transcription factors. Aberrant repression by leukemia fusion proteins such as PML-RAR, PLZF-RAR, AML-ETO and Stat5-RAR serves as prototypical examples in this regard. In all of these cases, chromosomal translocations result in fusion proteins which convert transcriptional activators into repressors. These constitutively repress genes important for hematopoietic differentiation via recruitment of HDACs (Gelmetti et al., 1998; Grignani et al., 1998; Guidez et al., 1998; He et al., 1998; Lin et al., 1998; Lutterbach et al., 1998; Wang et al., 1998; Hildebrand et al., 2001; Maurer et al., 2002). Similar events could also contribute to pathogenesis in other types of cancer. Mammalian histone deacetylases can be divided into three subclasses (Gray and Ekström, 2001). HDACs 1, 2, 3 and 8, which are homologs of the yeast RPD3 protein, constitute class I. HDACs 4, 5, 6, 7, 9 and 10 are related to the yeast Hda 1 protein and form class II. Recently, mammalian homologs of the yeast Sir2 protein have been identified forming a third class of deacetylases. All of these HDACs apparently exist in the cell as subunits of multiprotein complexes. In particular, class I and II HDACs have been shown to interact with the transcriptional corepressors mSin3, N-CoR and SMRT, which recruit HDACs to transcription factors (Alland et al., 1997; Heinzel et al., 1997; Laherty et al., 1997; Nagy et al., 1997; Huang et al., 2000; Kao et al., 2000). Currently, only limited information is available about the isoenzyme-specific or redundant biological functions of these HDACs. Class II HDACs have been shown to translocate from the cytoplasm to the nucleus in response to external stimuli (McKinsey et al., 2000). In particular, HDAC9 acts as a signal-responsive suppressor of the transcriptional program governing cardiac hypertrophy and heart failure (Zhang et al., 2002). Class I HDACs are constitutively nuclear and play important roles in dynamic gene regulation. The essential biological function of class I HDACs is emphasized by the finding that targeted deletion of the HDAC1 gene leads to early embryonic lethality in mice, possibly due to a proliferation defect upon unrestricted expression of the cell cycle inhibitors p21 and p27 (Lagger et al., 2002). Since recruitment of HDACs leads to transcriptional repression, inhibitors of this enzymatic activity can reverse aberrant repression and lead to re-expression of genes inducing differentiation. Therefore, HDAC inhibitors are considered as candidate drugs in cancer therapy (Krämer et al., 2001; Marks et al., 2001; Melnick and Licht, 2002). The benefit of these enzyme inhibitors has been established by many in vitro experiments, experimental therapy and ongoing clinical trials (Warrell et al., 1998; Melnick and Licht, 2002). However, many HDAC inhibitors such as trichostatin A (TSA) do not exhibit isoenzyme selectivity and are of limited therapeutic value due to poor bioavailability in vivo as well as toxic side-effects at high doses. Recently, we discovered that the well-tolerated antiepileptic drug valproic acid (VPA) is a class I selective HDAC inhibitor (Göttlicher et al., 2001). This activity can be distinguished from its therapeutically exploited antiepileptic activity (Göttlicher et al., 2001; Phiel et al., 2001). Since VPA has been used clinically for over two decades, the pharmacology and side-effects of this drug have been studied in detail. As expected for HDAC inhibitory compounds, VPA induces differentiation of carcinoma cells, transformed hematopoietic progenitor cells and leukemic blasts from acute myeloid leukemia (AML) patients. Moreover, tumor growth and metastasis formation are significantly reduced in animal experiments (Göttlicher et al., 2001). Interestingly, VPA was also reported to have beneficial effects for patients suffering from neuroblastomas and glioblastomas even before its HDAC inhibitory properties were established (Driever et al., 1999). During our analysis of VPA effects on HDACs, we discovered that VPA but not TSA triggers proteasome-mediated degradation of HDAC2. Thus, VPA appears to act as an isoenzyme-selective downmodulator of HDAC2 at therapeutically useful concentrations by both inhibiting HDAC catalytic activity and inducing specific degradation of HDAC2. Results Reduction of HDAC2 protein levels by VPA treatment We investigated whether HDAC inhibitors would not only inhibit activity but also affect expression of HDACs. We found a significant