Synergistic inhibition of APC/C by glucose and activated Ras proteins can be mediated by each of the Tpk1–3 proteins in Saccharomyces cerevisiae

Melanie Bolte, Patrick Dieckhoff, Cindy Krause{dagger}, Gerhard H. Braus and Stefan Irniger

Institute of Microbiology and Genetics, Georg-August-University, Grisebachstr. 8, D-37077 Göttingen, Germany

Correspondence
Stefan Irniger
sirnige{at}gwdg.de


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Proteolysis triggered by the anaphase-promoting complex/cyclosome (APC/C) is essential for the progression through mitosis. APC/C is a highly conserved ubiquitin ligase whose activity is regulated during the cell cycle by various factors, including spindle checkpoint components and protein kinases. The cAMP-dependent protein kinase (PKA) was identified as negative regulator of APC/C in yeast and mammalian cells. In the yeast Saccharomyces cerevisiae, PKA activity is induced upon glucose addition or by activated Ras proteins. This study shows that glucose and the activated Ras2Val19 protein synergistically inhibit APC/C function via the cAMP/PKA pathway in yeast. Remarkably, Ras2 proteins defective in the interaction with adenylate cyclase fail to influence APC/C, implying that its function is regulated exclusively by PKA, but not by alternative Ras pathways. Furthermore, it is shown that the three PKAs in yeast, Tpk1, Tpk2 and Tpk3, have redundant functions in regulating APC/C in response to glucose medium. Single or double deletions of TPK genes did not prevent inhibition of APC/C, suggesting that each of the Tpk proteins can take over this function. However, Tpk2 seems to inhibit APC/C function more efficiently than Tpk1 and Tpk3. Finally, evidence is provided that Cdc20 is involved in APC/C regulation by the cAMP/PKA pathway.


Abbreviations: APC/C, anaphase-promoting complex/cyclosome; MAPK, mitogen-activated protein kinase; PKA, protein kinase A

{dagger}Present address: Max-Planck-Institut für biophysikalische Chemie, Molekulare Entwicklungsbiologie, Am Fassberg, D-37077 Göttingen, Germany.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The anaphase-promoting complex/cyclosome (APC/C) is a highly conserved multisubunit complex which contains ubiquitin ligase activity (Peters, 2002; Zachariae & Nasmyth, 1999). APC/C is essential for two major events during mitosis: the separation of sister chromatids at the metaphase/anaphase transition and the exit from mitosis. The crucial role of APC/C in metaphase is ubiquitination of securins. In the budding yeast Saccharomyces cerevisiae, proteolytic destruction of securin Pds1 allows the separase Esp1 to become active (Amon, 2001; Nasmyth, 2002). Esp1 then cleaves the cohesin subunit Scc1 and thereby triggers sister chromatid separation. Important targets for the exit from mitosis are B-type cyclins, whose degradation leads to the inactivation of cyclin-dependent kinases (Morgan, 1999). Many other substrates of APC/C ubiquitin ligase have been identified, including the polo kinase Cdc5, kinesins, spindle-associated proteins and regulators of DNA replication (Harper et al., 2002; Peters, 2002; Zachariae & Nasmyth, 1999).

APC/C activity is cell cycle regulated. It is kept inactive during S-, G2- and early M-phase, turned on during metaphase and then remains active throughout late M-phase and during the subsequent G1-phase. A variety of regulatory proteins of APC/C have been identified in the last few years. Most is known about the two proteins Cdc20 and Cdh1 (Peters, 2002). Recent data demonstrated that they function as substrate recognition proteins, which target substrates to the APC/C core complex (Hilioti et al., 2001; Pfleger et al., 2001; Schwab et al., 2001; Vodermaier, 2001). An important feature is the temporal control of APC/C activation by Cdc20 and Cdh1 (Harper et al., 2002; Peters, 2002). Cdc20 always precedes Cdh1 in binding and activation during mitosis. Both WD40 proteins have fundamental functions in controlling APC/C during mitosis. They are the targets of the spindle checkpoint, either directly or indirectly (Gardner & Burke, 2000). Factors of the spindle assembly checkpoint directly bind and inhibit Cdc20 in response to defects in the integrity of the mitotic spindle or in the bipolar attachment of kinetochores. A checkpoint monitoring the orientation of the mitotic spindle indirectly inhibits the association of Cdh1 with APC/C and thereby delays cell division.

Further important regulatory proteins of APC/C are protein kinases, such as the cyclin-dependent kinase Cdk1 and polo kinase (Nigg, 2001). Both kinases were shown to trigger phosphorylation of specific APC/C subunits, known as Apc1, Cdc16, Cdc23 and Cdc27 (Golan et al., 2002; Rudner & Murray, 2000). It was recently shown that either of these kinases is capable of activating APC/C, but both of them are required for efficient APC/C activation (Golan et al., 2002).

