INTRODUCTION

Top Previous Next References

The purpose of this review is to summarize the current knowledge of signalling pathways in the yeasts for nonspecialists, particularly for fungal biologists who are beginning studies in this area. This review has been written to describe both how these pathways function and how they were figured outhow the components in these different pathways were identified, how they were ordered, and what kinds of assays are used in such studies.

I describe several pathways in both Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast), juxtaposing pathways used for the same response in an attempt to point out similarities and differences in the use of highly conserved components in different organisms. Several reasons have motivated me as a fungal biologist to do this. I am interested in cell-cell interactions between fungal cells and between fungal and plant cells. Because these interactions are likely to involve diverse signals and response pathways, an understanding of the different pathways that operate in the yeasts may be directly relevant to understanding the interactions of filamentous fungi with their environment and their hosts. The literature in the area of signalling is vast. I have therefore tried to extract some of the most important lessons to make this review accessible to nonspecialists, although I hope that specialists will also find it useful. Because information about signal transduction pathways in filamentous fungi is fragmentary, in many cases I have interspersed nuggets of information on this topic in the relevant sections describing the yeast pathways.

Several important general issues, which provide a framework for examining the different pathways, are as follows. (i) A given cell contains multiple pathways, each of which responds to a distinct signal that is transduced to give a specific response. Because the central component of these pathways, the mitogen-activated protein kinase (MAPK) cascade (see below), is highly conserved, the cell must have mechanisms for preventing inappropriate communication between pathways. One mechanism for preventing such cross talk involves scaffold or sequestering proteins. (ii) A given signalling component can be used in more than one pathway within the same cell to respond to different signals. This observation raises the question of how the specificity of the response is regulated in such cases. Specificity may involve modulation of the activity of a transcription factor by its association with different accessory proteins. (iii) Comparisons of pathways, for example, used for response to stress will illustrate that in one organism a pathway responds to only one stress signal whereas in another organism the same components are used to respond to a multitude of stress signals. We are then confronted with the question of how multiple input signals are integrated. One possibility is the use of a different sensor for each different signal. (iv) Different organisms use the same machinery to respond to the same signal, but some of the components of the machinery may be used differently. (v) The receptors used in different pathways are of different types: G-protein-coupled (serpentine or seven-transmembrane) receptors, His-Asp phosphorelay sensors, and a novel class of integral membrane proteins. In each case, a central issue (often unsolved) is how they communicate with downstream components. (vi) The responses elicited by the different signals are numerous and in many cases are common to all eukaryotes. In some cases, the response of a pathway to a given signal may serve to coordinate different processes, for example, osmolarity and mitosis or nutritional status and meiosis. (vii) A given pathway may link two different types of phosphorylation cascades. (viii) Many examples of redundancy of components are provided by the different pathways. (ix) Lastly, filamentous fungi may use some of the machinery used in the mating response in yeasts to regulate the intricacies of hyphal growth.

I first present a general introduction to the core of the signalling pathways, the MAPK cascade. I then describe the pathways that sense osmolarity in both yeasts and follow with a description of the pheromone response pathway. As part of the latter section, I discuss regulation of filamentous growth in the Basidiomycete fungi by pheromones and receptors. I also present information on the role of various signalling components in the life cycle of filamentous fungi. Each section has been written in a self-contained manner so that the reader can read the sections separately.

In eukaryotic cells, the MAPK cascade module is a key element in mediating the transduction of many signals generated at the cell surface to the nucleus (Fig. 1). Three protein kinases that are highly conserved in all eukaryotes make up this module: MAPK (also known as extracellular signal-regulated kinase [ERK]), MAPK kinase (MAPKK, also known as mitogen-activated, ERK-activating kinase [MEK]), and MAPK kinase kinase (MAPKKK, also known as MEK kinase [MEKK]). I will refer to them in this review as MAPK, MAPKK, and MAPKKK, respectively. Sequential activation of these kinases by phosphorylation lies at the heart of transduction of the signal through this kinase module. MAPK is activated by the dual-specificity serine/threonine tyrosine kinase MAPKK, and it is in turn activated by the serine/threonine kinase MAPKKK (reviewed in references 160, 175, and 183). The latter becomes activated in response to a signal generated by an input. Thus, input signals lead to activation of the MAPK cascade, which then generates output signals (Fig. 1).

View larger version (17K): [in this window] [in a new window]   FIG. 1.   MAPK module. (A) The MAPK module lies at the heart of many signalling pathways in eukaryotes. An input signal leads to activation of the MAPK cascade, which then generates an output response. (B) The MAPK cascade consists of MAPKKK (MEKK), MAPKK (MEK), and MAPK (ERK) (see the text for details). A receptor is activated in response to an extracellular signal, which leads to activation of the MAPK cascade (see the text for details). The activated MAPK phosphorylates substrates, some of which include transcriptional activators, resulting in altered patterns of gene expression and protein activity.

