identification and preliminary characterization of p31, a new pstaire-related protein in fission...

. 13: 727–734 (1997)

Identification and Preliminary Characterization of p31, aNew PSTAIRE-related Protein in Fission Yeast

SYLVIE TOURNIER1, YANNICK GACHET1 AND JEREMY S. HYAMS1*1Department of Biology, University College London, Gower Street, London WC1E 6BT, U.K.

Received 4 June 1996; accepted 23 December 1996

One of the defining characteristics of the catalytic subunit of the cyclin-dependent protein kinases (cdks) is theso-called PSTAIRE motif. Western blots of fission yeast cytosolic extracts using a monoclonal antibody againstthe PSTAIRE peptide revealed two bands at 34 kDa (p34cdc2) and 31 kDa (p31). Polyclonal antibodies to theC-terminus of p34cdc2 or to the full-length protein recognized the 34 kDa band but not p31. Overexpression of thecdc2+ gene resulted in the increase of the 34 kDa band but not p31. Like p34cdc2, the level of p31 revealed no obviouscell cycle regulation but the protein was present in spores where p34cdc2 was barely detectable. p31 expression wasunaffected by removal of either phosphate or ammonium from the growth medium, although the level of p34cdc2 wasreduced in the absence of phosphate. p31 was not associated with cyclin B, nor was it adsorbed to p13suc1 Sepharosebeads, two characteristics of p34cdc2. p31 did, however, interact with p15, the starfish homologue of p13suc1. p31 waspresent in cells in which cdc2+ was replaced by its budding yeast homologue CDC28. When fission yeast cytosolicextracts were subjected to gel filtration chromatography, p31 eluted in two peaks, one at approximately 100 kDa, theother at approximately 30 kDa. We conclude that p31 is a novel fission yeast PSTAIRE protein and therefore,potentially, a new cdk. ? 1997 by John Wiley & Sons, Ltd.

Yeast 13: 727–734, 1997.No. of Figures: 7. No. of Tables: 0. No. of References: 29.

— p31; cdc2; PSTAIRE; fission yeast; Schizosaccharomyces pombe; cell division cycle

INTRODUCTION

Mammalian cells possess an extended family ofcdc2-related protein kinases, now referred to ascyclin-dependent kinases or cdks (Meyerson et al.,1993). Together with their regulatory cyclin sub-units, the cdks regulate the critical events of thecell division cycle. A conserved feature of the cdksis the PSTAIRE motif near the N-terminus, theregion involved in cyclin binding (Jeffery et al.,1995). In the yeasts, Saccharomyces cerevisiae andSchizosaccharomyces pombe, organisms that haveplayed a major role in defining the molecular basisof cell cycle regulation, the cell cycle is driven by asingle cdk, the product of the CDC28 gene inbudding yeast and cdc2+ in fission yeast (Forsburgand Nurse, 1991). Versatility is imparted to thisapparently simple level of organization by the factthat a single cdk can associate with multiple cyclins(Nasmyth, 1993). Budding yeast possesses a

number of CDC28-related genes but only one ofthese, PHO85, contains the PSTAIRE sequence(Toh-e et al., 1988) and is detected by an antibodyraised against a PSTAIRE peptide (Espinoza et al.,1994). PHO85 has greater than 50% identity toCDC28, and is associated with three cyclins, theproducts of the PHO80, PCL1 and PCL2 genes(Espinoza et al., 1994; Measday et al., 1994;Kaffman et al., 1994). As with CDC28, PHO85/PHO80 complexes are associated with a regulatorysubunit, PHO81 (Schneider et al., 1994). PHO85was originally identified as a gene concerned withthe regulation of phosphate metabolism but therecent findings strongly imply a cell cycle role. Onepossibility is that PHO85 provides a link betweenthe cell cycle and the nutritional state of the cell.In fission yeast, only one cdc2-related kinase has

been identified. The gene crk1+/mop1+ is thehomologue of the vertebrate cdk-activating kinase(cak; Damagnez et al., 1995; Buck et al., 1995). Inmammals the catalytic subunit of cak is cdk7,*Correspondence to: Jeremy S. Hyams.

