Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by...

transcript

Cell Biology International 31 (2007) 1160e1172www.elsevier.com/locate/cellbi

Changes in expression of cell wall turnover genes accompany inhibitionof chromosome segregation by bovine protein kinase C a expression

in Saccharomyces cerevisiae

Jason A. Sprowl a, David J. Villeneuve b, Baoqing Guo b, Andrew J.M. Young c,Stacey L. Hembruff b, Amadeo M. Parissenti b,c,d,*

a Department of Biology, Laurentian University, Sudbury, Ontario, Canadab Tumour Biology Research Program, Regional Cancer Program, Sudbury Regional Hospital, Sudbury, Ontario, Canada

c Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canadad Division of Medical Sciences, Northern Ontario School of Medicine, Sudbury, Ontario, Canada

Received 2 January 2007; revised 12 March 2007; accepted 23 March 2007

Abstract

Expression of bovine PKCa in Saccharomyces cerevisiae results in growth inhibition, which is strongly augmented upon addition of phorbolesters. To investigate the nature of this PKC-induced inhibition of cell growth, wildtype and bovine PKCa-expressing yeast cells were examinedby flow cytometry and by fluorescence microscopy after staining with propidium iodide. Upon expression and activation of the mammalian PKCisoform, cells accumulated in the G2/M phase of the cell cycle and exhibited impaired chromsome segregation. In some instances, PKC expres-sion and activation was accompanied by a defect in septum formation between mother and daughter cells. cDNA microarray analysis revealed 4genes (CTS1, DSE1, DSE2, and SVS1) that changed expression in both a PKCa- and phorbol ester-dependent manner. These findings were con-firmed by quantitative real-time PCR. Three of these genes are involved in cell wall turnover and are regulated by a single transcription factor(Ace 2) that localizes to daughter cell nuclei after cytokinesis. Taken together, these observations suggest that expression and activation of bo-vine PKCa in yeast cells repress growth by inducing an accumulation of cells in G2/M, likely through an impairment of chromosome segre-gation, cytokinesis, and septum formation. Moreover, when these observations are taken in the context of previously published observationswith various yeast null mutants, we propose that bovine PKCa may directly or indirectly activate a subunit of the PP2A phosphatase complex(cdc55), which is a component of the mitotic spindle checkpoint.� 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Keywords: Microarray analysis; PKC; Yeast; Growth inhibition; Cell wall

Abbreviations: PKC, protein kinase C; PMA, phorbol-12 myristate-13

acetate; Q-PCR, quantitative reverse-transcription polymerase chain reaction;

YEp51, vector for the galactose-induced expression of genes in S. cerevisiae

with no cDNA insert; pYECNa, vector for the galactose-induced expression

of a bovine PKCa; cDNA in S. cerevisiae; APC, anaphase-promoting com-

plex; OD600, optical density at 600 nm; PBS, phosphate-buffered saline; PI,

propidium iodide.

* Corresponding author. Office of the Chair in Cancer Research, Regional

Cancer Program, Sudbury Regional Hospital, 41 Ramsey Lake Road, Room

32032, Sudbury, Ontario P3E 5J1, Canada. Tel.: þ1 705 522 6237; fax: þ1

705 523 7326.

E-mail address: aparissenti@hrsrh.on.ca (A.M. Parissenti).

1065-6995/$ - see front matter � 2007 International Federation for Cell Biolog

doi:10.1016/j.cellbi.2007.03.033

1. Introduction

The study of PKC structure and function in mammaliancells is a difficult task, given the genetic complexity of mam-mals, the presence of multiple PKC isoforms, and the extensivecross-talk that occurs amongst numerous signaling pathways(Coussens et al., 1986; Knopf et al., 1986; Housey et al.,1987; Parker et al., 1989; Slater et al., 2001). In some instances,these problems can be circumvented by the expression of mam-malian PKC isoforms in a much simpler, genetically-accessibleeukaryotic system such as the budding yeast Saccharomyces

1161J.A. Sprowl et al. / Cell Biology International 31 (2007) 1160e1172

cerevisiae. A wide variety of mammalian proteins have beensuccessfully expressed in yeast and disruptions of specific bio-chemical pathways in yeast can often be complemented byexpression of the corresponding mammalian protein homo-logue. For example, Erk1 (MAP Kinase) defects in yeast canbe complemented by the expression of mammalian ERK1 ho-mologues (Errede and Levin, 1993). Our laboratory and othershave expressed a number of mammalian PKC isoforms inSaccharomyces cerevisiae using a yeast expression plasmidwhere the PKC cDNA is under the tight control of a galac-tose-inducible promoter.

The yeast strain used in these studies (strain 334) possessesmutations that block both galactose catabolism ( gal1) andglucose repression of the GAL promoter (reg1e501). Thisprevents the hydrolysis of the inducing agent (galactose)and enables the galactose-inducible promoter to function inthe presence of the preferred carbon source (glucose). Mam-malian PKCs expressed in yeast have functional characte-ristics similar to those found in mammalian cells (Riedelet al., 1993a,b,c; Goode et al., 1994; Shieh et al., 1996;Parissenti et al., 1996b; Keenan et al., 1997; Parissentiet al., 1999). For example, classical PKC isoenzymes ex-pressed in yeast can be activated by phorbol esters (Riedelet al., 1993a,b). In addition, long term application of phorbolesters to bovine PKCa-expressing yeast cells results in thedownregulation of the expressed enzyme, similar to findingsin mammalian cells (Riedel et al., 1993b). Thus, the yeastS. cerevisiae appears to contain all the necessary componentsof mammalian PKC signaling pathways, including an endog-enous yeast enzyme (Pkc1p) encoded by the yeast gene pkc1,which is essential for growth in hypotonic media (Levin andBartlett-Heubusch, 1992).

The mechanism by which Pkc1p regulates yeast cell growthappears to involve its ability to activate a MAP kinase cascadeconsisting of Bck1, Mkk1, Mkk2, and Slt2 (the yeast MAPkinase) (Herskowitz, 1995; Livneh and Fishman, 1997). Thisincludes the protein’s ability to stimulate Slt2p phosphoryla-tion in a multiprotein complex at actin patches known to beassociated with sites of polarized growth (Sheu et al., 1998).The yeast Pkc1p has strong structural homology to mamma-lian PKCs, including two C1-like domains, a C2 domain, a ki-nase domain, a V5 region involved in subcellular localization,and two HR1 domains in the N-terminal region that appears tointeract with G proteins (Mellor and Parker, 1998). The GTP-binding protein Rho1p binds to yeast PKC1p and activates itsactivity (Nonaka et al., 1995; Kamada et al., 1996). PKC1deletion mutations induce cell cycle arrest (Levin et al.,1990; Simon et al., 1991), an osmotic stability defect (Levinand Bartlett-Heubusch, 1992) and enhanced mitotic recombi-nation (Huang and Symington, 1994).

We have found that the galactose-induced expression ofvarious mammalian PKC isoforms in yeast (along with treat-ment of the cells with PKC-activating phorbol esters) resultsin the induction of variety of cellular phenotypes. These in-clude a dramatic reduction in cell growth (Riedel et al.,1993a; Parissenti et al., 1999), gross alterations in cell mor-phology (Riedel et al., 1993a; Parissenti et al., 1996a, 1999),

Caþþ dependence for cell viability (Riedel et al., 1993b),and enhanced uptake of extracellular Caþþ (Riedel et al.,1993a,b, 1999). The growth inhibitory phenotype induced byPKC in yeast appears to be the result of a defect in cell sepa-ration after mitosis, since phorbol ester-treated cells formelongated multicellular structures that cannot be disrupted bysonication (Parissenti et al., 1999).

There is also considerable evidence for PKC’s involvementin growth promotion and growth inhibition in mammaliancells (Livneh and Fishman, 1997). Specific PKC isoformsare activated upon the treatment of cells with growth factors(Nishizuka, 1992), where they lead to the activation of down-stream kinases such as Raf, MEK, and ERK (Cai et al., 1997;Carroll and May, 1994; Kolch et al., 1993; Sozeri et al., 1992).In contrast, PKC activation by PMA actually inhibits growthin rat 3Y1 cells and vascular smooth muscle cells by blockingDNA synthesis (Ohno et al., 1994; Huang and Ives, 1987).Growth modulation by PKC (both positive and negative) cantake place during the G1 or G2/M phases of the cell cycle(Zhou et al., 1993; Thompson and Fields, 1996; Watanabeet al., 1992; Jackson and Foster, 2004). Given the wide varietyof roles for multiple PKC isoforms in regulating cell growth,the simple eucharyote S. cerevisiae may prove to be an excel-lent experimental system to uncover PKC-dependent mecha-nisms involved in cellular growth control that are sharedbetween lower and higher eucharyotic cells.

