Polo-like kinase 1 phosphorylates and regulates Bcl-xL during pironetin-induced apoptosis

Abstract

Bcl-xL, an anti-apoptotic Bcl-2 family member protein, contributes to the resistance against chemotherapies such as tubulin-binder treatment in many human tumors. Although Bcl-xL is phosphorylated after tubulin-binder treatment, the role of the phosphorylation and its responsible kinase(s) are poorly understood. Here, we identified Plk1 (polo-like kinase 1) as a Bcl-xL kinase. Same location of Bcl-xL and Plk1 was revealed by immunocytochemical analyses at M-phase in situ. Plk1 phosphorylates Bcl-xL in vitro, and we identified Plk1 phosphorylation sites in Bcl-xL. When all of these phosphorylation sites were substituted to alanines, the anti-apoptotic activity of the Bcl-xL mutant against the apoptosis induced by pironetin, but not against ultraviolet-induced apoptosis, was increased. These observations suggest that Plk1 is a regulator of Bcl-xL phosphorylation and controls the anti-apoptotic activity of Bcl-xL during pironetin-induced apoptosis.

Introduction

Apoptosis, a physiological cell suicide mechanism, is an important role for the homeostasis, development and the removal of superfluous cells (Wyllie et al., 1980). The deregulation of apoptosis induces many intractable diseases, such as cancer, AIDS (acquired immune deficiency syndrome) and neuronal disease (Reed, 2002). The Bcl-2 family proteins are the principal regulators of apoptosis, and are classified into two groups, anti-apoptotic members (that is, Bcl-2, Bcl-xL Bcl-w and Mcl-1) and pro-apoptotic members (that is, Bax, Bad, Bid and Bcl-xS). All Bcl-2 family proteins contain one or more BH (Bcl-2 Homology) regions that are necessary for their function and oligomerization. The quantitative balance between anti- and pro-apoptotic Bcl-2 family proteins determines whether a cell will live or die (Chao and Korsmeyer, 1998; Tsujimoto and Shimizu, 2000; Cory and Adams, 2002).

Bcl-xL is a 29-kDa mitochondrial membrane protein that inhibits apoptosis induced by various stimuli, such as ultraviolet (UV) irradiation, anti-tumor drug treatment and growth factor withdrawal (Boise et al., 1993). It has been reported that Bcl-xL is overexpressed in many human tumors, and contributes to their treatment resistance during chemotherapy (Cory and Adams, 2002). Bcl-xL contains four regions of BH and has a flexible loop domain between BH4 and BH3. This loop region is required for the post-translational modifications of Bcl-xL (Chang et al., 1997). In addition, the BH1 and BH2 regions of Bcl-xL are required for heterodimerization with Bax to prevent apoptosis (Cheng et al., 1996).

Bcl-2 family proteins are modified post-translationally, and their function, localization and protein–protein interaction are controlled by the modifications (Schinzel et al., 2004; Simizu et al., 2004; Tamura et al., 2004). For example, the anti-apoptotic function of Bcl-xL is regulated by phosphorylation at Ser62 (Basu and Haldar, 2003) and deamidation at Asn52 and Asn66 (Deverman et al., 2002). Although Bcl-xL is found to be phosphorylated by Hsp90 (heat shock protein 90) inhibitor-sensitive kinase(s), there are inconsistent results as to whether the proposed kinase(s) can actually phosphorylate Bcl-xL (Poruchynsky et al., 1998; Du et al., 2005). Upon the treatment with tubulin-binder, Bcl-xL is phosphorylated, but the role of the phosphorylation and its responsible kinase(s) remain poorly understood.

Plk1 (polo-like kinase 1), a serine/threonine protein kinase, is involved in multiple events during the mitosis, such as centrosome maturation, spindle assembly and chromosome segregation (Barr et al., 2004). In humans, there are four Plk family members, which contain an N-terminal catalytic domain and a C-terminal polo-box domain that contributes to the phospho-dependent substrate recognition (Elia et al., 2003). It has been reported that Plk1 is also overexpressed in many human tumors; therefore, Plk1 has attracted attention as a molecular target for anti-tumor drugs (Gumireddy et al., 2005). Recently, our group has shown that Plk1 is stabilized by Hsp90 (Simizu and Osada, 2000), and phosphorylates somatic type Wee1, leading to the degradation of Wee1 through ubiquitin–proteasome pathway (Watanabe et al., 2004, 2005). Although many substrates of Plk1, including Wee1 (Watanabe et al., 2004, 2005), tubulins (Feng et al., 1999) and Myt1 (Nakajima et al., 2003), are already known, the identification of additional substrates will enhance our general understanding of the important roles played by Plk1.

In this study, we found that Plk1 phosphorylates Bcl-xL at 13 Ser/Thr residues in vitro. When all 13 phosphorylation sites were mutated to alanines, cells expressing the mutant became more resistant to pironetin-induced apoptosis than those expressing wild-type Bcl-xL. Thus, Plk1 controls the anti-apoptotic activity of Bcl-xL through the phosphorylation.

