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Signaling pathways forfission yeast sexualdifferentiation at a glance

Yoko Otsubo1 and MasayukiYamamoto1,2,*1Kazusa DNA Research Institute, Kazusa-kamatari,Kisarazu, Chiba 292-0818, Japan2Department of Biophysics and Biochemistry,Graduate School of Science, University of Tokyo,Hongo, Tokyo 113-0033, Japan

*Author for correspondence

(yamamoto@biochem.s.u-tokyo.ac.jp)

Journal of Cell Science 125, 2789–2793

� 2012. Published by The Company of Biologists Ltd

doi: 10.1242/jcs.094771

Cellular differentiation is controlled largely

by specific gene expression programs. In the

fission yeast Schizosaccharomyces pombe,

the high mobility group (HMG) protein

Ste11 is the main transcription factor

responsible for the switch from cellular

proliferation to sexual differentiation

(Sugimoto et al., 1991; Mata and Bahler,

2006). Ste11 activates many genes that

are required for the initiation of sexual

differentiation, including the mating-type

genes and mei2, the latter of which encodes

a master regulator of meiosis. Expression

of ste11 is under the control of several

signaling pathways, which respond to

external factors, such as nutrition, stresses

and mating pheromones. They include the

cyclic adenosine monophosphate (cAMP)

pathway, the target of rapamycin (TOR)

complex 1 (TORC1) pathway and the two

mitogen-activated protein kinase (MAPK)

pathways (Yamamoto et al., 1997; Otsubo

and Yamamoto, 2010). Recently, it has

become apparent that phosphorylation of

the C-terminal domain (CTD) of RNA

polymerase II (Pol II) by CTD kinase I

(Ctdk1) specifically affects the expression

of ste11 (Coudreuse et al., 2010; Sukegawa

et al., 2011). It has also been shown that the

meiotic regulator Mei2, the expression of

which is governed by Ste11, enhances

expression of ste11 through a positive-

feedback loop (Sukegawa et al., 2011).

These new discoveries, together with

previous findings, illustrate that a number

of signal transduction pathways orchestrate

the regulation of ste11 expression in

response to intricate environmental

changes. In this Cell Science at a

Glance article, we summarize the current

knowledge of these signaling pathways,

and in the accompanying poster we present

a comprehensive scheme showing the

regulation of ste11 expression.

(See poster insert)

Cell Science at a Glance 2789

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Ste11 – a key transcription factor forsexual differentiationFission yeast is a unicellular

microorganism, which proliferatestypically in the haploid state, and thehaploid cell assumes one of the two

mating types, h+ (P) or h2 (M). Undernutrient-rich conditions, cells proliferatethrough the mitotic cell cycle. When

starved of nutrients, in particularnitrogen, the cells begin sexualdifferentiation; cells arrest in G1 phase,

and conjugation proceeds between h+ andh2 cells to give rise to h+/h2 zygotes.These resulting diploid zygotes initiatemeiosis and form asci, each of which

contains four haploid spores (see Poster).

Many genes have to be regulated inan organized manner to enable sexual

differentiation. Ste11 activates a largenumber of genes that are required formating and meiosis. The target genes

identified to date, such as mei2, and themating-type genes mat1-P and mat1-M,have nearly identical ten-base motifs (59-TTCTTTGTTY-39, the so-called TR box)

in their 59-upstream region (Sugimotoet al., 1991; Mata and Bahler, 2006).There are five TR boxes in mei2, and two

TR boxes in both mat1-P and mat1-M. Theste11 gene itself also has a TR box in its59-upstream region, and Ste11 appears to

activate its own transcription throughthis TR box (Kunitomo et al., 2000).Disruption of ste11 causes complete

sterility of the cell (Sugimoto et al.,1991), indicating that Ste11 is a keytranscription factor for the sexualdifferentiation of fission yeast.