A further protein kinase regulating APC/C activity is cAMP-dependent protein kinase (also termed protein kinase A or PKA). By using purified mammalian APC/C, it was shown that PKA directly phosphorylates the subunits Apc1 and Cdc27 in vitro (Kotani et al., 1998). In contrast to Cdk1 and polo kinase, PKA-mediated phosphorylation inhibits APC/C activity. In vitro ubiquitination assays revealed that the addition of purified PKA blocked the ability of APC/C to catalyse the formation of polyubiquitin chains on cyclin B (Kotani et al., 1999). These studies also showed that PKA may affect the binding of Cdc20 because Cdc20 failed to bind APC/C pre-incubated with PKA.

In budding and fission yeast, a direct phosphorylation of APC/C subunits by PKA has not yet been shown. Nevertheless, genetic data strongly implicated yeast PKA as negative regulator of APC/C, similar to the situation in mammalian cells. A variety of fission and budding yeast mutants defective in APC/C subunit genes were suppressed by reducing cAMP levels or PKA activity (Anghileri et al., 1999; Irniger et al., 2000; Yamada et al., 1997; Yamashita et al., 1996). It was shown that the addition of cAMP caused cell cycle arrest in mitosis, both at the metaphase/anaphase transition and in telophase (Anghileri et al., 1999). Many yeast APC/C subunits contain multiple PKA consensus phosphorylation sequences (Kennelly & Krebs, 1991). The Apc1 subunit for example contains 28 of these motifs.

In budding yeast, the cAMP/PKA pathway can be activated either by the addition of glucose to cells grown on poor carbon sources or by the activation of Ras proteins (Broach, 1991; Thevelein & de Winde, 1999). Recent data showed that the glucose signal is not transmitted by Ras proteins to adenylate cyclase Cyr1, as previously thought. Instead, a G-protein-receptor system, consisting of the receptor Gpr1 and the G{alpha} protein Gpa2, stimulates adenylate cyclase in response to glucose addition (Colombo et al., 1998).

Activation of the small GTP-binding proteins Ras1 and Ras2 is catalysed by Cdc25, a protein that promotes the exchange of GDP with GTP on Ras (Broek et al., 1987; Robinson et al., 1987). Ras-GTP stimulates adenylate cyclase and thereby induces an increase in cAMP levels and activation of PKA (Toda et al., 1985). In budding yeast, this kinase is encoded by three separate genes, TPK1, TPK2 and TPK3 (Toda et al., 1987). In the presence of cAMP the inhibitory protein Bcy1 releases the catalytic subunits which are then able to phosphorylate their target proteins (Broach, 1991; Thevelein & de Winde, 1999).

Consistent with the findings that PKA negatively regulates APC/C, we have previously shown that glucose medium and activation of Ras signalling is lethal for mutants defective in APC/C function (Irniger et al., 2000). Mutations in APC/C subunit genes, such as apc10-22 or cdc27-1, were suppressed either by decreasing Ras activity or by growth on the poor carbon source raffinose. In contrast, a constitutively activated RAS2Val19 allele or shifts to glucose medium were deleterious to these mutants. In this study, we show that glucose and activated Ras2Val19 synergistically cause APC/C inhibition and that Tpk1, Tpk2 and Tpk3 apparently have overlapping functions in this process.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast strains and plasmids.
Yeast strains used in this study are derivatives of the Saccharomyces cerevisiae W303 strain (MATa ade2-1 trp1-1 can1-100 leu2-3,12 his3-11,15 ura3 GAL psi+) and are listed in Table 1. All strains obtained from a different background were backcrossed at least four times to W303 strains to make them congenic. Plasmids containing the RAS2Val19, RAS2Val19Gly41 and RAS2Val19Asn45 alleles on the centromeric plasmid YCplac33 (Mösch et al., 1999), the TPK1, TPK2 and TPK3 genes on the high-copy plasmid pRS426 (Mösch et al., 1999) and the STE11-4 allele (Stevenson et al., 1992) cloned into the centromeric plasmid YCp50 were previously described. TPK genes expressed from the inducible GAL1 promoter were constructed by PCR amplification of the corresponding genes and subsequent fusions to the GAL1 promoter on the 2µ plasmid YEplac195. The GAL–CDC20 gene fusion was isolated from a GAL cDNA library (unpublished results).


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Table 1. Yeast strains used in this study

 
For deletion of a single TPK gene (TPK1, TPK2 or TPK3), a deletion cassette containing the kanamycin resistance gene, kan-R, from the Escherichia coli transposon Tn903 flanked by the 5'-region and the 3'-region of the TPK gene, was transformed into yeast strains. Transformants were selected on YEPD+Geneticin plates and restreaked on YEPD+Geneticin plates. Deletions in wild-type, apc10-22 and cdc27-1 strains were verified by Southern hybridization.

