Proc. Natl. Acad. Sci. USAVol. 84, pp. 4012-4016, June 1987Biochemistry

A protein kinase antigenically related to pp6Ov-src possibly involvedin yeast cell cycle control: Positive in vivo regulation by sterol

(cholesterol/ergosterol/oncogenes/celI proliferatlon/phosphatidylinositol)

CHARLES DAHL*t, HANS-PETER BIEMANNt, AND JEAN DAHL*tDepartments of tChemistry and tCell and Developmental Biology, Harvard University, Cambridge, MA 02138

Communicated by Konrad Bloch, February 19, 1987

ABSTRACT The effects of ergosterol, yeast's natural ster-ol, on cell cycling and a protein kinase antigenically related topp6O' were examined in a sterol auxotroph ofSaccharomycescerevisiae. Sterol-depleted cells accumulate in an unbudded, G1state. Cell budding and proliferation are reinitiated uponaddition of nonlimiting ergosterol or cholesterol with traceergosterol, whereas cholesterol or trace ergosterol alone is lesseffective. Stimulation of a protein kinase associated withimmune complexes of yeast protein and anti-pp60v1 shows apositive correlation with exit from the G1 phase followingergosterol addition. Ergosterol-stimulated cells also demon-strate an increase in phosphatidylinositol kinase activity. Thedata suggest that hormonal levels of ergosterol (effectiveconcentration, 1 nM) participate in a signaling processassociated with a protein kinase possibly involved in yeast cellcycle control.

In the budding yeast Saccharomyces cerevisiae nutritionalsignals normally control cell growth and division. When yeastcells stop growing in response to nutritional deprivation theyarrest in the unbudded, G1 phase of the cell cycle (1). Theisolation of many temperature-sensitive cell division cycle(cdc) mutants has led to a better understanding of theregulation of yeast cell proliferation and has resulted in thedescription of a G1-phase "start" event that is controlled bya number of genes (2). One of these genes, CDC28, has beencloned and sequenced (3). The CDC28 gene product shareshomology with a number of mammalian protein kinases,among these a vertebrate cAMP-dependent protein kinase, aswell as viral oncogenes such as v-mos and v-src (3). TheCDC28-encoded protein has now been shown to possess aprotein kinase activity, as has a related cdc2+-encodedprotein in the fission yeast (4, 5). Moreover, a decrease in theactivity ofthe cdc2-encoded protein correlates with cell cyclearrest upon nitrogen starvation in Schizosaccharomycespombe (5). cAMP-dependent protein kinase and the RASgene product have also been implicated in growth control inyeast (6). Interestingly, an association has been demonstrat-ed between the activities of the pp60vsrc tyrosine kinase andphosphatidylinositol (PtdIns) kinase in Rous sarcoma virus-transformed chicken embryo fibroblasts (7). Although therole of the polyphosphatidylinositol phosphates in yeastphysiology has yet to be determined (8-10), we have ob-served a marked ergosterol-mediated stimulation of theturnover of these lipids that correlates with an enhanced rateof cell proliferation (11).

Studies on the role of sterols in living cells have led us topostulate that sterols serve a multifunctional role in cells:one, fulfilled by the major amount of the sterol with amodicum of structural specificity, is that of a bulk membranecomponent that ensures the structural integrity of the mem-

brane; and a second, distinguished by its sterol structuralspecificity and by the very small amount of sterol required,is of a regulatory nature (11-13). This notion stems from theobservation that in sterol auxotrophs two sterols that areinsufficient to support good growth alone can, when suppliedtogether, sustain near maximal growth rates. In yeast ergos-terol, being the natural yeast sterol, appears to be the optimalsterol for the more structurally specific regulatory role as wellas the bulk role. On the other hand, cholesterol or cholesta-nol, which differ structurally from ergosterol and are notsynthesized by yeast, can provide the bulk sterol for mem-brane biogenesis but are less effective, in the case ofcholesterol (14), or ineffective, in the case ofcholestanol (15),in the regulatory role.Here our objective is to analyze the regulatory role of