Ste11 function is suppressed in multipleways during mitotic growth. Transcriptionof ste11 is reduced under nutrient-rich

conditions (Sugimoto et al., 1991). Ste11 isactive only in the G1 phase; in theremaining phases of the cell cycle, Ste11

is phosphorylated by Cdk, which preventsit from binding to DNA (Kjaerulff et al.,2007) (see Poster). In addition, Pat1 kinase(also known as Ran1) phosphorylates

Ste11 in vegetative cells (Li and McLeod,1996). The 14-3-3 protein Rad24 bindsto the phosphorylated form of Ste11

and inhibits its accumulation in thenucleus (Kitamura et al., 2001). As Ste11stimulates the expression of ste11 itself,

this nuclear exclusion contributes toreducing further the level of ste11

expression (Qin et al., 2003) (see Poster).

Ste11 is also subject to destruction throughthe ubiquitin–proteasome pathway duringvegetative growth (Kitamura et al., 2001;

Kjaerulff et al., 2007). Furthermore,negative control of Ste11 by the RNA-

binding protein Nrd1 (also known asMsa2) at the translation level has beenreported (Tsukahara et al., 1998; Oowatari

et al., 2011).

Upon nitrogen starvation, expression ofste11 is increased dramatically (Sugimoto

et al., 1991). This is brought about by thecooperation of four major signalingpathways, the cAMP pathway, the

TORC1 pathway, the mating pheromone-responsive MAPK pathway and the stress-responsive MAPK pathway (see Poster).We will focus on these signaling pathways

one by one in the sections below.

The cAMP pathwayMany cell systems use cAMP as animportant second messenger in the

regulation of adaptation to externalconditions. In fission yeast, depletion ofthe carbon source results in a rapid

reduction in the level of intracellularcAMP. Depletion of the nitrogen sourcealso reduces the cAMP level, but in thiscase, it takes hours to reduce the cAMP

level by half (Mochizuki and Yamamoto,1992; Isshiki et al., 1992). The decrease incAMP leads to inactivation of cAMP-

dependent protein kinase (Pka) (Maedaet al., 1990), which induces gluconeogenesisand sexual differentiation in fission yeast

(Hoffman and Winston, 1991; Yamamotoet al., 1997).

When there is a sufficient supply of

nutrients, the a-subunit of a heterotrimericG-protein (Gpa2), is activated by theseven-transmembrane protein Git3, a

putative glucose receptor (Isshiki et al.,1992; Welton and Hoffman, 2000;Hoffman, 2005). Activated Gpa2stimulates adenylate cyclase Cyr1 (also

known as Git2), which catalyzes theproduction of cAMP from ATP(Kawamukai et al., 1991). At high

concentrations, cAMP associates withCgs1, the regulatory subunit of Pka(DeVoti et al., 1991). This allows the

catalytic Pka subunit Pka1 to be releasedfrom the inhibitory Cgs1 subunit and toexert its kinase activity (Maeda et al.,

1994). Pka1 represses the expression ofste11 through phosphorylation andinactivation of the zinc-fingertranscription factor Rst2 (Kunitomo et al.,

2000; Higuchi et al., 2002; Matsuo et al.,2008; Gupta et al., 2011) (see Poster).

Rst2 activates transcription of ste11 bybinding to the STREP motif (59-CCCCTC-39) in its 59-upstream region. Fission yeast

cells lacking Rst2 grow mitotically, butcannot express ste11 in sufficient amountsto initiate sexual differentiation (Kunitomo

et al., 2000). The sterility of rst2-defectivecells can be counteracted with an artificialoverexpression of ste11 (Kunitomo et al.,

2000). Thus, Rst2 is a transcription factorthat regulates the expression of ste11

specifically through mediating cAMP

signaling.

In addition to regulating Ste11 at the

transcriptional level, it has been observedthat the Pka pathway also inhibits thenuclear accumulation of Ste11 (Valbuena

and Moreno, 2010).

The TORC1 pathwayThe TORC1 pathway, like the cAMPpathway, promotes vegetative growth and

suppresses sexual differentiation when it isactive (Otsubo and Yamamoto, 2010). Itdownregulates expression of ste11 in the

presence of nutrients, in particular in thepresence of nitrogen (see Poster). TORC1contains Tor2, one of the two TOR kinasesidentified in fission yeast, which is

essential for vegetative growth (Weismanand Choder, 2001). Analysis oftemperature-sensitive tor2 mutants has

shown that they arrest at G1 phase of thecell cycle in nutrient medium if they areshifted to the restrictive temperature

(Alvarez and Moreno, 2006; Uritani et al.,2006; Weisman et al., 2007; Matsuo et al.,2007). Furthermore, ste11 is expressed atthe restrictive temperature in these cells,

irrespective of the nutritional conditions,and results in the initiation of ectopicsexual differentiation in these cells

(Alvarez and Moreno, 2006; Matsuo et al.,2007; Valbuena and Moreno, 2010).