The signal that leads to activation of the MAPK cascade is perceived by a variety of types of receptors: G-protein-coupled seven-transmembrane receptors, His-Asp phosphorelay sensors, receptor-tyrosine kinases, and integral membrane proteins (reviewed in reference 73). In the yeasts, serpentine receptors, His-Asp phosphorelay sensors, and integral membrane protein sensors have been identified. The budding yeast genome does not code for any receptor-tyrosine kinases (reviewed in reference 83). The target of activation of the MAPK cascade module is often a transcription factor (reviewed in references 76 and 208), although other targets have also been identified. Phosphorylation of a transcription factor by the MAPK can in principle increase its binding affinity, activation ability, or cellular location, resulting in increased transcription of target genes. Inactivation of the MAPK cascade by dual-specificity phosphatases and by tyrosine phosphatases is one mechanism for attenuation of the signal and adaptation to the response (reviewed in references 82 and 146).

In S. cerevisiae, independently acting MAPK cascades exist that regulate response to osmotic stress, pheromones, perturbations of cell wall integrity, spore formation, and pseudohyphal growth (reviewed in reference 73). In S. pombe, fewer pathwaysthose for response to osmotic stress and pheromoneshave been characterized. I will discuss all of the above pathways except the sporulation pathway.

The components of the MAPK cascade module are related to other kinases by virtue of their catalytic domain, which consists of approximately 250 to 300 amino acid residues. They catalyze the transfer of gamma phosphate from ATP to a hydroxyl residue on Ser and Thr or on Thr and Tyr (reviewed in reference 68).

The catalytic domain of all kinases can be subdivided into 12 conserved domains, within which 12 residues are invariant or highly conserved. These residues must thus be critical for activity of the kinase (reviewed in reference 68). Analyses of mutations of these highly conserved residues have been crucial in elucidation of the functional requirement of the kinase activity. The conserved primary structure of the catalytic domains suggests that they may fold into similar three-dimensional structures. The crystallographic structures of the catalytic domains of two mammalian MAPKs, ERK2 and p38, have demonstrated many similarities in their topology. Despite these similarities, differences were found in the ATP and substrate binding sites and in the phosphorylation lip (214, 232; see also reference 68). These variations in the topology may be important for the specificity of recognition by the activating MAPKK and for recognition of substrate.

MAPKs are activated by phosphorylation on two closely spaced Thr and Tyr residues (Thr-X-Tyr) found in catalytic subdomain VIII; X can be Pro, Gly, or Glu. Mutation of the X residue does not appear to affect activation by MAPKK (see reference 214 and references therein). The MAPKKs are activated by phosphorylation on two closely spaced serine residues or serine and threonine residues in domain VIII.

The MAPKKKs have a large N-terminal noncatalytic region and a C-terminal catalytic domain. The noncatalytic domain appears to be autoinhibitory, as inferred from the fact that deletion of this region causes constitutive activation of the kinase in the absence of a stimulus (see, for example, references 25 and 127). The mechanism by which MAPKKKs become activated remains to be elucidated and is the subject of current intensive studies. Conformational changes brought about by interaction with another protein or by self-dimerization or removal of a protein that inhibits the activation of the catalytic domain may be involved in such activation.

These introductory comments should help the reader put into perspective the MAPK cascade as we "tour the pathways."

Glycerol plays an important role in the adaptation of both budding and fission yeast cells to increased external osmolarity. As both S. cerevisiae and S. pombe cells encounter hyperosmotic conditions, they increase their synthesis of glycerol, which leads to an increased internal glycerol concentration and thus to an increased internal osmolarity, which compensates for the elevated external osmolarity. S. cerevisiae and S. pombe cells unable to produce glycerol are unable to grow on hyperosmotic medium (2, 156). Thus, glycerol appears to be the major osmolyte used by the yeasts. Glycerol accumulation is in part due to increased activity of glycerol-3-phosphate dehydrogenase, encoded by GPD1 in S. cerevisiae and gpd1 in S. pombe (156). This increased activity results from activation of the MAPK cascade. Adaptation to high osmolarity is mediated by the HOG (high-osmolarity glycerol) pathway in S. cerevisiae and the Sty1 (suppressor of tyrosine phosphatase) pathway in S. pombe. In both cases, the MAPK cascade is activated in response to hyperosmolarity and leads to increased expression of a number of target genes.

