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First published online 11 July 2006
doi: 10.1242/jcs.03033
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Research Article |
1 Laboratório de Microbiologia, Centro de Estudos de Química Orgânica, Fitoquímica e Farmacologia da Universidade do Porto (CEQOFFUP), Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
2 Centro de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal
3 Laboratório de Farmacologia, Centro de Estudos de Química Orgânica, Fitoquímica e Farmacologia da Universidade do Porto (CEQOFFUP), Faculdade de Farmácia, Universidade do Porto, Rua Aníbal Cunha 164, 4050-047 Porto, Portugal
* Author for correspondence (e-mail: mcortereal{at}bio.uminho.pt)
Accepted 3 May 2006
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Summary |
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, stimulate S. cerevisiae acetic-acid-induced apoptosis essentially through a mitochondrial ROS-dependent pathway. However, their co-expression with Bcl-xL reveals a PKC-isoform-dependent modulation of Bcl-xL anti-apoptotic activity. A yeast pathway homologue to the mammalian SAPK/JNK is responsible for acetic-acid-induced Bcl-xL phosphorylation that is differently modulated by PKC isoforms. The data obtained suggest conservation of an ancient mechanism of apoptosis regulation in yeast and mammals and offer new insights into mammalian apoptosis modulation by PKC isoforms.
Key words: Apoptosis regulation, Bcl-xL, Mammalian PKC isoforms, Yeast
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Introduction |
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). Recently, it has emerged that the apoptosis modulation-associated proteins are regulated through phosphorylation by different protein kinases, such as protein kinase C (PKC) (Gutcher et al., 2003). PKC is a family of serine/threonine kinases with at least ten isoforms grouped into three subfamilies based on their structure and cofactors required for activation: the conventional or classical (
, ßI, ßII and 
), the novel (, 
, 
and 
) and the atypical (
and 
/
) isoforms (Spitaler and Cantrell, 2004). Regulated by distinct means and presenting different tissue distribution patterns, PKC isoforms have deserved particular attention owing to their involvement in the regulation of prominent apoptotic members, such as proteins of the Bcl-2 family (Deng et al., 2000; Kornblau et al., 2000; Musashi et al., 2000). The Bcl-2 family, consisting of anti-apoptotic (such as Bcl-2 and Bcl-xL) and pro-apoptotic (such as Bax and Bad) members, function at a point in the cell death pathway that dictates whether or not cells are committed to die (Reed, 1997; Antonsson and Martinou, 2000). Therefore, owing to both inhibitory and stimulatory effects on Bcl-2 proteins, and consequently on apoptosis (Gutcher et al., 2003), PKC isoforms have been recognised as potential targets for therapeutic modulation (Deng et al., 2000; Kornblau et al., 2000; Gutcher et al., 2003).
Among the several isoforms, PKC-, -, - and -, expressed in the majority of tissues, have been considered as key players in apoptosis (Musashi et al., 2000; Gutcher et al., 2003). In general, classical and atypical PKCs appear to be associated with cell survival, whereas novel PKCs are associated with apoptosis stimulation (Gutcher et al., 2003). However, the function carried out by individual PKC isoforms in apoptosis remains to be clarified (Gutcher et al., 2003; Hofmann, 2004). Such information could lead to the identification of PKC isoforms that might be targeted to obtain a selective modulation of apoptosis (Gutcher et al., 2003). Yet, for that purpose independent analysis of the role of each isoform must be carried out (Musashi et al., 2000; Hofmann, 2001; Gutcher et al., 2003), a goal that is difficult to achieve with mammalian cells, as a result of the co-existence of several PKC isoforms in the same cell.
In the present work, we propose a new methodological approach to address the issues raised above. We have previously shown that expression of individual PKC isoforms in Saccharomyces cerevisiae allows the study of the modulatory activity of several compounds on individual isoforms (Saraiva et al., 2003; Saraiva et al., 2004). Furthermore, several authors have demonstrated that yeast undergoes apoptotic cell death similar to that seen in mammalian cells (reviewed by Madeo et al., 2002a; Madeo et al., 2004; Ludovico et al., 2005).
The present study reveals that PKC-, -, - or - stimulates S. cerevisiae acetic-acid-induced apoptosis. Moreover, it was shown that PKC isoforms modulate the Bcl-xL anti-apoptotic activity differently through interference with the phosphorylated or dephosphorylated forms of Bcl-xL. These results support the interpretation that control of mammalian apoptosis by PKC-, -, - and - can be attributed to their distinct modulation of Bcl-xL and possibly of other apoptotic proteins.
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Results |
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, -, - or -, -, - or - in yeast does not induce cell death (Saraiva et al., 2003; Saraiva et al., 2004). Therefore, to study whether these isoforms interfere with the apoptotic pathway in S. cerevisiae, acetic acid was selected as an inducer of apoptosis (Ludovico et al., 2001; Ludovico et al., 2002).