It has been shown that Ste11 targetgenes, including ste11 itself, are

upregulated by loss of Tor2 anddownregulated by overexpression of Tor2(Matsuo et al., 2007; Valbuena and

Moreno, 2010). Tor2 can physicallyinteract with Ste11 (Alvarez and Moreno,2006), and Ste11 accumulates in thenucleus if Tor2 is inactivated (Valbuena

and Moreno, 2010). Thus, TORC1 mightalso inhibit sexual differentiation bycontrolling the localization of Ste11, such

as Pka and Pat1 kinase, in addition toregulating the level of ste11 transcription.

The mating pheromone-responsiveMAPK pathwayThe sexual differentiation of fission yeastis also under the control of matingpheromones (Yamamoto et al., 1997),

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which are short peptides necessary to elicitconjugation.

In fission yeast, the mat1 locus on

chromosome II determines the matingtype of the cell. If this locus contains theP sequence (mat1-P), the cell is h+ (P); if

the locus contains the M sequence (mat1-

M), the cell is h2 (M). Both mat1-P andmat1-M consist of two transcription units,

mat1-Pc and mat1-Pi (also known as mat1-

Pm), and mat1-Mc and mat1-Mi (alsoknown as mat1-Mm) (Kelly et al., 1988).

Transcription of mat1 is upregulated bySte11 through binding to its 59-upstreamTR-boxes (Sugimoto et al., 1991).

Upon nitrogen starvation, Ste11

activates the transcription of mat1-Pc inh+ cells and of mat1-Mc in h2 cells. Theproducts of these genes then elicit the

expression of the respective mating-type-specific genes. The P-specific genesinclude map2, which encodes the mating

pheromone P-factor, and map3, whichencodes the M-factor receptor. M-specificgenes include mfm1, mfm2 and mfm3,which all encode the mating pheromone

M-factor, and mam2, which encodes theP-factor receptor (Tanaka et al., 1993;Kitamura and Shimoda, 1991; Davey,

1992; Imai and Yamamoto, 1994;Kjaerulff et al., 1994). Subsequently, P-factor, a simple peptide of 23 amino acids

is secreted from h+ cells, and M-factor, apeptide of nine amino acids that isfarnesylated and carboxymethylated at the

C-terminus, is secreted from h2 cells (Imaiand Yamamoto, 1994; Davey, 1992).

P-factor binds to the P-factor receptorMam2 on the surface of h2 cells, whereas

M-factor binds to the M-factor receptorMap3 on the surface of h+ cells (Tanaka etal., 1993; Kitamura and Shimoda, 1991;

Toda et al., 1991). Upon pheromonebinding, the two receptors dock to thesame Ga protein, Gpa1, and activate it.

Activated Gpa1 then stimulates a MAPKcascade, comprising Byr2 (MAPKKK),Byr1 (MAPKK) and Spk1 (MAPK)(Nadin-Davis and Nasim, 1988; Wang

et al., 1991b; Gotoh et al., 1993; Neimanet al., 1993). The Byr2–Byr1–Spk1 MAPKcascade enhances expression of ste11 and

of additional genes required for mating andmeiosis in response to the matingpheromones (Aono et al., 1994; Kjaerulff

et al., 2005; Ozoe et al., 2002; Xue-Franzen et al., 2006). The MAPKKKByr2 is also stimulated by Ras1, the

fission yeast homologue of theoncoprotein RAS (Fukui et al., 1986;Nadin-Davis and Nasim, 1988; Nadin-

Davis et al., 1986; Wang et al., 1991b).The activity of Ras1 is positively

controlled by the GDP-GTP exchangefactor (GEF) Ste6, and is negativelyregulated by the GTPase-activatingprotein Gap1 (Hughes et al., 1990;

Hughes et al., 1994; Imai et al., 1991;Wang et al., 1991a). Ste11 activatestranscription of ste6 (Hughes et al.,

1994). Thus, the pheromone signalingappears to be reinforced and firmlyestablished by a positive-feedback

mechanism that involves expression ofSte11, the pheromone-sensing machinery,and activation of Ras1 (see Poster). It hasbeen suggested that Ste11 is a direct target

of Spk1 MAPK (Kjaerulff et al., 2005).