Activation of the Hog1 MAPK cascade by two different sensors. The Hog1 MAPK cascade (Fig. 2) consists of Ssk2 and Ssk22 (MAPKKK), Pbs2 (MAPKK), and Hog1 (MAPK) (16, 20, 127). Hog1 is activated by phosphorylation by Pbs2 on two residues (Thr174 and Tyr176) that reside in the catalytic domain and are conserved among all MAPKs. Pbs2 is activated by phosphorylation on two residues (Ser514 and Thr518) within the catalytic domain that are also conserved among MAPKKs (see "MAPK cascade nodule and signal transduction" above). Activation of Pbs2 can occur by two different branches (Fig. 2), both sensing hyperosmolarity and each acting independently of the other (127, 128). One branch (the Sln1 branch) involves a His-Asp phosphorelay system; the other (the Sho1 branch) employs a putative transmembrane osmosensor (Fig. 2) (128, 166). The Sln1 branch activates two redundant MAPKKKs (Ssk2 and Ssk22). These MAPKKKs, like other MAPKKKs, contain a large noncatalytic region that has been proposed to play a negative regulatory role in activation of the catalytic domain. A homolog (MTK1) of the Ssk2 and Ssk22 MAPKKKs has been recently identified and shown to mediate activation of the stress pathway in mammalian cells. Given that the similarity of these proteins extends to the noncatalytic regulatory region, it is possible that upstream regulators are also conserved (201).

View larger version (26K): [in this window] [in a new window]   FIG. 2.   Hyperosmotic stress response in S. cerevisiae and S. pombe. Response to hyperosmolarity is mediated by the HOG pathway in S. cerevisiae (left) and by the Sty pathway in S. pombe (right). Ptp2 is a phosphatase involved in down regulation of Hog1. Pyp2 is a phosphatase involved in down regulation of Sty1. Arrows indicate activation; lines with bars indicate inhibition. The dashed rectangle around Ypd1 indicates that the protein is present but not phosphorylated (see Fig. 5). See the text for details.

View larger version (19K): [in this window] [in a new window]   FIG. 3.   Multiple roles of the MAPKKK Ste11. The MAPKKK Ste11 can be activated by different signals resulting in different outputs. It can be activated by pheromones in the pheromone response pathway, resulting in mating (stippled arrows); a signal generated by nitrogen starvation in the pseudohyphal growth pathway, leading to morphological changes and growth properties of the cells (black arrows); and hyperosmolarity via Sho1 in the HOG pathway, resulting in the stress response (gray arrows). The mechanism by which activated Ste11 is prevented from inappropriately activating other pathways may involve scaffold proteins (see the text for details).

Osmosensing involves a multistep phosphorelay system related to the histidyl-aspartyl phosphorelay systems of bacteria. Yeast has coupled two different types of phosphorylation cascades in the high-osmolarity signal transduction pathway. Above, I described phosphorylation through the MAPK cascade, and now I describe phosphorylation in the histidyl-aspartyl (His-Asp) phosphorelay system and how this phosphotransfer affects activation of the MAPK cascade. First, I present a brief overview of His-Asp phosphorelay systems (also known as two-component systems) in bacteria (52).

View larger version (12K): [in this window] [in a new window]   FIG. 4.   Phosphorelay system of the HOG pathway. A phosphorelay system consisting of three proteins, Sln1, Ypd1, and Ssk1, activates the HOG pathway. Sln1, the osmosensor, contains both a histidine kinase domain (black rectangle) and a receiver domain at its C terminus (hatched rectangle). Upon signal sensing, Sln1 autophosphorylates His576. Ssk1 contains a receiver domain (hatched rectangle) and is the activator of the MAPKKKs Ssk2 and Ssk22. Arrows indicate direction of phosphotransfer. Mutation of the His or Asp residues blocks phosphotransfer and results in constitutive activation of the pathway, suggesting that phosphorylation prevents activation of the HOG MAPK cascade. See the text for other details.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   High osmolarity inhibits phosphotransfer in the phosphorelay system and causes activation of the HOG MAPK cascade. Normal osmolarity conditions (left) stimulate phosphotransfer and result in phosphorylation of Ssk1. Phosphorylated Ssk1 cannot activate the MAPK cascade, and consequently there is no response. High osmolarity (right) inhibits Sln1 by an unknown mechanism. Phosphotransfer is blocked, and unphosphorylated Ssk1 is proposed to activate the MAPK cascade, resulting in the stress response. Arrows indicate activation; lines with bars indicate inhibition. The dashed rectangle around the MAPK cascade (left) indicates that it is inactive. The dashed rectangle around Ypd1 (right) indicates that the protein is present but not phosphorylated. See the text for details.