Double deletions of TPK genes were constructed starting from the single deletion strains. The tpk-deletion cassette contains on both sides of the kan-R marker gene a loxP recombination sequence from the bacteriophage P1 (Güldener et al., 1996). By recombination of both loxP sequences, the kan-R sequence was removed from the genome and the strain regained sensitivity to Geneticin. Afterwards another tpk deletion cassette was transformed into the single deletion strain and the kan-R gene served as selection marker for the second tpk deletion. Plasmid pSH47 (Güldener et al., 1996) containing the Cre recombinase under control of the inducible GAL1 promoter and a URA3 selection marker was transformed in a first step into tpk single deletion strains. Induction of the GAL1 promoter resulted in the expression of the Cre recombinase, which performed recombination of the loxP sequences. Loss of the kan-R marker gene was verified by selection for Geneticin-sensitive transformants.

Genetic techniques and media.
Standard genetic techniques were used for manipulating yeast strains. To test synthetic phenotypes, the corresponding haploid strains were crossed resulting in diploids, which were sporulated, and then tetrads were analysed by dissection. Only tetrads producing four germinating spores were used for the analysis of genetic interaction.

When yeast cells were grown in complete medium, YEP medium (2 % bactopeptone, 1 % yeast extract, 0·005 % adenine sulfate) supplemented with 2 % glucose (YEPD), 2 % galactose (YEP+Gal) or 2 % raffinose (YEP+Raf) was used. For the selection of plasmid-containing strains, cells were grown in minimal medium, a synthetic medium containing 0·8 % yeast nitrogen base and 50 µg ml-1 each of uracil and adenine, supplemented with amino acids and 2 % glucose or 2 % raffinose (Rose et al., 1990).

Growth conditions and cell cycle arrest.
Prior to the incubation of mutant strains at elevated temperature on agar plates, the cells were always pre-incubated at 25 °C for 12–18 h. Prior to cell cycle arrest in liquid medium, cultures were pre-grown to OD600 0·3–0·6 at 25 °C. When a gene was expressed from the inducible GAL1 promoter, cells were pre-grown in medium containing raffinose as the sole carbon source. The GAL1-10 promoter was induced by the addition of 2 % galactose. To arrest cells in G1 phase with {alpha}-factor pheromone (Nova Biochem), 5 µg {alpha}-factor ml-1 was added. For prolonged {alpha}-factor treatments, additional {alpha}-factor was added after every 120 min to prevent a drop in the {alpha}-factor concentration.

Immunoblotting.
Whole-cell extracts for immunoblotting were prepared as previously described (Surana et al., 1993). Immunoblotting was performed using the enhanced chemiluminescence detection system (ECL, Amersham). Clb2 and Cdc28 antibodies were used in 1 : 1000 and 1 : 2000 dilutions, respectively. MYC and HA antibodies were both used in 1 : 100 dilutions.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Glucose and activated Ras2 proteins synergistically inhibit APC/C function
In previous experiments we showed that glucose or activated Ras2 proteins are deleterious for temperature-sensitive mutants in APC/C subunit genes (Irniger et al., 2000). Since it was reported earlier that glucose activates adenylate cyclase independently of Ras proteins (Colombo et al., 1998; Thevelein & de Winde, 1999), we tested whether this model may also apply to the regulation of APC/C. We argued that if the glucose signal and Ras proteins acted via separate pathways on adenylate cyclase, then the simultaneous activation of both pathways may have a synergistic effect on adenylate cyclase, PKA activity and inhibition of APC/C.

To test this model, apc10-22 mutants were transformed with a centromeric plasmid containing either the constitutively activated RAS2Val19 allele or, as control, the empty vector YCplac22 (TRP1 marker). Transformants were pregrown at 25 °C in minimal medium lacking tryptophan (-Trp) and containing raffinose as sole carbon source. Then cells were streaked to fresh -Trp plates containing either glucose or raffinose. apc10-22 mutants containing RAS2Val19 were viable at 28 °C on raffinose plates, but non-viable on glucose plates, displaying severe growth defects even at 25 °C (Fig. 1). apc10-22 cells carrying the control plasmid were viable under these conditions. apc10-22 RAS2Val19 cells were non-viable on raffinose plates at 34 °C, a temperature tolerated by apc10-22 mutants carrying the control plasmid. Both strains were non-viable on glucose medium at this temperature. These findings show that both glucose and activated Ras proteins interfere with viability of apc10-22 mutants. The lethality of apc10-22 RAS2Val19 cells at 28 °C demonstrates that glucose and the activated Ras2 protein synergistically reduce the viability of apc10-22 mutants. Similar findings were observed for cdc27-1 mutants (data not shown). Thus, glucose and activated Ras2 have a combined effect on APC/C function.