The stress-responsive MAPKpathwayFission yeast has another MAPK pathwaythat controls expression of ste11, the

stress-responsive Sty1 (also known asSpc1) MAPK pathway (Yamamoto et al.,1997). Sty1 is the fission yeast homologueof mammalian MAPK p38. It responds to

diverse stimuli, including heat shock, highosmolarity, oxidative stress and nutritionalstarvation (Shiozaki and Russell, 1995;

Millar et al., 1995; Shiozaki and Russell,1996; Degols et al., 1996). When cells areexposed to stress, the response regulator

protein Mcs4 associates with theMAPKKK Wis4 (also known as Wak1)and probably also with the MAPKKK

Win1, and upregulates MAPKKKactivity. Activated Wis4 and Win1phosphorylate the MAPKK Wis1, whichin turn phosphorylates Sty1 (Shiozaki and

Russell, 1995; Millar et al., 1995;Samejima et al., 1998; Shieh et al.,1998b). It has been indicated that Sty1

phosphorylation is also regulated bythe Pyp1 tyrosine phosphatase and aMAPKKK-independent pathway that

might involve the Cdc37 chaperone(Samejima et al., 1997; Shiozaki et al.,1998; Shieh et al., 1998a; Tatebe andShiozaki, 2003). Once activated by this

phosphorylation, Sty1 accumulates in thenucleus and phosphorylates severalsubstrates, including a bZIP transcription

factor Atf1 (also known as Gad7)(Shiozaki and Russell, 1996; Wilkinsonet al., 1996; Gaits et al., 1998). Atf1

associates with another bZIP protein, Pcr1,and binds to DNA as a heterodimer (Kanohet al., 1996) (see Poster). It has been shown

that activated Sty1 is recruited to thepromoters of stress-induced genes towhich Atf1 binds (Reiter et al., 2008;

Sanso et al., 2011). Wis1, Sty1 and Atf1are essential for appropriate G1 arrest and

the initiation of sexual differentiationunder nitrogen starvation (Shiozaki andRussell, 1996; Kanoh et al., 1996). Theexpression of ste11 in response to nitrogen

starvation is impaired in cells that aredefective in wis1, sty1, atf1 or pcr1

(Shiozaki and Russell, 1996; Kanoh et

al., 1996; Watanabe and Yamamoto,1996), suggesting a role for the stress-responsive MAPK pathway in the

induction of ste11 expression.

Considerable progress has been maderecently in understanding how theSty1 MAPK pathway regulates ste11

expression. It has become clear that thephosphorylation of Ser-2 residues on theheptad repeats of Pol II CTD by CTDK-I is

essential for the upregulation of ste11

transcription (Coudreuse et al., 2010;Sukegawa et al., 2011). Interestingly,

CTDK-I is dispensable for vegetativegrowth, and the main biological role ofthe Ser-2 phosphorylation appears to

be to activate transcription of ste11.Furthermore, the stress-responsive MAPKcascade is crucial for the activation ofCTDK-I under nitrogen starvation

(Sukegawa et al., 2011). It has also beenrevealed that activated Mei2 protein, whichswitches the cell cycle from a mitotic to a

meiotic one, can stimulate this pathwaythrough the MAPKKKs Wis4 and Win1and, hence, contribute to increased

transcription of ste11. Although the wayin which Mei2 activates MAPKKKsremains unclear, the feedback loop thatinvolves the activation of Wis4 and Win1

by Mei2, which in turn activates the Wis1–Sty1–CTDK-I cascade leading to CTDphosphorylation and Ste11 expression,

appears to be important in reinforcing thedecision of the cell to commit to meiosis(Sukegawa et al., 2011) (see Poster).