We found that expression of PKC-, -, - or - isoforms caused a marked increase in the percentage of dead cells compared with yeast transformed with the empty vector (Fig. 1A). This increase in cell death was accompanied by an increase in the number of TUNEL-positive cells (Fig. 1B), but not by an increase of the number of PI-positive cells (Fig. 1C). This indicates that the additional cell death resulting from expression of PKC isoforms is also apoptotic. Indeed, DNA degradation with preservation of plasma membrane integrity is a characteristic marker of apoptotic death in mammalian (Vermes et al., 2000) and in yeast cells (Madeo et al., 1997).
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, -, - or - expression on mitochondrial reactive oxygen species (ROS) production in acetic-acid-induced apoptosis was also studied using the mitochondria-selective probe MitoTracker Red CM-H2XRos. Fig. 2 shows the flow cytometric analysis of a representative experiment with cells treated in the absence (0 mM) and with 300 mM acetic acid. A very low ROS production was detected in yeast transformed with the empty vector (YEplac181 or YEp51 for PKC-) after treatment with 300 mM acetic acid (Fig. 2, compare dark-grey fill at 0 and 300 mM). Expression of each of the four PKC isoforms tested had very little effect on ROS production in cells not exposed to acetic acid, but resulted in the appearance of a second subpopulation displaying higher mean fluorescence intensity, indicative of increased ROS production in cells treated with 300 mM acetic acid (Fig. 2, compare grey line at 0 mM with 300 mM). This effect was particularly marked for PKC- and - and very small for PKC-. We confirmed, by epifluorescence analysis, that the subpopulation with increased mean fluorescence intensity displayed a bright red fluorescence localised in mitochondria (not shown).
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The possibility that PKC-, -, - or - expression interferes with the activity of the yeast caspase-related protease (Yca1p) during acetic-acid-induced apoptosis was also tested. Since in vivo yeast labelling with the caspase inhibitor FITC-VAD-FMK has been attributed to caspase activation (Madeo et al., 2002b; Herker et al., 2004), this activation was initially measured by flow cytometry of FITC-VAD-FMK stained cells. For yeast transformed with the empty vector (YEplac181 or YEp51 for PKC-), only about 20-30% of FITC-positive cells were found for the maximum acetic acid concentration tested (400 mM; see Fig. 3A). For this acetic acid concentration, expression of PKC isoforms did not increase the percentage of FITC-positive cells found with yeast transformed with the empty vector. The only exception occurred with cells expressing PKC- where a small increase in this percentage was observed (see Fig. 3A). Since caspase activation was only detected for the highest acetic acid concentration tested, and because a recent work reports that FITC-VAD-FMK binds nonspecifically to PI-positive cells, claiming that staining with this fluorogenic caspase substrate is subject to artefacts (Wysocki and Kron, 2004), additional tests to evaluate the possibility of nonspecific staining, were carried out. Thus, we investigated the effect of the nonfluorescent caspase inhibitor Z-VAD-FMK on the viability of acetic-acid-treated yeast cells expressing PKC-, for which the highest percentage of FITC-positive cells had been obtained. Although significant, Z-VAD-FMK only caused a decrease of 
16% in the percentage of dead cells (Fig. 3B). A similar value could be obtained by subtracting the percentage of PI-positive cells from the percentage of FITC-positive cells expressing PKC- after treatment with 400 mM acetic acid. This suggests that the real percentage of cells with caspase activation only corresponds to 16%. We also carried out a cell-free fluorimetric assay to assess caspase activity in yeast expressing PKC-, after treatment with 400 mM acetic acid. No caspase activation was detected for the three fluorogenic caspase substrates used (Fig. 3C). Non-detection of caspase activation by the in vitro fluorimetric assay is consistent with the low percentage of caspase activation detected by the other assays performed. Together these results suggest that stimulation of acetic-acid-induced apoptosis by PKC isoform is not associated with an enhancement of Yca1p activity.
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Mammalian PKC-, -, - or - differently modulate Bcl-xL anti-apoptotic effects in S. cerevisiae acetic-acid-induced apoptosis
First, acetic-acid-induced cell death in yeast expressing Bcl-xL and in yeast transformed with the two empty vectors (pOW4 and YEplac 181 or YEp51) was compared. For all acetic acid concentrations tested, Bcl-xL expression caused a significant reduction in the acid-induced yeast cell death compared with yeast transformed with the empty vectors (Fig. 4A). This reduction was accompanied by a decrease in the percentage of TUNEL-positive cells, particularly for 350 mM acetic acid (Fig. 4B) and by a marked decrease in ROS production (Fig. 5, compare grey lines with dark-grey fill at 300 mM acetic-acid-treated cells).