Results

Bcl-xL was phosphorylated by Plk1 at 13 Ser/Thr residues in vitro

For the following three reasons, we focused on Plk1 as a Bcl-xL kinase that is responsible for the Bcl-xL phosphorylation at M-phase and upon the treatment with tubulin binder. (1) Plk1 is activated and upregulated in M-phase (Barr et al., 2004). (2) Plk1 is one of the client proteins of Hsp90 (Simizu and Osada, 2000), and phosphorylation of Bcl-xL is inhibited by the treatment with Hsp90 inhibitor (Poruchynsky et al., 1998). (3) Plk1 interacts with tubulins (Feng et al., 1999). Thus, we first examined whether Plk1 can phosphorylate Bcl-xL in vitro. As shown in Figure 1a, Plk1 phosphorylated Bcl-xL in vitro, but did not readily phosphorylate Bcl-2. To identify the phosphorylation site(s) of Bcl-xL, we constructed a series of deletion mutants (Figure 1b). However, all of the Bcl-xL deletion mutants were phosphorylated by Plk1, implying that Bcl-xL was phosphorylated at multiple sites by Plk1. We had tried to identify the phosphorylation site(s) of Bcl-xL by mass spectrometry, but we were not able to detect the ion peak(s) phosphorylated (data not shown). Thus, we constructed mutant forms of Bcl-xL, in which all the Ser and Thr residues (30 residues) were replaced with Ala residues (designated as Bcl-xL/30A), and then, one by one, each Ala residue was replaced with the original Ser or Thr. As overexpression of Bcl-xL/30A inhibited UV irradiation-induced apoptosis to the same degree as wild-type Bcl-xL in HT1080 cells (data not shown), the Bcl-xL/30A protein seems to maintain its original functions sufficiently to identify the phosphorylation site(s) of Bcl-xL in vitro. As shown in Figure 1c, the 13 residues (Ser23, Ser28, Thr35, Ser43, Ser49, Ser56, Thr69, Ser72, Ser73, Ser74, Thr109, Ser122 and Thr190) of Bcl-xL were phosphorylated by Plk1 in vitro. Among them, Thr190 was the most highly phosphorylated residue, and serine residues at 43, 49, 72 and 73 were phosphorylated at relatively high levels. Thr190 is the sole phosphorylation site in the BH2 domain, whereas most of other phosphorylated residues were in the flexible loop domain (Figure 1d). To confirm these results, we made point mutants of Bcl-xL, in which one or some phosphorylation sites were simultaneously mutated to alanines (Figure 1e). Although singly mutated Bcl-xL proteins were phosphorylated at a similar level as the wild type, the phosphorylation levels of Bcl-xL/3A and Bcl-xL/5A, in which the strongly phosphorylated residues shown in Figure 1c (that is, Ser43/Ser49/Thr190 and Ser43/Ser49/Ser72/Ser73/Thr190, respectively) were simultaneously mutated to alanines, were slightly lower than those of the wild type. The Bcl-xL/13A, in which all 13 phosphorylation sites were replaced with Ala, could not be phosphorylated in the same way as Bcl-xL/30A (Figure 1e), suggesting that all of the phosphorylation sites might be determined. These findings indicated that Bcl-xL was phosphorylated by Plk1 at the 13 Ser/Thr residues in vitro. Next, we tried to detect the Bcl-xL phosphorylation in vivo. Phosphorylation of Bcl-xL at Ser62 was already reported and detected as mobility shift (Basu and Haldar, 2003). We also detected pironetin-induced Ser62 phosphorylation of Bcl-xL (Figure 2a), but cannot detect the mobility shift by the phosphorylation of 13 Ser/Thr residues. To detect the phosphorylation of Bcl-xL by Plk1, we examined two-dimensional western blotting. There were many spots of Bcl-xL in pironetin-treated cells. Among them, at least one spot was clearly disappeared in pironetin-treated Bcl-xL/13A-overexpressing cells, as compared with wild-type Bcl-xL (Bcl-xLwt) (Figure 1f). The spot was not detected in pironetin-untreated Bcl-xLwt-overexpressing cells from other experiments, suggested that Bcl-xL might be phosphorylated by Plk1 in pironetin-treated cells.

Figure 1
figure1figure1

Bcl-xL was phosphorylated by Plk1 at 13 Ser/Thr residues in vitro. (a) Bcl-xL, but little Bcl-2, was phosphorylated by Plk1 at multiple sites in vitro. An in vitro kinase assay was carried out using GST-Plk1 as enzyme and Bcl-xL, Bcl-2, Bcl-xLΔ1-30, Bcl-xLΔ31-60, Bcl-xLΔ61-90, Bcl-xLΔ91-125, Bcl-xS and Bcl-xLΔ186-212 as substrates. The protein levels of all His6-tagged substrates used in the assay were monitored by Coomassie brilliant blue (CBB) staining. (b) A schematic diagram of Bcl-xL deletion mutants. The His6-tag is denoted by a hatched box. Each Bcl-xL and Bcl-2 substrate lacked the transmembrane region (amino acid residues 213–233, 219–239, respectively) for expression in Escherichia coli. (c) The 13 residues of Bcl-xL were phosphorylated by Plk1 in vitro. An in vitro kinase assay was carried out using GST-Plk1 and several mutant forms of Bcl-xL, in which all Ser and Thr residues (total 30 residues) were replaced with Ala residues, later the Ala residues were replaced with the original Ser or Thr one by one. (d) A schematic diagram of Bcl-xL phosphorylation sites by Plk1. The transmembrane region (TM; residues 213–231) is denoted by a black box, and the BH domains (residues 4–24 for BH4, residues 86–100 for BH3, residues 129–148 for BH1, and residues 180–195 for BH2) are designated by hatched boxes. (e) The Bcl-xL/13A significantly reduced the phosphorylation by Plk1. An in vitro kinase assay was carried out using GST-Plk1 and several point mutants of Bcl-xL. 3A: S43A, S49A and T190A; 5A: S43A, S49A, S72A, S73A and T190A; 13A: S23A, S28A, T35A, S43A, S49A, S56A, T69A, S72A, S73A, S74A, T109A, S122A and T190A. (f) Detection of 1 of 13 Bcl-xL phosphorylation in cells. HT1080-Bcl-xLwt and Bcl-xL/13A cells were treated with pironetin (100 nM, 24 h). Cells were lysed and cell lysates were analysed by two-dimensional western blotting using the anti-Bcl-xL antibody.