To better understand how PKC regulates cell growth, wemonitored cell cycle progression, chromosome division, chitindistribution, gene expression, and cell morphology upon PKC-induced inhibition of yeast cell growth. The results of thesestudies suggest that bovine PKCa induces a delay in cell cycleprogression at M phase, accompanied by unequal chromosomedivision, impaired cell septum formation, and changes in theexpression of cell wall turnover genes. Based on these and pre-viously published observations, we propose that bovine PKCamay directly or indirectly activate a component of the mitoticspindle checkpoint, namely the cdc55 subunit of the PP2Aphosphatase complex.

2. Materials and methods

2.1. Culturing of yeast cells

Yeast cells (strain 334) and its transformants were propagated in Leu-

medium to maintain selection for the expression plasmid, which also encodes

for an enzyme complementing the Leu-mutation. To prepare 500 ml of Leu-

liquid medium, 1 g of Leu-dropout mixture, 0.85 g of yeast nitrogen base

without amino acids (Difco Laboratories, Livonia, MI), and 2.5 g of ammo-

nium sulphate were added to 500 ml of distilled water, and the resulting me-

dium adjusted to a pH of 5.6 and autoclaved. For solid Leu-medium, 7.5 g

of Difco agar was also added to the medium before autoclaving. A filter-

sterilized solution of 20% glucose (w/v) was also prepared as the carbon

source, and added to liquid or agar medium (after autoclaving) at a final con-

centration of 2% (w/v). Frozen stocks of yeast cells transformed with a plas-

mid coding for the galactose-inducible expression of bovine PKCa in yeast

(pYECNa) or an identical plasmid lacking the PKC cDNA insert (YEp51)

were streaked onto Leu-plates and incubated at 30 �C until single colonies

could be isolated. Three single isolates were then propagated for subsequent

studies.

1162 J.A. Sprowl et al. / Cell Biology International 31 (2007) 1160e1172

2.2. Measurement of PKC expression in yeastcells by immunoblotting experiments

The level of expression of bovine PKCa in yeast cells transformed with the

expression plasmid YEp51, with or without the bovine PKCa cDNA insert was

assessed by standard immunoblotting approaches using a monoclonal antibody

specific for rat brain PKCa (M4, Upstate Biotechnology, Lake Place, NY).

Blocking of non-specific binding sites was accomplished by supplementing

the primary or secondary antibody solutions with 5% skim milk powder and

0.1% Tween 20. The primary antibody was used at a 1:1000 dilution, while

the secondard antibody was used to probe the membrane at a 1:10,000 dilution.

Visualization of bands on immunoblots was by chemiluminescence using

Amersham ECL substrates.

2.3. Monitoring of yeast cell proliferation

Clonal isolates of yeast cells transformed with either YEp51 or pYECNa

were grown at 30 �C in 5 ml of liquid Leu-medium with vigorous shaking. The

growth of these clones was measured by serially diluting overnight cultures to

an OD600 of 0.10 in Leu- medium containing 2% glucose. Bovine PKCa

expression was then induced in some cultures by the addition of 2% galactose,

in the absence or presence of 1 mM PMA. OD600 measurements of all cultures

were taken at regular time intervals until saturation was reached, or no growth

was evident at the end of the experiment.

2.4. Assessment of cellular DNA content by flow cytometry

To determine whether growth inhibition by PKC correlated with the accu-

mulation of cells at a specific point in the cell cycle (as determined by DNA

content), YEp51- or pYECNa-transformed yeast cells were grown overnight

in Leu-medium supplemented with 2% glucose and 2% galactose until growth

saturation was achieved. This permitted strong induction of PKC-expression

in pYECNa-transformed cells and permitted the majority of cells to accumulate

in the G1 phase of the cell cycle. The cultures were then diluted in identical me-

dia to an OD600 of 0.1, with or without the addition of 1 mM PMA. Cells were

then monitored for growth by optical density measurements at various time

points. At the identical time points, an aliquot of cells was removed such that

1,000,000 cells could be assessed for DNA content by flow cytometry after PI

staining. Cells were collected by centrifugation at the indicated time points

and fixed by resuspension in 1 ml of 75% ethanol for 2 h. After fixation, cells

were harvested again by centrifugation and resuspended in 0.5 ml of PI solution

for a minimum of 1 h. The stained cells were harvested by centrifugation and re-

suspended in 0.5 ml of PBS prior to flow cytometric analysis. For each sample,

approximately 2 � 104 cells were analyzed using a Beckman Coulter Epics�

Elite flow cytometer. Fluorescence intensity upon stimulation with an argon-

ion laser at 488 nm was measured using the PMT4 channel (625DL filter) and

plotted against cell number. The overlay graphs were generated by the flow

cytometer to show the DNA profile change over time.

2.5. Visualization of cells after calcofluoror propidium iodine staining

The cell wall structure of pYECNa- or YEp51-transformed yeast cells un-

der various conditions was observed by fluorescence microscopy under vari-

ous conditions using calcofluor staining, an agent which binds to chitin within

the cell wall. Yeast cells from an exponentially growing culture were diluted

in 10 ml of Leu-/glucose medium supplemented with 2% galactose or both

1 mM PMA and 2% galactose. The starting optical density at 600 nm

(OD600) was approximately 0.002, except for pYECNa-transformed cells

treated with both galactose and PMA. These cells were diluted to an

OD600 of approximately 0.006 to account for the subsequent growth inhibi-

tion. Samples were incubated for approximately 15 h at 30 �C until an

OD600 of 0.2 was reached. Cells were harvested by centrifugation at

3000 � g for 5 min, washed in 500 mL of phosphate-buffered saline (PBS),

and resuspended in 500 mL of PBS. To 100 mL of cells, 10 mL of calcofluor

(1 mg/mL) was added and the samples incubated at room temperature for

1 h in the dark on a rotator. Each sample was washed 3 times using

500 mL of PBS, resuspended in 10 mL of PBS, and placed on a glass slide

for visualization by fluorescence microscopy using appropriate filters. To ob-

serve the subcellular distribution of genetic material, pYECNa- or YEp51-

transformed yeast cells were stained with the DNA-binding dye propidium

iodide (PI) and visualized by fluorescence microscopy. Cells were prepared

as mentioned above though fixed in 500 mL of 100% methanol for 1 h with

shaking, harvested again, and resuspended in 500 mL of PI solution

(0.1 mg/ml PI in a buffer composed of 0.1% sodium citrate, 0.3% NP-40,

and 0.1 mg/ml RNAse A). Cells were then washed twice with 500 mL of

PBS, and resuspended in 100 mL of PBS. Ten mL of the cell suspension

were then placed on a glass slide for visualization by fluorescence micros-

copy using appropriate filters. Number of cells per field were counted (large

buds counted as separate cells) as well as red fluorescence spots.

2.6. Preparation of RNA from yeast cells

Cells transformed with pYECNa or YEp51 were grown in liquid culture

under the various culture conditions describe above, expect that 10 mL of

medium were used. From a starting OD600 of 0.05, YEp51-transformed cells

under all culture conditions were grown until an OD600 of 0.8 was reached. A

similar approach was taken for pYECNa-transformed cells in absence of

PMA though in the presence of galactose alone. For cells transformed with

pYECNa and incubated with both PMA and galactose, the starting OD600 was

0.1, since cells become growth inhibited after PKC induction. Incubation lasts

approximately 8 h, which appears to mark a difference in cell growth in response

to PKC activity (Fig. 2A). After adjusting for differences in cell density amongst

the cultures, 10 ml of culture at an OD600 of 0.8 (1.6 � 108 cells) were harvested

by centrifugation for 5 min at 3000 � g. The supernatant was removed, after

which RNA isolation was conducted using a Gentra Yeast RNA isolation kit

(Gentra Systems, Minneapolis, MN) using the methods provided by Gentra.

2.7. Assessment of RNA quantity and quality

The amount of RNA in each sample was assessed by measuring the absor-

bance of a diluted sample at 260 nm using standard procedures. RNA quality

was determined by running the samples on denaturing agarose gels and stain-

ing of the gel with ethidium bromide. Only samples with no evidence of RNA

hydrolysis (two clearly distinct 28S and 18S ribosomal RNA bands) were used

in subsequent experiments.