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, -, - or - on the observed Bcl-xL anti-apoptotic effect, Bcl-xL and individual mammalian PKC isoforms were co-expressed in S. cerevisiae. Afterwards, the effect of co-expression on cell survival, evaluated by c.f.u. counts (Fig. 4A), DNA degradation by TUNEL staining (Fig. 4B) and mitochondrial ROS production by MitoTracker Red CM-H2XRros staining (Fig. 5) was assessed. The results showed that PKC-, -, - and - interfered with the Bcl-xL anti-apoptotic effect, but in different ways. The protective effect of Bcl-xL in acetic-acid-induced apoptotic death was completely abolished when PKC- was co-expressed with Bcl-xL (Figs 4, 5). On the other hand, the effects obtained with co-expression of PKC- and Bcl-xL on acetic-acid-induced yeast apoptotic death were close to those obtained with yeast co-transformed with the two empty vectors, suggesting that co-expression of PKC- and Bcl-xL neutralises the effect caused by each protein when individually expressed. On the contrary, co-expression of PKC- or - with Bcl-xL caused a marked enhancement of the Bcl-xL anti-apoptotic effects (Figs 4, 5). In the cell death assays, a pronounced reduction in acetic-acid-induced yeast cell death was obtained for co-expression of both isoforms. Values close to those obtained with 0 mM acetic acid were found with 250 and 300 mM acetic acid for PKC-, or with 300 and 350 mM acetic acid for PKC- (Fig. 4A). Surprisingly, a better survival than that obtained with untreated control, was observed with 250 mM acetic acid for PKC- (Fig. 4A). The percentages of apoptotic cells assessed by TUNEL assay, obtained with 300 and 350 mM of acetic acid were also close to that obtained with 0 mM acetic acid, especially for PKC- (Fig. 4B). Co-expression of PKC- and Bcl-xL practically abolished ROS production induced by 300 mM acetic acid (Fig. 5, third row; compare black lines at 0 and 300 mM). With PKC- and Bcl-xL co-expression, ROS production was also lower than that obtained with cells only expressing Bcl-xL (Fig. 5, fourth row; compare black line with grey line at 300 mM acetic-acid-treated cells). Together these data show a PKC-isoform-dependent modulation of Bcl-xL anti-apoptotic effects in S. cerevisiae acetic-acid-induced apoptosis.
Mammalian PKC-, -, - or - co-expression in S. cerevisiae differently interferes with acetic-acid-induced Bcl-xL phosphorylation
Treatment of yeast expressing Bcl-xL with 300 mM acetic acid caused the appearance of a second, slow-migrating, Bcl-xL band at 32 kDa. Furthermore, the intensity of the two Bcl-xL bands was dependent on the co-expressed PKC isoform (Fig. 6A). Co-expression with PKC- strongly reduced the intensity of the fast-migrating Bcl-xL band, whereas co-expression with PKC- or - strongly reduced the intensity of the slow-migrating band. Co-expression with PKC- only caused a slight decrease in the intensity of the fast-migrating Bcl-xL band.
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; Du et al., 2005), a dephosphorylation reaction using the -protein phosphatase (-PPase) was carried out. As shown in Fig. 6B, the -PPase treatment of protein extracts obtained from yeast expressing Bcl-xL and exposed to 300 mM acetic acid completely abolished the 32 kDa band but did not affect the 29 kDa band. This confirms that the slow-migrating Bcl-xL band corresponds to a phosphorylated form of Bcl-xL (P-Bcl-xL).
JNK inhibitor II reduces acetic-acid-induced cell death and abolishes Bcl-xL phosphorylation in yeast expressing Bcl-xL, but not in yeast co-expressing PKC- and Bcl-xL
Recently, the role of c-Jun N-terminal kinase (SAPK/JNK) signalling pathway on taxol- or 2-methoxyestradiol-triggered Bcl-xL phosphorylation was demonstrated using the potent cell-permeable JNK inhibitor II, which eliminated Bcl-xL phosphorylation and reduced concomitant cell apoptosis (Basu and Haldar, 2003).
In order to assess whether a yeast pathway homologue to the mammalian SAPK/JNK pathway was involved in Bcl-xL phosphorylation induced by acetic acid, the JNK inhibitor II was used before exposure to acetic acid. Pre-treatment of yeast cells expressing Bcl-xL with 20 µM of JNK inhibitor II for 8 hours rendered the cells more resistant to acetic-acid-induced cell death and led to the elimination of the Bcl-xL phosphorylated form (Fig. 7, YEplac181 + Bcl-xL and YEp51 + Bcl-xL). By contrast, for yeast co-expressing PKC- and Bcl-xL, pre-treatment of cells with 20 µM of JNK inhibitor II for 8 hours did not affect either acid-induced cell death nor the Bcl-xL phosphorylated form (Fig. 7, PKC- + Bcl-xL). The results support the existence of a yeast pathway homologue to the mammalian JNK pathway responsible for acetic-acid-induced Bcl-xL phosphorylation. Furthermore, they suggest that PKC- is also involved in Bcl-xL phosphorylation by a pathway independent of the yeast homologue to the mammalian JNK pathway.
