INTRODUCTION

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Despite their placid appearance, cells of the yeast Saccharomyces cerevisiae possess rapidly responding, highly complex signaling pathways. These pathways allow yeast cells to quickly adapt to a changing environment, a critical attribute for a nonmotile species. Prominent among yeast signaling pathways are the mitogen-activated protein kinase cascades (169, 249). These generally contain three protein kinases that act in series: a MAP kinase kinase kinase (MAPKKK or MEKK), a MAP kinase kinase (MAPKK or MEK), and a MAP kinase (MAPK) (66, 71, 290). Thus, when the cascade is activated, the MEKK phosphorylates the MEK, which in turn phosphorylates the MAPK. The MAPK cascades, found in animals (71, 290), plants (173), and fungi (118, 169), often regulate transcription factors by MAPK-mediated phosphorylation. Many extracellular and intracellular signals modulate transcription of specific genes through activation or inhibition of MAPK cascades.

Our understanding of the S. cerevisiae MAPK pathways is more complete than that of MAPK pathways in other organisms. Extensive genetic and biochemical analysis plus the complete sequencing of its genome has revealed that S. cerevisiae contains five MAPKs on five functionally distinct cascades (Fig. 1) (179). Four of these pathways, the mating pathway, the filamentation-invasion pathway, the cell integrity pathway, and the high-osmolarity growth pathway, are present in growing cells. The Smk1p MAPK, part of the spore wall assembly pathway, is not present in growing cells but appears during sporulation and regulates that developmental process. Another type of yeast, the fission yeast Schizosaccharomyces pombe, contains a set of MAPK cascades that have some similarity to those in S. cerevisiae. Although this review is focused on S. cerevisiae MAPK pathways, some similarities and, more importantly, differences between two related MAPK pathways in these two evolutionarily diverged yeasts are discussed. In this review, S. cerevisiae cells will be called yeast or budding yeast and S. pombe cells will be called fission yeast.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   MAPK cascades of S. cerevisiae. There are four MAPK pathways in vegetatively growing yeast and one, the spore wall assembly pathway, which is expressed only in sporulating yeast. Nomenclature for yeast genes and their products is as follows: STE20, gene name; ste20, recessive mutation; ste20, deletion (usually null) mutation; and Ste20p, protein product of STE20. The question marks indicate that a protein kinase has not yet been identified for this step in a cascade. Note that each cascade has a unique MAPK. In addition, certain protein kinases act in more than one pathway: the MEK Ste7p (two pathways), the MEKK Ste11p (three pathways), and the upstream MAPK cascade activator kinase Ste20p (two pathways). The arrows represent known or postulated steps in signal transduction; see the text for details.

The biochemical mechanisms mediating signal transduction among the three types of kinases in MAPK cascades are fairly well understood (397). MEKK has a regulatory domain at the NH₂ terminus and a protein kinase domain at the COOH terminus. When activated, MEKK phosphorylates both a serine and a threonine residue in a conserved domain in the NH₂-terminal portion of MEK. The phosphorylated and now activated MEK then phosphorylates MAPK on a threonine and a tyrosine residue, separated by a single amino acid, within the activation loop (199) of the conserved kinase domain, thereby activating the kinase activity.

Different classes of MAPKs exist in yeast and also in mammals. These can be classified by the pathways in which they participate and by the identity of the amino acid between the Thr and Tyr in the activation loop: Glu, Pro, or Gly in mammals, and Glu, Gly, or Asn in yeast. For example, the ThrGlyTyr MAPKs such as yeast Hog1p or mammalian p38 are found in stress-activated pathways and the ThrGluTyr MAPKs such as yeast Fus3p or mammalian ERK1 are found in growth factor-activated pathways (230, 397). Although the amino acid between the Thr and Tyr can be used to classify different MAPKs, other regions of the conserved protein kinase domain appear to play a more dominant role in determining the specificity of interactions with the upstream MEK and downstream substrates (49).

Despite a wealth of information on the MAPK cascade itself, there are many unsolved problems concerning this signaling device. The way in which the known upstream activators act on the cascade is still unclear. Identification of new target proteins for the MAPKs and novel activators of the MAPK pathways is still continuing. MAPK cascades appear to exist in cytoplasmic macromolecular complexes with other proteins that serve as scaffolds, anchors, or adaptors. Upon activation, MAPK or MEK is thought to move from the cytosol to the nucleus and phosphorylate target proteins such as transcription factors. How the cytoplasmic complexes of signaling proteins rearrange themselves during signaling to let MAPK or MEK go to the nucleus is not well understood. It is still unclear what determines the speed, magnitude, specificity, and duration of signaling through a MAPK cascade. The mechanisms by which signaling through MAPK pathways is integrated with that through other types of pathways is just starting to be studied. The yeast MAPK pathways are better characterized than those in other eukaryotes. The general principles of operation and the variations of this simple signaling cascade revealed in yeast and described here may thus help guide research on similar pathways in other eukaryotes. Each of the five yeast MAPK-containing pathways is discussed, starting with the mating-pheromone response pathway, the best understood of all eukaryotic MAPK pathways.