ergosterol in yeast cell proliferation by identifying keyregulatory enzymes whose activities are modulated by sterol.Our approach is to bring about controlled alterations in thesterol content of a yeast sterol auxotroph that result inGl-phase cell cycle arrest (sterol starvation) or G1 release andcell proliferation (sterol addition). This analysis has led to theidentification of a protein kinase having antigenic homologywith the transforming protein of the Rous sarcoma virus andwhose activity is positively regulated by a trace amount ofergosterol. To our knowledge, evidence for the existence ofa protein kinase possibly involved in yeast cell cycle control,whose activity is exquisitely sensitive in vivo to the presenceof ergosterol, has not been reported previously.

EXPERIMENTAL PROCEDURESYeast Strains and Growth Media. S. cerevisiae strain GL7

(MATa gal2/ergl2-1/hem3-6) was graciously supplied byDavid Sprinson (Columbia University, New York) (16). Awild-type strain (MATa his4-619) and a cdc28-1 strain (MATacdc28-1/tyrl) were provided by C. Holm (Harvard Univer-sity, Cambridge, MA). L. Hereford (Dana-Farber CancerInstitute, Boston, MA) supplied the cdc28-4 strain (MATacdc284/ade2/his4/lys2/trpl). GL7 was grown at 30°C or24°C on a rotary shaker in minimal medium consisting of0.67% Difco yeast nitrogen base and 2% glucose supplement-ed with 20 jig ofmethionine per ml, 50 ,ug of oleic acid per ml,and 0.025-1 ,ug of sterol per ml (11). Sterols and fatty acidswere added in Brij 58/ethanol, 1:4 (wt/wt). Other strainswere cultured at 24°C on minimal medium consisting of0.67%yeast nitrogen base, 2% glucose, 20 ,ug of the required aminoacids per ml, and 50 ,ug of adenine per ml. Cell number andbudding were determined in a hemocytometer after treatingthe cells with 0.1% methylene blue followed by brief soni-cation. Viable cell counts were also performed by platingcells on complex medium containing 1% yeast extract, 2%peptone, 2% glucose, 1.5% agar, 50 ,ug of oleic acid per ml,and 1 ,ug of ergosterol per ml.

Abbreviation: PtdIns, phosphatidylinositol.*To whom correspondence should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Membrane Preparation. All procedures were performed at40C. Cells were harvested, washed twice with 20 mMTris HCl, pH 7.5/0.25 M sucrose, and stored at -80'C. Thethawed cell pellets were suspended in buffer A [20 mMTris-HCl, pH 7.5/0.25 M sucrose/2 mM EDTA/2 mMEGTA/0.5 mM phenylmethylsulfonyl fluoride/50 mMNaF/5 mM dithiothreitol/100 AtM leupeptin (Sigma)/0.1 mgof aprotinin per ml (Sigma)]. Cells were disrupted in a Frenchpress at 20,000 psi (1 psi = 6.89 kPa), and lysates were clearedof cell debris by centrifugation at 5000 x g for 10 min. Themembrane fraction was pelleted at 100,000 x g for 60 min.Membranes were suspended in buffer B (50mM Tris HCl, pH7.5/20% glycerol/1 mM dithiothreitol/0.1 mM EDTA/0.5mM phenylmethylsulfonyl fluoride/50 mM NaF) to a proteinconcentration of 5 mg/ml and stored at -80°C. Proteinconcentrations were determined by the procedure of Lowryet al. (17) using bovine serum albumin as a standard.