downregulation of HDAC2 protein levels in cells treated with the carboxylic acids VPA or butyrate, whereas other HDAC inhibitors such as TSA and MS-27-275 failed to induce this effect (Figure 1A). Reduction of protein levels to ∼30% of untreated cells is found 24 h after VPA exposure and persists for at least 48 h (Figure 1B). The delayed response suggests the involvement of intermediary steps such as induction of protein expression. In murine F9 teratocarcinoma and human embryonic kidney HEK293T cells (Figure 1B and C), as well as in 14 additional cell lines (data not shown), a time- and dose-dependent reduction in HDAC2 protein levels upon VPA treatment is apparent. Protein levels of HDAC1 and HDAC3 were not reduced but even showed a transient induction in some experiments (Figure 1B). Furthermore, VPA treatment does not cause a reduction in protein levels of HDACs 4, 5 and 8 (data not shown). VPA doses required for reduction of HDAC2 protein levels are similar to those required for inhibition of HDAC enzymatic activity and clear effects are detected at 0.5–1 mM (Figure 1C). In HEK 293T cells which tolerate higher doses of VPA a more rapid and slightly more pronounced reduction is found if VPA concentrations are increased to 5 mM. Treatment with TSA for up to 48 h does not reduce HDAC2 levels in either F9 or HEK293T cells (Figure 1D). A significant reduction of HDAC2 protein levels is also found in vivo after treatment of mice with VPA (Figure 1E). Figure 1.VPA but not TSA leads to reduction of HDAC2 protein levels. (A) K562 human erythroleukemia cells were treated for 24 h as indicated with the HDAC inhibitors VPA (1.5 mM), TSA (100 nM), butyrate (1.5 mM) or MS-27–275 (5 μM). Amounts of HDAC2 protein were determined by western blot analysis of whole-cell extracts. Actin protein levels were determined to verify equal loading of samples. (B) F9 mouse teratocarcinoma or HEK293T human embryonic kidney carcinoma cells were exposed to 1 mM VPA for the indicated periods of time. Protein levels of HDAC2 as well as HDAC1, HDAC3 and actin were determined by western blot analysis. (C) The dose-dependent reduction of HDAC2 protein levels was determined in F9 or HEK293T cells after exposure to VPA for 30 or 24 h, respectively. (D) Time course analyses in F9 and HEK293T cells confirmed that TSA (100 nM) does not affect the amount of HDAC2 protein. (E) Reduction of HDAC2 protein levels after treatment of mice with VPA was tested by western blot analysis of tissue extracts and immunohistochemistry. Similar results were obtained in at least two sets of independent experiments. Download figure Download PowerPoint Induction of proteasomal degradation of HDAC2 Under conditions leading to a reduction in HDAC2 protein levels, no reduction of HDAC2 mRNA levels was found in F9 and HEK293T cells (data not shown). This finding suggests that VPA affects the rate of protein synthesis or degradation. HDAC2 protein synthesis rates with and without VPA pretreatment for 24 h were compared by pulse labeling with [35S]methionine in F9 or HEK293T cells. No substantial difference in HDAC2 synthesis rate between control or VPA-treated cells was found (Figure 2A). Figure 2.VPA induces degradation of HDAC2 protein. Synthesis and degradation rates of HDAC2 were determined by [35S]methionine labeling followed by chase analyses. [35S]HDAC2 was detected by HDAC2-specific immunoprecipitation, SDS–PAGE and autoradiography. (A) Pulse labeling for 1 h was performed in F9 and HEK293T cells, which were precultured for 24 h in the absence or presence of 1 mM VPA (F9) or 1.5 mM VPA (HEK293T). (B) For pulse–chase analysis, cells were cultured for 24 h either in the absence or presence of 1 mM VPA (F9) or 1.5 mM VPA (HEK293T) and labeled with [35S]methionine for an additional hour without or with VPA. After removal of [35S]methionine and addition of non-labeled methionine, the elimination of radiolabeled HDAC2 was followed over a period of 6 h. A pulse–chase analysis was also performed in non-pretreated HEK293T cells to which VPA was added only at the time when the chase was started [row marked (+)]; VPA at time of chase, triangles. Efficiency of immunoprecipitations was confirmed by proving depletion of HDAC2 from the precipitation supernatants. Comparable loading between lanes was controlled by Coomassie staining of gels for the amounts of antibodies. Similar results were obtained in at least a second independent experiment. (C) HDAC3 was also precipitated