Figure 2
figure2

Ser62 of Bcl-xL was phosphorylated after pironetin treatment by c-Jun N-terminal kinase (JNK)-related and other kinase(s), but not Plk1. (a) Ser62 of Bcl-xL was phosphorylated by JNK-related and other kinase(s) in situ. HT1080-Neo, HT1080-Bcl-xLwt and HT1080-Bcl-xL/S62A cells were treated with or without pironetin (100 nM) and/or SP600125 (10 μM) for 24 h. Cells were lysed and cell lysates were analysed by western blotting using the indicated antibodies. (b) Plk1 could not phosphorylate Ser62 of Bcl-xL. HT1080-Bcl-xLwt cells were transfected with luciferase siRNA or Plk1 siRNA. After transfection, cells were treated with or without pironetin (100 nM, 24 h) and analysed by western blotting using the indicated antibodies.

Ser62 of Bcl-xL was phosphorylated after pironetin treatment by c-Jun N-terminal kinase-related and other kinase(s), but not Plk1

It has been reported that the Ser62 residue of Bcl-xL is phosphorylated after tubulin-binder treatment by the c-Jun N-terminal kinase (JNK) pathway and that the phosphorylation of Bcl-xL on Ser62 is a trigger for taxol-induced apoptosis (Basu and Haldar, 2003). Thus, it is possible that the phosphorylation of Bcl-xL on Ser62 residue plays a pivotal role of tubulin binder-induced apoptosis, such as pironetin. Later, we examined whether Plk1 is also involved in the Ser62 phosphorylation or not. As shown above, in the wild-type Bcl-xL-expressing HT1080 cells (HT1080-Bcl-xLwt), Bcl-xL was phosphorylated at Ser62 after pironetin treatment, and the JNK inhibitor SP600125 partially reduced the level of pironetin-induced phosphorylation of Bcl-xL at Ser62 and Plk1 upregulation, indicating that a JNK-related kinase and some other kinases are responsible for the Ser62 phosphorylation and Plk1 upregulation (Figure 2a). Then, we investigated the effect of Plk1 knockdown on the Ser62 phosphorylation. It is reported that Plk1 depletion induces G2/M-phase arrest and apoptosis in cancer cells (Liu and Erikson, 2003). To avoid the effect against cell cycle arrest and apoptosis by Plk1 depletion, we treated pironetin immediately after the transfection of Plk1 small interfering RNA (siRNA). Upon depletion of Plk1 by the siRNA, the Plk1 expression level was decreased to an undetectable level and cells were arrested at the G2/M phase of the cell cycle (Figure 2b and data not shown). Depletion of Plk1 resulted in G2/M arrest, thereby leading to the induction of Ser62 phosphorylation of Bcl-xL without pironetin treatment. Moreover, despite Plk1 depletion by siRNA, pironetin treatment induced the Ser62 phosphorylation of Bcl-xL (Figure 2b). Considering these results in conjunction with those of the in vitro kinase assay (Figure 1c), we concluded that Plk1 is not involved in the Ser62 phosphorylation after pironetin treatment.

Same location of Bcl-xL and Plk1 in cells

Next, we analysed the location of Plk1 and Bcl-xL in cells. For this purpose, we established HT1080-derived cell lines that stably express 3HA-tagged Bcl-xL and FLAG-tagged Plk1 (designated as HT1080-3HA-Bcl-xL-FLAG-Plk1 cells). The expressions of these tagged proteins were observed in HT1080-3HA-Bcl-xL-FLAG-Plk1 cells, but not in control vector-transfected cells (data not shown). Immunocytochemistry of HT1080-3HA-Bcl-xL-FLAG-Plk1 cells revealed the distribution of 3HA-Bcl-xL and FLAG-Plk1 on the nucleus during normal mitosis (Figure 3). A similar distribution of each protein was observed using different antibodies and endogenous level in HT-29 (colon adenocarcinoma) cells (Figure 3 and data not shown). We observed the mitotic cells that showed chromatin condensation. These cells were overexpressed as endogenous or exogenous Plk1 in the nucleus as it is generally known. These observations indicated that these proteins localize in the nucleus during normal mitosis in cells.

Figure 3
figure3

Same location of Bcl-xL with Plk1 in cells. Subcellular localization of Bcl-xL and Plk1. HT1080-3HA-Bcl-xL-FLAG-Plk1 cells were stained with Hoechst 33258 (for nucleus; blue), anti-FLAG or anti-Plk1 (green) and HA probe or anti-Bcl-xL antibody (red). Plk (Z; Zymed), Bcl-xL (C; Cell Signaling), Plk1 (C; Calbiochem), Bcl-x (B; BD Biosciences).

