175
Signal transduction by MAP kinase cascades in budding yeast
Francesc Posas, Mutsuhiro Takekawa and Haruo Saito∗
Budding yeast contain at least four distinct MAPK (mitogen
activated protein kinase) cascades that transduce a variety
of intracellular signals: mating-pheromone response,
pseudohyphal/invasive growth, cell wall integrity, and high
osmolarity adaptation. Although each MAPK cascade
contains a conserved set of three protein kinases, the
upstream activation mechanisms for these cascades are
diverse, including a trimeric G protein, monomeric small
G proteins, and a prokaryotic-like two-component system.
Recently, it became apparent that there is extensive sharing
of signaling elements among the MAPK pathways; however,
little undesirable cross-talk occurs between various cascades.
The formation of multi-protein signaling complexes is probably
centrally important for this insulation of individual MAPK
cascades.
Addresses
Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney
Street, Boston, MA 02115, USA
∗e-mail: haruo [email protected]
Current Opinion in Microbiology 1998, 1:175–182
http://biomednet.com/elecref/1369527400100175
 Current Biology Ltd ISSN 1369-5274
Abbreviations
GS
1,3-β-D-glucan synthase
HOG
high osmolarity glycerol response
MAPK
mitogen activated protein kinase
MAPKK
MAPK kinase
MAPKKK MAPKK kinase
PKC
protein kinase C
Introduction
MAPK (mitogen activated protein kinase) cascades are
modular signaling units composed of three protein kinases: a MAPKKK (MAPK kinase kinase) activates a
cognate MAPKK (MAPK kinase) by phosphorylating
specific serine/threonine residues; the MAPKK in turn
activates a specific MAPK by phosphorylating specific
tyrosine and threonine residues [1,2]. MAPK cascades are
found both in higher and lower eukaryotic organisms,
including yeasts, plants, and mammals [3]. The upstream
mechanisms that activate MAPK cascades are diverse
and in many cases remain unclear. An activated MAPK
phosphorylates specific effector molecules, which include
nuclear transcription factors as well as cytosolic proteins.
The tandem arrangement of three kinases in each
MAPK cascade serves several important purposes. First, a
three kinase cascade amplifies the input signal, because
one activated kinase at one level may phosphorylate
and activate many kinase molecules at the next level.
Second, this arrangement confers an acutely sigmoidal
responsiveness (hypersensitivity) to signaling pathways:
MAPK will not be easily activated by weak signals but
when prodded by a stimulus above threshold it will be
activated decisively [4]. Third, this arrangement allows
integration of distinct upstream signals.
Each organism has multiple distinct MAPK cascades
that transduce different signals. For example, budding
yeast (Saccharomyces cerevisiae) has at least four different
MAPK cascades: the mating-pheromone pathway; the
pseudohyphal/invasive growth pathway; the protein kinase C (PKC) pathway; and the osmoregulatory pathway.
Recently, it has become apparent that a common element
can be used in more than one signal transduction pathways
(for example, in the case of the Ste11 MAPKKK, as
many as three pathways); however, cross-talk between
pathways is averted, probably because of the formation of
multi-component signaling complexes. In this article, we
review these and other recent findings in the yeast MAPK
cascades.
Mating-pheromone pathway
The yeast mating-pheromone response is one of the best
characterized signal transduction pathways in eukaryotic
organisms. Most of the components involved in this
pathway, from the transmembrane pheromone receptors
to the nuclear transcription activators and cell cycle
regulators, have been identified and characterized [5–7].
The receptors for the α and a mating factors (Ste2 and
Ste3, respectively) interact and activate a heterotrimeric
G protein, which is composed of Gα (Gpa1), Gβ (Ste4),
and Gγ (Ste18) subunits (see Figure 1a). When activated,
the dissociated Gβ–Gγ complex transduces signals through
Ste20 (a protein kinase of the PAK [p21-activated protein
kinase] family) and Ste5 (a scaffold protein) to the
mating-pheromone MAPK cascade. Ste5 forms the core
of a signaling complex that contains the Ste11 MAPKKK,
the MAPKK Ste7, and the Fus3/Kss1 MAPKs [8–11].