Mutations that turn the pathway on or offuse of epistasis relationships to order pathway components. In this section, I provide some examples of how components of the pathway have been identified, in some cases by genetic analysis and in others by inference from nucleotide sequence analysis. Genetic analysis relies on two key types of mutations: mutations that turn the pathway off and mutations that turn the pathway on in the absence of stimulus. There is no good reporter gene (see the next section) for measuring the activation of the HOG pathway, unlike the situation for the pheromone response pathway. Activation of the HOG pathway is assayed biochemically by determining tyrosine phosphorylation of the MAPK Hog1 with commercially available antiphosphotyrosine antibodies on proteins immobilized on a nitrocellulose membrane after a given treatment (see, for example, reference 165). Growth on plates with high osmoticum (1.5 M sorbitol or 0.9 M NaCl) is used in conjunction with the above assay. This plate assay is also used to screen for osmosensitive (Osm^s) mutants in the identification of pathway components.

Stress response element. Heat shock and osmotic stress induce synthesis of an overlapping set of proteins (211, 212; see also reference 77). Genes induced by osmotic stress, for example CTT1 and GPD1, contain a response element in their promoter region that appears to mediate the response to osmotic stress (129, 181). The stress response element (STRE) can also mediate the induction of transcription by other types of stresses, for example heat shock, nitrogen starvation, and oxidative stress, which are independent of the HOG pathway. Because this element mediates both HOG-dependent and HOG-independent induction, the use of STRE-reporter fusions has not proved very useful in analysis of the HOG pathway. Use of a transposon library (177) or of the more recently developed "gene microarrays" (186) should facilitate the identification of Hog1 targets and reporter genes for this pathway.

Fission yeast cells respond to osmotic stress via the Sty1 MAPK pathway (which is also called Spc1 and Phh1) (Fig. 2). Studies of this pathway have led to some important findings which clearly distinguish it from the HOG pathway of S. cerevisiae. First, in contrast to the HOG pathway, which is activated only by hyperosmotic stress, the Sty pathway is also activated by oxidative and heat shock stresses, nutrient limitation, and anisomycin (a protein synthesis inhibitor) (43, 222). Thus, in this respect the S. pombe pathway resembles the p38 and SAPK/JNK stress-activated pathways of mammalian cells (57, 67, 108). Activation of the Sty pathway by multiple stress signals raises the question whether different sensors are used for the different stress signals. Second, the Sty pathway integrates stress sensing with control of mitosis, a very important finding which may shed light on how the extracellular environment regulates mitosis (138, 190). Third, the Sty pathway utilizes a transcription factor with similarity to the transcription factor activated in mammalian cells in response to stress (189, 222). Therefore, the similarity of the pathway to that of mammalian cells extends to the transcriptional activator. Fourth, the Sty pathway links stress signalling with control of sexual differentiation (43, 97, 200). The S. pombe transcriptional activator governs the expression of target genes involved in the stress response and in the initiation of meiosis (189, 200, 222). Thus, the Sty pathway not only regulates stress responses but also integrates this response with two processes fundamental to all eukaryotes: control of mitosis and initiation of meiosis (Fig. 2 and 6).

View larger version (19K): [in this window] [in a new window]   FIG. 6.   Multiple stress conditions activate the Sty1 MAPK cascade, resulting in coordination of the stress response with mitosis and meiosis. The Sty pathway can be activated by osmotic, heat, and oxidative stress, by nutritional limitation (black arrows above rectangle), and by anisomycin (not shown). Osmotic stress not only activates the stress response (black arrow below rectangle) but also controls cell size at time of mitosis (open arrow below rectangle). In response to nutritional limitation, the stress response is activated, resulting in expression of ste11, which initiates sexual differentiation (stippled arrow below rectangle).

The components of the Sty1 MAPK pathway are homologous to those of the HOG pathway of S. cerevisiae. The MAPK cascade module of the Sty1 pathway (Fig. 2) consists of the MAPK Sty1, which is activated by phosphorylation on the Thr171 and Tyr173 residues by the MAPKK Wis1 (138, 178a, 190). This kinase in turn is activated on Ser469 and Thr473 (178a, 187, 188) by the MAPKKK Wak1 (also known as Wik1 and Wis4). The MAPKKK in turn appears to be activated by Mcs4 (187, 188) (Fig. 2), which has amino acid sequence similarity to Ssk1. It has been proposed that a His-Asp phosphorelay system similar to that regulating the HOG pathway is involved in the response to high osmolarity in S. pombe, although a histidine kinase has yet to be identified (187, 188).