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Fig. 1. Glucose and the activated RAS2Val19 allele have a combined lethal effect on apc10-22 mutants. apc10-22 mutants carrying either a centromeric plasmid with the RAS2Val19 gene or an empty vector plasmid (YCplac22, containing the TRP1 marker) were pregrown on minimal medium lacking tryptophan and containing raffinose as carbon source (MM-Trp+Raf). Cells were then streaked on MM-Trp plates containing either raffinose or glucose and incubated for 2·5 days at the indicated temperatures. A wild-type strain was used as control.

 
Inhibition of APC/C by activated Ras2 requires the PKA pathway
We next asked whether activated Ras proteins mediate their effect on APC/C by cAMP/PKA or whether other pathways may be involved. Ras1 and Ras2 activate two different signalling pathways, the cAMP/PKA pathway and the mitogen-activated protein kinase (MAPK) pathway consisting of Cdc42/Ste20/Ste11/Ste7/Kss1/Ste12 proteins (Mösch, 2000). For invasive growth of yeast, activation of either of these pathways by Ras was sufficient, indicating that cAMP/PKA and MAPK signalling had redundant functions in transmitting the signal from Ras to the effectors (Mösch et al., 1999).

To test whether APC/C inhibition by activated Ras may also be transmitted by both of these pathways, we used RAS2 alleles which contained, in addition to the activating mutation in the Val19 codon, second-site mutations at codons 41 and 45. Exchanges of Pro41 to Gly and Asp45 to Asn were shown to cause defects in the binding and activation of adenylate cyclase, but these proteins were still able to activate the MAPK pathway (Mösch et al., 1999). Thus, Ras2Val19 is able to activate both pathways, but Ras2Val19Gly41 and Ras2Val19Asn45 functions are restricted to the MAPK pathway. Centromeric plasmids containing RAS2Val19, RAS2Val19Gly41 or RAS2Val19Asn45 genes, or no insert, were transformed into a wild-type strain and into the apc mutant strains apc10-22 and cdc27-1. The plasmid-carrying strains were grown at 34 °C or 30 °C, respectively, on -Trp minimal medium with raffinose as the sole carbon source. In contrast to mutant cells carrying RAS2Val19, apc10-22 and cdc27-1 transformants with the double mutations in the RAS2 genes were viable under these conditions (Fig. 2a, b). apc10-22 mutants containing RAS2Val19Gly41 or RAS2Val19Asn45 were also viable on glucose medium at 28 °C and therefore do not display the synergistic phenotype observed when glucose and activated Ras2Val19 protein were combined (Fig. 2c). We conclude that Ras2 proteins which are defective in binding adenylate cyclase do not affect APC/C function. Thus, the signal from Ras appears to be transmitted through cAMP and PKA. In contrast to signalling for the induction of invasive growth, the MAPK pathway is apparently unable to replace the cAMP/PKA pathway.



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Fig. 2. Activated RAS2 alleles defective in stimulating adenylate cyclase are not lethal for apc10-22 and cdc27-1 mutants. (a, b) Centromeric plasmids carrying either the RAS2Val19, RAS2Val19Gly41 or RAS2Val19Asn45 gene, or the empty plasmid YCplac22 (containing TRP1 as selectable marker), were transformed into a wild-type strain and the apc10-22 and cdc27-1 mutant strains. Transformants were pregrown on MM-Trp+Raf medium at 25 °C, then streaked onto fresh plates and shifted to semi-permissive temperatures: 34 °C for apc10-22 mutants (a) and 30 °C for cdc27-1 mutants (b). Plates were incubated for 2·5 days. (c) Transformants of apc10-22 mutants were streaked onto MM-Trp plates containing either glucose or raffinose and incubated at 28 °C for 2·5 days. (d) Wild-type and apc10-22 mutants were transformed with centromeric plasmids containing the STE11-4 allele or the empty plasmid YCplac50 (with URA3 as selectable marker). Transformants were pregrown on MM-Ura+Raf at 25 °C, streaked onto fresh plates containing either glucose or raffinose and incubated at 34 °C for 2·5 days.

 
Activation of the MAPK pathway does not influence apc mutants
To further elucidate whether the MAPK pathway may cause APC/C inhibition, we transformed apc10-22 mutants with plasmids carrying a hyperactive STE11 allele, STE11-4 (Stevenson et al., 1992), on the centromeric plasmid YCp50. The Ste11 protein kinase is one component of the MAPK signalling cascade. We argued that APC/C inhibition caused by highly active MAPK signalling would interfere with viability of apc mutants, as seen for Ras2Val19. Transformants carrying plasmids with either the STE11-4 allele or no insert were tested on minimal medium containing raffinose as the sole carbon source. We found that apc10-22 containing STE11-4 produced colonies on raffinose plates, similar to mutant strains with the empty vector (Fig. 2d). Mutant cells containing either of the plasmids were non-viable on glucose plates. Thus, APC/C is apparently neither positively nor negatively affected by activation of the MAPK cascade.

