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1 Graduate School of Comprehensive Human Sciences, Center for Tsukuba Advanced Research Alliance, and ERATO Environmental Research Project, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba 305-8577, Japan
2 Tohoku University Biomedical Engineering Research Organization, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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A correlation between GATA-2 expression and cellular proliferation/survival has been suggested from a broad range of analyses. In particular, in Gata2–/– yolk sacs, the proliferation of colony forming cells was impaired and these cells became apoptotic. This phenotype was restored in Gata2-p53 double knockout mice (Tsai et al. 1997), suggesting that GATA-2 may antagonize p53-mediated apoptosis in proliferating hematopoietic cells. Analysis using an in vitro differentiation system of embryonic stem (ES) cells revealed that exogenous GATA-2 expression induces proliferation of erythroid lineage cells (Kitajima et al. 2002). In quail yolk-sac hematopoiesis, the retinoid-BMP-4-GATA-2 signaling pathway appears to control progenitor cell survival, with a lack of this signal pathway resulting in a significant reduction in erythroid cells (Ghatpande et al. 2002). We prepared green fluorescent protein (GFP) knock-in mice, in which the GFP gene was inserted into the Gata2 gene (Minegishi et al. 2003). The fluorescence signal from GFP was detected in proliferating hematopoietic progenitor cells in culture but not in the quiescent cells (NS and MY, in preparation). In the preadipocyte/adipocyte system and in regenerative muscles, GATA-2 has been shown to be a marker of immature proliferating cells (Musaro et al. 1999, 2001; Tong et al. 2000). In contrast, exogenous GATA-2 expression showed negative effects in the proliferation of myeloid cells and normal bone marrow stem/progenitor cells (Heyworth et al. 1999; Persons et al. 1999; Ezoe et al. 2002). Although the positive and negative roles of GATA-2 are thus described, the molecular mechanisms linking GATA-2 expression with cellular proliferation and/or survival remain to be clarified.
It has been well documented that both synthesis and degradation processes determine the expression level of individual proteins and proteins contain signals that determine their final destinations. Rapid protein degradation is particularly important for the accurate and succinct regulation of steady state protein levels of many key regulatory proteins. Indeed, a number of transcription factors (Salghetti et al. 2001; Freiman & Tjian 2003; Lipford & Deshaies 2003; Nie et al. 2003), especially those involved in hematopoietic cell differentiation (Li et al. 2003; Zhang et al. 2003; Kanei-Ishii et al. 2004), are under the control of rapidly acting protein degradation machinery. Proteins regulating proliferation, cell-cycle progression and apoptosis are also found to turn over rapidly (Naujokat & Hoffmann 2002; Busino et al. 2004; Pickart 2004; Yang et al. 2004; Zhang et al. 2004). One of the major regulatory protein degradation pathways is the ubiquitin-proteasome pathway, in which the poly ubiquitination of target protein is the trigger for degradation (Schnell & Hicke 2003; Pickart 2004).
In this study, we addressed how the transcription factor GATA-2 turns over in cells. We found that GATA-2 protein degrades rapidly in hematopoietic and neuroblastoma cell lines. We also found that ultraviolet (UV)-C (254 nm) irradiation of the cells induces instability of the GATA-2 protein, thereby severely repressing its expression level. GATA-2 is degraded through the ubiquitination-proteasome pathway and three regions (or degrons) that appear to be important for this degradation were identified, two of which reside within the transcription activation domains. These degrons contain putative protein modification motifs, through which cellular signals can regulate the stability of GATA-2 in hematopoietic cells.
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Results |
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In order to address how GATA-2 is degraded inside cells, we examined any changes in the endogenous GATA-2 protein level after cycloheximide treatment in an immunoblot analysis with anti-GATA-2 antibody. As shown in Fig. 1A, GATA-2 rapidly disappeared in P815 cells following cycloheximide treatment, while ß-actin expression was practically unchanged. Densitometric analyses of three independent GATA-2 immunoblots, which were normalized with the band intensity of ß-actin immunoblots, showed that the half-life of endogenous GATA-2 in P815 cells was 27.7 ± 6.1 min (Fig. 1C). This short half-life of GATA-2 is remarkable and similar to those of the other rapid turnover proteins: for instance, p21 (Bendjennat et al. 2003; Bloom et al. 2003; Jin et al. 2003), p53 (Zilfou et al. 2001), Nrf2 (Itoh et al. 2003), Skp2 and Cks1 (Bashir et al. 2004). We also examined the half-life of GATA-2 in 32D cells, a mouse leukemia-derived and cytokine-dependent cell line, and in a C1300 mouse neuroblastoma cell line. Determination of the half-life for GATA-2 in each cell line is shown as a line on the semilogarithmic plot. The results showed that GATA-2 was degraded rapidly in both cell lines, though with a half-life longer than that in P815 cells and there was a moderate fluctuation in the half-life determination of GATA-2 (Fig. 1D,E).
