Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb

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Abstract

Expression of oncoprotein c-Myb oscillates during hematopoiesis and hematological malignancies. Its quantity is not only regulated through transcriptional control but also through the ubiquitin–proteasome pathway, accompanied by phosphorylation, although the mechanisms are poorly understood. In this report, we tried to identify an E3 ubiquitin ligase, which targets c-Myb for ubiquitin-dependent degradation. We found that an F-box protein, Fbw7, interacted with c-Myb, which is mutated in numerous cancers. Fbw7 facilitated ubiquitylation and degradation of c-Myb in intact cells. Moreover, depletion of Fbw7 by RNA interference delayed turnover and increased the abundance of c-Myb in myeloid leukemia cells concomitantly, and suppressed the transcriptional level of γ-globin, which receives transcriptional repression from c-Myb. In addition, we analysed sites required for both ubiquitylation and degradation of c-Myb. We found that Thr-572 is critical for Fbw7-mediated ubiquitylation in mouse c-Myb using site-directed mutagenesis. Fbw7 recognized the phosphorylation of Thr-572, which was mediated by glycogen synthase kinase 3 (GSK3). In consequence, the c-Myb protein was markedly stabilized by the substitution of Thr-572 to Ala. These observations suggest that SCFFbw7 ubiquitin ligase regulates phosphorylation-dependent degradation of c-Myb protein.

Introduction

c-Myb is a transcription factor expressed at high levels in immature progenitors of all hematopoietic lineages. It is associated with the regulation of proliferation, differentiation and survival (Oh and Reddy, 1999). c-Myb can also act as an inhibitor of terminal differentiation of both erythroidblastic and myeloblastic leukemia cell lines (Selvakumaran et al., 1992; Bies et al., 1995). It is downregulated during terminal differentiation to mature blood cells (Ramsay et al., 1986). Ablation of c-Myb expression and/or activity by gene targeting or antisense/dominant-negative strategies has shown that c-Myb is essential for fetal liver hematopoiesis, erythroid and myeloid bone marrow colony formation and T-cell development (Friedman, 2002; Emambokus et al., 2003; Lieu et al., 2004; Vegiopoulos et al., 2006). On the other hand, overexpression of c-Myb in c-Myb−/− ES cells prevented the terminal differentiation of erythrocytes and megakaryocytes and completely abolished B-lymphocyte development (Sakamoto et al., 2006). These observations indicate that c-Myb has multiple cellular roles during normal hematopoiesis and that appropriate levels of the c-Myb protein are strictly defined at distinct differentiation steps of each hematopoietic cell lineage.

An elevated c-Myb expression has been reported in many cases of acute myeloblastic and lymphoblastic leukemias (Slamon et al., 1986; Siegert et al., 1990), but the mechanisms underlying such an increase are unclear. As many studies have suggested the therapeutic potential of targeting c-Myb, it is expected that a knowledge of the mechanisms underlying the enhanced expression in hematological malignancies will be valuable for the development of cancer therapy. The c-Myb protein has a short half-life of 30 min in vivo (Feiková et al., 2000), and the 26S proteasome functions as a major degradation pathway for the rapid breakdown of c-Myb in hematopoietic cells (Bies and Wolff, 1997; Feiková et al., 2000), although the details, including that of the E3 ligase for degradation, have not been cleared. Because a precise regulation of the cellular amount of the c-Myb protein is critical for hematopoietic cell growth and differentiation, the molecular mechanisms of c-Myb degradation should be elucidated.

The E3 ubiquitin ligase component of the enzyme cascade that mediates ubiquitin–protein conjugation is responsible for target specificity (Hershko and Ciechanover, 1998). The SCF complex consists of four components: the invariable subunits, Skp1, Cul1 and Rbx1, and the variable F-box protein that serves as a receptor for target proteins and thereby determines target specificity (Ohta et al., 1999). Among the many F-box proteins that have been identified, Fbw1/β-TrCP, Skp2 and Fbw7 have been well characterized and have been shown to control the abundance of proteins. Fbw1 ubiquitylates β-catenin and IκB, and Skp2 ubiquitylates various proteins, including transcriptional factors, Tob1, b-Myb and c-Myc (Hatakeyama et al., 1999; Kitagawa et al., 1999; Charrasse et al., 2000; Hiramatsu et al., 2006; Nakayama and Nakayama, 2006). Fbw7 also ubiquitylates various proteins, such as cyclin E, Notch1, c-Myc, SREBP, c-Jun and SRC-3 (Koepp et al., 2001; Strohmaier et al., 2001; Wu et al., 2001, 2007; Welcker et al., 2004; Yada et al., 2004; Minella and Clurman, 2005; Sundqvist et al., 2005; Wei et al., 2005). It has been proposed that these three E3 ligases often recognize the distinct consensus amino acid (aa) sequences in their targets, especially the consensus phospho-binding motif for Fbw7, termed Cdc4 phosphodegron (CPD), which is found in many substrates. We observed that there are three consensus recognition sequences for Fbw1, Skp2 and Fbw7, respectively, in the c-Myb protein. Furthermore, it is also known that Fbw7-mediated degradation of the substrates is triggered by glycogen synthase kinase 3 (GSK3) (Welcker and Clurman, 2008).

The c-Myb protein is phosphorylated by several kinases such as Nemo-like kinase (NLK), GSK3 and so on. Stability of the c-Myb protein is influenced by phosphorylation. The pathway activated by Wnt-1 signaling has been reported to promote c-Myb ubiquitin proteasome-dependent degradation through NLK-mediated phosphorylation at multiple sites (Kanei-Ishii et al., 2004a, 2004b). It has also been reported that inhibition of the phosphatidylinositol 3-kinase/Akt/GSK3β pathway enhances the stability of c-Myb (Corradini et al., 2005). Moreover, there are several reports that have tried to identify the critical phosphorylation site(s) for ubiquitin proteasome-dependent degradation of c-Myb, although it is still unclear as to which aas are essential for both phosphorylation and degradation (Bies and Wolff, 1997; Bies et al., 1999, 2000; Kanei-Ishii et al., 2004a, 2004b; Corradini et al., 2005).