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Discussion |
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). Furthermore, S. cerevisiae has also been used as an adequate model to study the modulatory activity of several compounds on individual PKC isoforms (Saraiva et al., 2003; Saraiva et al., 2004). Although S. cerevisiae harbours a single PKC enzyme structurally similar to mammalian PKC isoforms, Pkc1p, with a key role in signalling pathways, none of the mammalian isoforms are functionally complementary to the yeast Pkc1p (Perez and Calonge, 2002). Additionally, it was demonstrated that Pkc1p does not respond to typical mammalian PKC modulators (Shieh et al., 1996; Keenan et al., 1997; Saraiva et al., 2003; Saraiva et al., 2004). The results presented here support the view that S. cerevisiae is a valuable cell model to study the role of each mammalian PKC isoform in apoptosis.
Expression of mammalian PKC-, -, - or - stimulates S. cerevisiae acetic-acid-induced apoptosis
Here it is shown that expression of PKC-, -, - and - causes a pronounced stimulation of the acetic-acid-induced apoptotic cell death and of DNA fragmentation, with preservation of plasma membrane integrity. Therefore, this additional cell death observed when a PKC isoform is expressed is also apoptotic. The observed stimulation of mitochondrial ROS production in cells expressing individual PKC isoforms, after treatment with acetic acid, is consistent with the involvement of a mitochondrial-dependent pathway in S. cerevisiae acetic-acid-induced apoptosis (Ludovico et al., 2002). It should be stressed that, in the present study, a higher resistance to acetic-acid-induced apoptosis is detected in transformed yeast leading to the use of acetic acid at concentrations higher than those used by Ludovico et al. (Ludovico et al., 2001; Ludovico et al., 2002). This greater resistance to apoptosis is particularly evident in the ROS assay where 300 mM acetic acid does not cause ROS production in yeast transformed with the empty vector. Madeo et al. (Madeo et al., 2002b) have already reported a higher resistance to apoptosis for S. cerevisisae transformed with YEp52.
Based on genetic evidence, Madeo et al. (Madeo et al., 2002b) reported that the yeast caspase-related protease (Yca1p) is an executor of acetic-acid-induced apoptosis in S. cerevisiae. However, only 20-30% of caspase-positive cells were now detected by flow cytometry in the population undergoing apoptosis induced by 400 mM acetic acid. In addition, the interference of expression of PKC isoforms in this reduced Yca1p activation is rather restricted, an observation that was confirmed by the small increase in survival with the inhibitor z-VAD-FMK. These results are consistent with an apoptotic pathway induced by acetic acid, in which the role of yeast caspase seems less relevant compared with other apoptotic stimuli like oxidative (Madeo et al., 2002b) or hyperosmotic (Silva et al., 2005) stresses. Therefore, stimulation of S. cerevisiae acetic-acid-induced apoptosis by PKC isoform expression seems to involve an Yca1p-independent pathway. Nevertheless, since it can be expected that the yeast genome contains other metacaspases besides Yca1p, which might not cleave the fluorogenic caspase substrates used in this study, we cannot preclude the hypothesis that the observed effect of PKC isoform on S. cerevisiae acetic-acid-induced apoptosis is mediated through a caspase-dependent process.
Because expression of PKC-, in contrast to the other PKC isoforms tested, does not cause a prominent stimulation of mitochondrial ROS production, we proposed that different mechanisms might lie behind the observed stimulation of acetic-acid-induced apoptosis. It is conceivable that under the conditions tested, PKC-, - or - expression triggers an oxidative DNA-damage-dependent pathway, whereas PKC- expression induces a preferential DNA-damage apoptotic pathway, which seems to be ROS independent. Actually, in mammalian cells there is increasing evidence that the key targets for PKC- are located in the nucleus, such as DNA-dependent protein kinase (DNA-PK) and nuclear lamin B (Bharti et al., 1998; Gutcher et al., 2003; Martelli et al., 2004). Ectopic expression of the catalytically active form of PKC- failed to induce apoptosis in cells deficient in DNA-PK (Bharti et al., 1998). In yeast, the DNA-PK components, Ku70 and Ku80, are required for DNA-damage response and have functions similar to their mammalian counterparts (Burhans et al., 2003). Whether stimulation of yeast apoptosis by PKC- is mediated by phosphorylation of the yeast orthologues of DNA-PK and consequent enhancement of DNA damage is a question that deserves further study.
