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First published online 11 July 2006
doi: 10.1242/jcs.03033

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Specific modulation of apoptosis and Bcl-xL phosphorylation in yeast by distinct mammalian protein kinase C isoforms

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

View larger version (24K): [in a new window]   Fig. 1. Expression of mammalian PKC-, -, - or - in S. cerevisiae stimulates acetic-acid-induced apoptosis. Yeast transformed with the empty vector, YEplac181 or YEp51 (black) or yeast expressing PKC-, -, - or - (grey) were incubated with acetic acid (300, 350 or 400 mM) for 1 hour at 30°C. (A) Cell death experiments. Percentage of dead cells was determined by c.f.u. counts, considering as 0% dead cells (100% survival) the number of c.f.u. obtained after 1 hour incubation with no acetic acid. Data are the mean ± s.e.m. of three independent experiments with six replicates. (B) TUNEL staining and (C) Propidium iodide (PI) staining. Cells treated in the absence of acetic acid (0 mM) were used as negative control. Data are the mean ± s.e.m. of two independent experiments; means correspond to counts of at least 600 cells per sample. Significant differences from those obtained with the empty vector are indicated by *P<0.05; **P<0.001 (unpaired Student's t-test).

View larger version (18K): [in a new window]   Fig. 2. Stimulation of S. cerevisiae acetic-acid-induced apoptosis by expression of mammalian PKC-, - or - but not PKC- isoform is associated with significant ROS production. ROS production by mitochondria was detected by flow cytometry, using MitoTracker Red CM-H2XRos. Overlays of red fluorescence histograms were obtained with yeast treated with 0 and 300 mM acetic acid: black fill, yeast transformed with the empty vector (YEplac181 or YEp51); light-grey line, yeast expressing PKC-, -, - or -. Cells treated in the absence of acetic acid (0 mM) were used as a negative control. Data represent one of two independent experiments.

View larger version (21K): [in a new window]   Fig. 3. Stimulation of S. cerevisiae acetic-acid-induced apoptosis by expression of mammalian PKC-, -, - or - is not mediated by yeast caspase activation. (A) Flow cytometric analysis. Percentage of cells with active caspases obtained with yeast transformed with the empty vector (YEplac181 or YEp51) and with yeast expressing PKC-, -, - or -, after 1 hour treatment with 400 mM acetic acid. Treated cells were labelled with 50 µM FITC-VAD-FMK for 25 minutes at 30°C and analysed by flow cytometry. Cells treated in the absence of acetic acid (0 mM) were used as negative control. Data represent one of two independent experiments. (B) Effect of the caspase inhibitor Z-VAD-FMK on the survival of yeast expressing PKC- treated with 400 mM acetic acid. Before treatment with acetic acid, cells were pre-treated without (control) or with 100 µM Z-VAD-FMK for 2 hours at 30°C. The percentage of dead cells was determined by c.f.u. counts, as in Fig. 1. Data are the mean ± s.e.m. of two independent experiments with six replicates. Significant differences from those obtained with the empty vector are indicated by *P<0.05 (unpaired Student's t-test). (C) Fluorimetric analysis. Cell extracts obtained from yeast expressing PKC- and treated with 0 mM (negative control) or 400 mM acetic acid, were incubated with 50 µM of the fluorogenic caspase substrates Ac-DEVD-AMC, Ac-IETD-AMC or Ac-VEID-AMC. Recombinant caspase 3 (for Ac-DEVD-AMC), caspase 6 (for Ac-VEID-AMC) and caspase 8 (for Ac-IETD-AMC) were used as positive control. Aminomethylcoumarin (AMC) release was monitored for 90 minutes at 30°C in a spectrofluorimeter. Caspase activity is expressed in arbitrary fluorescence units (FU)/minute. Data are the mean ± s.e.m. of two independent experiments.

View larger version (21K): [in a new window]   Fig. 4. Expression of Bcl-xL abrogates S. cerevisiae acetic-acid-induced apoptosis. Co-expression of mammalian PKC-, -, - or - differently modulate Bcl-xL effects in S. cerevisiae acetic-acid-induced apoptosis. Yeast co-transformed with the empty vectors, pOW4 and YEp51 or pOW4 and YEplac181 (black), expressing Bcl-xL (pale grey) and co-expressing Bcl-xL and PKC-, -, - or - (dark grey) were incubated with acetic acid for 1 hour at 30°C. (A) Cell death experiments. Percentage of dead cells was determined by c.f.u. counts as in Fig. 1. Data are the mean ± s.e.m. of three independent experiments with six replicates. (B) TUNEL staining. Cells treated in the absence of acetic acid (0 mM) were used as negative control. Data are the mean ± s.e.m. of two independent experiments; means correspond to counts of at least 600 cells per sample. Significantly differences from those obtained with yeast co-transformed with the empty vectors and with yeast only expressing Bcl-xL are indicated by *P<0.05 and #P<0.05 (unpaired Student's t-test), respectively.