Protein Kinase Assay. Membranes were extracted in bufferB containing 1% sodium deoxycholate for 30 min at 4°C andcleared by centrifugation in a Microfuge for 7 min. Deoxy-cholate membrane extracts (0.1 mg ofprotein) adjusted to 0.5ml with Hermann's buffer (1% Nonidet P40/0.5% sodiumdeoxycholate/100 mM NaCl/10 mM Tris.HCl/1 mM EDTA,pH 7.2) containing 0.5 mM phenylmethylsulfonyl fluorideand 0.1 mg of aprotinin per ml were immunoprecipitated withantiserum from rabbits immunized with a bacterially pro-duced p60 (18) and adsorbed onto Staphylococcus aureus.After washing four times with Hermann's buffer and oncewith 20 mM Tris HCl, pH 7.5/0.5 M NaCl, the S. aureus-immune complexes were suspended in 20 mM Tris HCl, pH7.5/10 mM MgCl2/0.67 mg of casein per ml/10 ,M [-32P]ATP (108 cpm/nmol) in a total volume of 30 Al. Reactionsproceeded for 20 min at 30°C. After adding NaDodSO4/poly-acrylamide solubilizing buffer, phosphoproteins were ana-lyzed by polyacrylamide gel electrophoresis (10% polyacryl-amide) (19). Labeled casein bands were located by autoradi-ography and excised, and radioactivity was quantitated byscintillation counting.

PtdIns Kinase Assay. Reaction mixtures for PtdIns kinasecontained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.1 mMsodium orthovanadate, 0.5 mM PtdIns, 0.1% Triton X-100, 5mM [y-32PJATP (105 cpm/nmol), and 25 1Lg of membraneprotein in a final volume of 0.1 ml. Incubations wereperformed at 30°C for 10 min. Reactions were stopped with1.9 ml of chloroform/methanol, 2:1 (vol/vol), and 0.4 ml of1 M HCl. After dispersal in a Vortex, the upper layer wasdiscarded and the lower layer was reextracted with 1 ml ofchloroform/methanol/l M HCl, 3:48:47 (vol/vol). Thepolyphosphatidylinositol phosphates were separated on thin-layer plates and quantitated as described (11).

Materials. Ergosterol (Sigma) was recrystallized by themethod of Bills and Honeywell (20). Cholesterol was urifiedby recrystallization from ethanol and acetone. [y- P]ATPwas supplied by New England Nuclear. Anti-pp60v`s im-mune serum and S. aureus were the generous gifts of R. L.Erikson (Harvard University).

RESULTSEffect of Sterol on Gl-Phase Arrest and Release. When S.

cerevisiae strain GL7 (16), a sterol and unsaturated fatty acidauxotroph, was shifted in midlogarithmic phase from minimalmedium containing ergosterol (1 ,ug/ml) to sterol-free medi-um, cells underwent approximately one round ofdivision andthen cell division ceased. The percentage of budded cellsdropped from 60% to 13% (Fig. 1). As a control, a parallelculture was starved for methionine. Upon methionine dep-rivation cell proliferation ceased after one round of divisionand cells accumulated in a G1 or unbudded state. The viabilityof cells starved for ergosterol or methionine for 21 hr was 94%

10

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02 20--

0 2 4 6 8 10 12 14 16 18 20 +2 +4 +6 +8

Incubation Time (h)

FIG. 1. Effect of sterol starvation and readdition on budding andproliferation. A midlogarithmic-phase culture growing in minimalmedium containing ergosterol at 1 Ag/ml was collected on 0.45-,umMillipore filters, washed once with sterol- or methionine-free medi-um, and resuspended to a concentration of 1.5 x 106 cells per ml inergosterol-free medium (A) or methionine-free medium (a). Afterincubation for 21 hr at 30'C the sterol-free medium was supplementedwith ergosterol at 1 ,ug/ml (A&) or 0.1 ,g/ml (v), cholesterol at 1 ,g/ml(o), or cholesterol at 1 tug/ml with ergosterol at 0.1 Ag/ml (0).Methionine-free medium was supplemented with methionine at 20Ag/ml (o). (A) Cell proliferation. (B) Cell budding.