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In contrast, the half-life of GATA-1 appeared to be much longer than that of GATA-2. When we determined the half-life of GATA-1 in DS19 mouse erythroleukemia cells, cycloheximide treatment only marginally influenced the GATA-1 expression level (Fig. 1B). The half-life of GATA-1 was apparently longer than 6 h. These results thus unveiled that, whereas GATA-1 is a stable protein, GATA-2 is a short-lived protein that turns over rapidly.
Proteasome-dependent degradation of GATA-2
To clarify the mechanisms of GATA-2 degradation, we treated cells with the proteasome inhibitors. Simultaneous treatment with 10-µM MG132 or clasto-Lactacystin counteracted the inhibitory effect of cycloheximide and GATA-2 accumulated within the P815 cells (Fig. 2A). In the immunostaining experiments of C1300 cells, the staining intensity of anti-GATA-2 antibody in Fig. 2B was weak and varied from cell to cell (Fig. 2B, green). Treatment of MG132 or clasto-Lactacystin intensified the nuclear staining of GATA-2 in a large number of cells (Fig. 2D,E), while the cycloheximide treatment weakened the staining of GATA-2 (Fig. 2C). These results suggest that GATA-2 is degraded through the proteasome-dependent degradation pathway in hematopoietic and neuroblastoma cells and that the intranuclear expression level of GATA-2 is regulated by this turnover.
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In our search for cellular conditions stimulating the degradation of GATA-2, we found that UV-C irradiation of cells provokes the rapid degradation of GATA-2. After 30-J UV-C irradiation, the GATA-2 protein level in P815 cells started to decrease within 15 min (Fig. 3A), while in contrast the mRNA expression level of GATA-2 was unchanged (Fig. 3B). Induction of the p53-mediated apoptotic pathways by this UV-C irradiation was evident, as Perp, one of the target genes of p53 (Attardi et al. 2000; Chao et al. 2000; Ihrie et al. 2003), was induced within 4 h following UV-C irradiation (Fig. 3C), and the percentage of total cells which were apoptotic increased to 34% after 10 h (data not shown).
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Consistent with that observed in P815 cells, UV-C irradiation also induced instability in the GATA-2 protein in both 32D and C1300 cells (Fig. 4A). The UV-C irradiation significantly decreased the expression level of GATA-2 in P815 cells within 5 min, but had no additive effect on the decline of GATA-2 expression at 30 min after cycloheximide treatment (Fig. 4B). The half-life of GATA-2 was not shortened in P815 cells (Fig. 4C). These observations indicate that the UV-C treatment affects acutely the GATA-2 expression level and suggest that the activation of the protein degradation pathway may constitute the primary cause of the reduction in GATA-2 after UV-C irradiation. The preventive effect of MG132 (Fig. 4A,D) further supports the notion that the proteasome-dependent protein degradation pathway plays an important role in the rapid turnover of the GATA-2 protein.
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To identify the regulatory region or degron responsible for GATA-2 degradation, we carried out structure-function analyses on the GATA-2 protein. Plasmid vectors expressing several domains of GATA-2 as fusion proteins of yeast GAL4-DNA binding domain (GBD) were constructed (Fig. 5A). These plasmids were transfected into 293T cells along with the green-fluorescent-protein (GFP) expression plasmid and Luciferase reporter plasmid as an internal and loading control. Cells were harvested 48 h after transfection, with or without MG132 treatment for the last 12 h, and total cell lysates were prepared for immunoblotting. GBD-G2 (1–70) and GBD-G2 (412–480) fusion proteins were only faintly detectable in 293T cells without MG132 treatment, whereas GBD-G2 (69–149) and GBD-G2 (256–410) fusion proteins were stably expressed in these cells (Fig. 5B). The MG132 treatment significantly restored the expression of GBD-G2 (1–70), GBD-G2 (153–256), and GBD-G2 (412–480) fusion proteins, but not of GBD-G2 (69–149) and GBD-G2 (256–410) fusion proteins and control GFP. Intriguingly, in the luciferase reporter assay with the same GBD fusion plasmids both the GBD-G2 (1–70) and GBD-G2 (412–480) fusion proteins showed significant transcriptional activity (Fig. 5C), indicating that these two regions retain transactivation activity.