2.8. DNA microarray analysis

To identify changes in gene expression that accompany PKC-induced

growth inhibition in yeast, we conducted microarray studies using our recently

developed optimal approaches for the performance, analysis, and verification

of cDNA microarray experiments. Three independent RNA preparations

were isolated from YEp51-transformed and pYECNa-transformed yeast cells

treated with 2% galactose and from identical cells treated with both 2% galac-

tose and 1 mM PMA. As described in a previous study (Villeneuve et al.,

2006), RNA preparations were reverse transcribed yielding cDNA preparations

that were labelled with either Cy3- or Cy5-conjugated deoxyribonucleoside

triphosphates.

2.9. Array hybridization

The labeled cDNAs were allowed to hybridize to microarray slides as de-

scribed previously (Villeneuve et al., 2006) except that lifter slips (25 �60 mm; Erie Scientific, Portsmouth, NH) were placed on 6.4K yeast genome mi-

croarray slides (University Health Network Microarray Centre, Toronto, ON,

Canada) prior to hybridization.

2.10. Analysis of yeast microarray data

Hybridized and washed arrays were scanned on an Axon 4000B dual laser

scanner (532 nm/633 nm wavelengths). The voltage across the photomultiplier

tubes was adjusted until the intensity ratio of the red and green acquisition histo-

grams was between 0.9 and 1.1 (when all signals above 200 fluorescence units

were included in the analysis). The fluorescence intensities for each feature

(spot) on the array were determined using GenePix Pro array software (version

3.0) from Axon Instruments, using the gal file for the yeast microarrays supplied

by the University Health Network Microarray Centre (Toronto, ON). The

GenePix result files (.gpr) were imported into Microsoft Excel worksheets

(.xls). The data from duplicate spots on the 3 independent array experiments

(1 with reverse fluor) were corrected for background and normalized using a pre-

viously described method from our laboratory (Villeneuve and Parissenti, 2004).

2.11. Verification of microarray data by Q-PCR

The changes in gene expression identified by cDNA microarray analysis

were confirmed by Q-PCR using a modification of our recently published

method (Hembruff et al., 2005). Three independent samples of RNA from

YEp51- or pYECNa-transformed yeast cells treated with either 2% galactose

or 2% galactose and 1 mM PMA were prepared as described above for use in

confirmatory Q-PCR experiments. In these experiments, equivalent amounts of

RNA were assessed for quality by staining with ethidium bromide after dena-

turing agarose gel electrophoresis and contaminating DNA removed by diges-

tion with DNase I. Reverse transcription was carried out as previously

described (Hembruff et al., 2005) followed by the addition of 5 ml of the

cDNA mixture to gene-specific primers (at a final concentration of 300 nM),

and 12.5 ml of 2X SYBR Green I mix for amplification. A 5 point standard

curve was constructed for each gene.

Four replicate Q-PCR reactions were performed on an ABI Prism 7900 HT

instrument for each of the pooled cDNA samples described above. Reaction pa-

rameters included an incubation at 95 �C for 10 min (to activate the AmpliTaq

gold), and 40 cycles of (95 �C for 15 s, 55 �C for 15 s and 72 �C for 30 s) for the

melting, annealing, and elongation phases of the reaction, respectively. The PCR

product for each gene-specific amplification was melted after the reaction to en-

sure that only one melting temperature was obtained at the expected temperature

(suggesting only one PCR product). The mean amount of a particular cDNA in

each sample was then determined using ABI Prism gene quantification software

with Cln2 serving as the reference gene. (We found that Cln2 expression was

both galactose- and PMA-independent in most experiments.) The fold change

in expression induced by PMAwas calculated using the formula: fold change ¼Z/A, where Z ¼ the mean quantity of the cDNA of interest in pYECNa or

YEp51-transformed cells (treated with galactose in the absence of presence of

PMA), divided by the mean quantity of the Cln2 cDNA in identical cells treated

in the same manner. In contrast, A ¼ the mean quantity of the cDNA of

pYECNa-transformed cells treated with galactose divided by the mean quantity

of the Cln2 cDNA in identical cells treated with galactose. The gene-specific ol-

igonucleotide primers used for confirmation of the array data by Q-PCR were:

DSE1: Forward Primer, 50-AAAATCTACCGTATCAACGTCGATT-30, Reverse

Primer, 50-CCATGGCGAAACC-TTCAAAATA-30; DSE2: Forward Primer, 50-ACTCTCAACTAACTTTGGCGTCATC-30, Reverse Primer, 50-TAGTTCTTG

CTATAGTGGACCCTGTTT-30; CTS1: Forward Primer, 50-TAAGAAC-TTT

GTTTGCCGAAGGT-30, Reverse Primer, 50-AAGCATCCGGGTATGGACA

TT; SVS1: Forward Primer, 50-CGGAATCCGCCAACGA-30, Reverse Primer,

50-CCGATTCGGTT-ACTGTAGAAGCA; CLN2: Forward Primer, 50-TTACG

GGACCAAGCCAAATT-30, Reverse Primer, 50-ACAACCGCCCCAGTTTTA

GC-30.

3. Results

3.1. Verification of expression of bovinePKCa in yeast cells

In order to identify the PKC-dependent changes in gene ex-pression accompanying cell growth inhibition in yeast, we firstsought to confirm that yeast cells transformed with the induc-ible bovine PKCa expression vector (pYECNa) express theenzyme in a galactose-dependent manner. We also sought to

verify that cells transformed with the same expression vectorlacking the PKCa cDNA insert (YEp51) have no detectablelevels of PKCa protein. With these objectives in mind, deter-gent-soluble extracts of YEp51- and pYECNa-transformedyeast cells (grown in the absence or presence of galactose)were prepared as described previously (Parissenti et al.,1999). Proteins in the extract were then resolved by SDS poly-acrylamide gel electrophoresis, transferred to nitrocellulosemembranes, and probed with a PKCa-specific antibody asdescribed in Section 2. As shown in Fig. 1, yeast cells trans-formed with pYECNa and incubated with galactose did expr-ess a protein recognized by the PKCa antibody, which wasconsistent with the molecular weight of PKCa (w85 kDa).Purified preparations of baculovirus-expressed PKCa proteinwere also recognized by the PKCa antibody using this ap-proach, with a molecular weight similar to that of the yeast-expressed enzyme. In contrast, pYECNa-transformed cellsincubated in the absence of galactose showed little expressionof PKC. Control cells transformed with YEp51, as expected,showed no evidence of PKCa expression at identical levelsof galactose in the medium. Together, the data confirms thesuccessful galactose-inducible expression of bovine PKCa inyeast cells using the expression vector pYECNa.

3.2. Supression of growth by expression and activation ofbovine PKCa along with accumulation of cells in G2/M

In order to establish a direct correlation between PKCa ex-pression, growth inhibition, and other cellular phenomena, wefirst compared the growth of yeast cells transformed with eitherYEp51 or pYECNa in the presence of galactose, with or with-out the addition of PMA. Glucose was used as the carbonsource and growth was monitored over time by measuring tooptical density of the culture at 600 nm wavelength (OD600).As exhibited in Fig. 2A, control yeast cells transformed withthe expression vector YEp51 exhibited rapid growth in thepresence of both glucose and galactose (open circles). Addition

Fig. 1. Galactose-induced expression of bovine PKCa in S. cerevisiae. Deter-

gent-soluble protein extracts were prepared from yeast cells transformed with

either the expression vector YEp51 or an identical vector in which the coding

sequence for bovine PKCa was ligated downstream of the galactose-inducible

promoter. Extracts were prepared from cells incubated in the absence or pres-

ence of galactose (Gal). The proteins in the extract were resolved by polyacryl-

amide gel electrophoresis, transferred to nitrocellulose, and the membrane

probed with a PKCa-specific monoclonal antibody as described in Section 2.

A purified preparation of baculovirus-expressed PKCa was used as a positive

control. The mobilities of two molecular weight standards at 100 kDa and

72 kDa are depicted to the left of the immunoblot.

of the phorbol ester PMA to the medium had little effect ongrowth (closed circles). In contrast, pYECNa-transformedcells grew more slowly in the presence of glucose and galactose(open squares), likely due to the basal activity of the expressedbovine PKCa. When galactose-treated pYECNa-transformedcells were incubated with PMA (closed squares), a furtherreduction in growth was observed. The effect of PMA on cellgrowth was likely the result of activation of the expressedPKC, since PMA had no corresponding effect on YEp51-transformed cells grown in the presence of galactose.