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Co-expression of mammalian PKC-, -, - or - modulates the anti-apototic activity of Bcl-xL in S. cerevisiae
Together, the aforementioned results show that all the PKC isoforms tested cause a similar pro-apoptotic effect when expressed in yeast, in contradiction with that described for mammalian cells. In mammalian cells, PKC-, -, - and - are considered major isoforms in apoptosis and have distinct influences on apoptosis regulation (Musashi et al., 2000; Hofmann, 2001; Gutcher et al., 2003). Many proteins are known to be involved in the regulation of apoptosis, and the members of the Bcl-2 family are recognised central regulators, dictating the fate of the cell either to live or die (Reed, 1997; Antonsson and Martinou, 2000). Several authors have suggested that the inhibitory and stimulatory influences of PKC isoforms on apoptosis can be mainly due to regulation, through phosphorylation, of proteins of the Bcl-2 family (Deng et al., 2000; Kornblau et al., 2000; Musashi et al., 2000). The absence in yeast of endogenous proteins of the Bcl-2 family may be a possible explanation for the similar pro-apoptotic effects caused by PKC-, -, - or - expression in S. cerevisiae.
To test this hypothesis we co-expressed in yeast the PKC isoform with Bcl-xL, an anti-apoptotic protein of the Bcl-2 family, and evaluated the effect of this co-expression in S. cerevisiae acetic-acid-induced apoptosis. Expression of Bcl-xL in S. cerevisiae results in an increase in cell survival after treatment with acetic acid, accompanied by a reduction in DNA degradation and mitochondrial ROS production. These results demonstrate an anti-apoptotic cytoprotective effect of Bcl-xL in acetic-acid-induced yeast cell death and add new evidence pointing to the involvement of this mode of cell death in an apoptotic pathway. It was previously shown that Bcl-xL inhibits yeast apoptotic death induced by other apoptotic stimuli, such as hydrogen peroxide, menadione and heat that, like acetic acid, induce ROS production (Chen et al., 2003). The anti-apoptotic effect exhibited by Bcl-xL expression in yeast cells parallels that described for mammalian cells (Antonsson and Martinou, 2000). Together, these data further reinforce the idea that a bona fide cell death program occurs in yeast and that pathways downstream of Bcl-xL are conserved between yeast and higher eukaryotes, as suggested by Burhans et al. (Burhans et al., 2003). In addition, the occurrence of an anti-apoptotic effect of Bcl-xL in this unicellular eukaryote corroborates the use of yeast as a tool to unravel the complexities of the function of the Bcl-2 members in apoptosis.
We then evaluated if PKC-, -, - and - interfered with the Bcl-xL anti-apoptotic activity. The results obtained reveal an isoform-dependent influence of PKC on the Bcl-xL anti-apoptotic activity. Co-expression of PKC- or - with Bcl-xL completely abolishes the Bcl-xL anti-apoptotic activity, whereas co-expression of PKC- or - with Bcl-xL causes a marked enhancement of the Bcl-xL anti-apoptotic activity.
These results reinforced the hypothesis of a distinct mechanism for acetic-acid-induced apoptosis stimulation by PKC-. In fact, PKC-, - or -, which elicited a pronounced stimulation of mitochondrial ROS production, also interfered markedly with the mitochondrial anti-apoptotic protein Bcl-xL. Consistently, PKC-, which only caused a slight stimulation of mitochondrial ROS production, displayed a reduced effect on this mitochondrial protein. Furthermore, they corroborate the interpretation that the different influences of mammalian PKC isoforms on apoptosis can be due to distinct modulation of apoptotic members of the Bcl-2 family by each isoform (Gutcher et al., 2003).
A yeast pathway homologue to the mammalian SAPK/JNK is responsible for acetic-acid-induced Bcl-xL phosphorylation that is differently modulated by PKC isoforms
Using a cell-free phosphatase assay we detect, for the first time, the existence of a phosphorylated form of Bcl-xL after treatment with acetic acid in yeast expressing this protein. These results suggest that phosphorylation of Bcl-xL by a stress-response kinase-signalling pathway hinders the anti-apoptotic function of Bcl-xL and permits yeast cells to die by acid-induced apoptosis, as previously reported for mammalian cells (Fan et al., 2000; Basu and Haldar, 2003).
Moreover, we find that the abolishment of the Bcl-xL anti-apoptotic effect by PKC- co-expression is accompanied by a pronounced decrease of the Bcl-xL dephosphorylated form, and that the remarkable increase in the Bcl-xL anti-apoptotic effect by PKC- or - co-expression is accompanied by a pronounced decrease of the Bcl-xL phosphorylated form. Therefore, a possible explanation for the distinct modulation of Bcl-xL anti-apoptotic activity by each PKC isoform can be ascribed to a PKC-isoform-dependent modulation of Bcl-xL phosphorylation. These data further reinforce the hypothesis of a distinct mechanism for acetic-acid-induced apoptosis stimulation by expression of PKC-, as discussed above. In fact, PKC-, - or -, which elicited a pronounced stimulation of mitochondrial ROS production also interfered markedly with Bcl-xL, an anti-apoptotic protein primarily targeted to the mitochondria (Polcic and Forte, 2003). By contrast, PKC- caused only a slight stimulation of mitochondrial ROS and consistently displayed a reduced effect on that protein.