Yeast cells can exist as either haploid or diploid cells. Haploid cells of the opposite mating type (a or ) can mate, i.e., fuse and form a diploid. This process is stimulated by the release of small peptide mating pheromones, a-factor from MATa cells and -factor from MAT cells, that act on cells of the opposite mating type to prepare that cell for mating. Cellular responses to mating pheromone include polarized growth toward a mating partner, cell cycle arrest in G₁, and increased expression of proteins needed for cell adhesion, cell fusion, and nuclear fusion. A pheromone-activated signaling pathway that includes a MAPK cascade (Fig. 2) helps mediate many of these responses. Pheromone binds to and activates a seven-transmembrane domain receptor that in turn is thought to induce the dissociation of a heterotrimeric G protein (32, 99, 194, 316). As described below, the liberated G(Ste4p)-G(Ste18p) complex then activates downstream proteins Ste5p and Ste20p, and these in turn stimulate the Ste11p-Ste7p-Fus3p MAPK cascade. The MAPK Fus3p phosphorylates several downstream targets, e.g., Far1p, Dig1p, Dig2p, and Ste12p, that mediate various responses required for successful mating.

View larger version (41K): [in this window] [in a new window]   FIG. 2.   Pheromone response pathway of S. cerevisiae. The line with arrows connecting G to the GG indicates the ability of the protein subunits to form a complex in the absence of pheromone. , activation; , inhibition (these connections do not necessarily mean direct physical interactions). Proteins are labeled without the p suffix (e.g., Ste5 instead of Ste5p) to improve the legibility of the figure. See the text for details of signal transduction between different proteins on the pathway.

G activation of the mating-pheromone MAPK cascade is mediated primarily by Ste5p (51, 235, 446, 487) and Ste20p (236, 237, 499). Other proteins, such as Ste50p (387, 502) and Bem1p (204, 234, 243, 270), may also play a role in transducing signals from the G Ste4p, but their functions are not essential. Ste5p and Ste20p appear to be necessary and limiting for MAPK cascade activation by G (3, 61, 164, 218, 235). The way in which these proteins cooperate is not yet understood, but for each mediator there is some information about the transduction mechanism.

Ste5p, a scaffold for the MAPK cascade. Organization of signal transduction along pathways commonly involves scaffold, anchoring, or adapting proteins (364). Several lines of evidence argue that Ste5p is a scaffold protein for the pheromone-activated MAPK cascade. Ste5p associates with MEKK Ste11p, MEK Ste7p, and MAPK Fus3p (also MAPK Kss1p) in the two-hybrid system (61, 187, 287, 383) and in coprecipitation experiments (61, 218). Although these interactions could occur independently or indirectly, two observations suggest that Ste5p is a scaffold. First, Ste5p has separate binding sites for the different protein kinases (61, 187), and second, Ste5p appears to exist in a high-molecular-weight complex with these kinases (61), which has a high-specific-activity Fus3p kinase (62, 218). The sites of interaction of Ste5p with different protein kinases have been identified. Analysis of point mutations and deletions that block specific interactions (61, 187) showed that a single Ste5p polypeptide of 917 residues has separate sites required for binding Ste11p (residues 463 to 514), Ste7p (residues 744 to 895), and Fus3p (or Kss1p) (residues 241 to 336), respectively. Importantly, a mutation in a Ste11p binding site blocks signal transduction in the pathway, as revealed by a failure to complement the sterile phenotype of a ste5 strain, supporting a positive regulatory role for the scaffolding function of Ste5p in signaling (187).

(i) Oligomerization of Ste5p. Several studies have shown that Ste5p forms homo-oligomers in yeast. Because protein oligomerization has been implicated as a signal transduction mechanism in several systems (245, 322), this facet of Ste5p has received some attention. The existence of Ste5p oligomers was first suggested by observations of interallelic complementation of different ste5 mutants that did not complement a ste5 deletion on their own (505). The results of two-hybrid analysis and coprecipitation experiments confirmed the existence of Ste5p oligomers (127, 188, 505). Oligomerization of Ste5p does not require the MAPK cascade (505) and appears to be independent of mating pheromone (127, 505). Two domains of Ste5p, both located in the NH₂ terminus of the protein, mediate oligomerization (505). One domain (residues 335 to 586) overlaps the Ste11p-binding region, and the other (residues 139 to 239) contains a LIM (91, 404) or RING-H2 (40) domain. The LIM domain appears not to be essential for oligomerization, because ste5 mutants harboring deletions of the LIM domain still oligomerize efficiently based on two-hybrid analysis (505) and coprecipitation experiments (127). Two-hybrid analysis suggests that residues NH₂-terminal to the LIM domain may be essential for oligomerization (127, 283, 505).