Anti-apoptotic activity of Bcl-xL/13A was more resistant than that of Bcl-xL

Finally, we examined the effects of Bcl-xL phosphorylation at the 13 residues on the anti-apoptotic activity of Bcl-xL. To this end, we established HT1080-derived cell lines that stably express Bcl-xLwt or Bcl-xL/13A. The expression levels of each Bcl-xL were examined by western blotting analysis, indicating that Bcl-xLwt was comparably expressed with Bcl-xL/13A (Figure 4a). As shown earlier (Kondoh et al., 1999), treatment with 100 nM pironetin for 30 h disrupted cellular tubulin dynamics and induced apoptosis in approximately 60% of vector-transfected HT1080 cells (Neo) (Figure 4b). When Bcl-xLwt was stably expressed, the pironetin-induced apoptosis decreased to approximately 40%. Moreover, all the 13 Plk1 phosphorylation sites were mutated to alanines, and the apoptosis was significantly decreased to approximately 15%. However, such an increase in the anti-apoptotic activity of phosphorylation sites mutant of Bcl-xL could not be detected when apoptosis was induced by UV irradiation (Figure 4c). Indeed, Plk1 was upregulated during pironetin-induced apoptosis but downregulated during UV irradiation, suggesting that phosphorylation of Bcl-xL by Plk1 is not associated with the UV irradiation-induced apoptosis (Figure 4d). Finally, we observed the extent of apoptosis by western blot analysis of the cleaved Caspase-3 and its substrate poly(ADP-ribose)polymerase (PARP). Interestingly, although apoptosis was detected by flow cytometry analysis as a sub-G1 peak, no activation of Caspase-3 pathway was detected by western blot analysis in Bcl-xLwt- and Bcl-xL/13A-overexpressing cells (Figure 4e). These data suggested that Bcl-xLwt and Bcl-xL/13A can inhibit Caspase-dependent apoptosis, but pironetin-induced Caspase-independent cell death might not be suppressed by phosphorylated Bcl-xL. Therefore, we concluded that the anti-apoptotic activity of Bcl-xL in pironetin-induced apoptosis was controlled by its phosphorylation status. Collectively, our results suggest that Plk1 weakens the anti-apoptotic activity of Bcl-xL in certain specific types of apoptosis, such as pironetin-induced apoptosis.

Figure 4
figure4

The anti-apoptotic activity of Bcl-xL/13A was more resistant than that of Bcl-xL. (a) The expression levels of Bcl-xL and Bcl-xL/13A mutants in each clone cell line. HT1080-Neo, Bcl-xLwt and Bcl-xL/13A cells were lysed, and the total cell lysates were analysed by western blotting using anti-Bcl-xL or anti-Actin antibodies. (b) The effects of Bcl-xLwt and Bcl-xL/13A overexpressions on pironetin-induced apoptosis. HT1080-Neo (diamond), Bcl-xLwt (square) and Bcl-xL/13A (triangle) cells were treated with 100 nM pironetin. Dead cells were assessed by flow cytometry analysis as a sub-G1 peak until 30 h every 6 h. Values are the means±s.d. of quadruplicate determinations. (c) The effects of Bcl-xLwt and Bcl-xL/13A overexpressions on ultraviolet (UV) irradiation-induced apoptosis in HT1080 cells. Each cell line was untreated (open bars) or treated (hatched bars) with UV irradiation (200 J/m2, 40 h). Dead cells were assessed by flow cytometry analysis as a sub-G1 peak. Values are the means±s.d. of quadruplicate determinations. (d) Upregulation and downregulation of Plk1 by apoptosis-inducing stimuli. HT1080-Bcl-xLwt cells were treated with pironetin (100 nM, 24 h) or UV irradiation (200 J/m2, 40 h) and analysed by western blotting using the indicated antibodies. (e) The extent of apoptosis by western blot analysis on Caspase-dependent pathway. Each cell was untreated or treated with pironetin (100 nM, 24 h) and analysed by western blotting using the indicated antibodies.

Discussion

The bcl-x gene was originally identified as a bcl-2-related gene encoding two distinct proteins, Bcl-xL and Bcl-xS (Boise et al., 1993). Although there have been many reports on the anti-apoptotic function and expression levels of Bcl-xL, there have been few investigations into the phosphorylation of Bcl-xL. Bcl-xL has attracted attention as a molecular target for anti-tumor drugs, as it is overexpressed in many human tumors, and has been shown to contribute to the resistance to the effects of anti-tumor compounds (Cory and Adams, 2002; Oltersdorf et al., 2005). Here, we elucidated the control of the anti-apoptotic function of Bcl-xL by phosphorylation.