A transcription factor with similarity to mammalian ATF2 is the target of the MAPK cascade. A transcriptional activator has not been identified for the S. cerevisiae HOG pathway. In contrast, in S. pombe, the transcriptional activator Atf1 (also known as Gad7) is downstream of the Sty1 MAPK (189, 200, 222) (Fig. 2). Atf1 is a bZIP transcription factor (references 96 and 200 and references therein), highly homologous to mammalian ATF-2 (which is involved in the mammalian stress response). It is phosphorylated in a Sty1- and Wis1-dependent manner and appears to be a direct substrate for Sty1 (189, 200, 222). Several genes are known to be induced by different stress conditions in a Sty1-dependent manner (references 1, 43, 97, 138, 187, 189, 200, and 222 and references therein [detailing the identification and characterization of the genes]): pyp2 (encoding a tyrosine phosphatase), gpd1 (encoding glycerol-3-phosphate dehydrogenase), ctt1 (encoding catalase), fbp1 (encoding fructose-6-phosphatase), ste11 (encoding a high mobility group [HMG] transcription factor), and tps1 (encoding trehalose-6-phosphate synthase). Expression of these genes, not surprisingly, has also been shown to be Atf1 dependent (200, 222), and some evidence exists suggesting that Atf1 directly interacts with ATF-like binding sites in the promoter region of gpd1 (222).

Links between the Sty pathway and cell size control at mitosis. Understanding how eukaryotic cells coordinate the response to the extracellular environment and progress through the cell cycle is a key question, about which we have little information. Studies of the S. pombe Sty pathway may provide important insights into how this might occur. In this section, after a synopsis of fission yeast growth and mitotic cycle, I present some of the observations that have led to the proposal that the Sty pathway links the control of cell cycle progression and the response to stress (for more details see references 138 and 190).

Identification and order of some of the pathway components. As in the analysis of the HOG pathway, the use of mutations that turn the pathway off or on has been extremely useful in ordering the components of the Sty pathway. One way in which the Sty1 pathway can be activated constitutively is by expressing genes under the control of the inducible nmt promoter. In the presence of thiamine, the promoter is repressed, whereas in its absence, the promoter is activated, resulting in high-level expression of the gene under its control. Overexpression of mcs4 in this manner causes lethality; this lethality can be suppressed by mutations in wak1, wis1, and sty1, indicating that these genes are probably downstream of mcs4 (188). The order of wak1 with respect to wis1 was inferred from the following analysis. As is the case with the MAPKKK of the HOG pathway, deletion of the N terminus of Wak1 causes constitutive activation of the pathway and leads to lethality. Tyrosine phosphorylation of Sty1 in the absence of stimulus was observed under these conditions and was dependent on a functional Wis1 protein. The lethality of N-wak1 can be suppressed by mutations in wis1 but not in mcs4, indicating that wis1 is most probably downstream of wak1 and that mcs4 is most probably upstream of wak1 (187). These and other results support the order Mcs4-Wak1-Wis1-Sty1 (references 178a, 187, and 188 and references therein). Other observations (see references 187 and 188 for details) have led to the proposal that Wis1 may be activated by an independent pathway, by analogy to the HOG pathway of budding yeast (but see reference 178a for an alternative view).

Integration of the response to stress with sexual development. The Sty pathway has proven to be involved not only in regulation of the stress response and mitosis (see above) but also in the initial step leading to sexual differentiation (Fig. 2; also see Fig. 11). Nitrogen starvation, which is required for conjugation and sporulation (see the section on the pheromone response pathway in S. pombe, below), induces the Sty stress response. One consequence of this activation is the transcriptional induction of ste11 (97, 200) (Fig. 2). This gene encodes a transcription factor required for the expression of genes necessary for the initiation of sexual development (199). Thus, an output of the Sty1 pathway, Ste11, subsequently participates in the transcription of genes which then activate the pheromone response pathway (Fig. 2; also see Fig. 11).

In contrast to the HOG pathway, which responds to hyperosmolarity, the PKC pathway is activated by low osmolarity. It is also activated in response to a variety of stimuli: nutrient sensing, thermal stress, and pheromones (38, 40, 95, 136) (Fig. 7). It is thought that a major role of the PKC pathway in response to these stimuli is to maintain cell integrity by controlling cell wall assembly and perhaps membrane assembly.

View larger version (23K): [in this window] [in a new window]   FIG. 7.   The PKC pathway controls cell integrity. The PKC MAPK cascade is activated in response to heat and low osmotic stress and nutrient limitation. Hcs77 is a putative mechanosensor that is proposed to sense membrane stretch. Pkc1 is proposed to activate a branched pathway (one branch is the MAPK and the other is hypothetical). Rlm1 appears to be a target for Mpk1. Solid arrows indicate activation; the dashed arrow is speculative. See the text for details.