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1-74 mutant was much higher than wild-type GATA-2 and was not influenced significantly by MG132 treatment (Fig. 6B, lanes 3 and 4). The expression levels of the 154-256, 1-235, and 441-480 mutants were also higher than wild-type GATA-2 (Fig. 6B, lanes 7–12). These results are consistent with those from the GBD-GATA-2 fusion protein analysis, in that the expression of GBD-GATA-2 fusion proteins containing G2 (1–70), G2 (153–256), and G2 (412–480) regions were unstable, but their accumulation restored by the MG132 treatment (see Fig. 5). Thus, multiple degrons or protein degradation regulatory elements exist in GATA-2. Ubiquitination of GATA-2
Given that GATA-2 degradation is proteasome dependent, we surmised that there might be ubiquitin-conjugated forms of GATA-2. To address this point, 293T cells were transfected with expression plasmids for Flag-tagged wild-type GATA-2 or 1-235 along with a poly histidine (His)-tagged ubiquitin expression plasmid. Total cell extracts were prepared from the transfected and MG132-treated 293T cells, and the GATA-2 proteins were immunoprecipitated with anti-Flag antibody. Immunoblot analysis with anti-GATA-2 antibody demonstrated that both wild-type and 1-235 existed in the immunoprecipitates (Fig. 7A). The immunoblot analysis with both anti-ubiquitin and anti-GATA-2 antibodies demonstrated high molecular weight bands in the wild-type GATA-2 lanes of anti-Flag immunoprecipitates (Fig. 7A, lane 2), indicating that there were poly ubiquitinated forms of wild-type GATA-2 in the cells.
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1-235 were marginally detectable with the anti-GATA-2 antibody (Fig. 7A, lane 3 and Fig. 7B, lane 6). One explanation of the result is that one of the degrons may reside in the G2 (1–235) region of GATA-2, while the other explanation may be that epitopes for the GATA-2 antibody were masked due to the ubiquitin conjugation.
We also carried out immunoprecipitation-immunoblot analyses of 1-74, 153-256, and 440-480 mutants, which are deleting responsible regions for the rapid degradation (see Figs 5 and 6). Higher molecular weight bands were observed for all three GATA-2 mutants with anti-ubiquitin and anti-His tag antibodies (Fig. 7C). These results thus suggest that there may be multiple pathways leading to the GATA-2 degradation.
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Discussion |
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We surmise that the functional contributions of GATA factors may be categorized into two. One is a general function that is interexchangeable between the GATA factors, while the other is the specific function of individual GATA factors. For instance, the arrest of erythroid maturation caused by a hypomorphic Gata1 gene mutation (GATA-1.05 allele) can be rescued by the expressions of GATA-2 and GATA-3 under the control of the Gata1 gene hematopoietic regulatory domain (G1-HRD, Takahashi et al. 2000). A much more profound ablation of primitive hematopoiesis was found in Gata1–/y::Gata2–/– double knockout embryos than in Gata1–/y or Gata2–/– single knockout embryos (Fujiwara et al. 2004), suggesting the presence of overlapping functions of GATA-1 and GATA-2. In contrast, this study shows the difference in stability of the GATA-1 and GATA-2 proteins, the former being much more stable than the latter. Importantly, the non-finger regions of GATA-2 appear to contribute to its rapid turnover (see Fig. 8). It has been reported that in immature erythroid cells, death receptor signals and caspase degrade GATA-1, but not GATA-2 (De Maria et al. 1999). The cleavage site of caspase is also localized in the non-finger region. Thus, degradation of GATA-2 and GATA-1 proteins exploits different pathways and each contributes to the unique function of these factors.