PerspectivesAs discussed above, the regulation of ste11

expression is fairly complex and includes

the orchestration of a number of signalingpathways. Because expression of ste11 is ahallmark for commitment of a cell to

sexual differentiation, it is crucial for acell to check various internal and externalconditions and assess their balance before

making a final decision to express ste11,and to initiate sexual differentiation.Another remarkable feature of the

regulation of ste11 transcription is that itincludes several feedback loops to furtherenhance its transcription once it is

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initiated. This feedback mechanism isphysiologically important, as it would be

fatal for a cell to abandon thedevelopmental process after it has beeninitiated.

Although the accompanying poster

outlines the current knowledge ofregulatory systems that are relevant forste11 expression, we acknowledge that

many unresolved questions remain. Themolecular mechanisms are not yetsufficiently clear for any of the signaling

pathways described here. In addition, theexact way in which the signaling pathwayscooperate to monitor the nutritionalconditions remains elusive. Although the

cAMP–PKA pathway rapidly responds tothe availability of the carbon source, it alsode-escalates gradually after depletion of

the nitrogen source (Isshiki et al., 1992;Mochizuki and Yamamoto, 1992) andthis decrease appears to be important for

the initiation of sexual differentiation.However, how this gradual decrease,which is mediated by nitrogen depletion,

affects the initiation of sexualdifferentiation is currently unknown.

From the observations made to date, itappears that the TORC1 pathway more or

less directly detects and responds to thelack of a nitrogen source. As describedabove, this signal is also conveyed through

the stress-responsive MAPK pathway,presumably as a ‘stress of starving’.However, the Sty1 MAPK pathway does

not appear to upregulate expression ofste11 when nutrients other than nitrogenare limited (Shiozaki and Russell, 1996).Interestingly, specific transcription of a

Sty1-target gene cta3 under elevatedsalt concentrations is compromised inthe tup11-tup12 double-deletion mutant.

In the absence of the transcriptionalco-repressors Tup11 and Tup12, otherstresses induce the expression of cta3.

Thus, it is thought that Tup repressorsfunction as specificity factors by preventingthe induction of cat3 transcription in

response to inappropriate stresses (Greenallet al., 2002; Hirota et al., 2003). A similarmechanism might operate in the control ofste11 expression in response to nitrogen

starvation. Furthermore, it is noteworthy thatthe Sty1 pathway regulates transcription ofsome genes that are relevant for the cAMP–

PKA pathway (Stiefel et al., 2004; Davidsonet al., 2004), implicating that there is acomplex crosstalk between these pathways.

Finally, we would like to mention thatyet another signaling pathway that controlsexpression of ste11 might exist. TOR

complex 2 (TORC2), which containsTor1, the second TOR kinase in fissionyeast, is also required for sexualdifferentiation (Kawai et al., 2001;

Weisman and Choder, 2001). In contrastto the tor2 temperature-sensitive mutant,tor1 deletion mutants grow vegetatively,

but cannot arrest at G1 phase and entersexual differentiation under nitrogenstarvation (Kawai et al., 2001; Weisman

and Choder, 2001). Although little isknown with regard to the activation ofthe TORC2 pathway, it can be presumedthat it positively regulates expression of

ste11.

Further analysis of the regulation of

ste11 expression will certainly lead us to adeeper understanding of the mechanismsunderlying the switching from proliferation

to differentiation.

Acknowledgements

We thank Akira Yamashita for critical

reading of the manuscript.

Funding

The work of our laboratory was supported

by a Grant-in-Aid for Scientific Research

[grant number (S) 21227007 to M.Y.] from

the Japan Society for the Promotion of

Science and also in part by the Ministry of

Education, Culture, Sports, Science and

Technology Global Centers of Excellence

Program (Integrative Life Science Based on

the Study of Biosignaling Mechanisms)

[grant number A03; Program leader,

Yasushi Miyashita, University of Tokyo].

A high-resolution version of the poster is available for

downloading in the online version of this article at

jcs.biologists.org. Individual poster panels are available

as JPEG files at http://jcs.biologists.org/lookup/suppl/

doi:10.1242/jcs.094771/-/DC1.

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