In this paper, we identified Fbw7 as the responsible E3 ligase for c-Myb, and determined Thr-572 as a critical site for Fbw7-mediated degradation of c-Myb. It was also shown that phosphorylation of Thr-572 of c-Myb was required for the binding of Fbw7 and its degradation by Fbw7, and that GSK3 was involved in Thr-572 phosphorylation of c-Myb in intact cells. These investigations suggest that phosphorylation of Thr-572 by GSK3 participates in the Fbw7-mediated regulation of c-Myb.

Results

Fbw7 promotes ubiquitylation of c-Myb

It has been reported that c-Myb is degraded through a ubiquitin–proteasome pathway in a phosphorylation-dependent manner. We found possible CPD sequences for Fbw7 recognition in mouse c-Myb. To confirm whether an expression of Fbw7 facilitates ubiquitin conjugation to c-Myb, we performed an in vivo ubiquitylation assay after the detection of HA-ubiquitin-modified c-Myb using a double immunoprecipitation (IP). As the first immunoprecipitate might contain c-Myb and its associated proteins, after dissociation under denaturing conditions, c-Myb was immunoprecipitated as the only component again after immunoblotting (IB) with an anti-HA antibody to evaluate the ubiquitylation of only c-Myb. The result indicated that Fbw7 actually promoted the ubiquitylation of c-Myb (Figure 1a). This ubiquitylation activity of an Fbw7 target for c-Myb seemed to be remarkably strong, because the ubiquitylation ladder signal was also apparently detected in a straight IB of 3% input with an anti-c-Myb antibody. In many cases of E3 targets, ubiquitylation is detected only in immunoprecipitated substrates after IB with an anti-tag of Ub antibody; thus, it is not detected by straight IB with an antibody against substrates or their tags.

Figure 1
figure1

Enhancement of ubiquitylation of c-Myb by Fbw7. (a) HEK293 cells were transfected with expression constructs for Myc-tagged c-Myb, with or without FLAG-tagged Fbw7 and HA-tagged Ubiquitin (Ub) as indicated, and were then incubated with MG132. In order to precisely show the polyubiquitylation of c-Myb, cell lysates were subjected to double immunoprecipitation (IP) with antibodies against Myc or mouse IgG. The resulting precipitates, as well as the original cell lysates (input), were subjected to IB with anti-c-Myb or anti-FLAG antibodies. (b) HEK293 cells were transfected with expression constructs for FLAG-tagged E3 ligase with Myc-tagged c-Myb and HA-tagged Ub as indicated, and were then incubated with MG132. Cell lysates were subjected to IB with antibodies to c-Myb or FLAG.

The F-box proteins, Fbw1, Fbw7 and Skp2, which are receptors of the SCF complex, often recognize their targets in a phosphorylation-dependent manner. We also found possible consensus sequences for both Fbw1 and Skp2 recognition in c-Myb; therefore, we investigated whether these F-box proteins also promote ubiquitylation of c-Myb in vivo. Each FLAG-tagged F-box protein and HA-ubiquitin expression plasmids were co-transfected into HEK293 cells with a c-Myb expression plasmid. The cell extracts were then subjected to IB with antibodies to c-Myb. Only Fbw7 enhanced the ladder signal of c-Myb, whereas, in contrast, neither Fbw1 nor Skp2 could promote the ladder signal of c-Myb (Figure 1b). As shown in Supplementary Figure S1, the deletion mutant of Fbw7 that lacked seven WD40 repeat regions (ΔWD) could not bind to c-Myb, indicating that Fbw7 binds to c-Myb in a WD40 repeat-dependent manner. As the WD motif often comprises the substrate-recognition component of proteins, our results are compatible with earlier observations.

Facilitation of c-Myb degradation by Fbw7

To address the possibility that the SCFFbw7 ubiquitin ligase targets c-Myb for degradation, the effect of Fbw7 expression on the turnover of c-Myb was investigated by measuring c-Myb levels after blocking protein synthesis with cycloheximide (CHX). The coexpression of Fbw7 significantly facilitated the degradation of c-Myb protein (Figure 2). We ascertained that the CHX assay was suitable for the investigation of c-Myb degradation, and results could be compared with the data from a pulse-chase assay of metabolically radiolabeled proteins, using COS1 cells. As shown in Supplementary Figure S2, Fbw7-dependent degradation of c-Myb was confirmed by both the CHX assay and the pulse-chase assay.

Figure 2
figure2

Involvement of Fbw7 in c-Myb degradation. HeLa cells were transfected with Myc-tagged c-Myb in the absence or presence of FLAG-tagged Fbw7 in HeLa cells. For cycloheximide (CHX) analysis, cells were prepared at the indicated times of the chase incubation and were subjected to immunoblotting (IB) with antibodies against c-Myb, α-tubulin or FLAG. The percentage of c-Myb remaining after the various chase times was quantitated by image analysis. IB shows the representative data. The percent of c-Myb protein levels was calculated as the mean±s.d. from six independent experiments.

Fbw7 promoted ubiquitylation and degradation, not only of mouse c-Myb but also of human c-Myb (Supplementary Figure S3). These results indicated that Fbw7 targets c-Myb for degradation.

Depletion of Fbw7 stabilized c-Myb protein in K562 cells

To examine whether the abundance of endogenous Fbw7 affects c-Myb, we used RNAi to deplete Fbw7 in K562 cells using two small interfering RNAs (siRNAs), Fbw7-A and Fbw7-B. We confirmed the efficacy of the Fbw7 siRNA, using quantitative real-time–PCR. Both Fbw7-A and Fbw7-B reduced about 50% of the Fbw7 expression (Figure 3). The knockdown efficiency was reproducible and comparable with that in the earlier study (Welcker et al., 2004). The degradation of c-Myb in cells transfected with Fbw7 siRNAs was apparently delayed compared with that in cells transfected with a control siRNA, the half-life of which was about 20 min. These observations suggested that posttranslational regulation of endogenous c-Myb was mediated by Fbw7, and it was consistant with the results of in vivo degradation assay using transfected c-Myb and Fbw7 as indicated Figure 2.