(ii) G activation of Ste5p. G appears to activate the MAPK cascade through a direct interaction with Ste5p. Pheromone stimulates the binding of Ste4p to Ste5p (127), with Ste4p binding at the NH₂ terminus of Ste5p (487). Mutations in conserved cysteine residues of the RING-H2 domain block Ste4p binding (127, 188). These mutants are sterile and block pheromone-induced signal transduction (127, 188), although they still efficiently interact with Ste11p, Ste7p, and Fus3p (127, 188).

ste4 ste5

ste4 ste5

Ste20p regulation of the MAPK cascade. Upstream protein kinases that activate MAPK cascades have been identified in the pheromone response pathway, the filamentation-invasion pathway, and the cell integrity pathway. Ste20p is believed to be the upstream kinase that activates the MEKK Ste11p in the pheromone response pathway (236, 390). Ste20p also functions upstream of Ste11p in the filamentation-invasion pathway (257, 395). It is striking that Ste20p appears to have additional functions that are independent of MAPK cascade activation. These Ste20p functions include activation of myosin I function (238, 497, 498), adhesion of mating partners (239), and vegetative functions relating to budding (83) and cell elongation (396). Whether the separate functions of Ste20p are mediated by a single macromolecular complex or by separate protein complexes, each with a uniquely regulated Ste20p, remains unclear.

ste20 cla4

Cdc42p is required to orient the actin cytoskeleton to form a bud, to divide the cell during cytokinesis, and to form mating projections (1, 110, 254, 527). Cdc42p therefore interacts with a variety of different proteins that regulate actin cytoskeleton function. Cdc42p in cells exists in a dynamic equilibrium between the GDP-bound and GTP-bound forms. Exchange of GDP for GTP on Cdc42p is activated by Cdc24p (525), and the hydrolysis of the Cdc42p-bound GTP to GDP is predicted to be regulated by the GTPase-activating proteins (GAPs) Bem3p (525) and Rga1p (445). Cdc24p, like Cdc42p, is an essential protein required for polarized cell growth during bud formation and formation of mating projections during conjugation (60, 439).

Several observations suggest that Cdc42p plays an important role in the pheromone response pathway. Temperature-sensitive cdc24 or cdc42 mutants, when grown at a nonpermissive temperature, do not show an increase in FUS1-lacZ expression (FUS1 is a pheromone-induced gene [303, 466]) in response to pheromone treatment (435, 521) and cannot mate (393). Strains with the Cdc42-GAP Rga1p deleted show increased pheromone-induced transcription (445). Indeed, overexpression of a mutant Cdc42p locked in the GTP-bound state activates FUS1 expression (435, 521), even in a strain carrying a dominant negative mutant of the G Ste4p (435). The increased FUS1-lacZ expression in cells expressing an activated Cdc42p does require the presence of pheromone, suggesting that Cdc42p acts to modulate signaling by the pheromone response pathway.

Cdc42p appears to have multiple functions in the mating response, at least one of which does not involve the MAPK pathway. Yeast cells form mating projections in response to pheromone treatment. The growth of these projections is spatially oriented toward the source of pheromone and is therefore called chemotropic growth (416). This process involves activation of Cdc42p (335) but, importantly, does not require the protein kinases of the MAPK cascade (410). As discussed above, G Ste4p interacts with Ste5p and Ste20p and, by mechanisms yet unclear, activates the MAPK cascade. Ste4p also interacts with Cdc24p (335, 521), the guanine nucleotide exchange factor for Cdc42p. Mutations in Cdc24p that block the interaction with Ste4p also block chemotropic growth but have no effect on other responses to pheromone including MAPK cascade-mediated growth arrest and FUS1-lacZ expression (335). Because the function of Cdc24p is to activate Cdc42p and Cdc42p mediates polarized cell growth, the interaction of G with Cdc24p may provide a mechanism to locally activate Cdc42p and Cdc42p-dependent growth in the vicinity of pheromone-occupied receptors.