Using the samples monitored for growth, assessment forDNA content by flow cytometry after staining with PI (as de-scribed in Section 2) had been performed. As shown inFig. 2A, galactose-induced expression of bovine PKCa in yeastcells resulted in inhibition of growth that was further accentu-ated by the addition of the PKC-activating phorbol ester PMA.When cells during such growth experiments were assessed forDNA content by flow cytometry after PI staining (Fig. 2B), itwas possible to identify what effect the expression and

Fig. 2. Growth inhibition by PKCa in yeast cells associated with accumulation

of cells in G2/M. Overnight cultures of yeast cells transformed with either

YEp51 (circular symbols) or pYECNa (square symbols) were diluted to an

OD600 of 0.1 to permit assessment of growth by optical density (panel A)

and DNA content by flow cytometry after staining with propidium iodide

(panel B). Growth assessments were conducted in the absence (open symbols)

or presence (closed symbols) of 1 mM PMA.

activation of bovine PKCa had on cell cycle progression. Cellswith 1N of DNA content (peak of lower fluorescence) weredeemed to be in the G1 or G0 phase of the cell cycle, whereascells with 2N of DNA content (peak of higher fluorescence)were deemed to be in the G2 or M phase of the cell cycle. Atthe time of dilution from saturated culture (time ¼ 0 min),the predominant peak of fluorescence for all samples repre-sented cells in the G1 phase of the cell cycle, as expected. Dur-ing active growth in either the presence or absence of PMA, thepredominant peak of fluorescence for YEp51-transformed cellswas still represented by cells in G1. In contrast, transformationof cells with pYECNa and the resultant overexpression of bo-vine PKCa resulted in a significant reduction in the percentageof cells in G1 and a significant increase in the percentage ofcells in G2/M. Moreover, upon addition of PMA to the me-dium, the percentage of cells in G2/M increased even further,such that the majority of cells were in G2/M after 3 h ofgrowth. These findings illustrate dose-dependent relationshipsbetween the level of bovine PKCa activity and both the degreeof inhibition of yeast cell growth and the percentage of cells inthe G2 or M phase of the cell cycle. Moreover, these findingscorroborate PI staining experiments suggesting a defect incell cycle progression at G2/M related to aberrant chromosomedivision. Cells were only assessed for DNA content in the firstfew hours of growth since it has been established that long-termincubation of yeast cells expressing bovine PKCa with phorbolesters such as PMA results in downregulation of the enzyme(Riedel et al., 1993b).

3.3. Microscopic visualization of YEp51and pYECNa transformants

As mentioned previously, the presence of multicellularstructures in the population upon expression and activation ofbovine PKCa in yeast cells suggests that cell separation isinhibited by the mammalian PKC, when expressed in yeast.To assess whether expression and activation of bovine PKCaresults in defects in the segregation of genetic material duringmitosis, YEp51- and pYECNa-transformed cells grown ingalactose-containing medium, in the absence or presence ofPMAwere stained with the DNA-binding dye propidium iodideand visualized by bright field and fluorescence microscopy(Fig. 3). For each sample, a total of approximately 200 cellswere scored in order to determine the number of unbuddedcells, cells with small buds, cells with large buds, and cells inmitosis with or without successful chromosome segregation.In this scoring, cells with small buds were defined as cells earlyin S phase. Cells with large buds were defined as cells in late S/G2, where the daughter cells were still significantly smallerthan the mother cells. In contrast, cells in mitosis were definedas cells in which the mother and daughter cells were approxi-mately of equal size, where there was typically a clear divisionof nuclear material in YEp51-transformed cells (anaphasethrough telophase). As shown in Table 1, the percentage ofcells with small buds was very similar between YEp51-trans-formed cells (with or without PMA addition) and pYECNa-transformed cells in the absence of PMA. In contrast, the

Fig. 3. Staining of YEp51- and pYECNa-transformed yeast cells with propidium iodide. Yeast cells transformed with YEp51 (Control) or pYECNa (PKC) were

grown in Leu-medium containing 2% glucose and 2% galactose, in the absence or presence of 1 mM PMA. At mid-log phase, the cells were then stained with the

DNA-binding dye propidium iodide in the presence of RNAse, as described in Section 2. Cells were then visualized by bright field and fluorescence microscopy.

The propidium iodide staining intensity amongst the ‘‘cells’’ comprising the chain was extremely variable with one cell exhibiting the vast majority of bound

propidium iodide.

percentage of pYECNa-transformed cells with small buds de-creased significantly upon addition of PMA, consistent withcells accumulating in G2/M upon activation of PKCa expres-sion. Further supporting this hypotheis, the percentage of cellsin mitosis was dramatically increased only in pYECNa-trans-formed cells and only in the presence of PMA. This is consis-tent with the above flow cytometry data suggesting anaccumulation of pYECNa-transfected cells in the G2/M phaseof the cell cycle. The data depicted in Table 1 also suggests thatthe percentage of cells during mitosis without successful chro-mosome division also increased dramatically in pYECNa-transformed cells treated with PMA. Taken in context withthe budding and flow cytometry data, these findings stronglysuggest that expression and activation of bovine PKCa in yeastcells results in cell growth inhibition by inducing an accumula-tion of cells in mitosis, generally with impaired chromosomedivision and cell separation.

In order to observe whether septum formation had occurred,YEp51- and pYECNa-transformed cells in the presence ofgalactose and/or PMA were stained with the chitin-specificdye Calcofluor White. As shown using confocal microscopy(Fig. 4), chitin could be found throughout the cell peripheryin YEp51-transformed cells, consistent with the role of chitinas a structural component of cell walls. An intense band of Cal-cofluor staining could be observed between two dividing cells.This band was likely the ‘‘chitin ring’’, which is formed uponbud formation and helps maintain the integrity of the mother-bud neck, which is important for exchange between the motherand daughter cells and important for septum closure (Cabib andSchmidt, 2003). PKC-expressing cells (transformed withpYECNa) incubated in the absence of PMA had a similar ap-pearance. In contrast, PKC-expressing cells in the presence ofPMA formed ‘‘multicellular’’ structures with reduced or absentCalcofluor staining between some ‘‘cells’’. Such reduced

Calcofluor staining is suggestive of the absence of chitin ringsin these regions, which suggests a defect of cytokinesis whichcould also suggest a defect in chromosome segregation.

3.4. Identification of PKC-dependent genes inyeast by DNA microarray analysis

The analysis of gene expression upon induction and/or activa-tion of bovine PKCa in yeast may help provide further support forthe ability of bovine PKCa to module growth through its effectson cytokinesis and chromosome segregation. Consequently, we

Table 1

Effect of expression and activation of bovine PKCa on the percentage of yeast

cells in G2/M lacking DNA content

Gal. & PMA

pYECNa

Gal. & PMA

Cells counted 201 229 215 221

% unbudded cells 61.6 61.7 69.3 50.7

% of cells with

small buds (early S)

18.8 18.9 19.1 13.1

% of cells with large

buds (late S/G2)

13.1 11.9 6.5 7.2

% of cells in mitosis 6.6 7.5 5.1 29.0

% of cells in mitosis

without chromosome

segregation

2.2 2.5 2.3 24.9

Yeast cells transformed with either YEp51 or pYECNa were treated with 2%

galactose to induce bovine PKCa expression in pYECNa-transformed cells

(but not YEp51-transformed cells). The phorbol ester PMA was then added

to activate the expressed PKCa (if present), after which cells were stained

with the DNA-binding dye propidium iodide (PI) in the presence of RNAse.