Figure 3
figure3

Depletion of endogenous Fbw7 stabilized c-Myb protein in K562 cells. At 48 h after transfection of K562 cells with the indicated small interfering RNA (siRNA) for Fbw7, the cells were subjected to cycloheximide (CHX) analysis with antibodies against c-Myb or α-tubulin. The cell precipitate at CHX=0 min was also used to measure the abundance of mRNAs for Fbw7 and 18S rRNA as an internal control.

Depletion of Fbw7 promotes c-Myb-dependent transcriptional repression of γ-globin in K562 cells

Depletion of Fbw7 mRNA by Fbw7-A or Fbw7-B siRNA significantly induced the accumulation of c-Myb protein, whereas the mRNA levels of c-Myb, as well as those of GAPDH, did not significantly change (Figures 4a–d). This result was compatible with the result in c-Myb stability analysis (Figure 3). On the contrary, similar experiments using Skp2 siRNA showed no effect on c-Myb abundance in K562 cells (Supplementary Figure S4). This result strengthened the fact that Skp2 could not promote degradation of c-Myb. It has been reported that c-Myb inhibits γ-globin gene expression in K562 cells (Jiang et al., 2006). To examine the effect of ablation of Fbw7 on c-Myb-dependent transcriptional regulation, we measured the expression level of endogenous γ-globin using quantitative real-time–PCR. We found that depletion of Fbw7 resulted in a significant decrease in the abundance of γ-globin transcripts (Figures 4e, f). Finally, the amount of Fbw7 and γ-globin expression seemed to be positively related in K562 cells (Figure 4f). It proposed that the accumulation of c-Myb, induced by a depletion of Fbw7, influenced the transcriptional regulation of the target gene of c-Myb in K562 cells. We also found that the depletion of Fbw7 induced a shorter G1 phase in K562 cells (Supplementary Figure S5). These results strongly suggest that endogenous Fbw7 plays a role in the regulation of c-Myb as a pivotal E3 ligase for c-Myb degradation in intact cells.

Figure 4
figure4

Depletion of endogenous Fbw7 promoted transrepression of γ-globin in K562 cells. At 48 h after transfection of K562 cells with the indicated small interfering RNA (siRNA), c-Myb protein levels were analysed by immunoblotting with anti-c-Myb (a, b), and mRNA levels of c-Myb (c), GAPDH (d), Fbw7 (e), γ-globin and 18S rRNA were measured by quantitative real-time–PCR. These results shown are means±s.d. from indicated independent experiments. (f) The correlation of γ-globin gene expression with Fbw7 expression is shown. The data points included all values from K562 treated with siRNA for Fbw7.

Analysis of the c-Myb domain required for recognition and ubiquitylation by Fbw7

We tried to determine the regions of mouse c-Myb responsible for its ubiquitylation mediated by Fbw7. Myc-tagged c-Myb-deleted derivates were constructed as indicated in Figure 5a. These mutants were expressed in HEK293 cells in the absence or presence of Fbw7 to evaluate their Fbw7-mediated ubiquitylation. As shown in Figure 5a, all of the c-Myb mutants with various C-terminal truncations were dramatically reduced in Fbw7-dependent ubiquitylation in contrast to wild-type c-Myb. These results suggest that the critical sequences of c-Myb for ubiquitylation by Fbw7 are included in aa residues, 521–636.

Figure 5
figure5

Analysis of the required sites in c-Myb for Fbw7-mediated ubiquitylation. (a) Effect of C-terminal deletion on ubiquitylation of c-Myb by Fbw7. Schematic representation of mouse c-Myb mutants. DBS, DNA-binding sequences; TA, transactivation domain; Leu Zipper, leucine zipper; NRD, negative regulatory domain. HEK293 cells were transfected with HA-Ubiquitin and wild type (WT) or the deletion mutant of Myc-tagged c-Myb in the absence or presence of FLAG-Fbw7, and were then incubated with MG132 for 5 h. To detect ubiquitylation of c-Myb protein and expression of Fbw7, lysates were subjected to immunoblotting (IB) with antibodies against Myc and FLAG, respectively. Results of ubiquitylation of deleted c-Myb by Fbw7 were also summarized. (b) Consensus sequence for phosphorylation by glycogen synthase kinase 3 (GSK3) and Cdc4 phosphodegron (CPD) are indicated. In the C-terminal region of c-Myb, aa numbers indicates the sites of aa exchanged to Ala in the mutant c-Myb proteins which were used in (c). Four putative GSK3 sites (opened box) and two putative CPD sites (bold under bar) in the C-terminal region of mouse c-Myb are indicated. (c, d) HEK293 cells were transfected with HA-ubiquitin and either wild type or mutant c-Myb, in the absence or presence of FLAG-Fbw7. Ubiquitylation of c-Myb and expression of Fbw7 in cell lysates were determined by IB analysis with antibodies against Myc or FLAG.