Changes in cell wall composition can occur in response to growth medium, pheromones, cell fusion, cell cycle, and, in the case of pathogenic fungi, contact and growth within their host (136, 164, 230; see also reference 34). During apical growth of a yeast bud or a hypha in the cell cycle, the cell wall is in a dynamic state of change as new material is added at the growing point and subsequently modified to produce a structure capable of sustaining mechanical and chemical stress (reviewed in references 24 and 34). These modifications of the cell wall in response to diverse conditions entail activation of cell wall-synthesizing enzymes resident in the cell membrane and vectorial transport and exocytosis of vesicles that carry other wall and membrane components (reviewed in reference 34). The inability of a cell to modify its cell wall accordingly may result in a fragile cell wall and may lead to cell lysis, misshapen cells, or altered patterns of growth (for example, altered branching patterns or septum deposition).

Because one of the functions of the PKC pathway is to control the expression of genes encoding cell wall components and possibly also to control the vectorial transport of vesicles that may contain other components essential for cell integrity, this pathway is likely to be a major player during bud formation and hyphal extension. Studies of the PKC pathway in budding yeast should provide insights into how different input signals are integrated and mediate cell integrity.

Some notable features of the PKC pathway are as follows: (i) a small GTP binding protein (Rho1) controls the activity of the Pkc1 protein and also of glucan synthase, an enzyme involved in the synthesis of a major structural component of the fungal cell wall; (ii) a homolog of a phosphatidylinositol kinase regulates the activity of the Rho1 protein, perhaps linking phospholipid metabolism with cell wall synthesis and integrity in yeast; and (iii) a recently identified presumptive integral membrane protein may act as one of the sensors for the pathway.

PKC MAPK cascade and its activation. The MAPK cascade module that mediates the transduction of the signal generated by low osmolarity and heat stress consists of the MAPK Mpk1, two redundant MAPKKs, Mkk1 and Mkk2, and the MAPKKK Bck1 (Fig. 7) (38, 88, 89, 114, 115, 136, 206). The proposal for sequential activation by phosphorylation of the MAPK cascade is based on in vivo epistasis analysis and on structural relatedness to kinases in other pathways for which in vitro function has been established. The MAPK Mpk1 is tyrosine phosphorylated and activated in response to heat shock and hypotonic conditions in an Mkk1- Mkk2-, Bck1-, and Pkc1-dependent manner (40, 95, 230). Mpk1 tyrosine phosphorylation increases as osmolarity decreases from isotonic conditions. In contrast, as osmolarity increases, Hog1 tyrosine phosphorylation increases. Thus, these two MAPKs are activated by opposite osmolarity conditions (40).

(i) Activation by Pkc1. The MAPK cascade is activated by Pkc1 (Fig. 7), a serine/threonine protein kinase, although the mechanism of this activation is not known. Pkc1 contains several domains: a catalytic domain, a putative Ca²⁺ binding domain (C2 domain), a putative diacylglycerol (DAG) binding domain (C1 domain), and a pseudosubstrate domain. Thus, budding yeast Pkc1 resembles mammalian isoforms that require phospholipid, Ca²⁺, and DAG as cofactors for activation (118, 152). Mammalian protein kinase C enzymes (PKCs) govern cell growth, proliferation, differentiation, and other processes (reviewed in references 44 and 150). In mammalian cells, PKCs respond to extracellular signals through receptor-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP₃). DAG serves as a second messenger to activate PKC, and IP₃ functions to mobilize Ca²⁺ from intracellular stores (reviewed in reference 46). The pseudosubstrate domain is conserved in PKCs from yeast to mammalian cells and is proposed to maintain the PKCs in an inactive state in the absence of inducing signal (reviewed in reference 44). The fact that a mutation (Pkc1R398P) resulting in constitutive activation of yeast Pkc1 maps to the pseudosubstrate region supports this contention (152). Pkc1 is activated by Rho1 (see the section on Rho1 regulation of Pkc1 and glucan synthase, below).

mkk1 mkk2

(ii) Activation by pheromones. Mpk1 appears to be tyrosine phosphorylated and activated in response to pheromones (Fig. 7) (230). This phosphorylation is dependent on Bck1 and, surprisingly, also on Ste20, a protein kinase that activates the MAPK cascade of the pheromone response pathway (see the section on the pheromone response pathway in S. cerevisiae, below). Phosphorylation was observed 1 h after treatment with pheromone and was not dependent on Ste12. Pheromones induce the formation of a mating projection exhibiting highly polarized growth. Since Mpk1 has been implicated in polarized growth (proper chitin deposition, organization of cortical actin patches, and vectorial transport of vesicles), it is possible that pheromone induction of Mpk1 ensures cell wall integrity during projection formation (136, 230). In this context, it is worth noting that BCK1 mutants were identified in a synthetic lethal screen with mutants defective in SPA2, a gene required for projection formation during mating (38). The PKC pathway is necessary during projection formation but not during cell fusionan activated allele of Pkc1 blocks cell fusion (164). During cell fusion, localized degradation of cell walls occurs in the area of cell-cell contact. This degradation is essential to allow the fusion of cellular membranes to form a zygote. One possibility is that the PKC pathway is downregulated at this stage to allow cell fusion (see reference 164 for details).