These results support the model that APC/C inhibition caused by activated Ras signalling is mediated exclusively by the PKA pathway and not by the MAPK pathway.

Each of the Tpk1–3 proteins is sufficient for APC/C inhibition in response to glucose
Previous findings have shown that for some of the functions of yeast PKA, the Tpk1, Tpk2 and Tpk3 proteins are redundant, whereas other processes, such as the regulation of pseudohyphal growth, require one specific Tpk protein (Robertson et al., 2000; Robertson & Fink, 1998). To test the role of the three Tpk proteins in APC/C inhibition, we constructed apc10-22 and cdc27-1 mutants containing either single deletions or double deletions of TPK genes. We argued that if APC/C inhibition were mediated by a specific Tpk protein then a deletion of the corresponding gene would abolish lethality of apc mutations upon shift to glucose medium.

A cdc27-1 strain and cdc27-1 strains containing single tpk deletions (cdc27-1 tpk1{Delta}, cdc27-1 tpk2{Delta}, cdc27-1 tpk3{Delta}) or double tpk deletions (cdc27-1 tpk1{Delta} tpk2{Delta}, cdc27-1 tpk1{Delta} tpk3{Delta}, cdc27-1 tpk2{Delta} tpk3{Delta}) were incubated on YEP plates containing either glucose or raffinose at 30 °C, a semi-permissive temperature for cdc27-1 mutants. All cdc27-1 strains were viable at 30°C on raffinose medium, but failed to form colonies in the presence of glucose (Fig. 3a). Similarly, an apc10-22 mutant and each of the derivative tpk double deletions grew on raffinose medium, but were non-viable on YEPD at the semi-permissive temperature, 34°C (Fig. 3b). These results show that single or double deletions of TPK genes do not affect the viabilty of apc mutants. Thus, it appears that each of the TPK genes is sufficient for signal transmission from glucose to APC/C, suggesting that the three TPK genes fulfil a redundant function in this process.



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Fig. 3. One Tpk protein is sufficient for APC/C inhibition in response to glucose. (a) cdc27-1 mutant strains deleted for a single TPK gene (cdc27-1 tpk1{Delta}, cdc27-1 tpk2{Delta}, cdc27-1 tpk3{Delta}) or deleted for two TPK genes (cdc27-1 tpk1{Delta} tpk2{Delta}, cdc27-1 tpk1{Delta} tpk3{Delta}, cdc27-1 tpk2{Delta} tpk3{Delta}) were pregrown on YEP+Raf plates at 25°C, then streaked to fresh YEP+Glu or YEP+Raf plates and incubated at either 25°C or 30 °C for 2·5 days. (b) apc10-22 mutants deleted for two TPK genes (apc10-22 tpk1{Delta} tpk2{Delta}, apc10-22 tpk1{Delta} tpk3{Delta}, apc10-22 tpk2{Delta} tpk3{Delta}) were treated as in (a), but incubated at either 25 °C or 34 °C.

 
To further test the effect of glucose on apc mutants containing only a single TPK gene, cdc27-1 and cdc27-1 tpk double deletion strains were first pregrown in raffinose medium at 28 °C. Under these conditions, cdc27-1 mutants undergo cell cycle progression without obvious defects when using raffinose as carbon source. Cultures were split in two halves and either glucose or, as control, the poor carbon source galactose were added. We argued that glucose-induced inhibition of APC/C function would block cdc27-1 cells in mitosis. To monitor whether glucose causes cdc27-1 mutants to arrest in mitosis, {alpha}-factor was added and the cultures were further incubated at 28 °C. {alpha}-Factor induces cells to arrest as unbudded cells in G1-phase. We found that cdc27-1 as well as cdc27-1 strains containing only one TPK gene were blocked in mitosis upon glucose addition (Fig. 4a, b). After 3 h incubation in the presence of pheromone, 40–70 % of cells were still large-budded. In contrast, most cells arrested in G1-phase as unbudded cells in the cultures treated with galactose, similar to wild-type cells. In these cultures, the proportion of budded cells rapidly decreased after {alpha}-factor addition. Importantly, each of the cdc27-1 tpk double deletion strains shows a similar phenotype to the cdc27-1 strain upon glucose or galactose addition.