Activated Fus3/Kss1 MAPKs then induce the transcription
of specific genes by activating the transcription factor
Ste12 [12]. Activated Fus3 (but not Kss1) also arrests the
cell cycle at the G1/S transition by activating the inhibitor
of the Cdc28–Cln kinase, Far1 [13,14].
Major uncertainties remain concerning the roles of the
Gβ–Gγ complex, Ste5, and Ste20 in the activation of
the mating-pheromone MAPK cascade. In addition to
acting as a scaffold, Ste5 is also an active element in
transducing the mating-pheromone signal. The interaction
between Gβ protein and Ste5 seems essential for signal
transduction because Ste5 mutants that cannot bind Gβ
are unable to activate the mating-pheromone MAPK
cascade, even though such Ste5 mutant proteins still
bind all three kinases in the mating-pheromone MAPK
cascade ([15,16•]; E Elion, personal communication).
176
Cell regulation
Figure 1
a/α factors
(a)
(b)
Ste2/3
Cell membrane
GTP
?
Ras2
α
γ
β
Cdc42
Ste5
Ste20
Ste20
Ste11
MAPKKK
Ste11
Ste7
MAPKK
Ste7
Fus3
(Kss1)
MAPK
Kss1
Mating responses
Bmh1/2
Pseudohyphal and invasive
growth responses
Current Opinion in Microbiology
(a) Mating-pheromone pathway and (b) pseudohyphal/invasive growth pathway. (a) Binding of the α or a pheromones to their specific receptor
(Ste2 and Ste3, respectively) induces disassociation of the Gβ–Gγ heterodimer from the inhibitory Gα subunit of the trimeric GTPase. The
liberated Gβ–Gγ complex, together with the Ste20 kinase and the Ste5 scaffold protein, activates the MAP kinase cascade which is composed
of Ste11 (MAPKKK), Ste7 (MAPKK), and Fus3 (MAPK). Another MAPK, Kss1, may substitute for Fus3 under certain circumstances. Activated
Fus3 MAPK induces a set of mating-specific responses, including cell-cycle arrest at the G1/S interface, and expression of genes regulated by
the transcription factor Ste12. (b) The pseudohyphal/invasive growth pathway shares several components with the mating-pheromone pathway.
Its upstream activation mechanism, however, involves unique elements such as small G proteins (Ras2 and Cdc42) and 14-3-3 proteins (Bmh1
and Bmh2) that might regulate Ste20 activity. The upstream mechanism that activates Ras2 is unknown. Actived Kss1 (MAPK) regulates the
transcription factors Ste12 and Tec1. Although Ste12 is common to the two signaling pathways, a Tec1–Ste12 complex induces genes specific
to the pseudohyphal/invasive growth pathway.
The Gβ–Ste5 interaction may be a prerequisite for
Ste5–Ste5 oligomerization, which occurs during normal
signal transduction [16•,17•]. It is not known, however,
whether and how the Ste5 oligomerization leads to
activation of the mating-pheromone MAPK cascade.
The small GTP-binding protein Cdc42 was shown to
interact directly with Ste20 and mutant cells lacking
functional Cdc42 or Cdc24 — a GDP/GTP exchange factor
for Cdc42 — are defective in mating, suggesting that
Cdc42 is an upstream regulator of the mating-pheromone
MAPK cascade [18,19]. Ste20 mutants that no longer
bind Cdc42, however, are fully capable of activating the
mating-pheromone MAPK cascade, indicating that the
pheromone-induced activation of Ste20 is mediated by
another factor, perhaps the Gβ–Gγ complex [20•,21•]. The
role of Cdc42 in mating is probably to localize Ste20 at
shmoo tips in order to facilitate cell–cell adhesion during
conjugation. This role for Ste20–Cdc42 during conjugation
is most likely mediated by the SH3-domain protein Bem1,
which interacts with Ste5, Ste20 and actin [22,23].