Although the involvement of protein kinases in Bcl-xL phosphorylation, in response to distinct apoptotic stimuli, is reported in several studies with mammalian cells, the kinase(s) implicated still remain unclear (Du et al., 2005). Nevertheless, Jun N-terminal kinase (JNK) is a kinase responsible for phosphorylation of Bcl-xL, as well as of other anti-apoptotic members of the Bcl-2 family, in response to oxidative stress (Fan et al., 2000; Basu and Haldar, 2003). Consistently with the results obtained in prostate cancer cells treated with taxol or 2-methoxyestradiol (Basu and Haldar, 2003), pre-treatment of yeast cells expressing Bcl-xL with JNK inhibitor II renders the cells more resistant to acetic-acid-induced apoptosis and eliminates Bcl-xL phosphorylated form. These results support the idea that Bcl-xL phosphorylation disables its anti-apoptotic function. In addition, they suggest the existence of a pathway in yeast homologous to the mammalian SAPK/JNK pathway responsible for acetic-acid-induced Bcl-xL phosphorylation. The mammalian protein kinases p38 and SAPK/JNK are structurally and functionally homologous to Hog1p in yeast (Kültz et al., 1997). Therefore, it is expected that they share similar regulatory mechanisms (Takekawa et al., 1998) and it should be considered that Hog1p mediates the observed Bcl-xL phosphorylation in response to acetic acid treatment in yeast. Supporting this possibility, Hog1p activation has already been observed in response to weak carboxylic acid stress (Lawrence et al., 2004). By contrast, in yeast co-expressing PKC- and Bcl-xL, JNK inhibitor II does not interfere with either cell death or Bcl-xL phosphorylation. These results suggest that Bcl-xL phosphorylation can occur by two independent pathways and that PKC- can be involved in phosphorylation of Bcl-xL through a pathway independent of SAPK/JNK.
Du et al. (Du et al., 2005) proposed the existence in mammalian cells of a kinase and phosphatase system that may be operating in tandem, leading to a coordinated phosphorylation-dephosphorylation cycle that modulates Bcl-xL activity. However, the precise mechanisms for this modulation remain to be determined. Our study offers new insights into the role of phosphorylation on the modulation of the Bcl-xL function, identifying individual PKC isoforms as modulators of the phosphorylation-dephosphorylation of Bcl-xL when expressed in yeast. Indeed, we demonstrate that PKC- can phosphorylate Bcl-xL directly, or indirectly through a kinase other than the yeast homologue to the mammalian JNK, and the possibility of modulation of Bcl-xL dephosphorylation through a yeast phosphatase activation by PKC- and - is currently under investigation.
In conclusion, the results presented here point to conservation of targeted Bcl-xL phosphorylation in yeast and mammals and hence to the existence of a common ancient mechanism of apoptosis regulation. In the context of our findings and the recognised involvement of PKC on mammalian apoptosis regulation there is the interesting possibility that the isoforms studied preserve their functional role regarding Bcl-xL phosphorylation. In addition, the elucidation of the enzymology of Bcl-xL phosphorylation may contribute to understand the molecular mechanism of action of anti-neoplasic chemotherapeutic drugs (Du et al., 2005) and to design new strategies for apoptosis modulation. Finally, the genetically tractable yeast promises advances in the unravelling of apoptosis regulation, namely of the Bcl-2 members by individual PKC isoforms, towards a better exploitation of mammalian PKC as a therapeutic target.
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expression, the bovine PKC- cDNA was cloned into the YEp51 yeast expression plasmid. For PKC-, PKC- and PKC- expression, the rat PKC-, the mouse PKC- or PKC- cDNA, respectively, was cloned into the YEplac181 yeast expression plasmid. These plasmids contain the promoter GAL (GAL1-10; GAL10 in YEp51 and GAL1 in YEplac181) that is galactose inducible, and the LEU2 gene as a selectable marker. For Bcl-xL expression, the human Bcl-xL cDNA was cloned into the pOW4 yeast expression plasmid. This plasmid contains an ADH1 promoter for Bcl-xL expression and a URA3 gene as a selectable marker.
All the plasmids used were amplified in Escherichia coli DH5 and confirmed by restriction analysis.
Yeast strain, transformation and growth conditions
Saccharomyces cerevisiae CG379 [ ade5 his7-2 leu2-112 trp1-289 ura3-52 (Kil-O)] strain was used for the yeast expression studies. Yeast cells were transformed using the lithium acetate method (Ito et al., 1983). To ensure selection of transformed yeast, cells were routinely grown in a minimal selective medium, consisting of 2% (w/v) glucose, 0.67% (w/v) yeast nitrogen base with ammonium sulphate (Difco) and all the amino acids required for yeast growth (50 µg/ml), except leucine (for yeast only transformed with YEp51 or YEplac181 expression plasmids) or leucine and uracil (for yeast co-transformed with pOW4 and YEplac181 or YEp51 expression plasmids). Transformed yeast cells were previously grown in this medium at 30°C with continuous shaking (150 r.p.m.), to approximately 1 OD600 (Cary 1E Varian Spectrophotometer). Cells were afterwards collected by centrifugation, diluted to 0.05 OD600 in a 2% (w/v) galactose-selective medium (PKC transcription inducer) with 3% (v/v) glycerol (alternative carbon source) and grown to 0.5 OD600 (mid-log phase).