from HEK293T cell extracts. (D and E) Phosphoimager analyses of the experiments in (B) and (C) were quantitatively evaluated by subtraction of local background. The graphs show averages of two or three experiments (control, open symbols; VPA, filled symbols; VPA at time of chase, triangles) and were used for half-life determination. Standard deviations are shown if bars exceed symbol sizes. Download figure Download PowerPoint Protein half-life of HDAC2 was determined by pulse–chase analysis and was substantially decreased by pretreatment of cells with VPA (Figure 2B). In F9 cells, a decrease from 3 to 1.2 h was found and in HEK293T cells the change was from 6.8 to 2.4 h (Figure 2D). VPA had no effect on the HDAC2 protein degradation rate when added only at the time of chase (Figure 2B and D). This experiment provided additional evidence that the response of HDAC2 protein levels to VPA is indirect rather than being a direct response, for example, to a conformational change of HDAC2 upon interaction with VPA. Degradation of HDAC3 was not affected by VPA-pretreatment of HEK293T cells (Figure 2C and E), consistent with a lack of reduction in steady-state HDAC3 protein levels upon VPA treatment. To investigate whether HDAC2 degradation is due to either protease-dependent or proteasomal degradation, several inhibitors of proteases or the proteasome were applied in combination with VPA (Figure 3A). None of the protease inhibitors pepstatin A, leupeptin or ALLM had an effect on HDAC2 levels in the presence or absence of VPA (Figure 3A). Treatment of HEK293T cells with the proteasome inhibitors ALLN or MG-132 either abolished or significantly reduced VPA-induced degradation of HDAC2 (Figure 3A). Thus, increased proteasomal degradation by a mechanism involving synthesis of intermediary factors appears to be the most likely cause of VPA-induced degradation of HDAC2. Figure 3.VPA induces polyubiquitination and proteasome-dependent degradation of HDAC2. (A) HEK293T cells were treated for 24 h with VPA or left untreated. The protease inhibitors pepstatin A (20 μM), leupeptin (10 μM), or ALLM (20 μM), or the proteasome inhibitors ALLN (5 μM), or MG 132 (10 μM), were added at the same time as VPA. HDAC2 protein levels were determined by western blot analysis. (B) Precipitation of poly-ubiquitinated proteins with anti-HDAC2 antibody as well as high molecular weight anti-HDAC2-reactive proteins with anti-ubiquitin antibody was tested by immunoprecipitation analysis from extracts of HEK293T cells that had been left untreated or were treated with 1.5 mM VPA for 36 h. The proteasome inhibitors ALLN (25 μM) or MG 132 (20 μM) were added 4 h before cell harvest. The presence of ubiquitinated proteins in anti-HDAC2 immunoprecipitates is shown in the middle panel. The left panel shows the corresponding control experiment with preimmune serum instead of the anti-HDAC2 antibody. The right panel shows precipitation with an anti-ubiquitin or non-immune (pre) serum and detection of poly-ubiquitinated HDAC2 protein by western blot analysis. (C) Ubiquitinated HDAC2 was also precipitated from F9 cells that had been treated with 1 mM VPA in experiments comparable to those in (B). One representative example of two independent experiments is shown. Download figure Download PowerPoint The most common mechanism of targeting proteins for degradation by the proteasome depends on poly-ubiquitination (Hicke, 2001). Therefore, the presence and VPA-dependent induction of HDAC2 ubiquitination were tested. Immunoprecipitates generated with an anti-HDAC2 antibody were analyzed by western blot against ubiquitin for the presence of high molecular weight ubiquitinated proteins. Untreated cells contain only a small amount of ubiquitinated proteins that precipitate with anti-HDAC2 antibody (Figure 3B, middle panel). Pretreatment of cells with VPA alone reduced this signal, possibly due to reduced levels of HDAC2 at the time of analysis. The proteasome inhibitor ALLN alone had no significant effect. Only cotreatment with VPA and proteasome inhibitors dramatically increased the amount of precipitated high molecular weight ubiquitinated proteins. The signal is specific for immunoprecipitates formed with an antibody against HDAC2 since preimmune serum did not precipitate detectable amounts of anti-ubiquitin reactive proteins (Figure 3B, left panel). TSA has no effect on the amount of immunoprecipitated anti-ubiquitin-reactive material (data not shown). These