Pseudohyphal/invasive growth pathway
Diploid yeast cells undergo a dimorphic transition to
pseudohyphal (filamentous) growth when starved of
nitrogen [24]. In an apparently related phenomenon,
haploid cells grow invasively on rich media [25]. Both
pseudohyphal development and invasive growth require
a subset of the signaling components found in the
mating-pheromone pathway (i.e. Ste20, Ste11, Ste7, and
Ste12) (see Figure 1b). Thus, haploid yeast cells have two
signaling pathways that share many elements of the same
MAPK cascade. Several elements that are essential for the
mating-pheromone pathway are, however, not involved in
pseudohyphal/invasive growth: these include pheromone
receptors, heterotrimeric G protein, and the scaffold protein Ste5 [25]. Conversely, pseudohyphal/invasive growth
requires several elements that are not shared with the
mating pathway (e.g. Ras2, Cdc42, Bmh1, and Bmh2)
[25,26].
Dominantly active Ras2 (Ras2V19) or Cdc42 (Cdc42V12)
induces both filamentous growth and expression of the
filamentous-growth-specific reporter gene FG(TyA)::LacZ
but not the mating pathway reporter gene FUS1::LacZ
[27•]. In contrast, only FUS1::LacZ, but not FG(TyA)::LacZ,
is induced by the mating pheromone. Thus, even though
the expression of both reporter genes is dependent on
Ste20, Ste11, Ste7, and Ste12, there is no cross-talk be-
Signal transduction by MAP kinase cascades in budding yeast Posas, Takekawa and Saito
tween the two signaling pathways. Ras2 seems to activate
Ste20 through Cdc42 but the upstream mechanism that
activates Ras2 is not known. Filamentous growth requires
the 14-3-3 proteins Bmh1 and Bmh2, which most likely
modify Ste20 to allow signal transmission from Ras2/Cdc42
to downstream components [28]. In contrast, Bmh1 and
Bmh2 are not essential for the mating pheromone MAPK
cascade signaling.
Although the mating-pheromone pathway and the pseudohyphal/invasive growth pathway share several constituents, the two pathways appear to use different
MAPKs. Recent reports suggest that the Kss1 MAPK
regulates mainly filamentation and invasion, whereas
the Fus3 MAPK regulates mating [29••,30••]. Activated
Kss1 promotes invasive growth, partly by inhibiting
Dig1 and Dig2 (negative regulators of invasive growth)
[31,32]. Interestingly, however, both Fus3 and Kss1 also
have kinase-independent activities that inhibit invasive
growth [29••,30••]. Indeed, fus3 kss1 double mutants grow
invasively, indicating that their inhibitory function is as
important as their positive roles. As previously shown,
Kss1 can mediate mating-pheromone signaling in the
absence of Fus3 [33]. Under such conditions, however,
there is erroneous cross-talk in which mating pheromone
also activates filamentation-specific gene expression [29••].
Perhaps a key to this (loss of) mutual insulation is the
tethering protein Ste5. If Ste5 binds Fus3 preferentially
over Kss1, then signals emanating from the pheromone
177
receptor will be channeled preferentially to a complex
containing Fus3 but not Kss1. In the absence of Fus3,
Kss1 may have an access to Ste5, causing the observed
cross-talk. In contrast, 14-3-3 proteins (Bmh1/2) may route
the signal from Ras2 to Ste11 that is not complexed with
Ste5.
The pathogenic fungus Candida albicans can also switch
between unicellular and filamentous forms. This morphological switch is governed by a MAPK cascade homologous
to the S. cerevisiae pseudohyphal pathway [34,35]. The
finding that nonfilamentous Candida mutants are avirulent
may have important clinical implications [36].
Protein kinase C pathway
Yeast Pkc1 is a homolog of mammalian already defined
PKC and regulates a MAPK cascade composed of Bck1
(MAPKKK), Mkk1/2 (MAPKKs) and Mpk1 (MAPK) (see
Figure 2) [37]. Hypotonic shock and heat shock are known
to activate the PKC pathway [38,39]. Mutants in the
PKC signaling cascade undergo cell lysis because of a
deficiency in cell wall construction that is exacerbated at
high temperatures.