or 98%, respectively. After 120 hr, the viability of ergosterol-starved cells dropped to 55%, whereas methionine-starvedcells remained 84% viable.To examine the effect of sterol on the release of yeast from

the G1 phase, the starvation medium was supplemented withsterol after 21 hr (Fig. 1). Addition of ergosterol at 1 ,ug/mlresulted in a sharp increase in the portion of budded cells to80% within 2 hr and a subsequent rise in the rate of celldivision. Lowering the ergosterol concentration by a factor of10 to 0.1 ,tg/ml markedly reduced the effectiveness of thesterol as did substitution of cholesterol at 1 ,g/ml forergosterol. By contrast, combination of two insufficientsterols-i.e., ergosterol at 0.1 ,g/ml and cholesterol at 1,ug/ml-stimulated budding and cell division almost as wellas nonlimiting ergosterol alone. Both conditions for G1 arrestand release-i.e., ergosterol or methionine deprivation andreaddition-yielded similar results, although recovery fromergosterol starvation was much more rapid (Fig. 1).

Effect of Sterol-Mediated Cell Cycle Arrest and Release onpp6iV-Related Protein Kinase and Ptdlns Kinase. Immunecomplexes formed between antiserum prepared againstbacterially produced p60 and deoxycholate-solubilized yeastmembrane protein were found to incorporate 32p from [-32P]ATP into casein. The protein kinase activity was negli-gible in the normal serum control (Fig. 2). Phosphoamino acidanalysis (21) of an excised casein band showed the kinase tobe specific for serine and threonine (data not shown).Phosphotyrosine was not detected. The membrane fractionof yeast has been shown to possess protein kinase activity(22, 23).To correlate sterol-mediated cell cycle arrest with protein and

PtdIns kinase activities, cells were grown to midlogarithmicphase on cholesterol (1 ,g/ml) and then transferred to sterol-free medium. At the time of transfer the anti-pp60v's-Iim-munoprecipitable protein kinase was found to transfer 200 fmolof 32P to casein in 20 min per 0.1 mg of yeast protein (Fig. 3).

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"NI

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OL-

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FIG. 2. Protein kinase activity of immune complexes of deoxy-cholate-solubilized yeast membrane protein. Immune complexeswere prepared by using normal rabbit serum (NS) or increasingconcentrations of anti-pp60vsrc immune serum. The autoradiographshown above the histogram corresponds to the respective incorpo-rations of 32P into casein.

After 25 hr in sterol-free medium, cells had arrested in theunbudded, G1 phase and the protein kinase activity dropped bya factor of 8. In the same cells the PtdIns kinase activityincreased 25% from the value found in logarithmic-phase cells.When ergosterol (0.5 gg/ml) was added back to sterol-starvedcells the pp60vs"-related protein kinase increased >10-foldwithin 15 min of sterol addition. The PtdIns kinase activity wasenhanced by only 50%o. By contrast, cholesterol (0.5 tkg/ml), asterol shown to be less effective than ergosterol in stimulatingcell budding and growth (see Fig. 1), had little or no effect on therespective kinase activities within the first hour of steroladdition. As with cells arrested in G1 phase by ergosteroldeprivation, the pp60vsrc-related protein kinase activity inmethionine-starved cells was lowered by a factor of 5. How-ever, in contrast to the rapid recovery observed upon ergosteroladdition, the time required for the activity ofthe specific proteinkinase to reach the prestarvation level following the readditionof methionine was long-i.e., 5-6 hr (data not shown). This inturn correlated with the relatively slow recovery rates ofbudding and cell proliferation in methionine-supplemented cells(see Fig. 1). To explore further the relationship between thepp60v-src-related protein kinase and Gj-phase arrest, mem-branes from logarithmic-phase GL7 cells (62% budded) andcells arrested in G1 phase by a,-mating factor (3% budded) werecompared for their immunoprecipitable casein kinase activity.Again the activity associated with cells treated for 3 hr witha,-mating factor was lowered by a factor of 5 (data not shown).