Elucidation of the molecular mechanisms as to how GATA-2 is degraded inside cells and how protein modification contributes to this process are the immediate questions to be addressed. In this study, we identified the strongest degron in the G2 (1–70) N-terminal domain that also retained transactivation activity. The overlap of degrons with the transactivation domains has also been observed in other transcription factors, E2F1, Fos, Jun, c-Myc, and GCN-5 (Salghetti et al. 2000, 2001; Lipford & Deshaies 2003), and ubiquitin binding and proteasome activity appear to influence the transcription of target genes (Freiman & Tjian 2003; Lipford & Deshaies 2003; Schnell & Hicke 2003). For instance, when recruited to c-Myc target promoters, the SCFskp2 E3 ligase activates target gene transcription and promotes ubiquitination and proteasome-mediated degradation of c-Myc simultaneously (Lehr et al. 2003). In this case, it has been speculated that firing off a round of transcription would inevitably require a fresh molecule of activator (Lipford & Deshaies 2003). Therefore, it is possible that the degradation of GATA-2 may be also coupled with the transcription of target genes.
Interestingly, while the G2 (1–70) region does not contain any lysine residues that can be ubiquitinated, this region induces degradation of GBD-fusion proteins in transfected cells. One plausible explanation is that the G2 (1–70) region might regulate ubiquitin conjugation to lysine residues in the GBD. Actually, Cul1-Rbx1-Skp1-F box SCF complex, one of ubiquitin ligase complexes, has a highly elongated structure with segregated substrate-binding site (Skp2) and ubiquitin-binding site (Rbx1) located to opposite ends (Zheng et al. 2002), and the calculated distance from Skp2 to Rbx1 is long enough to assume that the ubiquitin ligase complex binding to one domain of the substrate would induce ubiquitination of lysine residues in the neighboring domain. Alternatively, the detection of ubiquitinated forms of 1-74 mutant in our analysis (Fig. 7) suggests that the function of G2 (1–70) in ubiquitin conjugation may be indirect through protein modification.
The protein degradation process is occasionally regulated by phosphorylation (Taylor et al. 2000; Brooks & Gu 2003; Nie et al. 2003; Kanei-Ishii et al. 2004). Indeed, GATA-2 protein contains multiple phosphorylation sites and was phosphorylated by MAP kinases (Towatari et al. 1995). In the immunoblotting analysis of 32D and C1300 cells, we noticed that there is a band slightly shifted from the wild-type GATA-2 position (Fig. 4A), suggesting the existence of a phosphorylated form of GATA-2. It is interesting to note that the G2 (153–256) region is rich in proline (P), glutamate (E), serine (S), and threonine (T) residues (43.3%; see Fig. 8) and that the GBD-G2 (153–256) protein produced faint and multiple bands in immunoblotting analysis, suggesting that the PEST sequence-like activity (Rechsteiner & Rogers 1996) of this region may contribute to the induction of GATA-2 protein instability.
Lysine residues are known to regulate the ubiquitin-proteasome degradation pathway, not only through their conjugation with ubiquitin, but also through their modification with small ubiquitin-related modifier (SUMO) or acetylation (Desterro et al. 1998; Hochstrasser 2001; Gronroos et al. 2002; Comerford et al. 2003; Freiman & Tjian 2003; Stelter & Ulrich 2003). Indeed, both the SUMOylation of GATA-2 (Chun et al. 2003) and acetylation of GATA-2 by p300 and GCN5 (Hayakawa et al. 2004) have been reported. However, we did not observe distinct bands that are consistent with SUMO-conjugated forms of GATA-2 in hematopoietic or neuroblastoma cells, although one consensus SUMO-conjugation motif resides within the G2 (153–256) region responsible for the protein instability (Hochstrasser 2001). The acetylated residues of GATA-2 were outside the three proposed degrons (Hayakawa et al. 2004). Nonetheless, it should be noted that GATA-2 is rich in serine, threonine and lysine residues, which are well conserved in human, mouse, chicken, Xenopus and zebrafish (Fig. 8). Therefore, we surmise that it is likely that the mechanisms inducing phosphorylation, SUMO-conjugation and acetylation of GATA-2 may have certain relation to GATA-2 protein degradation.
In this study, it was found that UV-C irradiation, which induces the p53-Perp apoptotic pathway (Attardi et al. 2000; Ihrie et al. 2003), triggers the down-regulation of GATA-2 expression. Our results are consistent with the proposition that GATA-2 counteracts the p53 pathways in hematopoietic cells as a survival factor. This concept is supported by the report of partial rescue of the apoptotic phenotype of GATA-2–/– hematopoietic cells by a loss of p53 activity (Tsai et al. 1997) and the findings that GATA-2 regulates the proteasome-dependent degradation of p21waf1 and p27kip1 (Ezoe et al. 2002). However, to identify the exact roles GATA-2 plays in cell survival and apoptosis, detailed analyses of the modulators of GATA-2 function as well as target genes of GATA-2 should be executed. A comprehensive understanding of GATA-2 expression seems to be of importance in clarifying the integrity of the differentiation, proliferation and survival of hematopoietic stem and progenitor cells.