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Fig. 4. One Tpk protein is sufficient for the mitotic arrest of cdc27-1 mutants. A wild-type strain, a cdc27-1 mutant strain and cdc27-1 mutants deleted for two TPK genes (cdc27-1 tpk1{Delta} tpk2{Delta}, cdc27-1 tpk1{Delta} tpk3{Delta}, cdc27-1 tpk2{Delta} tpk3{Delta}) were pregrown on YEP+Raf medium at 28 °C. Then, either 2 % glucose or 2 % galactose was added and cells were further incubated for 5 h at the same temperature. Subsequently, {alpha}-factor was added to arrest cells, which are able to complete mitosis, in G1 phase. (a) Percentage of budded cells at the indicated time points after {alpha}-factor addition. Values are means of three independent experiments. (b) Photographs of cells of the indicated strains grown on galactose or glucose medium before {alpha}-factor addition (left) and 5 h after {alpha}-factor addition (right). (c, d) Immunoblotting of Clb2 protein levels before (c) or after (d) {alpha}-factor addition using Clb2 antibodies. Cdc28 was used as loading control.

 
To test whether indeed APC/C-mediated proteolysis is impaired in the cdc27-1 tpk double deletion strains upon treatment with glucose, we determined protein levels of the mitotic cyclin Clb2, a substrate of APC/C. Clb2 levels were analysed by immunoblotting before and 3 h after pheromone treatment. Before {alpha}-factor addition, Clb2 was present in all cultures, but Clb2 protein levels were marginally higher in cdc27-1 mutant strains treated with glucose (Fig. 4c). After {alpha}-factor addition Clb2 levels dropped to low levels in wild-type cells and in cdc27-1 cells incubated in galactose medium. In contrast, cyclin levels remained high in cdc27-1 cells grown in glucose medium (Fig. 4d). In cdc27-1 tpk1{Delta} tpk3{Delta} cells, Clb2 levels were similar to those in cdc27-1 cells, but they were partially decreased in the other double tpk deletion strains. These results indicate that Tpk2 has a more potent function than Tpk1 and Tpk3 in APC/C regulation. However, Clb2 degradation was at least partially affected in each cdc27-1 strain with double tpk deletions in the presence of glucose. Thus, the inhibitory signal from glucose to APC/C is transmitted in each of these strains.

Taken together, these results show that one single Tpk protein is sufficient for APC/C inhibition in response to glucose and that Tpk2 may be particularly efficient in this process.

Overexpression of TPK genes is deleterious to apc mutants
To further test whether Tpk proteins may mediate APC/C inhibition with different efficiencies, we determined the effects of high levels of Tpk proteins on the viability of apc mutants. cdc27-1 and apc10-22 mutants were transformed with high-copy plasmids containing the TPK1, TPK2 or TPK3 gene. The viability of these mutants at elevated temperatures was determined on -Ura minimal medium containing raffinose (Fig. 5). Consistent with our data suggesting that Tpk2 efficiently inhibits APC/C (Fig. 4d), we found that overexpression of TPK2 caused a distinct reduction of the viability of apc mutants. In contrast, the TPK1-containing plasmid only marginally affected apc mutants. Remarkably, overexpressed TPK3 also efficiently interferes with the viability of these mutants. Previously, Tpk3 was shown to have a low catalytic activity, but this was apparently due to the poor expression of the TPK3 gene (Mazon et al., 1993). When present in high levels, Tpk3 also appears to have high catalytic activity and thereby efficiently inhibits APC/C function. Since TPK3 is only expressed to low levels in cells containing single copies of TPK genes, Tpk2 is apparently the most efficient Tpk protein in mediating APC/C inhibition in response to activation of the cAMP/PKA signalling pathway.



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Fig. 5. Different effects of overexpressing different TPK genes on cdc27-1 and apc10-22 mutants. cdc27-1 and apc10-22 mutants carrying either TPK1, TPK2, TPK3 or no insert in a high-copy plasmid (with URA3 as selectable marker) were streaked to minimal medium lacking uracil and containing raffinose as carbon source. Plates were incubated at (a) 30 °C or (b) 35 °C for 2·5 days. A wild-type strain was used as control.

 
Glucose affects APC/C independently of the regulatory protein Cdh1
Cdh1 and Cdc20 are regulatory proteins of APC/C and we addressed the question whether the cAMP/PKA pathway affects APC/C function by regulating these proteins. To find out whether PKA regulates APC/C activity via Cdh1, we first tested the ability of PKA to phosphorylate Cdh1. Cdh1 was previously shown to be phosphorylated by Cdk1 and this modification can be monitored by mobility shifts in immunoblots (Zachariae et al., 1998). In G1 cells, Cdk1 is inactive and Cdh1 is not phosphorylated. This allowed us to test whether Tpk proteins are able to trigger Cdh1 phosphorylation. We induced the expression of TPK1, TPK2 or TPK3 genes in {alpha}-factor-arrested G1 cells and analysed the mobility of Cdh1 by immunoblotting. In contrast to cycling cells, no slower-migrating forms of Cdh1 were detectable upon the expression of TPK genes to high levels, indicating that Cdh1 is not a target of Tpk proteins (Fig. 6a).