Mutants of Rho1 (a small GTP-binding protein) display
a cell lysis defect similar to that of mutants defective in
the PKC pathway. Furthermore, expression of an activated
allele of PKC1 or overexpression of wild-type PKC1
suppresses the cell lysis defect of rho1 mutants, suggesting
Figure 2
Hcs77
?
GS
complex
Rho1
Stt4
Pkc1
Cdc28–Cln1/2
Bck1
MAPKKK
Mkk1/2
MAPKK
Mpk1
MAPK
Cell cycle control and
cell wall construction
Current Opinion in Microbiology
PKC signaling pathway. A PKC homolog, Pkc1, may be regulated by several different proteins: the Cdc28–Cln1/2 complex during early bud
growth; Hcs77 upon heat shock; and Stt4 (a phosphatidylinositol 4-kinase), or Rho1 (small G protein) during cell wall morphogenesis. Pkc1
activates the MAP Kinase cascade composed of Bck1(MAPKKK), Mkk1/2 (MAPKKs), and Mpk1 (MAPK). Activated Mpk1 controls the cell cycle
and cell wall construction, perhaps by regulating several transcription factors (e.g. Rlm1 and SBF complex). Rho1 is also a positive regulator of
the 1,3-β-D-glucan synthase (GS) complex.
178
Cell regulation
that Rho1 has a role in the PKC pathway [40,41•]. It
was shown recently that GTP-bound Rho1 associates with
Pkc1 and confers upon Pkc1 the ability to be stimulated
by the co-factor phosphatidylserine [41•]. Rho1 is known
to be regulated by Rom1/2 (GDP/GTP exchange factors
specific to Rho1), Sac7 (a GTPase-activating protein for
Rho1), and Tor2 (a phosphatidylinositol kinase homolog
and an activator of Rom2) [42,43]. Thus, these molecules
may also have roles in the PKC pathway. Rho1, when
bound to GTP, is also a positive regulator of 1,3-β-D-glucan
synthase (GS) [44•,45•]. GS is a multi-enzyme complex
that catalyzes the synthesis of 1,3-β-linked glucan, a major
structural component of the yeast cell wall. Interestingly,
expression of Fks2, a component of GS complex, is
controlled by the PKC pathway [41•]. Thus, GTP-bound
Rho1 stimulates cell wall construction directly by activating GS and indirectly by stimulating the PKC pathway.
Mutants of the HCS77 gene exhibit similar phenotypes
as PKC pathway mutants and are partially suppressed
by overexpression of Pkc1 [46•]. Furthermore, hcs77
mutants are defective in heat shock induction of Mpk1
activity, suggesting that Hcs77 acts upstream of the PKC
MAPK cascade [46•]. As Hcs77 is a putative type I
transmembrane protein, it may act as a cell-surface sensor
for heat shock. The activation of the PKC pathway is
also partially dependent on the Cdc28–Cln1/2 kinase
[46•,47•]. Thus, the PKC pathway, which promotes bud
emergence and cell surface growth, may be activated
by the Cdc28–Cln1/2 kinase at the G1/S transition and
by Hcs77 upon heat shock or hypo-osmotic shock. The
phosphatidylinositol-4-kinase, Stt4, may also be involved
in the PKC pathway but its exact role is not clear [48].
The PKC pathway is also activated by mating pheromones.
This apparent cross-talk, however, is an indirect effect
of cell wall re-organization induced by the matingpheromone pathway [49]. The two MAPK cascades
are actually well insulated. For instance, under normal
conditions, the MAPKK in the mating pathway (Ste7)
cannot functionally substitute for the MAPKK in the PKC
pathway (Mkk1/2). A Ste7 mutant (Ste7P368) with elevated
activity, however, can suppress defects in the PKC MAPK
pathway if Ste5 is absent, suggesting that Ste5 is important
Figure 3
Extracellular hyperosomolarity
Cell membrane
Sho1
Sln1
SH3
Ypd1
Ssk1
Ste11
Ssk2/22
MAPKKK
Pbs2
MAPKK
Hog1
MAPK
Osmoadaptive
responses
Current Opinion in Microbiology
Osmoregulatory signaling pathway. Yeast have two independent osmosensors, Sho1 and Sln1. The Sho1 osmosensor contains four
transmembrane segments and a cytoplasmic SH3 domain that binds to the amino terminus of Pbs2 MAPKK. Activation of Pbs2 by Sho1
requires the Ste11 MAPKKK, a kinase that is also used in the mating-pheromone pathway and the pseudohyphal/invasive growth pathway.