Over 100 cells in G2/M were then scored in order to determine the percentage

of cells in which the mother and daughter cells did not have similar staining

(DNA content).

employed our recently optimized method of DNA microarrayanalysis (Villeneuve and Parissenti, 2004; Villeneuve et al.,2006) to identify PMA-dependent changes in gene expressionthat occurred in pYECNa-transformed cells but not in YEp51-transformed cells (see Section 2). As shown in Table 2, a surveyof the entire yeast genome by cDNA microarray analysis revealedonly 3 genes that exhibited reduced expression in response toPMA in galactose-treated pYECNa-transformed cells but notYEp51-transformed cells (under identical conditions). Thesegenes were CTS1, DSE1, and DSE2. The reductions in the expres-sion of these genes by PMA in pYECNa transformants incubatedin the presence of galactose were 3.89-, 2.95-, and 2.29-fold, re-spectively. In contrast, PMA reduced expression of DSE1 andDSE2 by only 1.01- and 1.30-fold, respectively, in YEp51 trans-formants under identical conditions (Table 2). CTS1 expressionvaried slightly by 1.04-fold in response to PMA in YEp51 trans-formants. The standard error in the data was small, likely due tothe averaging of data from multiple array experiments, half ofwhich were performed under ‘‘reverse fluor’’ conditions to elim-inate systematic error in the data associated with differential sta-bility of the Cy3 and Cy5 dyes. Only one gene showed increasedexpression upon PMA treatment specifically in pYECNa trans-formants incubated in the presence of galactose (SVS1). SVS1 ex-hibited a 2.86-fold increase in expression by PMA in pYECNatransformants in the presence of galactose (Table 2). This was sig-nificantly higher than the 1.18-fold reduction in SVS1 expressioninduced by PMA in YEp51 transformants in the presence of ga-lactose. Again, the difference in SVS1 expression betweenpYECNa and YEp51 transformants was highly significant withminimal error in the data.

When one examines the identities of the genes exhibitingaltered expression upon PMA-induced activation of expressed

Fig. 4. Staining of YEp51- and pYECNa-transformed yeast cells with calcofluor white. Yeast cells transformed with YEp51 or pYECNa were grown in Leu-

medium containing 2% glucose and 2% galactose, in the absence or presence of 1 mM PMA. At mid-log phase, the cells were then stained using the chitin-specific

dye calcofluor white, as described in Section 2. Cells were then visualized by bright field and fluorescence microscopy.

Table 2

Effect of bovine PKCa e

Gene Fluorescence (G

YEp51-transfect

cells (flu)

Biological role References

CTS1 1859.0 � 280 Chitin-hydrolyzing enzyme within the

cell wall; required for cell division;

transcribed by Ace2 within

the daughter nucleus

(Kuranda and Robbins,

1991; O’Conallain

et al., 1999)

DSE1 647.2 � 51.6 Transcribed by Ace2; DSE1 null

mutants defective in cell separation after

chromosome division; expression affects

sensitivity to cell wall-targeting drugs

(Colman-Lerner

et al., 2001;

Doolin et al., 2001)

DSE2 497.5 � 78.0 Transcribed by Ace2; Gene

product secreted by

daughter cell; Degrades cell wall,

allowing separation

from mother cell

(Colman-Lerner

et al., 2001;

Doolin et al., 2001)

SVS1 120.2 � 10.3 Ser/Thr rich protein; may play

a role in vanadate

resistance; Expression cell

cycle-regulated and associated

with the cell wall synthesis

(Nakamura et al.,

1995; Terashima

et al., 2002)

To identify changes in ge tivator PMA, RNA was isolated from YEp51- or pYECNa-transformed

cells grown in Leu-medi reverse transcribed to cDNAs in the presence of fluorescently labeled

oligonucleotides and the bed in Section 2. Of the genes representing the complete yeast genome,

only 4 (CTS1, DSE1, DS amount of hybridization associated with the binding of the fluorescently

labeled cDNA probes co in expression of the genes induced by PMA in YEp51- and pYECNa-

transformed cells is also

Sprowl

iologyInternational

31(2007)

1160e1172

xpression and activation on gene expression in yeast cells

Fluorescence

(Gal þPMA)

YEp51-transfected

cells (flu)

Fluorescence (Gal)

pYECNa-transfected

cells (flu)

Fluorescence

(Gal þPMA)

pYECNa-transfected

cells (flu)

Fold change

induced by PMA in

YEp51-transfected

Fold change

induced by PMA i

pYECNa-transfect

1932.9 � 347.3 2038.6 � 106.3 524.2 � 41.8 þ1.04 �3.89

637.8 � 46.1 1032.8 � 163.8 350.2 � 43.1 �1.01 �2.95

381.9 � 37.3 456.0 � 46.0 198.9 � 26.5 �1.30 �2.29

102.0 � 11.3 190.1 � 17.9 543.0 � 95.3 �1.18 þ2.86

ne expression that are induced specifically in bovine PKCa-expressing yeast cells upon treatment with the PKC ac

a containing 2% glucose and 2% galactose, in the absence or presence of PMA. The RNA preparations were then

labeled cDNAs used to probe microarrays containing PCR products representing the entire yeast genome as descri

E2, and SVS1) had altered expression specifically in pYECNa-transformed cells that was modulated by PMA. The

rresponding to these genes to the PCR products on the array is listed in fluorescence units (flu). The fold change

described, along with the biological role of the genes in yeast and appropriate references.

Fig. 5. Verification of changes in gene expression by Q-PCR. Each of the PKC-induced changes in gene expression identified by cDNA microarray analysis was

verified using Q-PCR. RNA was isolated from yeast cells transformed with pYECNa or YEp51 plasmids grown in Leu-medium containing 2% glucose and 2%

galactose, in the absence or presence of 1 mM PMA, as outlined in Section 2. The RNA was reverse-transcribed to cDNA and the cDNA amplified using appropriate

gene-specific primers in the presence of SYBR Green I. The expression of each gene was relative to the expression of the constitutively expressed gene CLN2.

Mean quantity values for each transcript obtained by Q-PCR were divided by the mean quantity values for CLN2 to normalize the expression (outlined in Section

2). The normalized ratio of expression between the gene of interest and CLN2 was then divided by the normalized expression of pYECNa treated with galactose.

This value is listed in brackets for each amplification plot under each condition. The dark line depicted in each plot represents the ‘‘crossover threshold’’ used to

quantify gene expression.

bovine PKCa in yeast, it is interesting to note that all fourgenes code for gene products that play a role in cell wall turn-over. This suggests that growth inhibition by PKC in yeastmay involve the ability of the enzyme to modulate (either di-rectly or indirectly) the expression of genes involved in cellwall turnover. This could then help explain the unique physicalcharacteristics of yeast cells expressing activated bovinePKCa (see Section 4).

3.5. Verification of microarray data by Q-PCR

The PKC-induced changes in gene expression identified inthe above cDNA microarray experiments were then subjectedto verification by Q-PCR using gene-specific primers as de-scribed in Section 2. The level of each transcript under variousconditions (Fig. 5) was expressed relative to that of a constitu-tively expressed gene CLN2 as it was found to be unchangedby microarray analysis (in parentheses). The mRNA levels of

CTS1, DSE1 and DSE2 (relative to CLN2) were indeed re-duced by 3.33-, 3.57-, and 2.94-fold, respectively. This is con-sistent with the changes in gene expression observed by cDNAmicroarray analysis (Table 2), where the genes exhibited re-duced expression by 3.89-, 2.95-, and 2.29-fold, respectively.In contrast, expression of CTS1, DSE1, and DSE2 (relativeto CLN2) increased 1.21-, 2.18-, and 1.12-fold in responseto PMA in galactose-treated YEp51-transformed cells. CLN2mRNA levels were found to be unchanged in response toPMA (Fig. 5). Q-PCR experiments also confirmed an increasein SVS1 expression in pYECNa-transformed cells upon addi-tion of PMA (2.03-fold). PMA actually slightly reduced ex-pression of SVS1 (by 1.17-fold) in control cells transformedwith YEp51. By comparing the magnitude and direction ofthe PMA-induced changes in gene expression, it was clearthat a high degree of concordance could be found betweenthe cDNA microarray and Q-PCR data. Both approaches indi-cated that CTS1, DSE1, and DSE2 gene expression were

reduced by PMA specifically in pYECNa transformants, whileSVS1 expression was increased.