Thr-572 in c-Myb is an essential site for recognition, ubiquitylation and degradation by Fbw7

It has been reported that ubiquitin-dependent degradation of c-Myb is regulated in a phosphorylation-dependent manner, and the Fbw7-mediated degradation of substrate is often triggered by the activation of GSK3. GSK3 phosphorylates serine and threonine residues, provided by another phosphoserine or phosphothreonine that is present four aa C-terminal to the site of phosphorylation, and is termed the ‘priming phosphorylation’ (Figure 5b, open box) (Cohen and Goedert, 2004). Reported CPD (Cohen and Goedert, 2004) is also indicated in Figure 5b, bold under bar. Assuming that both Asp and Glu can mimic priming phosphorylation, we noticed some possible GSK3 phosphorylation sites and CPD sequences within aa residues 521–636 of mouse c-Myb (Figure 5b, bottom panel). To determine whether phosphorylation of these domains is required for ubiquitylation by Fbw7, we prepared aa-substituted mutants of mouse c-Myb ((i)549/553A, (ii)556/560A and (iii+iv)568/572/576A), in which all Ser, Thr, Asp and Glu residues were replaced by Ala. We expressed each mutant c-Myb plasmid in the absence or presence of Fbw7 in HEK293 cells. All except the 568/572/576A mutant facilitated ubiquitylation in the presence of Fbw7, as well as in the presence of the wild type (Figure 5c). Then to determine whether Ser-568, Thr-572 or Glu-576 was required for ubiquitylation of c-Myb, we prepared three kinds of single-point mutants in which the Ser-568, the Thr-572 or the Glu-576 residue was substituted by Ala (S568A, T572A or E576A). The T572A and E576A mutants completely lost the capacity of enhanced ubiquitylation by Fbw7, whereas the S568A mutant retained it (Figure 5d). These results imply that both Thr-572 and Glu-576 residues are required for ubiquitylation by Fbw7. We reconfirmed the ablation of ubiquitylation of the T572A mutant using HeLa cells (Supplementary Figure S6).

Thr-572 of c-Myb is able to constitute a motif of the consensus of GSK3-phosphorylation, because it is presumable that Glu-576 should mimic priming phosphorylation for phosphorylation at Thr-572 by GSK3 kinase. To evaluate whether Thr-572 of c-Myb was phosphorylated in vivo, the phospho-specific antibody, which recognizes the phosphorylation of Thr-572 (αP-T572 c-Myb), was prepared. It was confirmed that it selectively recognizes and binds to the P-T572 peptide but not to the NP-T572 peptide (Supplementary Figure S7). We found that wild-type c-Myb, which was expressed in HEK293 cells treated with both phosphatase inhibitor and proteasome inhibitor, was detected by αP-T572 c-Myb antibody, but neither the T572A or the E576A mutant c-Myb was detected (Figure 6a). Furthermore, wild-type c-Myb-expressed HEK293 cells were incubated with or without Okadaic acid. The signal detected by the αP-T572 c-Myb antibody was extinguished without Okadaic acid (Figure 6b). These results suggested Thr-572 might be phosphorylated depending on mimic priming phosphorylation (Glu-576) in vivo. We further examined whether GSK3 kinase would phosphorylate Thr-572 in vitro. We prepared four types of synthetic peptides, T572-phosphorylated (P-T572), nonphosphorylated (NP-T572), Ser-568 replaced with Ala (568A) and both Ser-568 and Thr-572 replaced with Ala (568A/572A) (Figure 6c, bottom). The phosphorylation level of the 568A or P-T572 peptide was approximately one-half of that of NP-T572, whereas the 568A/572A peptide was not phosphorylated at all by GSK3 (Figure 6c, top). These indicated that no other site except S568 or T572 was phosphorylated by GSK3. As phosphorylated Thr-572 promotes the phosphorylation of Ser-568 as a priming site, it was speculated that the incorporation of 32P into the P-T572 peptide was because of the phosphorylation of Ser-568. Moreover, it was suggested that the NP-T572 peptide was phosphorylated at both T572 and S568, whereas the 568A peptide and P-T572 peptide were phosphorylated only at T572 and S568, respectively. Therefore, by subtracting the count of P-T572, the phosphorylation level on Thr-572 in the NP-T572 peptide was calculated. It is reasonable that its numerical value is equal to the phosphorylation level of 568A. Therefore, our data suggested that GSK3 could phosphorylate Thr-572. Next, the phosphorylation capacity of GSK3 on the wild-type or T572A mutant mouse c-Myb protein expressed in 293 cells was evaluated. As shown in Figure 6d, it was shown that only wild-type c-Myb incubated with GSK3 was recognized by the anti-p-T572 antibody. Finally, our data suggested that GSK3 could phosphorylate Thr-572.

Figure 6
figure6

Thr-572 of c-Myb is phosphorylated in vivo. (a) c-Myb, either wild type or mutant, was transiently expressed in HEK293 cells. In order to inhibit proteolysis and dephosphorylation of c-Myb, cells were treated with 20 μM MG132 and 20 nM Okadaic acid for 5 h before harvest. Cell lysates were prepared with lysis buffer, which contained phosphatase inhibitors, then subjected to immunoblotting (IB) analysis with antibodies against c-Myb or P-T572 c-Myb. (b) To reconfirm that Thr-572 of c-Myb is phosphorylated in vivo, HEK293 cells were transfected with wild-type c-Myb, again. Cells were treated with 20 μM MG132 in the absence or presence of 20 nM Okadaic acid for 5 h before harvest. Cell lysates were prepared with lysis buffer, which contained phosphatase inhibitors, then subjected to IB analysis with antibodies against P-T572 c-Myb, c-Myb or α-tubulin. (c) Phosphorylation of synthetic peptide by GSK3 kinase. Indicated synthetic peptides were incubated with [γ-32P]ATP in the absence or presence of glycogen synthase kinase 3 (GSK3) kinase for the indicated time as described in the Materials and methods section. The levels of incorporation of 32P are means±s.d. of results of triplicate assays. Each peptide sequence was indicated below. Opened boxes are putative GSK3 phosphorylation sites. (d) Wild-type c-Myb protein was phosphorylated by GSK3β in vitro. Myc tagged wild type or T572A mutant c-Myb or empty vector plasmid was transfected in HEK293 cells. Cells were treated with MG132 for 5 h starting at 43 h after transfection and then harvested. Cell lysates were prepared in the absence of phosphatase inhibitors, and subjected to immunoprecipitation with anti Myc antibody. The precipitates were incubated with or without recombinant GSK3β kinase at 30 °C for 30 min, and then subjected to immunoblot analysis. Mouse c-Myb phosphorylated Thr-572 and the total level of precipitated c-Myb was detected with P-T572 c-Myb antibody and anti c-Myb antibody, respectively.