(i) Activation of Pkc1. Rho1 belongs to the family of small GTP binding proteins which includes the Rho, Rac, and Cdc42 subfamilies. These proteins have GDP- and GTP-bound states and act as molecular switches regulating a variety of cellular processes (reviewed in references 172 and 203). The switch from one state to the other is controlled by two types of proteins: a guanine exchange factor (GEF), which promotes the transition from the GDP-bound form to the GTP-bound form, and a GTPase-activating protein (GAP), which promotes GTP hydrolysis and hence the transition from the GTP state to the GDP state. The GTP-bound form is usually the active form and interacts with target proteins. (Examples are known, however, in which both GTP- and GDP-bound forms have different functions [159].) Rho has been proposed to be involved in reorganization of the actin cytoskeleton in mammalian cells and, through this process, to affect cell morphology, motility, and cytokinesis (reviewed in references 172 and 203). In S. cerevisiae, Rho1 is localized at sites of active growth or secretionthe presumptive bud site, the bud tip, and the neck regionand has been proposed to play an important role in bud formation. Rho1 localization coincides with localization of actin patches during the cell cycle (229). Actin is proposed to be involved in the transport of vesicles to the actively growing regions. Whether Rho1 is involved in actin reorganization in yeast is not known at present. The similarity of phenotype of both pkc1(ts) and rho1(ts) mutants and their suppressibility by 1 M sorbitol led to the suggestion that both RHO1 and PKC1 act in the same pathway.

(ii) Rho1 activates glucan synthase. Yeast cell walls appear to be layered, with an inner, electron-transparent layer containing mainly glucan polymers and a fibrillar, electron-dense outer layer consisting mainly of mannoproteins. 1,3--Glucan polymers and mannoproteins are the major structural components of the yeast cell wall (reviewed in references 24, 34, and 87). In the filamentous Ascomycetes and in the Basidiomycetes, in addition to 1,3--glucan and mannoproteins, chitin is another major structural component (reviewed in reference 192). Synthesis of 1,3--glucan polymers is mediated by 1,3--glucan synthase (GS), a membrane-bound enzyme whose activity is stimulated by GTP. GS catalyzes the transfer of a glucosyl residue from UDP-glucose to a growing chain of 1,3--linked glucosyl residues (141; reviewed in references 24 and 87). Two genes, FKS1 and FKS2, encoding components of the catalytic moiety of GS have been identified in S. cerevisiae. Deletion of both FKS1 and FKS2 is lethal, whereas deletion of either is not, although the single mutants are very sensitive to cell wall inhibitors such as echinocandin. Both Fks1 and Fks2 exhibit a high degree of similarity at the amino acid level: 16 putative membrane-spanning domains, of which 6 comprise a block separated from the other 10 by a stretch of hydrophilic amino acids (50, 135). Because their structure resembles that of ATP binding cassette (ABC) transporters of bacteria (reference 50 and references therein), it is hypothesized that the glucan chain is extruded into the periplasmic space as it is synthesized (51).

(i) Rlm1, a transcriptional factor downstream of Mpk1. Rlm1 is a member of the MADS box of transcription factors, of which S. cerevisiae Mcm1 and mammalian serum response factor (SRF) are also members (218). The DNA binding domain of Rlm1 is most similar to that of mammalian MEF2 and appears to bind the same consensus sequence as MEF2 (47). The yeast genome contains a homolog of Rlm1, Smp1, which recognizes an extended version of the consensus recognized by Rlm1. The two proteins can form heterodimers (47). The C-terminal region of Rlm1, which may be the transcriptional activation domain, appears to be directly phosphorylated by Mpk1 in vivo and in vitro, suggesting that Mpk1 modulates the activation domain of Rlm1 (47, 217). Deletion of RLM1 or of SMP1 does not result in the cell lysis phenotype characteristic of deletion of the components of the PKC pathway. One possibility is that Mpk1 exerts its effects via targets in addition to Rlm1 and Smp1 (for details, see references 47 and 217).