4. Discussion

In this study, we provide evidence that expression and acti-vation of bovine PKCa in yeast cells results in strongly re-duced growth that correlates with the accumulation of cellsin G2/M with many cells exhibiting aberrant chromosome di-vision, impaired septum formation, or impaired cytokinesis.The PKC-dependent phenotype, as shown in Figs. 3 and 4, ap-pears to correlate with a defect in exit from mitosis. Interest-ingly, some cells upon calcofluor staining (Fig. 4) displayedshared cytoplasms between cells with the absence of a chitinring. Previous studies have shown that deletion of particularcomponents of the Mitotic Exit Network (MEN) also resultin the formation of filamentous like cell structures and disrup-ted cytokinesis (Park et al., 2003; Song and Lee, 2001). TheMEN is made up of various important proteins such asCdc15 and Cdc14 that work to activate the anaphase promot-ing complex (APC), which adds ubiquitin groups to specificproteins and targets them for degradation by the 26S proteo-some (King et al., 1995). In addition, it is responsible forthe formation of the septum ring between mother and daughtercells (Park et al., 2003), which is clearly not visible in somePKCa-expressing cells (Fig. 4). However, errors in the MENtypically yield chains of cells, with each cell possessing itsown genetic material. This was not observed in the PKCa-ex-pressing cells described in this study, which often showed de-fective chromosome segregation. It is well known thatactivated APC interaction with Cdc20 is capable of targetingPds1 (securin) for destruction. Since Pds1 interacts with andinhibits separin (Esp1), a protease capable of degradingScc1, this results in the freeing of sister chromosomes andchromosome segregation (Morgan, 1999). Therefore theAPC is of great importance for chromosome segregation dur-ing the transition from metaphase to anaphase. Esp1 releasetriggers the activation of the Cdc14 early anaphase promotingcomplex pathway (FEAR), causing Cdc14 to leave the nucle-olus, which may then aid in the activation of the MEN in orderto activate Sic1 and the APCcdh1 (Visintin et al., 1998). OnceSic1 and the APCcdh1 become activated, mitotic cyclins suchas those involved in the Clb2-Cdc28 complex are targetedfor degradation in order to exit from mitosis (Bardin andAmon, 2001). Pathways involved in the MEN and FEAR net-work are depicted in Fig. 6. It is upon disruption of particularmitotic exit network proteins (such as Cdc14 and Cdc15) thatchained cells with disrupted cytokinesis are formed (Parket al., 2003). Therefore, PKCa phosphorylation likely acti-vates a regulator of both the MEN and FEAR network, butalso plays a role in blocking chromosome segregation. Thisdisruption in chromosome segregation may involve, for exam-ple, activation of a component of the mitotic spindlecheckpoint.

In an attempt to identify a potential target for bovine PKCa,an optimized method of cDNA microarray analysis was used.The expression of 4 genes was found to be altered upon

expression and activation of bovine PKCa in yeast cells. Onegene (CTS1) is downregulated by PKC and codes for an endo-chitinase required for cell wall breakdown and cell separationafter mitosis (Kuranda and Robbins, 1991). Disruption ofCTS1 expression in yeast cells has been found to result in de-fective cell separation (O’Conallain et al., 1999). AnotherPKC- and phorbol ester-dependent gene identified in our studywas DSE1 (Dietrich et al., 1997). This gene codes for ‘‘daugh-ter cell-specific protein 1’’ (DSE1), which upon knockoutresults in disrupted cytokinesis after chromosome division(Colman-Lerner et al., 2001). In addition, its sister geneDSE2 was also downregulated upon expression of bovinePKCa activity. Its gene product is also a daughter cell-specificprotein, and is similar to the glucanases that degrade the cellwall from the daughter side to permit separation from themother cell (Doolin et al., 2001; Colman-Lerner et al., 2001).Interestingly, all three of the downregulated genes describedabove are controlled by a single transcription factor (Ace2).This transcription factor acts in late M phase and localizes tothe daughter cell nucleus during mitotic exit (Colman-Lerneret al., 2001). However, a PKC-dependent inhibition of Ace2function could not be solely responsible for the PKC-dependentgrowth inhibition, since previous studies have shown that Ace2null mutants form chains of cells but are not inhibited forgrowth (Laabs et al., 2003). Also, analysis of the Ace2 se-quence by the NetPhosK 1.0 Server did not identify potentialPKCa phosphorylation sites. Therefore while all three genes(CTS1, DSE1, and DSE2), if downregulated, could possiblyresult in defective or inhibited cytokinesis, they cannot fullyaccount for the phenotypes induced by PKCa expression andactivation in yeast. Ace2 also regulates two additional genes,ENG1 (Baladron et al., 2002) and SCW11 (Doolin et al.,2001) which aid in the breakdown of the cell wall, though thesetwo genes showed only minor reductions in expression in ourmicroarray studies. In this study, SVS1 expression was upregu-lated by expression and activation of bovine PKCa. SVS1 codesfor a Ser/Thr-rich protein whose expression is highly regulatedduring cell cycle progression and whose activity plays a rolein vanadate resistance and is highly cell cycle-regulated(Nakamura et al., 1995; de Lichtenberg et al., 2003). Thoughlittle is known about this protein, a recent study showed thatdeletion of Swm1, which codes for a subunit of APC, resultedin a 2.7-fold increase in SVS1 gene expression and a failure ofcells to separate (Ufano et al., 2004). However, similar to Ace2null mutants, the Swm1 null mutants possessed phenotypes notobserved in our PKCa-expressing cells, including septumformation and changes in the expression of a wide variety ofadditional genes besides DSE1, DSE2, CTS1, and SVS1 (Ufanoet al., 2004). Therefore, it is possible that without the activationof the APC or Sic1, cdc28-clb2 activity could lead to Ace2phophorylation and changes in the expression of CTS1,DSE1, DSE2, and SVS1.

The data obtained by cDNA microarray analysis likely re-flect bovine PKCa-induced changes in gene expression thatoccur as a result of inhibition of the MEN or FEAR networks.It is possible that a regulator of these networks may be directlyor indirectly altered by bovine PKCa activity, such as the

Fig. 6. Pathways involved in the regulation of SVS1, CTS1, DSE1, and DSE2 expression in yeast cells. The cell wall degradation genes CTS1, DSE1, and DSE2 are

all regulated by the transcription factor Ace2, which is only able to enter the nucleus in its dephosphorylated form. Ace2 phosphorylation is controlled by the

activity of the Clb2/Cdc28 complex, whose activity is, in turn, regulated through input from Swe1, Sic1 and the anaphase promoting complex (APC). Swm1

is a subunit of the APC complex. Swm1 knockouts have been shown to increase SVS1 protein levels, suggesting that SVS1 levels are negatively regulated by

ubiquitination and subsequent proteolysis by the APC complex. Bovine PKCa may inhibit yeast cell growth by phosphorylating a regulator of the mitotic exit

network (MEN) and the Cdc14 early anaphase release (FEAR) network, such as Cdc55 which would result in a chromosome segregation defect as well as inac-

tivation of cdc14, which in turn would result in higher activity of the Clb2/Cdc28 complex.

cdc55 subunit of the PP2A phosphatase complex (Wang andBurke, 1997). The PP2A phosphatase inhibits the activity ofAPCcdc20, Cdc14, and Swe1 [a negative regulator of Clb2-Cdc28 activity (Yellman and Burke, 2006)]. Inhibition ofa negative regulator of Clb-Cdc28 activity would then resultin enhanced phosphorylation of Ace2 and blocked access ofthis transcription factor to the nucleus. The PP2A complexwould also be expected to inhibit chromosome segregationby deactivating the APCcdc20 complex and preventing its de-struction of Pds1 (Shirayama et al., 1999). Pds1 would thenbe free to inhibit the separase Esp1, blocking the separationof sister chromosomes (Ciosk et al., 1998). Hyperativationof Cdc55 by PKCa could possibly explain each phenotype ob-served during this study. Interestingly, analysis of the Cdc55primary sequence by NetPhosK 1.0 Server at http://www.cbs.dtu.dk/services/NetPhosK/identified with a high threshold(0.85) two likely PKC-specific phosphorylation sites withinthe protein. Therefore it is possible that bovine PKCa couldphosphorylate and activate Cdc55 activity.

Given that expression of mammalian PKC in yeast cells re-sults in changes in the expression of genes involved in cell wallturnover (which do not exist in mammalian cells), one may feelthat little insight can be gained through study of mammalianPKC function in the yeast Saccharomyces cerevisiae. However,

several observations suggest that there is strong conservation ofPKC signalling pathways involved in cellular growth controlacross lower and higher eucaryotes. As stated previously,PKC functions upstream of the MAP kinase pathway in bothyeast and mammalian cells and deletion of the MAP kinasegene Slt2 in yeast can be rescued by transformation with a plas-mid coding for the mammalian homolog of Slt2 (Errede andLevin, 1993). Moreover, PDK1 homologs are able to activatethe MAP kinase pathway in yeast (Inagaki et al., 1999). Per-haps the strongest evidence of a compatibility between yeastand mammalian PKC-dependent signalling pathways is the ob-servation that a yeast expression plasmid coding for mamma-lian PKCh can be used to rescue yeast Pkc1 deletion mutants(Nomoto et al., 1997). Interestingly, PKCh actually inhibitedyeast cell growth when expressed in yeast cells containingfunctional Pkc1p (Saraiva et al., 2002, 2003) (similar to our ob-servations with bovine and human PKCa). Thus, gene dosageor genetic background may also affect the ability of mamma-lian PKCs to modulate yeast cell growth.

