Institute of Microbiology and Genetics, Georg-August-University, Grisebachstr. 8, D-37077 Göttingen, Germany
Correspondence
Stefan Irniger
sirnige{at}gwdg.de
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ABSTRACT |
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Present address: Max-Planck-Institut für biophysikalische Chemie, Molekulare Entwicklungsbiologie, Am Fassberg, D-37077 Göttingen, Germany.
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INTRODUCTION |
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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
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.
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METHODS |
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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 1218 h. Prior to cell cycle arrest in liquid medium, cultures were pre-grown to OD600 0·30·6 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 -factor pheromone (Nova Biochem), 5 µg
-factor ml-1 was added. For prolonged
-factor treatments, additional
-factor was added after every 120 min to prevent a drop in the
-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.
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RESULTS |
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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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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. 2
a, 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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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 Tpk13 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, cdc27-1 tpk2
, cdc27-1 tpk3
) or double tpk deletions (cdc27-1 tpk1
tpk2
, cdc27-1 tpk1
tpk3
, cdc27-1 tpk2
tpk3
) 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. 3
a). 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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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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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 (Myc18Cdc20; Shirayama et al., 1998
) was transformed with high-copy plasmids containing either GALTPK1 or GALTPK2 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 GALCDC20 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.
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DISCUSSION |
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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 78 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.
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ACKNOWLEDGEMENTS |
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Received 17 October 2002;
revised 31 January 2003;
accepted 19 February 2003.
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