CCC 0749–503X/97/080727–08 $17.50? 1997 by John Wiley & Sons Ltd

which contains the cyclin-binding sequenceNRTALRE. The corresponding peptide in crk1+/mop1+ is DISALRE, which is also similar to thePSSALRE motif of cdk5 (Lew et al., 1994). Wehave found that fission yeast cytoplasmic extractscontain two bands that react with a monoclonalantibody raised against the peptide EGVP-STAIREISLLKE (Yamashita et al., 1991), cdc2itself and a second species at 31 kDa (p31). Likecdc2, p31 associates with the cyclin-dependentkinase inhibitor p21Cip1 when this is heterolo-gously expressed in Sz. pombe (Tournier et al.,1996). We demonstrate here that p31 is a novelfission yeast PSTAIRE protein and therefore,potentially, a new cdk.

MATERIALS AND METHODS

Yeast strains and cell cultureThe wild-type Sz. pombe strains used in this

study were 972 h" or 975 h+. The strain over-expressing cdc2 was h" cdc2::LEU2 ura4 leu1-32ade6.216 containing the plasmid pcdc2, which car-ries the strong promoter of the fission yeast adhgene (Draetta et al., 1987). The strain SP611 is h"

cdc2::LEU2 ura4 Leu1-32 ade6.216 pCDC28-2(Booher and Beach, 1987). pCDC28-2 is pIRT1,which carries a fragment bearing the CDC28 genefrom S. cerevisiae. These strains were all kindlyprovided by Dr D. Beach (Cold Spring HarborLaboratory). The S. cerevisiae strain was S288c.Fission yeast cells were cultured in supplementedminimal medium (EMM) or in yeast extractmedium (YE) as described in Nurse (1975) andAlfa et al. (1993). In some experiments cells wereincubated for 12 h at 25)C in EMM lacking eitherphosphate or ammonium chloride. Spore isolationand germination were performed using the wild-type strains 972h" and 975h+ as previously de-scribed (Moreno et al., 1991). For synchronouscultures, 972h" cells were grown in YE at 30)Cto a cell density of 5#106 cells/ml and subjectedto centrifugal elutriation using a Beckman JE-6centrifuge (Mitchison, 1988). Small cells wereremoved and grown at 30)C in YE. Samplesfor protein extracts, cell number estimation andseptation index were taken at 20-min intervals.

Preparation of cytosolic extracts from Sz. pombeand S. cerevisiaeExponential cultures of yeast cells were har-

vested by centrifugation then broken in the

presence of an equal amount of cold 50 ìm glassbeads and an equal volume of lysis buffer (50 m-Tris pH 7·4, 250 m-NaCl, 50 m-sodium fluor-ide, 5 m-EDTA, 0·1 m-sodium orthovanadate,1 m-DTT and 0·1% Triton X100) containingthe following protease inhibitors: 0·1 m-PMSF,20 ìg/ml leupeptin, 20 ìg/ml aprotinin, 20 ìg/mlpepstatin A, 20 ìg/ml tosyl -lysine chloromethylketone and 20 ìg/ml tosyl phenylalanine chloro-methyl ketone. The samples were vortexed for6#30 s at 4)C. The soluble protein fraction wasrecovered by centrifugation for 15 min at 14 000 g.Proteins were dissolved in Laemmli sample bufferfor SDS–PAGE analysis (Laemmli, 1970).

Immunoprecipitation and affinity depletionImmunoprecipitations were carried out by incu-

bating 1 mg of protein extract in 1 ml lysis bufferwith 10 ìl of cyclin B antibody for 2 h at 4)C on arotator. Following incubation, samples weretreated with 50 ìl of a 10% protein A–Sepharosesuspension (Pharmacia LKB Biotechnology Inc.).In some experiments, extracts were directly treatedwith 50 ìl of p13suc1 beads or p15cdk-BP beads andincubated for 1 h at 4)C on a rotator. The beadpellets were then washed three times with lysisbuffer and two times with 50 m-Tris–HCl, pH7·5, containing antiproteases. Proteins bound tothe beads were dissolved in Laemmli sample bufferfor SDS–PAGE analysis (Laemmli, 1970).