Cycloheximide Does Not Inhibit Ergosterol Stimulation ofpp6Ovsrc Related Protein Kinase. Cycloheximide rapidlyblocks the synthesis of yeast cell proteins (24). To explore thepossibility that ergosterol exerts its stimulatory effect onpreexisting protein, cells starved for ergosterol were treatedwith cycloheximide S min before the addition of eitherergosterol or cholesterol. As shown in Fig. 4, ergosterol wasequally as effective in stimulating protein kinase in cellstreated with cycloheximide as in untreated cells. Cholesterol,on the other hand, had no effect in either case. The ergos-terol-mediated stimulation of PtdIns kinase, which wasalways found to be 1.5-fold or less, was also unchanged bycycloheximide treatment (data not shown).The pp6Ov"-Related Protein Kinase Is Probably Not the

Protein Encoded by CDC28. Since the data suggested that thepp60V-Src-related protein kinase may be required for traversalofG1 phase, the question arose whether the protein kinase wewere measuring was identical to the CDC28-encoded proteinkinase described by Reed et al. (4). To investigate this

*+1 +2 +3Incubation Time (h)

FIG. 3. Effect of sterol starvation and readdition on cell budding,proliferation, PtdIns kinase, and anti-pp60v'-"immunoprecipitableprotein kinase. A midlogarithmic-phase culture growing in minimalmedium containing cholesterol at 1 ,g/ml was harvested on 0.45-,mMillipore filters, washed, resuspended in sterol-free medium (A) to aconcentration of 3.3 x 106 cells per ml, and incubated for 25 hr at30°C. The culture medium was then supplemented with ergosterol at0.5 ,ug/ml (A) or cholesterol at 0.5 ,g/ml (o). After a further 15-, 30-,or 60-min incubation, cells were harvested and stored at -80°C priorto membrane preparation. (A) Cell proliferation. (B) Cell budding. (C)Ptdlns kinase activity (100% is 0.26 nmol of 32p in Ptdlns-4-phosphate per min per mg of protein). (D) Anti-pp60v-sIcim-munoprecipitable protein kinase activity (the Inset shows theautoradiograph of 32P-labeled casein corresponding to each point onthe graph).

possibility, antiserum to p60 was used to probe cdc28ts strainsfor a temperature-sensitive protein kinase. Immunoprecipi-tates from GL7, wild-type, and two cdc28ts mutant strainsgrown at the permissive temperature (24°C) were assayed forcasein kinase activity at 24°C and at the restrictive temper-ature, 37°C (Table 1). The activity at 24°C from all strainsincreased over a 2-fold range in the order cdc28-4 < cdc28-1< wild type < GL7. At 37°C the protein kinase activities fromGL7, wild type, and cdc28-1 were reduced by 50%, whereasthat of cdc28-4 was slightly more stable and fell by only 20%.Thus, the antiserum used for these studies precipitates akinase that may be distinct from the CDC28-encoded proteinsince the protein kinase activity from several temperature-sensitive cdc28 mutant strains is known to be morethermolabile than from wild type (4).

Ergosterol Stimulates Protein Kinase at a Concentration rooLow to Support Budding Growth in the Absence of a BulkSterol. If sterol performs a dual role in yeast, one regulatoryand another structural, one might predict that sterol inamounts too small to support net membrane biogenesis maystill be able to fulfill a regulatory function. To test thispossibility, ergosterol-starved, Gl-arrested cells were ex-

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.' .c 50CGiG)a}

-en -o o-() 40Co DIc E

o ° 300o sCo E0Q 0 200C~Jc -1Q- * 100N-

None Erg Erg ChcCHX

Additions to Medium

ol CholCHX

FIG. 4. Effect of cycloheximide on the ergosterol stimulation ofthe anti-pp6 '-"-immunoprecipitable protein kinase. A midlogarith-mic-phase culture growing in minimal medium containing ergosterolat 1 1ug/ml was harvested on 0.45-gim Millipore filters, washed,resuspended in sterol-free medium to a cell concentration of 3.5 x106, and incubated at 30'C for 25 hr. Cultures (8 x 106 cells per ml)were then treated with cycloheximide (CHX) at 50 ,Ag/ml for 5 minbefore adding ergosterol (Erg) at 0.25 Ag/ml or cholesterol (Chol) at0.25 ,ug/ml. After a further 15-min incubation cells were harvestedand stored at -80'C.