To investigate whether ubiquitylation of c-Myb by Fbw7 depends on GSK3 kinase activity, we attempted to perform an in vivo ubiquitylation assay with or without GSK3 inhibitors. Both the inhibitors, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (Type II) and 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), inhibited the ubiquitylation of c-Myb by Fbw7 in a dose-dependent manner (Figure 7a). Both GSK3 inhibitors also delayed the turnover of c-Myb in the presence of Fbw7 in the CHX assay (Supplementary Figure S8). We further performed a CHX assay to investigate the necessity of kinase for the degradation of c-Myb by Fbw7. GSK3β K85R, which is the dominant-negative form because of its binding ability to c-Myb (data not shown), was coexpressed with c-Myb and Fbw7 in HeLa cells. GSK3β K85R delayed the turnover rate of c-Myb in the presence of Fbw7 (Figure 7b), whereas NLK K155M, which is also a dominant-negative form (Kanei-Ishii et al., 2004a), faintly affected c-Myb degradation (data not shown).

Figure 7
figure7

Fbw7 recognized phosphorylation of Thr-572, which could be mediated by glycogen synthase kinase 3 (GSK3). (a) GSK3 inhibitors repressed c-Myb ubiquitylation which was mediated by Fbw7. HEK293 cells were transfected with FLAG-Fbw7, HA-Ub and c-Myb, as indicated, and were treated with the indicated GSK3 inhibitors for 24 h, and were incubated in the presence of MG132 during the final 5 h. Cell lysates were subjected to immunoblotting (IB) with antibodies to c-Myb or Fbw7. (b) GSK3 was involved in stability of c-Myb protein. HeLa cells were transfected with c-Myb in the absence or presence of FLAG-Fbw7 with or without a dominant negative kinase, GSK3βK58R. For the cycloheximide (CHX) analysis, cells were prepared at the indicated times of the chase incubation and subjected to IB with antibodies against c-Myb or αTubulin. The percentage of c-Myb remaining after the various chase times was quantitated by image analysis. Percent of c-Myb protein levels were calculated as the mean±s.d., from three independent experiments. (c) Depletion of GSK3β inhibited phosphorylation of Thr-572. HEK293 cells were transfected with c-Myb expression plasmid and control or GSK3β of siRNA oligonucleotides. After 43 h transfection, cells were treated with Okadaic acid and MG132 for 5 h. Cell lysates were prepared and subjected to IB analysis with antibodies against P-T572 c-Myb, c-Myb, GSK3β or α-tubulin. (d) Interaction of Fbw7 with a synthetic peptide was analysed by an in vitro ‘pull down’ assay. HEK293 cells were transfected with FLAG-Fbw7. The cell lysates were subsequently subjected to a ‘pull-down’ assay with beads conjugated with nonphosphorylated (NP-T572) peptide or phosphorylated Thr-572 (P-T572) peptide corresponding to aa 567–577 of c-Myb. To detect FLAG-Fbw7 bound to peptides, the resulting precipitates and 5% of the input cell lysates were subjected to IB analysis with antibodies against FLAG. (e) T572A mutant was resistant to Fbw7-promoted degradation. HeLa cells were transfected with c-Myb, either wild type or T572A, in the absence or presence of FLAG-Fbw7. For the CHX analysis, the cells were prepared at the indicated times of the chase incubation and subjected to IB with antibodies against c-Myb, α-tubulin or FLAG. The percentage of c-Myb remaining after the various chase times was quantitated by image analysis. Immunoblots show the representative data, and the percentage of c-Myb protein levels was calculated as the mean±s.d. from three independent experiments.

Next, we examined whether depletion of GSK3 affected the phosphorylation of Thr-572 in intact cells. As shown in Figure 7c, it was found that phosphorylation of Thr-572 was inhibited by the depletion of GSK3β. Therefore, it was suggested that GSK3 was involved in Thr-572 phosphorylation of c-Myb in intact cells. To determine whether phosphorylation of Thr-572 of c-Myb is required for the binding of Fbw7, we tested the ability of synthetic peptides to interact with Fbw7 in vitro. Fbw7 expressed in HEK293 cells apparently interacted with the P-T572 peptide, whereas the interaction with the NP-T572 peptide was completely abolished (Figure 7d). Moreover, we performed a CHX assay using the T572A mutant in HeLa cells. In the absence of Fbw7, the T572A mutant was stable similar to wild-type c-Myb, whereas the coexpression of Fbw7 promoted the degradation of wild-type c-Myb but not that of the T572A mutant (Figure 7e). These results suggested that phosphorylation of Thr-572 of mouse c-Myb by GSK3 could be one of the key events for the ubiquitylation and degradation mediated by SCFFbw7.

Discussion

It has been reported that the c-Myb protein is phosphorylated at multiple sites and degraded by a ubiquitin–proteasome pathway (Bies et al., 2000, 2001; Kanei-Ishii et al., 2004a, 2004b; Corradini et al., 2005). In this study, we investigated the mechanism of c-Myb degradation. We have indicated that Fbw7 promotes the ubiquitin-dependent degradation of c-Myb. Moreover, we found that phosphorylation of Thr-572 of mouse c-Myb is critical for Fbw7-mediated ubiquitylation, followed by proteosomal degradation of c-Myb, and GSK3 might play a crucial role in it. It has been reported that the Fbw7-mediated degradation of its substrates, such as cyclin E, c-Myc, SREBP, c-Jun and SRC3, is likely triggered by the activation of GSK3 (Welcker and Clurman, 2008). The CPD motif contains a central phosphorylated serine/threonine immediately followed by proline, and a phosphorylated serine/threonine or a negatively charged aa is needed in +4 (Figure 5b). The aa sequence surrounding Thr-572 of c-Myb coincides with both GSK3-phosphorylation sequence and CPD. Thr-572 in mouse c-Myb is not conserved in human c-Myb, although we indicated that human c-Myb was also targeted for degradation by Fbw7. There are Fbw7-phosphodegron-like sequences, such as Ser-600, and many putative GSK3 phosphorylation sites. Corradini et al. (2005) reported that the GSK3β pathway promoted the degradation of human c-Myb, although it has not been identified as to which residues that were phosphorylated by GSK3 correlated with protein stability. To identify a critical site for the degradation of human c-Myb, we generated many point mutants including Ser-600, as well as several putative GSK3 phosphorylation sites and various deletion mutants of human c-Myb, and evaluated their susceptibility for the ubiquitylation and degradation promoted by human Fbw7. Single-point mutants did not show severe defects in their Fbw7-mediated ubiquitylation and degradation (data not shown). Moreover, both the C-terminal and the N-terminal portions in human c-Myb were required for ubiquitylation by Fbw7 (data not shown). These results suggest that multiple phosphorylation sites may be involved in Fbw7-mediated degradation of human c-Myb.