data suggest that VPA treatment induces ubiquitination of one or several proteins precipitated with an antibody against HDAC2. However, this assay does not distinguish whether HDAC2 itself or proteins associated with HDAC2 are poly-ubiquitinated. To obtain evidence for direct ubiquitination of HDAC2, an anti-ubiquitin antibody was used for immunoprecipitation and western blots were probed with an anti-HDAC2 antibody. A high molecular weight band migrating slower than unmodified HDAC2 was detected, which is consistent with the presence of poly-ubiquitinated HDAC2 (Figure 3B, right panel). Similar to the result of the anti-HDAC2 precipitation, the intensity of the HDAC2-reactive signal was increased in the anti-ubiquitin precipitation only when cells had been treated with both VPA and a proteasome inhibitor. However, mono- and oligo-ubiquitinated forms of HDAC2 could not be detected, possibly due to the small amount of material that can be precipitated and/or insufficient sensitivity of the assay. Similar results were obtained from experiments in F9 cells (Figure 3C). In addition, an increase in high molecular weight material is already seen only after treatment with proteasome inhibitor, suggesting that HDAC2 is also to some extent ubiquitinated in the absence of VPA. In addition to those bands seen in HEK293T cell extracts, F9 cells show prominent bands consistent with mono- and oligo-ubiquitinated HDAC2 in both immunoprecipitations. Similar results were obtained when HEK293T cells were transfected with a His6-tagged form of ubiquitin and Ni2+-NTA–agarose was used for precipitation (data not shown). Under these conditions, bands that are consistent with the mobility of mono- or oligo-ubiquitinated HDAC2 are also detectable in HEK293T cells. Identification of the ubiquitination machinery for HDAC2 Ubiquitin-conjugating and -ligating enzymes involved in the modification of HDAC2 were identified by two approaches. A systematic search for VPA-inducible genes in F9 cells by suppression subtractive hybridization (SSH) had revealed that expression of the E2 ubiquitin-conjugating enzyme Ubc8 (the ortholog of yeast Ubc4/Ubc5) is induced by VPA (M.Golebiewski, unpublished data). This was confirmed by real-time RT–PCR analysis (Figure 4A) and northern blot analysis of murine F9 and human HEK293T cells using species-specific probes for murine Ubce8 and its human homolog UbcH8, respectively (Figure 4B). Subsequently, the term Ubc8 will refer to either the human or the murine form, depending on which cell type is used. Although the induction of the E2 ligase Ubc8 could account for the increased degradation of HDAC2, it could not explain the lack of an effect in TSA-treated cells since Ubc8 is also induced by TSA at least as efficiently as by VPA (Figure 4A). Ubc8 is also induced at the protein level and, again, there is no substantial difference between VPA and TSA (Figure 4C). Figure 4.The HDAC inhibitors VPA and TSA induce expression of the E2 ubiquitin-conjugating enzyme Ubc8, whereas the E3 ligase RLIM is differentially regulated by TSA and VPA. (A) F9 cells were treated for 17 h with 1 mM VPA (V) or 100 nM TSA (T) and expression levels of Ubce8 mRNA were determined by real-time RT–PCR assuming a 1.5-fold amplification per cycle. The amplicon was part of the coding region of the Ubce8 mRNA. Results were normalized for GAPDH expression. Average values ± range of two independent experiments, each with triplicate determinations. (B) Northern blot analysis of 5 μg of poly(A)+ mRNA from F9 or HEK293T cells treated for the indicated times with 1 mM VPA (F9 cells) or 1.5 mM VPA (HEK293T cells) was performed to confirm RT–PCR results. Probes were derived from the 3′-UTR of murine Ubce8 or the 5′ coding region of human UbcH8. Phosphoimager analysis was performed for quantitative evaluation and relative values for Ubc8 mRNA abundance normalized for GAPDH are presented below the panels. One of two experiments with similar results is shown. (C) Ubce8 protein levels were determined by western blot analysis in total extracts of F9 cells that had been treated for 24 h as indicated. (D) HEK293T cells were treated for 24 h with VPA, TSA or ALLN (2.5 μM) as indicated. Abundance of the E3 ubiquitin ligase RLIM was determined by western blot analysis. Actin protein levels were determined to verify equal loading. Download figure Download PowerPoint The second approach to finding potential ubiquitin ligases for HDAC2 relied on the