View larger version (22K): [in a new window]   Fig. 5. Expression of Bcl-xL reduces S. cerevisiae acetic-acid-induced ROS production. Co-expression of PKC-, -, - or - differently modulates Bcl-xL effects on ROS production. ROS production by mitochondria was detected by flow cytometry, using MitoTracker Red CM-H2XRos. Overlay of red fluorescence histograms was obtained with yeast treated with 0 and 300 mM acetic acid. Dark grey fill, cells co-transformed with the empty vectors (pOW4 and YEp51 or pOW4 and YEplac181); light grey line, cells expressing Bcl-xL; black line, cells co-expressing Bcl-xL and PKC-, -, - or -. Cells treated in the absence of acetic acid (0 mM) were used as negative control. Data represent one of two independent experiments.

View larger version (22K): [in a new window]   Fig. 6. Mammalian PKC-, -, - or - co-expression in S. cerevisiae differentially interferes with acetic-acid-induced Bcl-xL phosphorylation. (A) Western blot analysis reveals two different Bcl-xL migrating bands in protein extracts obtained from yeast expressing Bcl-xL and treated with 300 mM acetic acid for 1 hour. The intensity of these two Bcl-xL bands is dependent on the PKC co-expressed isoform. Without acetic acid, only a single band of Bcl-xL at 9 kDa can be observed (see Fig. 8B); the lane corresponding to protein extracts obtained from yeast co-expressing PKC- and Bcl-xL was located in a different part of the same gel. (B) In vitro phosphatase treatment of protein extracts obtained from yeast expressing Bcl-xL and treated with 300 mM acetic acid for 1 hour. Yeast extract with 25 µg protein was incubated with 400 U -protein phosphatase (+-PPase), at 30°C for 30 minutes. Yeast extract without -PPase was used as a control. Western blot analysis showed that the slow-migrating Bcl-xL band (32 kDa) was completely abolished by -PPase, indicating that this band corresponds to a phosphorylated form of Bcl-xL (P-Bcl-xL). For western blot analysis, protein extracts were separated by 15% SDS-PAGE. For Bcl-xL detection an anti-Bcl-xL goat polyclonal antibody, followed by horseradish-peroxidase-conjugated rabbit anti-goat IgG, were used. For ß-actin detection, used as a loading control, membranes were stripped and then reprobed with an anti-actin rabbit antibody. Immunoblots were developed by enhanced chemiluminescence.

View larger version (33K): [in a new window]   Fig. 7. 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. Yeast cells expressing Bcl-xL (YEplac181 + Bcl-xL; YEp51 + Bcl-xL) and co-expressing PKC- and Bcl-xL (PKC- + Bcl-xL) were pre-treated in the presence of solvent (DMSO) or in the presence of 20 µM JNK inhibitor II for 8 hours followed by treatment with 0 and 300 mM acetic acid for 1 hour at 30°C. Percentage of dead cells was determined by c.f.u. counts as in Fig. 1. Data are the mean ± s.e.m. of five to eight independent experiments with six replicates. Significant differences from those obtained with DMSO are indicated by *P<0.05 (unpaired Student's t-test). For western blot analysis, protein extracts were separated by 15% SDS-PAGE. For Bcl-xL detection an anti-Bcl-xL goat polyclonal antibody, followed by horseradish peroxidase-conjugated rabbit anti-goat IgG, were used. For ß-actin detection, used as a loading control, membranes were stripped and then reprobed with an anti-actin rabbit antibody. Immunoblots were developed by enhanced chemiluminescence.

View larger version (65K): [in a new window]   Fig. 8. Expression of individual mammalian PKC isoforms or of Bcl-xL in S. cerevisiae is not affected by co-expression of both proteins. (A) Comparable levels of PKC isoform expression were detected in protein extracts obtained from yeast expressing PKC-, -, - or - (PKC-pOW4 lanes) and from yeast co-expressing the PKC isoform and Bcl-xL (PKC-Bcl-xL lanes), after incubation in 2% (w/v) galactose selective medium. Protein extracts obtained from yeast co-transformed with the empty vectors were used as negative controls (YEp51-pOW4 or YEplac181-pOW4 lanes). (B) Comparable levels of Bcl-xL expression were detected in protein extracts obtained from yeast expressing Bcl-xL (YEp51-Bcl-xL or YEplac181-Bcl-xL lanes) and from yeast co-expressing the PKC isoform and Bcl-xL (PKC-, -, - or --Bcl-xL lanes), after incubation in 2% (w/v) galactose selective medium. Protein extracts obtained from yeast co-transformed with the empty vectors were used as negative controls (YEp51/pOW4 or YEplac181-pOW4 lanes). Protein extracts (10-15 µg/lane) were separated on 15% SDS-PAGE followed by western blot analysis. For PKC isoform detection, a specific rabbit antibody to each PKC isoform, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG, was used. For Bcl-xL detection, anti-Bcl-xL goat polyclonal antibody, followed by horseradish peroxidase-conjugated rabbit anti-goat IgG, was used. For ß-actin detection, used as a loading control, membranes were stripped and then reprobed with an anti-actin rabbit antibody. Immunoblots were developed by enhanced chemiluminescence

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