Recently, the degradation of mouse c-Myb by Fbw7 was reported by Kanei-Ishii et al. (2008), wherein they also indicated the importance of the phosphorylation of c-Myb in the recognition by Fbw7. These results coincide with our findings. Although in the report, they did not identify the critical phosphorylation site for c-Myb degradation, they indicated that two kinds of multiple-site mutants of c-Myb, 15A (substitutions of the fifteen Ser/Thr residues to Ala) and N3A+C3A (substitutions of six Ser/Thr residues in the N-terminal and C-terminal to Ala), inhibited Fbw7-mediated degradation. As these mutants included the replacement of T572 with Ala, it did not contradict our result that the c-Myb protein was markedly stabilized by the substitution of Thr-572 to Ala. We originally found that phosphorylation of Thr-572, which could be mediated by GSK3, enhanced the interaction between c-Myb and Fbw7, and promoted c-Myb degradation by Fbw7, whereas Kanei-Ishii et al. (2008) indicated that the c-Myb/Fbxw7α interaction was enhanced by NLK phosphorylation of c-Myb. Thr-572 constitutes a part of the consensus motif of GSK3 phosphorylation, while it is also the presumable NLK recognition site (S/T-P). As we found that phosphorylation of Thr-572 was not detected in the E576A mutant, we speculated that Thr-572 might be phosphorylated depending on mimic priming phosphorylation (Glu-576) in vivo. Moreover, we indicated that the depletion of GSK3β inhibited the phosphorylation of Thr-572. These results suggest that GSK3 could phosphorylate Thr-572.

It has been reported that degradation of b-Myb by ubiquitin-mediated proteolysis involves the Cdc34–SCFp45Skp2 pathway (Charrasse et al., 2000). As c-Myb has a Skp2 recognition consensus sequence (E-x-S/T-P-x-x-P), we speculated that c-Myb might also be one of the substrates of Skp2. Nonetheless, Skp2 scarcely promoted ubiquitylation of c-Myb (Figure 1b), and ablation of Skp2 did not affect the stability of c-Myb in K562 cells (data not shown). These results indicate that Skp2 is not an E3 ligase for c-Myb. As Skp2 is often overexpressed in human cancers and targets tumor-suppressor proteins such as p130, Tob1, p27Kip1 and p57Kip2, Skp2 is thought to be a potential oncogene. Whereas Fbw7 is frequently deleted or mutated in human cancers and is involved in the degradation of oncogenic proteins such as cyclin E, c-Myc and c-Jun, Fbw7 is thought to be a tumor-suppressor gene. These are consistent with our findings that Fbw7 targets c-Myb, which is one of the oncogene products. It is interesting that Fbw7 could not promote ubiquitylation of b-Myb (data not shown). It was suggested that c-Myb and b-Myb were regulated by different mechanisms.

It has been reported that Siah1, which is one of the E3 ligases induced by p53, promotes the degradation of c-Myb (Tanikawa et al., 2000). It was also indicated that Siah1 binds with the DNA-binding domain of c-Myb and suppresses c-Myb expression in a cell-type-dependent manner, which was observed in CV-1 cells but was not obvious in 293T cells. However, it was not determined whether Siah1 promoted the ubiquitylation-dependent degradation of c-Myb or whether it occurred in a phosphorylation-dependent manner. It might suggest that Siah1 and Fbw7 might regulate c-Myb stability through different mechanisms, although future studies are necessary to clarify this.

Feiková et al. (2000) earlier studied the subcellular localization of c-Myb degradation by the 26S proteasome. They detected the distribution of c-Myb in the nuclear but not in the cytoplasmic fraction, the experiments of which were performed in the absence of a 26S proteasome inhibitor. Therefore, the nucleus was identified as the site of c-Myb proteolytic breakdown. It has also been reported that c-Myb is localized in the nucleus, excluding in the nucleoli of MOLT4 cells. Three isoforms (α, β and γ) of Fbw7 are produced from mRNAs with distinct 5′ exons. Matsumoto et al. (2006) investigated the distribution of Fbw7 mRNA isoforms. Fbw7α mRNA was detected in all mouse tissues examined; in contrast, Fbw7β and Fbw7γ mRNAs were restricted to the brain and testis, and to the heart and skeletal muscle, respectively. From our preliminary data, it seems that neither Fbw7β nor Fbw7γ promotes a distinct ubiquitylation of c-Myb, in contrast to Fbw7α.