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Experimental procedures |
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Murine leukemia-derived P815 cells and C1300 neuroblastoma cells were cultured in RPMI 1640 medium (Sigma, St. Luis, MO, USA) containing 10% fetal bovine serum (FBS) and 5% CO2. The IL-3-dependent hematopoietic 32D cells, and Ba/F3 cells were cultured in RPMI 1640 medium containing 10% FBS and 10% WEHI3 conditioned medium. A human kidney epithelial cell line 293T was cultured in the DMEM (Sigma) with 10% FBS. The pcDNA3.0-based plasmids were constructed by insertion of the tagged cDNA sequences amplified by PCR. Transfection experiments and the luciferase-reporter analysis were performed using FUGENE 6 reagent (Roche Diagnostics Co., Indianapolis, USA) and a dual-luciferase reporter assay system (Promega, Madison, WI, USA). MG132 and clasto-Lactacystin were purchased from CALBIOCHEM and cycloheximide was purchased from Sigma, respectively.
Antibodies and immunoblotting
Anti-GATA-2 monoclonal antibody (RC1.1.1) was previously described (Minegishi et al. 2003). Anti-GATA-2 goat polyclonal antibody (c-20), anti-GATA-2 rabbit polyclonal antibody (H116), anti-Ubiquitin antibody (P4D1), and anti-histidine tag antibody (H15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Flag antibody (M2) was from Sigma. The SDS-polyacrylamide gel electrophoresis and immunoblotting analyses were performed as described (Minegishi et al. 2003). The data analysis was performed using GS-710 Calibrated Imaging Densitometer and VersaDoc3000 with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
Immunostaining of GATA-2
C1300 cells were cultured in glass-bottom dishes and fixed with 4% paraformaldehyde in PBS for 10 min. Fixed cells were then permeabilized with 0.5% Triton X-100 for 5 min. For blocking nonspecific binding, SuperBlock blocking solution in PBS (PIERCE, Rockford, IL, USA) containing 1 x blocking reagent for ELISA (Roche) and 5% FBS was used for washing and dilution of antibodies. Fluorescence Allexa-488-conjugated (Molecular-Probe, Eugene, OR, USA) secondary antibodies were analyzed with laser-confocal microscopy (Carl Zeiss, LSM5). Nuclear DNA was stained with TOPRO3 (Molecular-Probe).
Protein ubiquitination
Total cell extract was prepared from transiently transfected cells using M-PER reagent (PIERCE) containing a protease inhibitor cocktail (Complete, Roche Diagnostics, Mannheim) and mixed with SuperBlock blocking solution in PBS. Flag-tagged proteins were then purified with anti-Flag M2 affinity gel (Sigma). His-tagged proteins were purified using EzviewTM Red HIS-SelectTM HC Nickel Affinity Gel (Sigma) with 1 x RIPA buffer containing 350 mM NaCl, 30 mM Imidazole (Sigma) and protease inhibitor cocktail. Purified proteins were analyzed by immunoblotting with anti-GATA-2 (c-20) and anti-ubiquitin antibodies.
RNA extraction, Northern blotting and RT-PCR analysis
Total RNA was extracted from UV-irradiated p815 cells with ISOGEN (Nippon Gene, Toyama). Northern blotting and reverse transcription (RT)-PCR analysis were performed according to the methods previously described (Minegishi et al. 1998). Primers used were as follows: Perp sense primer (5'-ACGACATGCTGCGCTGCGGC-3'), Perp anti-sense primer (5'-TCAGGCTCTTCCTCCCACATTAGGCTGG -3'), HPRT sense primer (5'-GCTTGCTGGTGAAAAGGACC-3'), and HPRT anti-sense primer (5'-CAACTTGCGCTCATCTTAGGC-3').
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: masi{at}tara.tsukuba.ac.jp; nmine{at}tubero.tohoku.ac.jp
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Received: 9 March 2005
Accepted: 28 March 2005
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