previous identification of RLIM as a negative regulator of LIM homeodomain transcription factors (LIM-HDs). RLIM represses LIM-HD-dependent gene expression by several apparently complementary mechanisms, which include recruitment of the mSin3 corepressor (Bach et al., 1999) and E3 ubiquitin ligase activity towards the CLIM coactivators of LIM-HDs (Ostendorff et al., 2002). Since RLIM has been shown to interact with a corepressor in vivo, we speculated that it might also act towards other components of corepressor complexes, such as HDACs. The analysis of RLIM protein expression in cells treated with the HDAC inhibitors VPA or TSA revealed a differential response. VPA did not alter RLIM expression, whereas TSA reduced RLIM protein levels (Figure 4D, left panel). Steady-state mRNA levels of RLIM were not changed by any of the HDAC inhibitors (data not shown). Therefore, TSA is likely to affect either synthesis or stability of RLIM protein. The fact that a proteasome inhibitor prevents TSA-dependent downregulation of RLIM protein levels suggests that TSA, in contrast to VPA, induces proteasomal degradation of RLIM by an as yet unidentified mechanism (Figure 4D, right panel). Interestingly, Ubc8 serves as an E2-conjugating enzyme for RLIM so that both proteins could act in concert (Ostendorff et al., 2002). In order to demonstrate that the regulation of both Ubc8 and RLIM by HDAC inhibitors provides a plausible explanation for the selective downregulation of HDAC2, it is essential to show that they can act on HDAC2 as a substrate. The first indication for that came from the finding that RLIM can be coprecipitated with antibodies against HDAC2 (Figure 5A). RLIM does not coprecipitate with HDACs 1 and 3 (Figure 5A), which is likely to contribute to the selective degradation of HDAC2. We subsequently tested whether HDAC2 is a substrate for Ubc8- and RLIM-dependent ubiquitination by an in vitro ubiquitination assay using bacterially expressed proteins and in vitro translated HDAC2 as a substrate (Figure 5B). HDAC2 was ubiquitinated in vitro, as indicated by both the reduction in non-modified HDAC2 levels and the appearance of slower migrating proteins of the expected mobility of mono-, oligo- and poly-ubiquitinated HDAC2. Efficient ubiquitination was only found when both Ubc8 and RLIM were included in the reactions, but not if only ubiquitin and E1 enzyme were present. This assay appears to be specific, since the GATA-4 transcription factor and its cofactor FOG-2 were not ubiquitinated in a control experiment (data not shown). Figure 5.RLIM interacts with HDAC2 and induces poly-ubiquitination in vitro. (A) Interaction of HDACs 1, 2 and 3 with RLIM was tested by co-immunoprecipitation from whole-cell extracts of HEK293T cells that had been pretreated with the proteasome inhibitor ALLN (25 μM) for 4 h. Immunoprecipitations efficiently depleted HDACs from the cell extracts (not shown) and coprecipitated RLIM was detected by western blot analysis. Control immunoprecipitations were performed with non-immune serum. The left lane shows 5% of the extract used for immunoprecipitation. (B) In vitro translated [35S]methionine-labeled HDAC2 was incubated for 2 h in buffer with E1 ubiquitin ligase and ubiquitin only, or in reactions containing the recombinantly expressed E2 ubiquitin ligase Ubce8 without or with the E3 ligase RLIM. For control (left lane), the reaction was stopped immediately after addition of ubiquitin and E1 ligase. Loss of input HDAC2 and appearance of high molecular weight radiolabeled proteins were determined by SDS–PAGE followed by autoradiography. Download figure Download PowerPoint To test whether HDAC2 is also a substrate of Ubc8 and RLIM in the cell and whether the abundance of these enzymes is rate limiting for HDAC2 degradation, both enzymes were overexpressed by transient transfection. A rate-limiting role of Ubc8 for HDAC2 degradation could be confirmed since increasing amounts of transfected Ubc8 expression vector gradually decrease the abundance of HDAC2 in murine F9 or human HEK293T cells in the absence of VPA (Figure 6A). Interestingly, murine Ubce8 and human UbcH8 exhibit relevant differences since they are fully functional towards HDAC2 only if expressed in cells from their species of origin. Unchanged levels of β-actin and HDACs 1 and 3 (data not shown) indicate that the effect on HDAC2 is specific and not due to general protein degradati
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