Evidence in the literature also supports a role for PKC inregulating cell cycle progression after DNA replication. Forexample, overexpression of PKCd and PKCb-II isoformswithin mammalian cells can promote or inhibit cell growth, re-spectively, through changes that occur in the G2/M transition

of the cell cycle. PKCb-II appears to promote the G2/M tran-sition through the phosphorylation of mitotic lamin B and thebreakdown of the nuclear envelope (Goss et al., 1994; Thomp-son and Fields, 1996). Cells in which PKCd is overexpressedexhibit disrupted cytokinesis and multinucleation (Watanabeet al., 1992; Griffiths et al., 1996). Thus, these observationsand the findings of this study suggest that PKC isoformsmay regulate cell growth by affecting processes after DNAreplication that may possibly be conserved between yeastand mammalian cells.

In summary, this study provides evidence for PKC’s abilityto induce an accumulation of cells in G2/M, likely by blockingchromosome segregation, cytokinesis, and septum formation.While the precise mechanism by which bovine PKCa affectsthese processes remains to be elucidated, evidence presentedin this study strongly suggests that the mammalian enzymemay activate a regulator of the MEN and the FEAR network,such as the Cdc55 subunit of protein phosphatase 2A. This, inturn, results in reduced expression of Ace2-dependent genes(CTS1, DSE1, DSE2) and increased expression of SVS1.

Acknowledgements

This work was supported by a grant from the Canadian In-stitutes of Health Research (MOP-15037) to A.M.P. and bysupport funds from Ontario Research and Development Chal-lenge Fund and the Northern Cancer Research Foundation.

References

Baladron V, Ufano S, Duenas E, Martin-Cuadrado AB, del RF, Vazquez de

Aldana CR. Eng1p, an endo-1,3-beta-glucanase localized at the daughter

side of the septum, is involved in cell separation in Saccharomyces cere-

visiae. Eukaryot Cell 2002;1:774e86.

Bardin AJ, Amon A. Men and sin: what’s the difference? Nat Rev Mol Cell

Biol 2001;2:815e26.

Cabib E, Schmidt M. Chitin synthase III activity, but not the chitin ring, is re-

quired for remedial septa formation in budding yeast. FEMS Microbiol

Lett 2003;224:299e305.

Cai H, Smola U, Wixler V, Eisenmann-Tappe I, az-Meco MT, Moscat J,

et al. Role of diacylglycerol-regulated protein kinase C isotypes in

growth factor activation of the Raf-1 protein kinase. Mol Cell Biol

1997;17:732e41.

Carroll MP, May WS. Protein kinase C-mediated serine phosphorylation di-

rectly activates Raf-1 in murine hematopoietic cells. J Biol Chem 1994;

269:1249e56.

Ciosk R, Zachariae W, Michaelis C, Shevchenko A, Mann M, Nasmyth K.

An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at

the metaphase to anaphase transition in yeast. Cell 1998;93:1067e76.

Colman-Lerner A, Chin TE, Brent R. Yeast Cbk1 and Mob2 activate daughter-

specific genetic programs to induce asymmetric cell fates. Cell 2001;107:

739e50.

Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield MD, et al.

Multiple, distinct forms of bovine and human protein kinase C suggest

diversity in cellular signaling pathways. Science 1986;233:859e66.

de Lichtenberg U, Jensen TS, Jensen LJ, Brunak S. Protein feature based iden-

tification of cell cycle regulated proteins in yeast. J Mol Biol 2003;329:

663e74.

Dietrich FS, Mulligan J, Hennessy K, Yelton MA, Allen E, Araujo R, et al.

The nucleotide sequence of Saccharomyces cerevisiae chromosome V.

Nature 1997;387:78e81.

Doolin MT, Johnson AL, Johnston LH, Butler G. Overlapping and distinct

roles of the duplicated yeast transcription factors Ace2p and Swi5p. Mol

Microbiol 2001;40:422e32.

Errede B, Levin DE. A conserved kinase cascade for MAP kinase activation in

yeast. Curr Opin Cell Biol 1993;5:254e60.

Goode NT, Hajibagheri MA, Warren G, Parker PJ. Expression of mammalian

protein kinase C in Schizosaccharomyces pombe: isotype-specific induc-

tion of growth arrest, vesicle formation, and endocytosis. Mol Biol Cell

1994;5:907e20.

Goss VL, Hocevar BA, Thompson LJ, Stratton CA, Burns DJ, Fields AP. Iden-

tification of nuclear beta II protein kinase C as a mitotic lamin kinase.

J Biol Chem 1994;269:19074e80.

Griffiths G, Garrone B, Deacon E, Owen P, Pongracz J, Mead G, et al. The

polyether bistratene A activates protein kinase C-delta and induces growth

arrest in HL60 cells. Biochem Biophys Res Commun 1996;222:802e8.

Hembruff SL, Villeneuve DJ, Parissenti AM. The optimization of quantitative

reverse transcription PCR for verification of cDNA microarray data. Anal

Biochem 2005;345:237e49.

Herskowitz I. MAP kinase pathways in yeast: for mating and more. Cell

1995;80:187e97.

Housey GM, O’Brian CA, Johnson MD, Kirschmeier P, Weinstein IB. Isolation

of cDNA clones encoding protein kinase C: evidence for a protein kinase C-

related gene family. Proc Natl Acad Sci U S A 1987;84:1065e9.

Huang CL, Ives HE. Growth inhibition by protein kinase C late in mitogenesis.

Nature 1987;329:849e50.

Huang KN, Symington LS. Mutation of the gene encoding protein kinase C 1

stimulates mitotic recombination in Saccharomyces cerevisiae. Mol Cell

Biol 1994;14:6039e45.

Inagaki M, Schmelzle T, Yamaguchi K, Irie K, Hall MN, Matsumoto K. PDK1

homologs activate the Pkc1-mitogen-activated protein kinase pathway in

yeast. Mol Cell Biol 1999;19:8344e52.

Jackson DN, Foster DA. The enigmatic protein kinase Cdelta: complex roles

in cell proliferation and survival. FASEB J 2004;18:627e36.

Kamada Y, Qadota H, Python CP, Anraku Y, Ohya Y, Levin DE. Activation of

yeast protein kinase C by Rho1 GTPase. J Biol Chem 1996;271:9193e6.

Keenan C, Goode N, Pears C. Isoform specificity of activators and inhibitors

of protein kinase C gamma and delta. FEBS Lett 1997;415:101e8.

King RW, Peters JM, Tugendreich S, Rolfe M, Hieter P, Kirschner MW. A 20S

complex containing CDC27 and CDC16 catalyzes the mitosis-specific

conjugation of ubiquitin to cyclin B. Cell 1995;81(2):279e88.

Knopf JL, Lee MH, Sultzman LA, Kriz RW, Loomis CR, Hewick RM, et al.

Cloning and expression of multiple protein kinase C cDNAs. Cell 1986;46:

491e502.

Kolch W, Heidecker G, Kochs G, Hummel R, Vahidi H, Mischak H, et al. Pro-

tein kinase C alpha activates RAF-1 by direct phosphorylation. Nature

1993;364:249e52.

Kuranda MJ, Robbins PW. Chitinase is required for cell separation during

growth of Saccharomyces cerevisiae. J Biol Chem 1991;266:19758e67.

Laabs TL, Markwardt DD, Slattery MG, Newcomb LL, Stillman DJ,

Heideman W. ACE2 is required for daughter cell-specific G1 delay in

Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 2003;100:

10275e80.

Levin DE, Bartlett-Heubusch E. Mutants in the S. cerevisiae PKC1 gene dis-

play a cell cycle-specific osmotic stability defect. J Cell Biol 1992;116:

1221e9.

Levin DE, Fields FO, Kunisawa R, Bishop JM, Thorner J. A candidate protein

kinase C gene, PKC1, is required for the S. cerevisiae cell cycle 19. Cell

1990;62:213e24.