Cell fractionationSz. pombe nuclei were isolated according to

Gallagher et al. (1993). Briefly, a 2-litre mid-exponential phase culture of wild-type cells in MMmedium was harvested by centrifugation at 2900 gin a Beckman GS-6R centrifuge at 4)C and resus-pended in 80 ml 0·1 -Tris–HCl, pH 8·0, 0·1 -EDTA, 0·5% 2-mercaptoethanol at 30)C for 10min. Cells were pelleted at 1700 g for 3 min,washed once in protoplasting buffer (20 m-potassium phosphate, pH 6·5, 1 -sorbitol, 1 m-MgCl2) and then incubated at 37)C for 95 minwith slow shaking in 40 ml of protoplasting buffercontaining 1 mg/ml zymolyase 20T (SeikagakuKogyo Company). Novozym 234 (Novo BioLabs,Denmark) was added at 1 mg/ml after 40 min,by which time the cells had become highly osmoti-cally sensitive. Protoplasts were pelleted through2#20 ml sucrose cushions (0·2 -KCl, 0·8 -sucrose, 1 m-PMSF, 20 ìg/ml leupeptin, 3 ìg/mlpepstatin A), for 15 min at 600 g. Pellets were

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? 1997 by John Wiley & Sons, Ltd . 13: 727–734 (1997)

gently resuspended in 8 ml lysis buffer (20 m-potassium phosphate, pH 6·5, 1 m-MgCl2, 20%Ficoll 400-DL, 1 m-PMSF, 40 ìg/ml leupeptin,3 ìg/ml pepstatin A), then homogenized by14#5 s passes in a Silverson mixer-emulsifier(Silverson, London) at 4)C, followed by 10 strokesusing a teflon pestle in a 50 ml polycarbonatecentrifuge tube. The homogenate was centrifugedat 4000 g for 10 min and the supernatant collectedand centrifuged at 5000 g (MSE 21, 4)C). Thesupernatant (3 ml for each tube) was layered ontoa 9 ml Ficoll (Sigma) step gradient (3 ml#50%;3 ml#40%; 3 ml#30% in 20 m-potassium phos-phate at pH 6·5, containing 1 m-MgCl2, 1 m-PMSF, 40 ìg/ml leupeptin and 3 ìg/ml pepstatinA). The gradient was centrifuged at 58 000 g for 60min (Pegasus 65 centrifuge) using a 6#16·5 mlswing-out rotor at 4)C.

Polyacrylamide gel electrophoresis andimmunoblottingProtein extracts and proteins bound to affinity

beads were dissolved in Laemmli sample bufferand subjected to PAGE (Laemmli, 1970). Electro-transfer onto an immobilon membrane (Millipore)was performed as described by Harlow and Lane(1988) using a semi-dry transfer apparatus. Immu-noblots were probed using the following anti-bodies: anti-PSTAIRE monoclonal antibody(Yamashita et al., 1991); C8, a polyclonal antibodyraised against the carboxy-terminal part of Sz.pombe cdc2 (B. Ducommun, unpublished); G8, apolyclonal antibody raised against the full-lengthSz. pombe cdc2 protein (Draetta et al., 1987) wereall used at 1/1000 dilution. Anti-cyclin B antibody(Alfa et al., 1990) was used for immunoprecipita-tion experiments at 1/100 dilution. The corre-sponding preimmune serum was used at the samedilution. p13suc1 Sepharose beads were preparedaccording to Brizuela et al. (1987). p15cdk-BP was agenerous gift of L. Meijer (Azzi et al., 1994).Immunodetections were performed using goatanti-mouse horseradish peroxidase-conjugatedantibodies (Sigma) and the ECL-enhanced chemi-luminescence detection kit (Amersham) accordingto the manufacturer’s instructions.