posed to a low concentration of ergosterol alone or incombination with a 10-fold higher concentration of choles-terol (Fig. 5). Cells treated with either no sterol or onlyergosterol or cholesterol at the higher concentration servedas controls. We then determined the effect of sterol onprotein kinase and cell budding, the logic being that theprotein kinase measures the specific regulatory function ofergosterol, whereas budding is indicative of new membranesynthesis (25). Budding was determined when the differencebetween ergosterol- and cholesterol-supplemented cells wasmaximum-i.e., at 2 hr after sterol addition (see Figs. 1 and2). In cells treated with the higher concentration of ergoster-ol, protein kinase and budding were positive, whereas cellstreated with either no sterol or cholesterol alone at the higherconcentration were negative on both counts. The addition of1/10th the amount ofergosterol along with cholesterol, on theother hand, raised the level of both parameters to levelsapproaching those seen with nonlimiting ergosterol. Aspredicted, the small amount of ergosterol alone stronglystimulated the protein kinase but was less effective than thehigher concentration of ergosterol in supporting cell budding.Ergosterol exerted its stimulatory effect on the protein kinaseat a concentration <5 nM (data not shown).

DISCUSSIONTo explore the regulatory role of ergosterol in yeast cellproliferation we have determined the effect of sterol starva-

Table 1. Effect of temperature on pp601vsrc-related protein kinasefrom different yeast strains

pp60VS-rcimmunoprecipitable

casein kinase activityYeast strain 240C 370CGL7 304 172Wild-type 275 119cdc28-1 183 100cdc28-4 152 125

Immune complexes from cells grown at 240C were prepared andincubated for 30 min at the assay temperature (24°C or 37°C) beforestarting the reaction. The activity is expressed as fmol of 32pincorporated into casein per 20 min/0.1 mg of yeast membraneprotein.

LI-.c.s 500(n -

0 0o 4400

'o E

0 200Y o

100-E

Erg Chol(0.25) (0.25)

orno sterol

Cho(0.25)Erg

(0.025)

50

400

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20'O_20 'n

in10m- 10 IR

Erg(0.025)

Sterol Addition to Medium

(,ug/m )

FIG. 5. Correlation of anti-pp60v-src-immunoprecipitable proteinkinase activity and cell budding in sterol-starved cells treated withvarious sterols. A culture starved 25 hr for sterol as described in Fig.4 was supplemented with ergosterol (Erg) at 0.25 or 0.025 ,ug/ml,cholesterol (Chol) at 0.25 ,ug/ml, or cholesterol at 0.25 ug/ml withergosterol at 0.025 ,ug/ml, or no sterol. Protein kinase was assayedin membrane fractions from cells harvested 15 min after the additionof sterol.

tion and readdition on cell budding and proliferation. Yeastcells can sense impending starvation for certain nutrients,and growth is arrested in the G1 or unbudded phase of the cellcycle generally before "start" (2). Our results now show thatstarvation of GL7 for ergosterol also arrests proliferation inthe G1 phase. The readdition of ergosterol alone or togetherwith cholesterol stimulates budding and cell proliferation,whereas cholesterol alone is less effective.Evidence implicating protein kinases with cell cycle con-

trol in yeast is emerging from studies on temperature-sensitive cdc mutants (4, 5), mutants related to the cAMPpathway such as ras, cryl, and bcyl (6, 26, 27), andnutritionally induced cell cycle arrested cells (28). Using anantiserum raised against a bacterially produced p60, thetransforming protein of Rous sarcoma virus, we have showna striking correlation between the activity of an immunopre-cipitable protein kinase from yeast and ergosterol-mediatedcontrol of cell cycle arrest and release. The activity of thisprotein kinase is greatly reduced in ergosterol-starved, G1-arrested cells and rapidly stimulated upon the reintroductionof ergosterol. Cholesterol, on the other hand, is less effectivein stimulating the protein kinase activity as well as buddingand proliferation. Ergosterol-mediated control of the proteinkinase appears to be exerted on enzymes present in the cellat the time of sterol addition since cycloheximide, an inhib-itor of protein synthesis, does not eliminate the activation.