Tetzlaff et al. (2004) reported that Fbw7-null mice died around 10.5 dpc because of a combination of deficiencies in hematopoietic and vascular development and heart chamber maturation. Concomitantly, the accumulation of cyclin E and Notch1 proteins is found in Fbw7−/− embryos. It has been reported that Fbw7 targets Notch1, which transcriptionally controls c-Myc (Sharma et al., 2007), whereas transcription of Notch1 is positively regulated by c-Myb. The constitutive active signaling of Notch1 is involved in the pathogenesis of T-cell acute lymphoblastic leukemias (T-ALL) (Weng et al., 2004; O’Neil et al., 2007). c-Myc is also thought to be an important regulator for T-cell differentiation; furthermore, Fbw7 targets c-Myc as well as Notch1 (Onoyama et al., 2007). There are some reports regarding the mutation of the Fbw7 gene in T-ALL cells (O’Neil et al., 2007; Thompson et al., 2007). Fbw7 deficiency in patients might result in a pleiotropical influence on leukemia progression. Eventually, Fbw7 acts as a critical fail-safe against the premature loss of hematopoietic stem cells and the development of T-ALL. (Matsuoka et al., 2008). Lahortiga et al. (2007) tested the effect of inhibition of Notch1 combined with c-Myb siRNA treatment in T-ALL cell lines, which resulted in a strong inhibitory effect on proliferation and viability. They suggested that the highly constitutive expression of c-Myb is involved in malignant transformation by promoting the continuous proliferation of immature cells and by preventing further progression of relatively mature cells. Moreover, c-Myb expression is elevated in many cases of acute myeloblastic and lymphoblastic leukemia (Slamon et al., 1986; Siegert et al., 1990). It has been reported that somatic duplications of the c-Myb gene or somatic translocations of the c-Myb gene and the TCR locus were found in some human leukemias, and hence c-Myb was overexpressed in these situations (Clappier et al., 2007; Lahortiga et al., 2007). In addition, the abrogation of c-Myb degradation by Fbw7 would cause an increase in the protein level of c-Myb. We obtained the result that a depletion of Fbw7 induced an accumulation of c-Myb and shortened the G1 phase in K562 cells (Supplementary Figure S5). Taken together, this evidence suggested that the accumulation of c-Myb, accompanied by a defect in Fbw7 or overexpression of c-Myb caused by the above mechanisms, might be involved in the growth of some leukemia cells.

In this study, we show that c-Myb is downregulated by Fbw7 by the ubiquitin–proteasome pathway. It seems that the Fbw7 E3 ligase preferably targets the regulators of hematopoiesis, c-Myc, Notch1 and c-Myb, and as a result, Fbw7 is an important controller of substrate abundance for appropriate and sequential maturation of hematopoietic cells. Eventually, the combined inhibition of c-Myc, Notch1 and c-Myb, through the increase of Fbw7 expression, might be beneficial for treatment in T-ALL patients.

Materials and methods

Cell culture

HEK293 and HeLa cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. K562 cells (RIKEN) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum.

Antibodies and synthetic peptides

The antibodies used in this study were anti-Myc antibody 9B11 (Cell Signaling, Beverly, MA, USA), anti-Myc antibody 9E10 (Roche, Indianapolis, IN, USA), anti-FLAG antibody M2 (Sigma, Deisenhofen, Germany), anti-HA antibody 12CA5 (Roche), anti-c-Myb antibody 1–1 (Upstate, Lake Placid, NY, USA), anti-Fbw7 antibody H-300 (Santa Cruz, Santa Cruz, CA, USA), anti-GSK-3β antibody clone 7 (BD Biosciences Pharmingen, Heidelberg, Germany) and anti-αTubulin antibody DM1A (Sigma). Anti mouse c-Myb-phosphorylated Thr-572 polyclonal antibody (αP-T572 c-Myb) was raised against KLH-conjugated chemically synthesized phosphorylated Thr-572 (P-T572) peptide, corresponding to the CPD region of c-Myb (aa residues 567–577) (Peptide Institute Inc., Osaka, Japan). The anti-serum obtained from an immunized rabbit was purified using column chromatography conjugated with the P-T572 peptide. The affinity-purified αP-T572 c-Myb was then passed through a column packed with resin that was conjugated to a nonphosphorylated Thr-572 (NP-T572) peptide (aa 567–577 of c-Myb), which was chemically synthesized (Peptide Institute Inc.) to deplete contaminated antibodies, which can bind with nonphosphorylated antigen. The specific binding of the purified antibody to the P-T572 peptide was confirmed by enzyme-linked immunosorbent assay. The other synthetic peptides, 568A peptide and 568A/572A peptide, were also chemically synthesized (Peptide Institute Inc.).

Plasmids

Complementary DNAs encoding c-Myb wild type and its mutants were cloned into pcDNA3.1/Myc-His (Invitrogen, Karlsruhe, Germany). Expression plasmids of ubiquitin, Skp2, Fbw1 and the kinase dominant-negative mutant of GSK3β (pCGN-HA-Ub, pcDNA3-FLAG-Skp2, pcDNA3-FLAG-Fbw1 and pCGN-GSK3β-K85R) were described earlier (Kitagawa et al., 1999; Nakayama et al., 2000; Tanji et al., 2002). The expression plasmids of pCGN-HA-human-Fbw7 and pcDNA3-FLAG-mouse-Fbw7 were kindly provided by Keiichi Nakayama, Kyushu University. All deletion and point mutants of c-Myb or Fbw7 were constructed using standard recombinant DNA techniques.

Immunoprecipitation

For IP, cell lysates were incubated with 2 μg of antibodies and protein G+ Sepharose 4FF (GE Healthcare, Little Chalfont, Buckinghamshire, UK) at 4 °C for 1 h. Immunocomplexes were washed five times with lysis buffer. For double IP, the first immunocomplexes, which were prepared with anti-Myc antibody, were denatured by treatment with an SDS sample buffer at 100 °C for 8 min. Then ubiquitylated c-Myb was immunoprecipitated again with anti-Myc antibody. Immunoprecipitated samples, as well as the original cell lysates (input), were separated by SDS–polyacrylamide gel electrophoresis and transferred from the gel onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA), followed by IB. The proteins were visualized using an enhanced chemiluminescence system (Perkin Elmer, Waltham, MA, USA).

In vivo ubiquitylation assay

All plasmids were transfected into HEK293 or HeLa cells by the calcium phosphate method or Lipofectamine 2000 (Invitrogen), respectively. As described in earlier reports (Uchida et al., 2005), to induce the accumulation of poly-ubiquitylated c-Myb, cells were treated with the proteasome inhibitor, MG132 (20 μM), for 5 h starting at 43 h after transfection, and then harvested. Cell lysates were prepared with lysis buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 0.5% Triton X-100, 10 μg/ml each of antipain, pepstatin, E-64, leupeptin and trypsin inhibitor and 2.5 μg/ml of chymostatin) after IB analysis.