The second osmosensor, Sln1, is homologous to prokaryotic two-component signal transducers and contains two transmembrane segments,
a cytoplasmic histidine kinase domain and the phospho-accepting receiver domain. Sln1 is a part of a multistep phospho-relay composed of
three proteins (Sln1, Ypd1 and Ssk1) that regulates the osmoregulatory MAPK cascade. Under high osmotic stress conditions, Sln1 histidine
kinase is inactivated resulting in accumulation of unphoshorylated Ssk1, which activates the Ssk2/22 MAPKKKs, which in turn activate Pbs2.
Thus, Pbs2 integrates signals from two independent osmosensors. Activated Pbs2 then phosphorylates the Hog1 MAPK, which induces a set of
osmoadaptive responses, including glycerol synthesis.
Signal transduction by MAP kinase cascades in budding yeast Posas, Takekawa and Saito
179
to Sln1–Asp1144, then to Ypd1–His64, and finally to
Ssk1–Asp554. This four-step phospho-relay reaction has
been reconstituted in vitro using purified recombinant
proteins [57••]. The end product of the phospho-relay
is aspartate-phosphorylated Ssk1. Under normal osmotic
conditions, active Sln1 histidine kinase most likely maintains Ssk1 in the phosphorylated state whereas, at high
osmotic conditions, the Sln1 histidine kinase is inhibited,
leading to the accumulation of unphosphorylated Ypd1
and Ssk1. Unphosphorylated Ssk1 then activates the
redundant Ssk2 and Ssk22 MAPKKKs, which in turn
phosphorylate and activate the Pbs2 MAPKK, leading to
Hog1 MAPK activation [51,55,59•].
for preventing undesirable cross-talk between the two
pathways [50].
Osmoregulatory pathway
The fourth major yeast MAPK cascade regulates adaptation to hyperosmolarity (see Figure 3). Because a major
outcome of the activation of the osmoregulatory MAPK
pathway is elevated glycerol synthesis, this pathway is
frequently referred to as the HOG (high osmolarity
glycerol response) pathway [51,52]. The HOG pathway
is essential for survival in high osmolarity environments
[51,53]. In yeast, extracellular hyperosmolarity is detected
by one of two transmembrane osmosensors, Sln1 and
Sho1 [54,55]. Although the two osmosensors are not
structurally related, both activate the same MAPK, Hog1.
Sln1 — a homolog of prokaryotic two-component signal
transducers — contains two transmembrane segments, a
cytoplasmic histidine kinase domain, and a receiver
domain [56]. Sln1 transmits signals to the redundant Ssk2
and Ssk22 MAPKKKs, via the Sln1–Ypd1–Ssk1 multi-step
phosphorelay system [54,55,57••,58]. Ssk1 is also homologous to the receiver domains of prokaryotic two-component signal transducers [54]. The phospho-relay system is
initiated by the auto-phosphorylation of Sln1–His576 (see
Figure 4). This phosphate is then sequentially transferred
Activated alleles of the SLN1 gene nrp2 encode mutant
proteins with a high histidine kinase activity that, when
combined with a disruption mutation of the second
osmosensor Sho1, cause an osmosensitive phenotype
[60]. Sln1nrp2 mutant proteins may be locked in the
phosphorylated state and thus cannot activate the HOG
MAPK cascade. Interestingly, nrp2 mutants have another
phenotype, namely hyper-expression of genes regulated
by the transcription factor Mcm1. This latter phenotype
is independent of the presence or absence of either Ssk1
or the Hog1 MAPK, suggesting that Sln1 may have two
distinct and independent roles: regulation of the Hog1
Figure 4
Extracellular
sensor domain
Normal extracellular
osmotic conditions
Cell membrane
Histidine
kinase
Receiver
domain
D
H
P
Sln1
P
(a)
(c)
P
(b)
H
Ypd1
P
D
Ssk1
(d)
Regulation of the HOG
MAP kinase cascade
Current Opinion in Microbiology
Multistep phospho-relay reaction in the yeast two-component osmosensor. (a) Under normal extracellular osmotic conditions, Sln1
autophosphorylates a histidine (H) residue near its histidine kinase domain. (b) This phosphate is then sequentially transferred to the Sln1
carboxy-terminal receiver domain, (c) to a histidine residue in Ypd1, and (d) finally to an aspartic acid (D) in the Ssk1 receiver domain. Under
high osmotic stress conditions, Sln1 histidine kinase is inactivated, resulting in accumulation of unphosphorylated Ssk1, which activates the
Ssk2/Ssk22 MAPKKKs (see Figure 3). P, phosphorylation.