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Fig. 6. APC/C inhibition in response to glucose occurs independently of Cdh1, but may involve Cdc20. (a) Strains containing a GALL–HA3–CDH1 construct and carrying either a GAL–TPK1, GAL–TPK2 or GAL–TPK3 fusion or no insert on a 2µ plasmid (with URA3 as selectable marker) were pregrown in MM-Ura+Raf medium. {alpha}-Factor was added to arrest cells in G1 phase. Then galactose was added to induce expression of the corresponding fusion genes and cells were incubated for 2 h. Cdh1 protein was analysed by immunoblotting using the HA antibody. A cycling culture was used as control for the detection of phosphorylated Cdh1. The faster-migrating additional bands visible in lanes 4 and 5 are most likely degradation products. (b) cdh1{Delta}, apc10-22 and apc10-22 cdh1{Delta} strains were pregrown on YEP+Raf plates at 25 °C, streaked onto fresh YEP+Raf or YEP+Glu (YEPD) plates and then incubated at either 25 °C or 34 °C for 2·5 days. (c) A strain containing an N-terminally Myc18-tagged CDC20 gene and either a GAL–TPK1 or a GAL–TPK2 fusion or no insert on a 2µ plasmid (with URA3 as selectable marker) were pregrown in MM-Ura+Raf medium. Nocodazole was added to arrest cells in mitosis. Then galactose was added to express the corresponding fusion genes and cells were incubated for 2 h. Cdc20 protein was analysed by immunoblotting using the MYC antibody. (d) A cdc27-1 mutant carrying a centromeric plasmid containing a GAL–CDC20 fusion (with URA3 as selectable marker) and a centromeric plasmid containing RAS2Val19 (with TRP1 as selectable marker) were pregrown on minimal medium lacking uracil and tryptophan and containing raffinose at 30 °C. Cells were then streaked onto minimal medium lacking uracil and tryptophan and containing raffinose and galactose to induce CDC20 expression. Cells were incubated at 30 °C and photographed after 40 h incubation.

 
We tested the possible role of Cdh1 in PKA-mediated APC/C regulation by an alternative experiment, by constructing apc10-22 mutants lacking the non-essential CDH1 gene. We argued that if PKA regulates APC/C activity predominantly by Cdh1 phosphorylation, then a deletion of CDH1 would abolish the lethal effect of glucose on apc10-22 mutants. apc10-22 cdh1{Delta} mutants were tested with regard to their viability on glucose and raffinose medium. On glucose medium, apc10-22 and the double mutant apc10-22 cdh1{Delta} were unable to grow at 34 °C (Fig. 6b). Both strains were viable on YEP+Raf medium at this temperature. Thus, the absence of the regulatory protein Cdh1 has no influence on the viability of apc10-22 mutants on glucose or raffinose medium. These results indicate that inhibition of APC/C activity in response to glucose occurs independently of Cdh1.

Evidence for Cdc20 as potential target of the cAMP/PKA pathway
We next aimed to determine whether cAMP/PKA-mediated inhibition of APC/C function may involve the Cdc20 protein. Cdc20 protein levels are cell cycle regulated by transcriptional and post-transcriptional mechanisms (Harper et al., 2002). We first tested whether high PKA activity affects Cdc20 protein levels. A yeast strain containing an N-terminally Myc-tagged Cdc20 (Myc18–Cdc20; Shirayama et al., 1998) was transformed with high-copy plasmids containing either GAL–TPK1 or GAL–TPK2 fusions. Cells were then grown in raffinose medium and arrested in metaphase with the microtubule-depolymerizing drug nocodazole. In this period of the cell cycle, Cdc20 protein levels are normally high (Shirayama et al., 1998). To determine whether the overexpression of TPK1 or TPK2 genes affects Cdc20 protein levels, galactose was added and Cdc20 was analysed by immunoblotting (Fig. 6c). We found that Cdc20 protein levels remained the same under conditions of low or high PKA activity.

We then addressed the question whether Cdc20 function may be affected by PKA activity. We argued that if this were the case, then the inhibitory effect of activated Ras2Val19 on apc mutants may be reduced by the overexpression of CDC20. To test this, a cdc27-1 mutant containing both RAS2Val19 and GAL–CDC20 on centromeric plasmids was pregrown in raffinose medium at 25 °C, streaked onto either raffinose or galactose plates and incubated at 30 °C. Microscopic examination of cells showed that high levels of Cdc20 allowed many cells to form colonies (Fig. 6d), albeit distinctly more slowly than wild-type cells. Thus, high levels of Cdc20 partially suppress the inhibitory influence of activated Ras signalling on the viability of cdc27-1 mutants. These results provide evidence that the activation of the cAMP/PKA pathway affects APC/C, at least in part, via the Cdc20 regulatory protein.