Western blot
Cell extracts of about 109 cells were prepared in 50 µl lysis buffer containing 0.5% NP-40, 20 mM HEPES (pH 7.4), 84 mM KCl, 10 mM MgCl2, 0.2 mM EDTA, 0.2 mM EGTA, 1 mM DTT, 5 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin and 1 mM phenylmethylsulfonyl fluoride. Cell suspensions were mixed with the same volume of glass beads (0.45-0.5 mm diameter; Sigma) and lysed mechanically by six 30-second vortexing steps interrupted by cooling on ice for 1 minute. Cell lysates were then clarified by centrifugation at 20,000 g for 15 minutes at 4°C. The supernatant was collected and protein concentration was determined using a kit for protein quantification (Coomassie® Protein Assay Reagent Kit, Pierce). Protein lysates (10-15 µg/lane) were separated on 15% SDS-polyacrylamide gels (SDS-PAGE) and transferred to nitrocellulose membranes (Bio-Rad). Preblocking and immunostaining of blots were performed in Tris-buffered saline (TBS) containing 0.05% Tween 20 and 5% non-fat milk at room temperature. After preblocking for 1 hour, membranes were incubated with the primary antibody for 2 hours, followed by incubation with the secondary antibody for 1 hour. For PKC detection, membranes were incubated with a specific rabbit antibody to the individual mammalian PKC isoform: anti-PKC- (1:20,000 dilution); anti-PKC- (1:11,000 dilution); anti-PKC- (1:16,000 dilution) or anti-PKC- (1:20,000 dilution) (Sigma), followed by the horseradish-peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilution) (Sigma). For Bcl-xL detection, membranes were incubated with a Bcl-xL goat polyclonal antibody (1:300 dilution) (Santa Cruz Biotechnology), followed by the horseradish-peroxidase-conjugated rabbit anti-goat IgG (1:5000 dilution) (Zymed Laboratories). Membranes were stripped of bound antibody by incubating the strip in a buffer comprising 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM ß-mercaptoethanol at 50°C for 30 minutes, and then reprobing with an actin rabbit antibody (1:200 dilution) (Sigma) followed by the horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilution). Immunoblots were developed by enhanced chemiluminescence (ECL Western Blotting Analysis System; Amersham Pharmacia Biotech).
Western blot analysis showed that expression of the four PKC isoforms in S. cerevisiae, after incubation in galactose-selective medium, occurred at comparable levels (Fig. 8A, third lane). Expression of Bcl-xL was confirmed by the occurrence of a single band at 29 kDa (Fig. 8B). Expression of individual PKC isoforms or of Bcl-xL was not affected by co-expression of both proteins (Fig. 8A,B).
Cell death assays
Exponential phase transformed yeast cells, previously grown in 2% (w/v) galactose-selective medium, were harvested and suspended in Sabouraud Dextrose Broth Medium (Difco) containing 0, 250, 300, 350 or 400 mM acetic acid and with a final pH 3.0 (set with HCl). The treatments were carried out for 1 hour at 30°C with mechanical shaking (200 r.p.m.). In all the experiments, the extracellular pH did not change during incubations.
Yeast cells expressing Bcl-xL (YEplac181 + Bcl-xL; YEp51 + Bcl-xL) and co-expressing PKC- and Bcl-xL (PKC- + Bcl-xL) were pre-treated only in the presence of solvent (DMSO) or in the presence of 20 µM JNK inhibitor II (Calbiochem) for 8 hours followed by incubation with 300 mM acetic acid for 1 hour at 30°C.
Cell death was determined by colony forming unit (c.f.u.) counts after 2 days incubation at 30°C on Sabouraud Dextrose Agar (Difco) plates. No further colonies appeared after that incubation period. The percentage of dead cells was estimated considering 0% dead cells (100% survival) the number of c.f.u. obtained after 1 hour incubation in the absence of acetic acid (0 mM).
Propidium iodide and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) staining
Propidium iodide (PI) staining was used to monitor cell membrane integrity, as previously described (Ludovico et al., 2001). After treatment with acetic acid, 300 µl of yeast samples were taken and incubated with 1.5 µl of 1 mg/ml PI (Molecular Probes), for 10 minutes at room temperature. TUNEL staining, to detect DNA strand breaks, was performed using the In Situ Cell Death Detection Kit, Fluorescein (Roche Applied Science) and basically as described (Ludovico et al., 2001).