Further evidence supporting the notion that this proteinkinase is associated with traversal of the G1 phase derivesfrom the finding that the activity of the pp60vsrc-relatedprotein kinase is depressed in cells arrested in G1 phase bythree independent methods-i.e., ergosterol deprivation,methionine deprivation, and exposure of cells to a,-matingfactor. That this protein kinase is probably not the proteinkinase encoded by CDC28 is suggested by the observationthat antiserum prepared against p60 precipitates a caseinkinase from cdc28ts mutant strains that is not inactivated atthe nonpermissive temperature.An enzyme possibly associated with oncogene-encoded

tyrosine kinases in mammalian cells (7, 29)--i.e., PtdInskinase-is also stimulated slightly by ergosterol addition.This result supports those presented previously by us show-ing that the addition of ergosterol to S. cerevisiae GL7growing poorly on cholesterol stimulates the rapid turnover

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of polyphosphatidylinositol phosphates as well as increasedcell proliferation (11). The low level of stimulation of mem-brane-associated PtdIns kinase observed here is in agreementwith that seen in crude membrane preparations from Roussarcoma virus-transformed chicken embryo fibroblasts (30)and in whole cell lysates of polyoma-transformed NIH 3T3cells (31). This 2-fold level of activation is in sharp contrastto the >20-fold stimulation found for Ptdlns kinase activityassociated with middle tumor antigen immune complexesfrom transformed cells (31). Although increased turnover ofpolyphosphatidylinositol phosphates and subsequent pro-duction of two second messengers-i.e., diacylglycerol andinositol trisphosphate-is associated with the stimulation ofmammalian cells by growth factors, hormones, and neuro-transmitters (32), the role of polyphosphatidylinositol phos-phate turnover in yeast physiology remains undefined. More-over, the relationship between PtdIns kinase and thepp60V-src-related protein kinase reported here is not known.The 10-fold difference in magnitude between the stimulationsof the protein kinase and PtdIns kinase suggests, however,that the two enzymes are not identical.The rapid stimulation of a pp60v-src-related protein kinase

upon addition of trace ergosterol to sterol-depleted, G1-arrested cells implies that this kinase activity may be part ofa G1-phase signal transduction process that senses the avail-ability of sterol needed for growth and commits the cell to anew round of mitotic division. Furthermore, the notion thatsterol performs a dual role in cells-one regulatory andanother that ensures the structural integrity of membranes-derives support from the fact that ergosterol at a concentra-tion too low to support membrane biogenesis is still able toexert a stimulatory effect on the protein kinase. The resultspresented raise the possibility that yeast possess a high-affinity receptor for sterol that may be part of this signaltransduction process. This argument becomes more tenablewhen one considers that the effective concentration forergosterol in the culture medium-i.e., <5 nM-falls wellwithin the range for the action of steroid hormones. In thisregard the recent discovery in yeast of a steroid receptor andhormone previously found in mammalian cells-i.e., 17(3-estradiol-only serves to heighten the possibility of a func-tional sterol receptor system in this unicellular eukaryote (33,34).Taken together, the results reported here suggest that a

trace amount of the intact sterol molecule serves in yeast cellcycle control as a nutritional signal analogous to the action ofgrowth factor or hormonal signals in multicellular eukary-otes.

Note Added in Proof. We now find that a partially purified, mem-brane-associated protein kinase from GL7 is stimulated 2- to 3-foldin vitro by ergosterol at 1 nM. By contrast, the effective concentra-tion for stimulation by cholesterol is 1 ,uM-i.e., 1000-fold higherthan that for ergosterol.

We thank Konrad Bloch, Raymond Erikson, Bruce Demple,Connie Holm, and Steve Jones for constructive comments during thepreparation of the manuscript. This work was supported by a grantfrom the National Institutes of Health (HL-02477).

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