180
Cell regulation
MAPK and activation of the Mcm1 transcription factor
[60,61].
cilitate the recovery of the pathway to the pre-stimulation
state [65,66].
It is not known how changes in extracellular osmolarity
regulates the Sln1 cytoplasmic histidine kinase. Increased
extracellular osmolarity causes the outflow of water
through the plasma membrane, shrinkage of the cytoplasmic volume, and a separation of the plasma membrane and
the cell wall (plasmolysis). It is thus possible that the Sln1
extracellular region somehow monitors the distance of the
plasma membrane and the cell wall, perhaps by making
specific contact with a cell wall component. Another
possibility is that the shrinkage of cell volume causes
change in the plasma membrane itself, perhaps because of
decreased membrane tension, such changes perhaps being
detected by Sln1.
Conclusions
Sho1, the second osmosensor, has four predicted transmembrane segments and a carboxy-terminal cytoplasmic
region containing an SH3 domain [55]. An SH3 domain
interacts with a target protein containing a proline-rich
motif [62]. The physiologically relevant target of the
Sho1 SH3 domain is Pbs2. A mutant Pbs2 that has
a single amino acid substitution (Pro96→Ser) within a
proline-rich sequence (KPLPPLPVA) fails to interact
with Sho1 and is unable to transduce a signal from
the Sho1 osmosensor. The same Pbs2 mutant, however,
is fully capable of transducing signals from the Sln1
osmosensor [55]. Although Sho1 interacts directly with
the Pbs2 MAPKK, the activation of Pbs2 requires the
Ste11 MAPKKK [57••]. Thus, constitutively active Ste11
activates both the mating and osmoregulatory MAPK
cascades. Despite these findings, however, the two
pathways are normally well insulated from each other:
mating pheromones activate only the mating pathway, and
osmotic stress activates only the HOG pathway [57••].
As previously mentioned, Ste5 is a probable determinant
in the signaling specificity. Furthermore, Pbs2 may also
play an important role in determining specificity. As Pbs2
interacts with Sho1, Ste11, and Hog1, it may also have a
scaffold-like function [57••].
A comparison of the four MAPK cascades in S. cerevisiae
illustrates two important principles that are applicable
to other organisms including mammals. First, there exist
amazingly diverse ways to activate MAPK cascades;
activation of the first kinase in the cascade (MAPKKK)
may require a protein kinase (such as Ste20 or Pkc1),
or the interaction with another protein (for example
Ssk1). Furthermore, upstream elements may involve
trimeric and monomeric G proteins, two-component
sensors, cyclin-dependent kinases, and transmembrane
sensors (such as Hcs77 and Sho1). Second, though a
common signaling component may be shared by more
than one pathway, there is little, if any, cross-talk between
the pathways. For example, the Ste11 MAPKKK is
involved in three different pathways; mating-pheromone,
pseudohyphal/invasive, and osmoregulatory. Formation of
multiprotein complexes (signalosomes) is the probable
mechanism to ensure the specificity of each signaling
pathway but the way such a complex is formed may be
unique to each signaling pathway.
Acknowledgement
We thank Michel Streuli for his helpful comments on the manuscript.
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