Livneh E, Fishman DD. Linking protein kinase C to cell-cycle control. Eur J

Biochem 1997;248:1e9.

Mellor H, Parker PJ. The extended protein kinase C superfamily. Biochem J

1998;332(Pt 2):281e92.

Morgan DO. Regulation of the APC and the exit from mitosis. Nat Cell Biol

1999;1:E47e53.

Nakamura T, Namba H, Ohmoto T, Liu Y, Hirata D, Miyakawa T. Cloning and

characterization of the Saccharomyces cerevisiae SVS1 gene which en-

codes a serine- and threonine-rich protein required for vanadate resistance.

Gene 1995;165:25e9.

Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activa-

tion of protein kinase C. Science 1992;258:607e14.

Nomoto S, Watanabe Y, Ninomiya-Tsuji J, Yang LX, Nagai Y, Kiuchi K, et al.

Functional analyses of mammalian protein kinase C isozymes in budding

yeast and mammalian fibroblasts. Genes Cells 1997;2:601e14.

Nonaka H, Tanaka K, Hirano H, Fujiwara T, Kohno H, Umikawa M, et al. A

downstream target of RHO1 small GTP-binding protein is PKC1, a homo-

log of protein kinase C, which leads to activation of the MAP kinase cas-

cade in Saccharomyces cerevisiae. EMBO J 1995;14:5931e8.

O’Conallain C, Doolin MT, Taggart C, Thornton F, Butler G. Regulated nu-

clear localisation of the yeast transcription factor Ace2p controls expres-

sion of chitinase (CTS1) in Saccharomyces cerevisiae. Mol Gen Genet

1999;262:275e82.

Ohno S, Mizuno K, Adachi Y, Hata A, Akita Y, Akimoto K, et al. Activation of

novel protein kinases C delta and C epsilon upon mitogenic stimulation of

quiescent rat 3Y1 fibroblasts. J Biol Chem 1994;269:17495e501.

Parissenti AM, Kim SA, Colantonio CM, Snihura AL, Schimmer BP. Regula-

tory domain of human protein kinase C alpha dominantly inhibits protein

kinase C beta-I-regulated growth and morphology in Saccharomyces cere-visiae. J Cell Physiol 1996a;166:609e17.

Parissenti AM, Kirwan AF, Kim SA, Colantonio CM, Schimmer BP. Molecu-

lar strategies for the dominant inhibition of protein kinase C. Endocr Res

1996b;22:621e30.

Parissenti AM, Villeneuve D, Kirwan-Rhude A, Busch D. Carbon source-

dependent regulation of cell growth by murine protein kinase C epsilon

expression in Saccharomyces cerevisiae. J Cell Physiol 1999;178:216e26.

Park CJ, Song S, Lee PR, Shou W, Deshaies RJ, Lee KS. Loss of CDC5 func-

tion in Saccharomyces cerevisiae leads to defects in Swe1p regulation and

Bfa1p/Bub2p-independent cytokinesis. Genetics 2003;163:21e33.

Parker PJ, Kour G, Marais RM, Mitchell F, Pears C, Schaap D, et al. Protein

kinase Cda family affair. Mol Cell Endocrinol 1989;65:1e11.

Riedel H, Hansen H, Parissenti AM, Su L, Shieh HL, Zhu J. Phorbol ester ac-

tivation of functional rat protein kinase C beta-1 causes phenotype in yeast.

J Cell Biochem 1993a;52:320e9.

Riedel H, Parissenti AM, Hansen H, Su L, Shieh HL. Stimulation of calcium

uptake in Saccharomyces cerevisiae by bovine protein kinase C alpha.

J Biol Chem 1993b;268:3456e62.

Riedel H, Su L, Hansen H. Yeast phenotype classifies mammalian protein

kinase C cDNA mutants. Mol Cell Biol 1993c;13:4728e35.

Saraiva L, Fresco P, Pinto E, Sousa E, Pinto M, Goncalves J. Synthesis and in

vivo modulatory activity of protein kinase C of xanthone derivatives. Bio-

org Med Chem 2002;10:3219e27.

Saraiva L, Fresco P, Pinto E, Goncalves J. Isoform-selectivity of PKC inhibitors

acting at the regulatory and catalytic domain of mammalian PKC-alpha,

-betaI, -delta, -eta and -zeta. J Enzyme Inhib Med Chem 2003;18:475e83.

Sheu YJ, Santos B, Fortin N, Costigan C, Snyder M. Spa2p interacts with cell

polarity proteins and signaling components involved in yeast cell morpho-

genesis. Mol Cell Biol 1998;18:4053e69.

Shieh HL, Hansen H, Zhu J, Riedel H. Activation of conventional mammalian

protein kinase C isoforms expressed in budding yeast modulates the cell

doubling timeda potential in vivo screen for protein kinase C activators.

Cancer Detect Prev 1996;20:576e89.

Shirayama M, Toth A, Galova M, Nasmyth K. APC(Cdc20) promotes exit

from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5.

Nature 1999;402:203e7.

Simon AJ, Milner Y, Saville SP, Dvir A, Mochly-Rosen D, Orr E. The ide-

ntification and purification of a mammalian-like protein kinase C in the

yeast Saccharomyces cerevisiae. Proc R Soc Lond B Biol Sci 1991;243:

165e71.

Slater SJ, Seiz JL, Stagliano BA, Stubbs CD. Interaction of protein kinase C

isozymes with Rho GTPases. Biochemistry 2001;40:4437e45.

Song S, Lee KS. A novel function of Saccharomyces cerevisiae CDC5 in

cytokinesis. J Cell Biol 2001;152:451e69.

Sozeri O, Vollmer K, Liyanage M, Frith D, Kour G, Mark III GE, et al. Acti-

vation of the c-Raf protein kinase by protein kinase C phosphorylation.

Oncogene 1992;7:2259e62.

Terashima H, Fukuchi S, Nakai K, Arisawa M, Hamada K, Yabuki N,

Kitada K. Sequence-based approach for identification of cell wall proteins

in Saccharomyces cerevisiae. Curr Genet 2002;40(5):311e6.

Thompson LJ, Fields AP. betaII protein kinase C is required for the G2/M

phase transition of cell cycle. J Biol Chem 1996;271:15045e53.

Ufano S, Pablo ME, Calzada A, del RF, Vazquez de Aldana CR. Swm1p sub-

unit of the APC/cyclosome is required for activation of the daughter-spe-

cific gene expression program mediated by Ace2p during growth at high

temperature in Saccharomyces cerevisiae. J Cell Sci 2004;117:545e57.

Villeneuve DJ, Parissenti AM. The use of DNA microarrays to investigate the

pharmacogenomics of drug response in living systems. Curr Top Med

Chem 2004;4:1329e45.

Villeneuve DJ, Hembruff SL, Veitch Z, Cecchetto M, Dew WA,

Parissenti AM. cDNA microarray analysis of isogenic paclitaxel- and

doxorubicin-resistant breast tumor cell lines reveals distinct drug-specific

genetic signatures of resistance. Breast Cancer Res Treat 2006;96:17e39.

Visintin R, Craig K, Hwang ES, Prinz S, Tyers M, Amon A. The phosphatase

Cdc14 triggers mitotic exit by reversal of Cdk-dependent phosphorylation.

Mol Cell 1998;2:709e18.

Wang Y, Burke DJ. Cdc55p, the B-type regulatory subunit of protein phospha-

tase 2A, has multiple functions in mitosis and is required for the kineto-

chore/spindle checkpoint in Saccharomyces cerevisiae. Mol Cell Biol

1997;17:620e6.

Watanabe T, Ono Y, Taniyama Y, Hazama K, Igarashi K, Ogita K, et al. Cell

division arrest induced by phorbol ester in CHO cells overexpressing

protein kinase C-delta subspecies. Proc Natl Acad Sci U S A 1992;89:

10159e63.

Yellman CM, Burke DJ. The role of Cdc55 in the spindle checkpoint is

through regulation of mitotic exit in Saccharomyces cerevisiae. Mol Biol

Cell 2006;17:658e66.

Zhou W, Takuwa N, Kumada M, Takuwa Y. Protein kinase C-mediated bidi-

rectional regulation of DNA synthesis, RB protein phosphorylation, and

cyclin-dependent kinases in human vascular endothelial cells. J Biol

Chem 1993;268:23041e8.

Changes in expression of cell wall turnover genes accompany inhibition of chromosome segregation by...

Documents