Gel filtrationSz. pombe extracts (6 mg protein) were applied

to a gel filtration HR 100 column pre-equilibratedwith 50 m-Tris pH 8, 200 m-NaCl, 50 m-sodium fluoride, 0·1 m-sodium orthovanadate,

1 m-DTT, 0·1% Triton X100 and 0·1 m-PMSF.The column was maintained at 4)C and set at aflow rate of 0·5 ml/min and 2 ml fractions werecollected. Each fraction was precipitated by theaddition of 10% TCA final concentration. Follow-ing centrifugation at 10 000 g, the precipitatedprotein pellet was washed three times with ice-coldethanol and then dissolved in Laemmli samplebuffer. The column was calibrated with standardmolecular weight marker protein solution (Phar-macia), containing ribonuclease A (13·7 kDa),chymotrypsinogen A (25 kDa), ovalbumin(43 kDa) and BSA (65 kDa).

RESULTS

In numerous experiments we observed that West-ern blotting of fission yeast cytosolic extracts pre-pared in the presence of a cocktail of proteaseinhibitors revealed two bands that reacted with amonoclonal antibody to the so-called PSTAIREmotif (Yamashita et al., 1991), one at 34 kDa, thesecond at 31 kDa (p31) (Figure 1). Overexpressionof the cdc2+ gene greatly increased the intensity ofthe 34 kDa band, which we therefore identify asthe product of the cdc2+ gene (p34cdc2), but notp31 (Figure 1, left panel). Polyclonal antibodiesraised against the C-terminus of p34cdc2 (C8) or tothe full-length protein (G8) recognized the 34 kDaband but did not recognize p31 (Figure 1, rightpanel). Synchronous cultures prepared by cell cycleblock and release using the mutant cdc25.22(Booher et al., 1989) revealed that the level of p31,like that of p34cdc2, showed little cell cycle vari-ation (Figure 2). By contrast, the level of cyclin Brevealed the characteristic cell cycle fluctuation.However, when wild-type cells were mated andallowed to go through meiosis and form spores,p31 was clearly detectable even though the level ofp34cdc2 was markedly reduced (Figure 3). No ob-vious change in the expression of p31 was detectedin cytosolic extracts from cells grown in the ab-sence of either phosphate or ammonium chloride,although p34cdc2 expression was depressed in theabsence of phosphate (Figure 3).To confirm that p31 was a unique protein and

not a breakdown product of p34cdc2, cytosolicextracts were subjected to immunoprecipitationusing an antibody to p63cdc13, the fission yeastcyclin B (Booher et al., 1989). Whereas p34cdc2 waspresent in immune complexes with this antibody,p31 was undetectable (Figure 4A). Neither was itadsorbed by p13suc1 Sepharose, a reagent that

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efficiently precipitated p34cdc2 (Figure 4B). Con-versely, p31 was adsorbed by Sepharose conju-gated to the 15 kDa starfish homologue of p13suc1

(Azzi et al., 1994; Figure 4B). This reagent doesnot bind starfish cdc2 but interacts efficiently withp34cdc2 from Sz. pombe, albeit less efficiently thanp13suc1 (Figure 4B). We next investigated the pres-ence of p31 in cytosolic extracts prepared from astrain in which cdc2+ was replaced by the buddingyeast CDC28 gene. p31 was still present in thisstrain but not in control budding yeast extracts,which contained, as shown previously, twoPSTAIRE-positive bands, Cdc28 and the moreslowly migrating Pho85 (Espinoza et al., 1994;Figure 5). Finally, we examined whether p31 was

Figure 1. Western blotting by Sz. pombe cytosolic extracts withdifferent cdc2 antibodies. Left panel: different amounts of totalsoluble protein from wild-type cells or from a strain overexpressingcdc2t were loaded onto SDS–PAGE and analysed by Westernblotting using the PSTAIRE antibody. Right panel: 100 ìg ofcytosolic extract was separated by SDS–PAGE and analysed byWestern blotting using the PSTAIRE, C8 or G8 antibodies. Thearrowheads indicate the positions of cdc2 (34 kDa) and p31(31 kDa).

Figure 2. The level of p34cdc2 and p31 protein is constantthrough the cell cycle. Wild-type cells were synchronized byelutriation and the septation index and cell number determinedat 20-min intervals. For each time point, 50 ìg of total proteinextract was separated by SDS–PAGE and analysed by Westernblotting using PSTAIRE or cyclin B antibodies.