Cells incubated in the absence of acetic acid (0 mM) were used as negative control. The samples were observed under an Eclipse E400 fluorescence microscope (Nikon, Japan) equipped with a 100 W mercury lamp and appropriate filter setting.
Determination of ROS production
Reactive oxygen species (ROS) production by mitochondria was monitored by flow cytometry using a mitochondria-selective probe, MitoTracker Red CM-H2XRos (Molecular Probes), mainly as described (Ludovico et al., 2002). After treatment with acetic acid, about 107 cells/ml were harvested, washed once and resuspended in 1 ml phosphate-buffered saline solution (PBS, pH 7.4) and preloaded with 0.4 µg/ml dye for 20 minutes at 37°C. Cells incubated in the absence of acetic acid (0 mM) were used as a negative control.
Determination of caspase activity
Flow cytometric analysis: Detection of caspase activation was performed using the CaspACE, FITC-VAD-FMK In Situ Marker Kit (Promega). Briefly, after treatment with acetic acid, 106 cells were washed with PBS, resuspended in 100 µl staining solution containing 50 µM of FITC-VAD-FMK and incubated for 25 minutes at 30°C. After incubation, cells were washed once with PBS and resuspended in 1 ml PBS. Cells incubated in the absence of acetic acid (0 mM) were used as a negative control.
Cell death assay in the presence of a caspase inhibitor
In experiments with the nonfluorescent caspase inhibitor Z-VAD-FMK (Promega), cells were treated for 2 hours at 30°C with 100 µM of Z-VAD-FMK, before treatment with 300 mM acetic acid. Cell death was assessed as described above.
Fluorimetric analysis
Cell extracts were obtained as described for western blot. Caspase activity was determined by incubation of 25 µg protein lysate with 50 µM of the fluorogenic caspase substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin (Ac-DEVD-AMC), N-acetyl-Ile-Glu-Thr-Asp-aminomethylcoumarin (Ac-IETD-AMC) or N-acetyl-Val-Glu-Ile-Asp-aminomethylcoumarin (Ac-VEID-AMC) (Biomol) in 200 µl buffer containing 50 mM HEPES (pH 7.3), 100 mM NaCl, 10% sucrose, 0.1% CHAPS, and 10 mM DTT. Cells incubated in the absence of acetic acid (0 mM) were used as a negative control. The recombinant caspase 3 (for Ac-DEVD-AMC), caspase 6 (for Ac-VEID-AMC) and caspase 8 (for Ac-IETD-AMC) were used for testing the fluorogenic substrates and assay conditions (positive control). Aminomethylcoumarin (AMC) release was monitored for 90 minutes at 30°C in a SpectraMAX Gemini Fluorescence Plate Reader (Molecular Devices), using an excitation wavelength of 360 nm and an emission wavelength of 475 nm. Caspase activity was determined as the slope of the resulting linear regressions and expressed in arbitrary fluorescence units per minute.
Flow cytometric data acquisition and analysis
Flow cytometric analysis was performed in an Epics® XL-MCLTM (Beckman Coulter), flow cytometer equipped with an argon-ion laser emitting a 488 nm beam at 15 mW. Green fluorescence was collected through a 488 nm blocking filter, a 550 nm long-pass dichroic and a 525 nm band-pass filter. Red fluorescence was collected through a 560 nm short-pass dichroic, a 640 nm long-pass, and another 670 nm long-pass filter. 20,000 cells were analysed per sample at low flow rate. Data were analysed by WinMDI 2.8 software.
In vitro phosphatase treatment
Cell extracts of co-transformed yeast treated with 300 mM acetic acid, were prepared as described for western blot. Aliquots of 25 µg protein lysate were incubated with and without 400 units of -phosphatase (-PPase; New England BioLabs) in 25 µl phosphatase buffer containing 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT, 0.01% Brij 35 and 2 mM MnCl2, for 30 minutes at 30°C. The reaction was stopped by boiling for 5 minutes in Laemmli sample buffer. Cell extracts were afterward analysed by western blot as described above.
Statistical analysis
Data were analysed statistically using the SigmaStat 3.1 programme (SYSTAT Software, Inc.). Differences between means were tested for significance using the unpaired Student's t-test. P values of 0.05 or lower were considered statistically significant. Results are expressed as the mean ± s.e.m. of the indicated number of experiments.
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Acknowledgments |
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, to Dr Nigel Goode for YEplac181, YEplac181-PKC-, YEplac181-PKC- and YEplac181-PKC- and to Dr Charles Rudin for pOW4 and pOW4-Bcl-xL. We are also grateful to IZASA for the use of the Epics® XL-MCLTM (Beckman Coulter); to Joana Tavares for her help in some experiments; to Vitor Costa and Natércia Teixeira for providing some reagents; and to Elsa Bronze for technical advice in the phosphatase assay. We thank Fundação para a Ciência e a Tecnologia, (FCT, Lisbon, Portugal) (I&D No. 226/94 and 655) POCTI and FEDER for financial support. |
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