We demonstrated that 13 residues of Bcl-xL were phosphorylated by Plk1 in vitro (Figure 1). Among them, Thr190, the most strongly phosphorylated residue, is the only phosphorylation residue within the BH2 domain. According to the multiple alignment analysis of the BH2 domain, Thr190 is not conserved in other Bcl-2 family proteins containing the BH2 domain. However, in human Bcl-w, Bok and Caenorhabditis elegans Ced-9, Thr190 was replaced with acidic amino acid. Most of the phosphorylated residues in Bcl-xL, including Thr190, Ser43 and Ser49, are not conserved in Bcl-2, which may be the reason why Bcl-2 was not efficiently phosphorylated by Plk1 (Figure 1a). Thus, we suppose that the functional control by Plk1-mediated phosphorylation may be a specific mechanism for Bcl-xL. As shown in Figure 4d, we did not observe the reduction of Bcl-xL proteins on each apoptosis-inducing condition. We concluded that the difference of anti-apoptotic function between Bcl-xLwt and Bcl-xL/13A on pironetin-induced apoptosis was not because of the phospho- and ubiquitin/proteasome-dependent degradation but because of functional change by phosphorylation. The three-dimensional structures of several Bcl-2 family proteins have been determined by X-ray and nuclear magnetic resonance (Petros et al., 2004). On the basis of the structure of Bcl-xL (Muchmore et al., 1996), Bcl-xL has a hydrophobic pocket, which binds to the pro-apoptotic Bcl-2 family proteins and is formed by BH1, BH2 and BH3 domains (Petros et al., 2004). Thr190 was on the hydrophobic pocket, indicating that the phosphorylation of Bcl-xL at Thr190 might alter the interaction with pro-apoptotic Bcl-2 family proteins. Thus, we investigated the effect of Bcl-xL phosphorylation on the interaction between Bcl-xL and Bax by immunoprecipitation assay (Kawatani and Imoto, 2003), but no significant change was detected irrespective of the presence of phosphorylation (data not shown). Further studies will be needed to clarify the role of other pro-apoptotic Bcl-2 family proteins during pironetin-induced apoptosis.

Most of the sites for phosphorylation of Bcl-xL by Plk1 are located in the flexible loop domain. To identify the phosphorylation sites of Bcl-xL, we used several deletion mutants or point mutants of Bcl-xL in which most Ser and Thr residues were replaced with Ala (Figure 1). The loop domain in particular includes many Ser and Thr residues, and these are often close to each other. The following consensus sequences were previously proposed for Plk1-phosphorylated sites: -D/E/Q-X-S/T-Φ- and -D/E-X-S/T-Φ-X-D/E- (X, any amino acid; Φ, a hydrophobic amino acid) (Nakajima et al., 2003; Barr et al., 2004). However, according to our in vitro kinase assays, residues that do not fit into this consensus sequence were significantly phosphorylated. In addition, phosphorylation site(s) of several known substrates of Plk1 often do not fit into the proposed consensus sequences either (Barr et al., 2004). Therefore, we would like to suggest expanding the consensus sequence to the following, which can accommodate almost all the Bcl-xL phosphorylation sites: -S/T-D/E/Φ- and/or -D/E-X0–2-S/T- and/or -S/T-X0–2-D/E-.

The immunocytochemical analysis indicated that Bcl-xL and Plk1 were distributed in the nucleus during normal mitosis (Figure 3). For this reason as well, we assumed that same location of Bcl-xL and Plk1 might be controlled by mitosis-dependent Plk1 upregulation or activation. It is known that Bcl-xL is dominantly localized to the mitochondrial outer membrane (Kaufmann et al., 2003), and Plk1 is localized to centrosomes and kinetochores during the normal cell cycle (Barr et al., 2004). However, it has been reported that Bcl-xL can exist in the cytosol as a homodimer that binds the BH3 domain and transmembrane domain (Jeong et al., 2004), and some phosphorylated Bcl-xL on Ser62 also localized on the nucleus during vinblastine-induced apoptosis in PC12 cells (Cittelly et al., 2007). Moreover, transmembrane domain deleted Bcl-xL and Bcl-2, which cannot localize on the mitochondria, and maintained the anti-apoptotic activity (Tamura et al., 2004; Zheng et al., 2008), suggesting that Bcl-xL can suppress apoptosis other than localizing on the mitochondria, but the apoptosis suppression-detailed mechanisms of the nucleus Bcl-xL remain unknown.

It has been reported that Plk1 phosphorylates many mitosis-associated proteins and is essential for cell division. Several substrates of Plk1 have been identified, and some substrates have multiple phosphorylation sites, such as Nlp, Mklp2/Rabkinesin6 and APC/C (Barr et al., 2004). Thus, it may be a reasonable suggestion that Bcl-xL was phosphorylated by Plk1 at multiple sites. It is known that the polo-box of Plk1 is involved in phospho-dependent substrate recognition, and it has been reported that the optimal binding sequence for phospho-dependent binding between Plk1 and substrate is -P/F-Φ/P-Φ/A/Q-T/Q/H/M-S-pS/pT-P/X- (Elia et al., 2003). As for the Bcl-xL, we have found a similar sequence (66-N-G-A-T-A-H-S-S-S-L-D-76), but have not yet defined whether it is the phospho-dependent Plk1 binding site.

We identified the sites of Bcl-xL phosphorylation by Plk1 in vitro and found that Bcl-xL/13A, in which all 13 phosphorylation sites were replaced with Ala, was more resistant to pironetin-induced apoptosis than was Bcl-xLwt. We proposed the possible mechanisms of apoptosis concerning Plk1 and Bcl-xL phosphorylation (Figure 5a). Plk1 is upregulated through JNK, Bcl-xL is phosphorylated and weakened after pironetin treatment, and cells are subsequently induced Caspase-independent apoptosis. Indeed, treatment with SP600125 suppressed pironetin-induced apoptosis (data not shown). Thus, we speculated that treatment with pironetin of the cells resulted in Plk1 upregulation via JNK, thereby leading to the induction of phosphorylated Bcl-xL, which could inhibit Caspase-dependent but not Caspase-independent apoptosis (Figure 5a). However, treatment with Plk1 siRNA failed to suppress pironetin-induced apoptosis (data not shown). As Plk1 phosphorylates and regulates many mitotic regulators, thus, depletion of Plk1 alone resulted in the induction of apoptosis.