Figure 3. Effect of growth conditions on the level of p31 andp34cdc2. 100 ìg of total soluble protein from spores and fromwild-type cells grown in the absence of either phosphate orammonium was analysed by Western blotting using thePSTAIRE antibody. p34cdc2 is considerably less abundant inspores than in normal vegetative cells whilst the relative level ofp31 appears to be similar.

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increased in a strain overproducing crk1+/mop1+,the putative fission yeast homologue of cak(Damagnez et al., 1995; Buck et al., 1995). Westernblots of cytoplasmic extracts from this strain wereindistinguishable from wild type (data not shown).

All PSTAIRE proteins isolated thus far are cdksand hence associated with a cyclin subunit, thepossible exception being cdk5 whose 31 kDa as-sociated subunit falls outside of the accepted defi-nition of a cyclin (Lew et al., 1994). If p31 is a cdkit would be reasonable to expect that the proteinwould exist as part of a complex with at least oneother protein. Cytosolic extracts were thereforesubjected to gel filtration chromatography onSephacryl HR100. Both p34cdc2 and p31 wereeluted at approximately 100 kDa but p31 was alsoresolved in a second peak at approximately 30 kDa(Figure 6). Thus, p31 appears to exist in bothmonomeric and associated forms.To determine the subcellular localization of p31,

spheroplasts were prepared from wild-type cellsand homogenized. The homogenate was run into a30–50% step ficoll gradient which resolved threefractions: soluble proteins, which did not enter thegradient; an organelle fraction, which formed adiffuse band in the 30% ficoll phase; and a fractionenriched in nuclei, which formed a sharp band inthe 40% ficoll phase (Gallagher et al., 1993). Wholecells, the crude cytosolic extract and the threegradient fractions were separated by SDS–PAGEand immunoblotted with the PSTAIRE antibody.As can be seen in Figure 7, neither p34cdc2 nor p31were present in the soluble fraction but both wereenriched in the nuclei. The low level of both

Figure 4. p31 is not associated with cyclin B or p13suc1 but canassociate with p15cdk-BP. 1 mg of total protein extract was immu-noprecipitated with cyclin B or preimmune antibody (A) or pre-cipitated with either p13suc1 or p15cdk-BP (B) and analysed byWestern blotting using the PSTAIRE antibody.

Figure 5. p31 is present in cells deleted for cdc2+. Totalsoluble protein from wild-type strains of Sz. pombe and S.cerevisiae and from a Sz. pombe strain deleted for cdc2+ butcarrying a plasmid containing the S. cerevisiae CDC28 gene wasanalysed by Western blotting using the PSTAIRE antibody.

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proteins in the organelle fraction was probably dueto contaminating nuclei.

DISCUSSION

A major contribution to our understanding of cellcycle control mechanisms in mammalian cells wasthe identification of an extended family of cdc2-related protein kinases, now referred to as cyclin-dependent kinases (Meyerson et al., 1993). ThePSTAIRE motif was first recognized as a uniquefeature of the founding member of this classof protein kinases, the fission yeast cdc2+ gene

(Simanis and Nurse, 1986). Subsequently, it hasbeen demonstrated that divergence at this sitespecifies the cyclin partner to which a particularcdk can bind. Cyclin binding causes a reorien-tation of the PSTAIRE helix that results in aconformational change in the ATP binding site(Jeffrey et al., 1995).In Sz. pombe, both the G1/S and G2/M cell cycle

transitions are driven by cdc2+ (Forsburg andNurse, 1991). The homologous protein in buddingyeast is CDC28 but S. cerevisiae possesses a secondgene, PHO85, that contains the PSTAIRE se-quence (Toh-e et al., 1988). PHO85 was originallyidentified as a gene concerned with the regulationof phosphate metabolism but the recent findingsstrongly imply a cell cycle role. One possibility isthat PHO85 provides a link between the cell cycleand the nutritional state of the cell. Although thelist of CDC28-related kinases in budding yeastcontinues to grow, including now FUS3, KSS1,CTK1, KIN28 and SRB10, probing cytoplasmicextracts with an anti-PSTAIRE antibody revealsonly two species, CDC28 and PHO85 (Espinozaet al., 1994).In fission yeast only one cdc2-related kinase has