In vivo degradation assay

All plasmids were transfected into HeLa cells. A total of 24 h after transfection, each transfectant was divided into five culture dishes for the chase experiment, and after an additional 24 h, the cells were treated with 12.5 μg/ml of CHX for the indicated times. Cell lysates were subjected to IB. The intensity of the bands was quantitated using image analysis software Image Gauge 4.21 (Fujifilm, Tokyo, Japan), and the signal intensity of each c-Myb was normalized using the individual levels of α-tubulin.

RNA interference

K562 cells were transfected with siRNA oligonucleotides using HiPerFect transfection reagent (Qiagen, Hilden, Germany), according to the manufacturer's protocol. For the degradation assay, 6 h after transfection, each transfectant was divided into five culture dishes. After 42 additional hours, four culture dishes for the chase experiment were treated with 12.5 μg/ml of CHX for the indicated times. Cell lysates were subjected to IB. In case of depletion of Fbw7, the other dish was used for complementary DNA synthesis, so that the amount of transcripts could be calculated. The nucleotide sequences of siRNA for Fbw7 were 5′-GUGUGGAAUGCAGAGACUGGAGA-3′ (Fbw7-A) with 3′ dTdT overhangs and 5′-IndexTermAAUGAAAGCACAUAGAGUGCCAACU-3′ (Fbw7-B). Fbw7-A encodes the same sequences as does Fbw7–2, which has been reported earlier (Welcker et al., 2004).

Quantitative real-time–PCR analysis

Total RNA was isolated from cells with the use of Isogen (Wako, Osaka, Japan), and subjected to reverse transcription with random hexanucleotide primers and SuperScript Reverse Transcriptase II (Invitrogen). The resulting complementary DNA was subjected to quantitative real-time–PCR using the Rotor-Gene 3000 system (Corbett Research, NSW, Australia) and the SYBR premix Ex Taq kit (TaKaRa, Shiga, Japan). The amount of the transcripts of interest was normalized against that of 18S rRNA as an internal standard.

In vitro peptide phosphorylation assay

Final 1.468 mM of NP-T572 peptide, P-T572 peptide, 568A peptide or 568A/572A peptide was incubated with 500 units of recombinant GSK3 kinase (NEB) at 30 °C for the indicated times in a reaction buffer that contained 50 μM ATP and 0.6 μCi [γ-32P] ATP (6000 Ci/mmol), in a final volume of 20 μl. Reactions were terminated by the addition of 10 μl of 250 mM H3PO4. The peptides were trapped on P81 papers (Whatman, Maidstone, Kent, UK), which were washed six times with 75 mM H3PO4, and then monitored for radioactivity in a liquid scintillation counter, as described in the earlier report (Kitagawa et al., 1996).

In vitro protein phosphorylation assay

Myc-tagged wild type or T572A mutant of mouse c-Myb, or empty vector as a negative control, was transiently expressed in HEK293 cells. The cells were treated with 20 μM MG132 for 5 h, starting at 43 h after transfection, and then harvested. Cell lysates were prepared in the absence of phosphatase inhibitors but in the presence of proteasome inhibitors, and subjected to IP with an anti Myc antibody. The precipitates were incubated with or without recombinant GSK3β kinase (Carna Biosciences, Kobe, Japan) at 30 °C in a reaction buffer that contained 67 μM ATP for 30 min and then subjected to immunoblot analysis. Thr-572-phosphorylated c-Myb and the total level of precipitated c-Myb were detected with P-T572 c-Myb antibody and anti c-Myb antibody, respectively.

Analysis of phosphorylated level of exogenous c-Myb in vivo

HEK293 cells were transfected with a c-Myb expression plasmid and control or with GSK3β of siRNA oligonucleotides, using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. The nucleotide sequence of siRNA for GSK3β was 5′-IndexTermGUAAUCCACCUCUGGCUAC-3′ with 3′ dTdT overhangs (Yamamoto et al., 2007). A total of 43 h after transfection, the cells were treated with 20 nM Okadaic acid and 20 μM MG132 for 5 h. The cell lysates were subjected to IB.

In vitro ‘pull down’ assay

NP-T572 peptide and P-T572 peptide were conjugated to TOYOPEARL AF-Tresyl-650M affinity matrix (Tosoh, Tokyo, Japan) (Goto and Inagaki, 2007). Cell lysates containing FLAG-Fbw7 were incubated with the above peptide-conjugated gel for 30 min at 4 °C. The conjugated gel was washed four times with lysis buffer and the resulting precipitates were subjected to IB analysis.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

We thank Yojiro Kotake, Keiichi I Nakayama, Masaki Matsumoto, Akinobu Matsumoto and Issay Kitabayashi for providing plasmids and useful discussions, and Sayuri Suzuki, Tomomi Abe, Ning Liu, Harumi Shiratori, Daisuke Hiraoka, Daizo Ueno, Yasue Kirita, Tatsuya Kobayashi, Konomi Mizuguchi, Erina Nihashi, Syuhei Iizuka, Daisuke Ichikawa and Naohiro Takamoto for their technical assistance. This study was supported in part by grants from the Ministry of Education, Science, Sports, Culture and Technology of Japan (MK and KK), by a COE program of Hamamatsu University School of Medicine from the Ministry of Education, Science, Sports, Culture and Technology of Japan (MK).

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Correspondence to M Kitagawa.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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Kitagawa, K., Hiramatsu, Y., Uchida, C. et al. Fbw7 promotes ubiquitin-dependent degradation of c-Myb: involvement of GSK3-mediated phosphorylation of Thr-572 in mouse c-Myb. Oncogene 28, 2393–2405 (2009) doi:10.1038/onc.2009.111

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Keywords

  • c-Myb
  • ubiquitin-proteasome
  • Fbw7
  • phosphorylation
  • GSK3

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