Figure 5
figure5

Possible mechanism of apoptosis induction concerning Plk1 and Bcl-xL. (a) Treatment with pironetin of the cells overexpressing Bcl-xL results in upregulation of Plk1, and Bcl-xL is phosphorylated and weakened anti-apoptotic activity against Caspase-independent apoptosis, thereby the cells subsequently undergo apoptosis. (b) On the other hand, in ultraviolet-irradiated cells, Plk1 is downregulated, so Bcl-xL is not phosphorylated; thereby the cells overexpressing Bcl-xL are still resistant to apoptosis.

In contrast, Plk1 is downregulated and Bcl-xL is not phosphorylated after UV irradiation, and cells overexpressing Bcl-xL are still resistant to apoptosis. Indeed, it has already been reported that Plk1 mRNA and protein are downregulated after ionizing radiation (Ree et al., 2003), and UV irradiation decreases Plk1 protein (Chen et al., 2006). We also examined the effect of UV irradiation-induced apoptosis in wild-type and constitutive active mutant (S137D/T210D) of Flag-tagged Plk1-overexpressing HT1080-Bcl-xLwt cells. As the results, UV irradiation-induced apoptosis was enhanced by the transfection of Plk1 wild-type and constitutive active mutant, as compared with control vector-transfected cells (data not shown). These data supported that Plk1 inactivates Bcl-xL by phosphorylation. Thus, taken together, these findings suggest the possibility that Plk1 is a regulator of Bcl-xL phosphorylation and controls its anti-apoptotic function under certain specific conditions.

Materials and methods

Materials

Pironetin is an inhibitor of tubulin assembly, arrests cell cycle progression in M-phase and subsequently induces apoptosis (Kondoh et al., 1999). Pironetin covalently binds to Lys352 of α-tubulin and inhibits the interaction of tubulin heterodimers (Usui et al., 2004). A JNK inhibitor, SP600125, was purchased from Tocris Cookson Inc. (Ellisville, MO, USA).

Plasmid constructs

Human bcl-xL cDNA or 3HA-bcl-xL cDNA were subcloned into the pCI-neo vector (Promega, Madison, WI, USA) or the pRSET C vector (Invitrogen, Carlsbad, CA, USA). Human full-length Plk1 cDNA or FLAG-Plk1 cDNA was subcloned into the pZeoSV2 (+) vector (Invitrogen). The all point mutant or deletion mutant expression vectors were constructed using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA) (Simizu et al., 2005). The nucleotide sequences were checked by the dideoxynucleotide chain-termination procedure using an automated sequencer (Applied Biosystems, Foster City, CA, USA).

Cell culture and establishment of stable Bcl-xL- and Plk1-overexpressing cell lines

A human tumor cell line, HT1080 (fibrosarcoma), was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum at 37 °C in an incubator containing 5% CO2. The HT1080 cells were transfected with wild-type Bcl-xL (designated as Bcl-xLwt) or mutant forms of Bcl-xL expression vectors using the FuGENE 6 transfection reagent (Roche Diagnostics AG, Basel, Switzerland), which was followed by 400 μg/ml G418 (Calbiochem, San Diego, CA, USA) selection. In addition, the HT1080 cells were transfected with FLAG-tagged Plk1 expression vector using the Effectene transfection reagent (Qiagen GmbH, Hilden, Germany), which was followed by 50 μg/ml Zeocin (Invitrogen) selection. Each stable transfectant was cloned by the standard limited dilution method.

Western blot analysis

The cells were lysed in lysis buffer (10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-KOH, 142.5 mM KCl, 5 mM MgCl2, 1 mM EGTA (ethylene glycol bis(β-aminoethylether)-N,N,N′,N′,-tetraacetic acid), 0.2% Nonidet P-40, 0.1% aprotinin, 2 mM phenylmethylsulfonyl fluoride and pH 7.2) at 4 °C. To detect the mobility shifts in the Bcl-xL protein due to phosphorylation, an SDS–polyacrylamide gel containing 15% acrylamide and 0.07% N,N′-methylenebisacrylamide was used for separation. Proteins were immunoblotted with anti-Bcl-xL antibody (mouse monoclonal; Oncogene Research Products, San Diego, CA, USA), anti-Plk Cocktail (mouse monoclonal; Zymed, South San Francisco, CA, USA), anti-Actin antibody (rabbit polyclonal; Sigma, St Louis, MO, USA), anti-PARP-1 antibody (mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-cleaved Caspase-3 (Asp175) antibody (rabbit polyclonal; Cell Signaling Technology, Beverly, MA, USA). To obtain phospho-Ser62 Bcl-xL-specific antibody, phospho-Ser62 peptide (CPSWHLADpSPAVNGAT) and Ser62 peptide (CPSWHLADSPAVNGAT) were synthesized and used for antigen and affinity purification.

In vitro kinase assay

Recombinant GST-Plk1 protein (activated by okadaic acid) was prepared as described earlier (Watanabe et al., 2004). The His6-tagged recombinant Bcl-xL and Bcl-2 proteins lacking the C-terminal transmembrane domain (amino acid residues 213–233 and 219–239, respectively) were expressed in Escherichia coli BL21 (DE3) cells. Removal of the transmembrane domain was necessary to produce soluble Bcl-xL and Bcl-2 proteins. Cells were lysed in lysis buffer (20 mM HEPES-NaOH, 100 mM NaCl, 8 M urea, 20 mM imidazole and pH 8.0). The proteins were purified using Ni-nitrilotriacetic acid affinity agarose (Qiagen) and desalted using a PD-10 desalting column (Amersham Biosciences, Uppsala, Sweden). The kinase assay was carried out in reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, 10 μCi [γ-32P]ATP, 20 μM unlabeled ATP and pH 8.0) at 30 °C for 30 min. The samples were resolved by SDS–PAGE and visualized by autoradiography.