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The activity of the anaphase-promoting complex/cyclosome (APC/C) is controlled during the cell cycle by a variety of positive and negative regulators (Harper et al., 2002; Peters, 2002; Zachariae & Nasmyth, 1999). One of these regulatory factors is cAMP-dependent protein kinase, also known as protein kinase A (PKA), which inhibits APC/C activity in yeast and mammalian cells.

In S. cerevisiae, PKA can be stimulated by a shift to glucose medium or by the activation of Ras proteins (Thevelein & de Winde, 1999). We have presented genetic data showing that glucose and dominantly active Ras2Val19 proteins, the equivalent to the oncogenic mammalian RasVal12, severely reduce the viability of apc mutants in a synergistic manner. We suggest that the expression of the RAS2Val19 allele in combination with growth on glucose medium causes an efficient activation of adenylate cyclase, resulting in enhanced PKA activity and potent inhibition of APC/C. Our findings are consistent with the model proposing that glucose stimulates adenylate cyclase independently of Ras1 and Ras2 (Colombo et al., 1998).

We also showed that APC/C inhibition in response to activated Ras2 proteins seems to occur exclusively via cAMP and PKA, but not by the MAPK pathway. Furthermore, we found that Tpk1, Tpk2 and Tpk3 have overlapping roles in APC/C inhibition, suggesting that each of the Tpk proteins is capable of taking over this function in response to a shift to glucose medium. However, our data provide evidence that Tpk2 performs this function more efficiently than Tpk1 or Tpk3 (Fig. 4d). This effect might be explained by the findings that these kinases have distinctly different catalytic activities (Mazon et al., 1993; Toda et al., 1987; Zähringer et al., 1998). Indeed, Tpk2 was shown to have higher catalytic activity than Tpk1 and Tpk3. For Tpk3, this is apparently due to its poor expression (Mazon et al., 1993). This is consistent with our data, showing that TPK3, similar to TPK2, mediates efficient APC/C inhibition when overexpressed (Fig. 5). In conclusion, Tpk proteins have overlapping functions in APC/C inhibition but obviously have different efficiences at normal expression levels.

Genetic and biochemical data suggest that PKA-mediated inhibition of APC/C is conserved in eukaryotes from yeast to mammals (Anghileri et al., 1999; Kotani et al., 1998, 1999; Yamada et al., 1997; Yamashita et al., 1996). In budding and fission yeast, it remains to be shown whether PKA directly phosphorylates APC/C subunits, as shown in vitro with the reconstituted mammalian APC/C (Kotani et al., 1998). Most yeast APC/C subunits contain consensus phosphorylation sites for PKA (Kennelly & Krebs, 1991). Remarkably, 28 potential sites were found in the Apc1 subunit, and 7–8 sites in three other subunits. An important task in the future will be to find out how PKA inhibits APC/C activity. In mammalian cells, it was shown that the activator protein Cdc20 was unable to bind APC/C when the complex was preincubated with PKA. Thus, PKA may inhibit APC/C function by modifying critical subunits required for the binding of Cdc20, thereby preventing its association with the core complex. Our results showing that high levels of Cdc20 partially suppress the inhibitory effect of RAS2Val19 (Fig. 6d) are consistent with such a model.

It will be an interesting task to elucidate which intra- or extracellular signals regulate PKA during mitosis. The growth medium appears to be one of these signals (Anghileri et al., 1999; Irniger et al., 2000). The availability of rich carbon sources such as glucose may cause a delay in the progression through mitosis, by activation of PKA and inhibition of APC/C. Such a model is consistent with the findings that daughter cells are born at larger cell size on rich medium (Alberghina et al., 1998). PKA-mediated inhibition of APC/C may also be a mechanism for the delay in mitosis during pseudohyphal growth (Kron et al., 1994; Rua et al., 2001). Other intra- or extracellular signals may be transmitted by Ras proteins. Taken together, the cAMP/PKA pathway represents a suitable system for the integration of multiple signals which are then communicated to the cell cycle machinery.


   ACKNOWLEDGEMENTS
 
We thank Hans-Ulrich Mösch and Maria Meyer for support and for providing plasmids and yeast strains. We acknowledge Ingrid Bahr for help with the figures. This work was supported by the Deutsche Forschungsgemeinschaft (grant IR 36/1-3), the Fonds der Chemischen Industrie and the Volkswagen-Stiftung.


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Received 17 October 2002; revised 31 January 2003; accepted 19 February 2003.



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