been identified, the predicted Sz. pombe homo-logue of cak (Damagnez et al., 1995; Buck et al.,1995). The crk1+/mop1+ gene product has a pre-dicted molecular weight of 38·5 kDa and is onlyweakly related to the peptide against which thePSTAIRE antibody is raised (DGIDISALREIK-FLRE compared to EGVPSTAIREISLLKE;Yamashita et al., 1991). More importantly, p31was not enriched in a strain in which crk1+ wasoverproduced. Thus we have identified a protein,p31, that we believe to be a new fission yeastPSTAIRE protein for the following reasons: (1)the protein is detected by an anti-PSTAIRE mono-clonal antibody (Yamashita et al., 1991) but not bytwo polyclonal antibodies to p34cdc2; (2) over-expression of cdc2 results in increased levels of p34but not p31; (3) p31 is present in cells in whichcdc2 has been replaced by CDC28; (4) p31 is notassociated with cyclin B; (5) unlike p34cdc2, p31was not retained by p13suc1 Sepharose beads, al-though, like p34cdc2, it was adsorbed by Sepharosebeads conjugated to p15, a p13suc1-related proteinfrom starfish; (6) p31 was present in spores wherecdc2 was detectable only at low level. Finally, likep34cdc2 (Gallagher et al., 1993), p31 is a nuclearprotein which shows little cell cycle regulation(Simanis and Nurse, 1986; Booher et al., 1989;Moreno et al., 1989).

Figure 6. Gel filtration chromatography of p31. Sz. pombe cellextract (6 mg protein) was applied to an HR 100 gel filtrationcolumn and 2 ml fractions were collected. Each fraction wasanalysed by Western blotting using the PSTAIRE antibody.The column was calibrated with standard molecular weightmarker protein solution (Pharmacia), containing ribonucleaseA (13·7 kDa), chymotrypsinogen A (25 kDa), ovalbumin(43 kDa) and BSA (65 kDa). p31 elutes at an apparent molecu-lar weight of 31 kDa (arrowhead) and in the excluded volumeof the column (fractions 9 and 10).

Figure 7. p34cdc2 and p31 are nuclear proteins. Wild-typefission yeast cells were fractionated and the different fractionsanalysed by Western blotting using the PSTAIRE antibody.Both p31 and p34cdc2 were substantially enriched in the nuclearfraction.

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Although none of these findings is individuallycompelling, taken together, they strongly suggestthat p31 is a new fission yeast PSTAIRE protein.All PSTAIRE proteins described to date are cdks.Preliminary gel filtration experiments indicate thatp31 exists as both an unassociated form andassociated with another protein in the cyclin sizerange. This heterogeneity, together with the lowabundance of p31, has so far precluded purifica-tion of the protein. Is p31 the fission yeast homo-logue of PHO85? PHO85 and its cyclin, PHO80,regulate the secretion of acid phosphatase in S.cerevisiae cells grown in low phosphate (Kaffmanet al., 1944). But PHO85 may have a second cellcycle role via its association with two other cyclins,PCL1 and PCL2 (Espinoza et al., 1994; Measdayet al., 1994). Although the structural gene for acidphosphatase in Sz. pombe is known (Yang andSchweingruber, 1990), little is known of its regula-tion. Whether p31 plays some role in this processor if it serves some quite distinct role awaits thecloning of the p31 gene and experiments to thisend are at hand.

ACKNOWLEDGEMENTS

We thank Yannick Gachet for assistance with thechromatographic characterization of p31, JackieHayles for much useful advice regarding theabsence of p34cdc2 in spores and David Beach,Bernard Ducommun, Laurent Meijer, JonathanMillar, Peter Piper and Masakane Yamashita forstrains and reagents. We also acknowledge theexcellent technical assistance of Vasanti Amin.

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identification and preliminary characterization of p31, a new pstaire-related protein in fission...

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