Two-dimensional electrophoresis

Cells were washed with ice-cold phosphate-buffered saline (PBS) twice and lysed in lysis buffer (40 mM Tris, 8 M urea and 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid)). Sonicated samples were centrifuged at 12 000 r.p.m. for 10 min. To remove the impurities, samples were purified by the two-dimensional Clean-Up Kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions. Samples were applied by rehydration onto immobilized pH gradient (IPG) strips (13 cm, pH 4–7, non-linear gradient; GE Healthcare) and subjected to isoelectrofocusing in an IPGphor IEF system (GE Healthcare). Strips were incubated in equilibration buffer (50 mM Tris-HCl, 6 M urea, 30% glycerol, 2% SDS and pH 8.8) containing 1% dithiothreitol for 15 min and thereafter incubated in the same buffer containing 2.5% iodoacetamide for 15 min. Strips were transferred to the tops of 15% polyacrylamide gels and carried out western blot analysis as described above.

siRNA transfection

The HT1080-Bcl-xLwt cells were transfected with siRNA (Nippon EGT, Toyama, Japan) against firefly luciferase (GL2; 5′-CGUACGCGGAAUACUUCGAdTdT-3′) or human Plk1 (5′-AGAUCACCCUCCUUAAUAdTdT-3′) by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Immediately after transfection, transfected cells were treated with or without pironetin and immunoblotted with appropriate antibodies as described above.

Cell staining

The cells grown on glass coverslips were fixed with 3.7% formaldehyde in PBS and incubated in 0.1% Triton X-100 in PBS for 5 min. After being washed with PBS, the cells were incubated with the appropriate first antibody for 30 min. We used anti-FLAG M2 (mouse monoclonal; Sigma), HA probe (rabbit polyclonal; Santa Cruz Biotechnology), anti-Plk Cocktail, anti-Plk1 (rabbit polyclonal; Calbiochem), anti-Bcl-x (mouse monoclonal; BD Biosciences, San Jose, CA, USA) and anti-Bcl-xL (rabbit polyclonal; Cell Signaling Technology). The samples were incubated with the appropriate fluoro-labeled secondary antibody for 30 min. Alexa Fluor 488- or Alexa Fluor 568-conjugated anti-mouse or anti-rabbit IgG antibodies (Molecular Probes, Eugene, OR, USA) were used as the secondary antibody. After being washed an additional three times, the samples were incubated with 2 μg/ml Hoechst 33258 (Wako, Osaka, Japan) for 5 min in the dark to stain the nucleus. The samples were then washed three more times with PBS and observed using a fluorescence microscope (Olympus, Tokyo, Japan).

Apoptosis detection

The cells were stained by propidium iodide solution (50 μg/ml propidium iodide, 0.1% sodium citrate and 0.2% Nonidet P-40), and apoptotic cells were detected by flow cytometry analysis (FACSort; Becton Dickinson, Franklin Lakes, NJ, USA) as a sub-G1 peak. Values are the means±s.d. of quadruplicate determinations.

References

  1. Barr FA, Silljé HH, Nigg EA . (2004). Polo-like kinases and the orchestration of cell division. Nat Rev Mol Cell Biol 5: 429–440.

  2. Basu A, Haldar S . (2003). Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis. FEBS Lett 538: 41–47.

  3. Boise LH, González-García M, Postema CE, Ding L, Lindsten T, Turka LA et al. (1993). bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74: 597–608.

  4. Chang BS, Minn AJ, Muchmore SW, Fesik SW, Thompson CB . (1997). Identification of a novel regulatory domain in Bcl-xL and Bcl-2. EMBO J 16: 968–977.

  5. Chao DT, Korsmeyer SJ . (1998). BCL-2 family: regulators of cell death. Annu Rev Immunol 16: 395–419.

  6. Chen J, Dai G, Wang YQ, Wang S, Pan FY, Xue B et al. (2006). Polo-like kinase 1 regulates mitotic arrest after UV irradiation through dephosphorylation of p53 and inducing p53 degradation. FEBS Lett 580: 3626–3630.

  7. Cheng EH, Levine B, Boise LH, Thompson CB, Hardwick JM . (1996). Bax-independent inhibition of apoptosis by Bcl-xL . Nature 379: 554–556.

  8. Cittelly DM, Nesic-Taylor O, Perez-Polo JR . (2007). Phosphorylation of Bcl-xL after spinal cord injury. J Neurosci Res 85: 1894–1911.

  9. Cory S, Adams JM . (2002). The BCL2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2: 647–656.

  10. Deverman BE, Cook BL, Manson SR, Niederhoff RA, Langer EM, Rosová I et al. (2002). Bcl-xL deamidation is a critical switch in the regulation of the response to DNA damage. Cell 111: 51–62.

  11. Du L, Lyle CS, Chambers TC . (2005). Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction. Oncogene 24: 107–117.

  12. Elia AE, Rellos P, Haire LF, Chao JW, Ivins FJ, Hoepker K et al. (2003). The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell 115: 83–95.