(ii) Genes for cell wall biosynthesis. The expression of several genes involved in cell wall biosynthesis is cell cycle regulated in a SWI4- and SWI6-dependent manner and peaks at the G₁/S boundary, which coincides with active apical growth and extensive wall remodelling (84). These genes are FKS1, KRE6, MNN1, VAN2, CSD2, and GAS1, which encode, respectively, a subunit of GS, a protein for 1,6--glucan synthesis, proteins involved in mannosylation, a protein involved in glycosylation, chitin synthase III, and a glycosylphosphatidylinositol (GPI)-anchored membrane protein (reference 84 and references therein). Not surprisingly, these genes contain cell cycle regulatory sites in their 5' regions (84). Pkc1 is not required for this cell cycle regulation but is required for a high constitutive level of expression of FKS1, CSD2, MNN1, KRE6, and GAS1 throughout the cell cycle. It has been proposed that the PKC pathway and Swi4-Swi6 regulate the synthesis of these genes in a coordinated manner and that this might explain the synthetic lethality observed for swi4 or swi6 and components of the PKC pathway (84).

Hcs77, a putative receptor that senses membrane stretch. HCS77 encodes a protein of 378 amino acid residues with a single putative transmembrane domain and an N-terminal signal sequence (65, 155). A number of observations indicate that it functions in the PKC pathway. Deletion of HCS77 results in a phenotype that is similar to the phenotype observed for deletion of components of the PKC pathwaya temperature-dependent cell lysis phenotype that can be remedied by osmotic stabilizers. hcs77 bck1 and hcs77 mpk1 double mutants exhibit the same phenotype as the single mutants, consistent with the view that they act in the same linear pathway (65). Of particular importance is the observation that heat induction of Mpk1 activity was reduced in the hcs77 mutant strain. Taken together, these results suggest that Hcs77 may be an upstream regulator of Pkc1. Because the phenotype of hcs77 is not as severe as that of pkc1, it can be argued that Pkc1 can be activated in other ways.

View larger version (17K): [in this window] [in a new window]   FIG. 8.   Proposed activation of the Rho1 module by Hcs77. Hcs77, a putative mechanosensor, may activate Tor2, a phosphatidylinositol kinase homolog. Tor2 is proposed to activate Rom2, a GEF for Rho1-GDP. Alternatively, Hcs77 could activate Rom2 directly or could inhibit Sac7, a GAP for Rho1-GTP. This inhibition would promote the Rho1-GTP state. Rho1-GTP activates Pkc1, glucan synthase, and presumably other substrates. Solid arrows indicate activation; dashed arrows are highly speculative. See the text for details.

PHEROMONE RESPONSE MAPK CASCADE FOR MATING, MEIOSIS, AND FILAMENTOUS GROWTH

Top Previous Next References

Pheromone Response Pathway in S. cerevisiae

The pheromone response pathway of S. cerevisiae mediates cell-cell interactions during mating. This pathway has been intensively studied for many years and is one of the best-understood signalling pathways in eukaryotes. Studies with the pheromone response pathway of budding yeast have provided a framework for understanding how mitogenic factors regulate cell cycle progression in mammalian cells. An overview of this pathway follows (Fig. 9). (i) Peptide pheromones secreted by cells interact with seven-transmembrane receptors and initiate the response. (ii) The receptors interact with a heterotrimeric G protein that transmits the signal to downstream components. (iii) Partially redundant MAPKs mediate the activation of a transcription factor. (iv) One of the MAPKs phosphorylates a cyclin-dependent kinase inhibitor (CKI), leading to cell cycle arrest. (v) A component of the response pathway appears to act as a scaffold that brings together the MAPK cascade components and may prevent cross talk between this and other pathways. (vi) Many of the target genes for this pathway are known and include genes necessary for cell and nuclear fusion and for cell cycle arrest.

View larger version (16K): [in this window] [in a new window]   FIG. 9.   Pheromone response pathway of S. cerevisiae. The pheromone response pathway is activated upon binding of a-factor or -factor (solid sphere) the to serpentine receptors Ste3 and Ste2, respectively. Ste5 is shown as the shaded rectangle that holds Ste11, Ste7, and Fus3 or Kss1 together. Solid arrows indicate activation; dashed arrows indicate that evidence is not conclusive; lines with bars indicate inhibition. See the text for details.

MAPK cascade and Ste5 scaffold. The MAPK cascade (Fig. 9) consists of the MAPKKK Ste11, which phosphorylates and activates the MAPKK Ste7 on two residues conserved among all MAPKKs. Ste7 in turn phosphorylates and activates the redundant MAPKs Fus3 and Kss1 on conserved Thr and Tyr residues (56, 59, 122, 148, 235).