  13. Feng Y, Hodge DR, Palmieri G, Chase DL, Longo DL, Ferris DK . (1999). Association of polo-like kinase with α-, β- and γ-tubulins in a stable complex. Biochem J 339: 435–442.

  14. Gumireddy K, Reddy MV, Cosenza SC, Nathan RB, Baker SJ, Papathi N et al. (2005). ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent. Cancer Cell 7: 275–286.

  15. Jeong SY, Gaume B, Lee YJ, Hsu YT, Ryu SW, Yoon SH et al. (2004). Bcl-xL sequesters its C-terminal membrane anchor in soluble, cytosolic homodimers. EMBO J 23: 2146–2155.

  16. Kaufmann T, Schlipf S, Sanz J, Neubert K, Stein R, Borner C . (2003). Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane. J Cell Biol 160: 53–64.

  17. Kawatani M, Imoto M . (2003). Deletion of the BH1 domain of Bcl-2 accelerates apoptosis by acting in a dominant negative fashion. J Biol Chem 278: 19732–19742.

  18. Kondoh M, Usui T, Nishikiori T, Mayumi T, Osada H . (1999). Apoptosis induction via microtubule disassembly by an antitumour compound, pironetin. Biochem J 340: 411–416.

  19. Liu X, Erikson RL . (2003). Polo-like kinase (Plk) 1 depletion induces apoptosis in cancer cells. Proc Natl Acad Sci USA 100: 5789–5794.

  20. Muchmore SW, Sattler M, Liang H, Meadows RP, Harlan JE, Yoon HS et al. (1996). X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death. Nature 381: 335–341.

  21. Nakajima H, Toyoshima-Morimoto F, Taniguchi E, Nishida E . (2003). Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate. J Biol Chem 278: 25277–25280.

  22. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA et al. (2005). An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435: 677–681.

  23. Petros AM, Olejniczak ET, Fesik SW . (2004). Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta 1644: 83–94.

  24. Poruchynsky MS, Wang EE, Rudin CM, Blagosklonny MV, Fojo T . (1998). Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 58: 3331–3338.

  25. Ree AH, Bratland Å, Nome RV, Stokke T, Fodstad Ø . (2003). Repression of mRNA for the PLK cell cycle gene after DNA damage requires BRCA1. Oncogene 22: 8952–8955.

  26. Reed JC . (2002). Apoptosis-based therapies. Nat Rev Drug Discov 1: 111–121.

  27. Schinzel A, Kaufmann T, Borner C . (2004). Bcl-2 family members: intracellular targeting, membrane-insertion, and changes in subcellular localization. Biochim Biophys Acta 1644: 95–105.

  28. Simizu S, Osada H . (2000). Mutations in the Plk gene lead to instability of Plk protein in human tumour cell lines. Nat Cell Biol 2: 852–854.

  29. Simizu S, Takagi S, Tamura Y, Osada H . (2005). RECK-mediated suppression of tumor cell invasion is regulated by glycosylation in human tumor cell lines. Cancer Res 65: 7455–7461.

  30. Simizu S, Tamura Y, Osada H . (2004). Dephosphorylation of Bcl-2 by protein phosphatase 2A results in apoptosis resistance. Cancer Sci 95: 266–270.

  31. Tamura Y, Simizu S, Osada H . (2004). The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria. FEBS Lett 569: 249–255.

  32. Tsujimoto Y, Shimizu S . (2000). Bcl-2 family: life-or-death switch. FEBS Lett 466: 6–10.

  33. Usui T, Watanabe H, Nakayama H, Tada Y, Kanoh N, Kondoh M et al. (2004). The anticancer natural product pironetin selectively targets Lys352 of α-tubulin. Chem Biol 11: 799–806.

  34. Watanabe N, Arai H, Iwasaki J, Shiina M, Ogata K, Hunter T et al. (2005). Cyclin-dependent kinase (CDK) phosphorylation destabilizes somatic Wee1 via multiple pathways. Proc Natl Acad Sci USA 102: 11663–11668.

  35. Watanabe N, Arai H, Nishihara Y, Taniguchi M, Watanabe N, Hunter T et al. (2004). M-phase kinases induce phospho-dependent ubiquitination of somatic Wee1 by SCFβ-TrCP. Proc Natl Acad Sci USA 101: 4419–4424.

  36. Wyllie AH, Kerr JF, Currie AR . (1980). Cell death: the significance of apoptosis. Int Rev Cytol 68: 251–306.

  37. Zheng JY, Tsai YC, Kadimcherla P, Zhang R, Shi J, Oyler GA et al. (2008). The C-terminal transmembrane domain of Bcl-xL mediates changes in mitochondrial morphology. Biophys J 94: 286–297.

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Acknowledgements

We thank Y Ichikawa and R Nakazawa (Bioarchitect Research Group, RIKEN) for the DNA sequencing, and I Kagawa, M Kumai, E Oka and F Sakai (Research Resources Center, RIKEN) for the peptide synthesis, phospho-Ser62-specific rabbit polyclonal antibody production and affinity purification. This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Chemical Biology Project (RIKEN). YT and ST are recipients of the Junior Research Associate fellowship of RIKEN.

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Correspondence to H Osada.

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Tamura, Y., Simizu, S., Muroi, M. et al. Polo-like kinase 1 phosphorylates and regulates Bcl-xL during pironetin-induced apoptosis. Oncogene 28, 107–116 (2009) doi:10.1038/onc.2008.368

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Keywords

  • apoptosis
  • Bcl-xL
  • phosphorylation
  • Plk1
  • pironetin

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