Bem1p, like Cdc42p, interacts with several proteins important for the function of the actin cytoskeleton in polarized growth (28, 57, 59, 110). Bem1p associates with actin and with the pheromone response pathway-signaling proteins Ste5p and Ste20p (243, 270). The Bem1p-bound Ste5p is complexed to the Ste11p-Ste7p-Fus3p MAPK cascade (270). Interaction of Ste20p with Bem1p is required for association of Ste20p with actin (243). The fraction of these signaling proteins associated with macromolecular complexes in the cell is considerable. At least half of the cellular Ste5p, Ste20p, and Bem1p localizes to a particulate fraction of the cell and remains there after extraction of membrane proteins with nonionic detergents (243). Bem1p interacts in cells with other signaling proteins: the Cdc42p guanine nucleotide exchange factor Cdc24p (298, 370); Far1p (270), a protein needed for pheromone-induced cell cycle arrest (55) (see below); and Boi1p and Boi2p (29, 298), proteins involved in the regulation of the Rho-type GTPase Rho3p and Rho3p-dependent growth-related processes.

Bem1p-associated proteins can have more than one function. For example, Far1p has two functional parts, a COOH-terminal domain required for chemotropism (107, 473) and an NH₂-terminal domain required for pheromone-induced cell cycle arrest (473). The observation that the MAPK cascade is required for cell cycle arrest (113) but not chemotropism (410) provides further confirmation that these are mechanistically separate responses to pheromone. The mechanism by which Far1p performs two very different functions is unknown. Thus, the multitude of interacting partners for Bem1p and their functional diversity raise the question whether a single Bem1p molecule can complex simultaneously with all potential partners or whether different Bem1p molecules form separate complexes with different protein partners.

So far, it has not been possible to detect an effect of pheromone on the extent of interaction between Bem1p, Ste20p, and Ste5p as assayed by coimmunoprecipitation experiments (243, 270). Thus, Bem1p might just simply tether the signaling pathway to the cytoskeleton. Bem1p does, however, facilitate signaling by the pheromone pathway. Deletion of BEM1 decreases the pheromone-induced transcription of FUS1 (204, 270). In addition, overexpression of BEM1 stimulates the kinase activity of the MAPK Fus3p (270) and suppresses the mating defect of a dominant negative STE4 mutant (234). These data suggest that Bem1p is involved not only in cross-linking the Ste5p-MAPK cascade complex to the cytoskeleton but also in transmitting signals to the MAPK cascade either directly or by facilitating its association with an upstream activator.

The pheromone-activated signaling pathway containing the Ste11p-Ste7p-Fus3p MAPK cascade is required for sending signals from the pheromone receptors in the plasma membrane to gene targets in the nucleus. There are no known second messengers relaying signals on the pathway. Therefore, some protein or protein complex must leave the cytoplasm and move across the nuclear membrane. In animal cells, MAPK moves from the cytoplasm into the nucleus following stimulation by growth factor (58). This movement involves dissociation of MAPK from its cytoplasmic complex with MEK (135). MEK appears to be in the cytoplasm and to remain there after growth factor treatment (522). However, more recent experiments suggest that MEK can also be induced to move from the cytoplasm to the nucleus following growth factor stimulation if its nuclear export signal (134) and catalytic site are inactivated by mutation (191). Disruption of the nuclear export signal in MEK strongly stimulates MEK-dependent morphological changes and malignant transformation (133). Thus, the apparent cytoplasmic localization of MEK in growth factor-stimulated cells may reflect transient nuclear entry followed by rapid export from the nucleus (133, 191). A leucine-rich sequence near the NH₂ terminus of MEK acts as the nuclear export signal (134); it is interesting that the yeast MEK Ste7p has a very similar sequence near its NH₂ terminus.

In the case of the yeast pheromone response pathway, it is still a mystery how the signal actually gets to the nucleus. Of the proteins on the MAPK cascade, the MAPK Fus3p appears to be present in the cytoplasm and nucleus (62). The MAPK Kss1p of the filamentation-invasion pathway (see below) is mostly in the nucleus (271). These MAPK locations change little after pheromone treatment. Due to their apparent low abundance, the locations of Ste11p and Ste7p in the cell have been more difficult to determine and are not known with certainty at present.

Ste5p does seem to change location after pheromone treatment, although whether nuclear entry of Ste5p is required for signaling has not yet been determined. At different times and under different conditions, Ste5p is alternatively found at or near the plasma membrane, in the cytoplasm, or in the nucleus. Microscopic analysis shows Ste5p to be present in both the cytoplasm and the nucleus in vegetatively growing cells (283). After pheromone treatment, Ste5p moves from the nucleus to the cytoplasm and becomes associated with the plasma membrane in mating projections (97, 283). Interaction of Ste5p with Ste4p is required for the association of Ste5p with the plasma membrane (97). The association of Ste5p with the plasma membrane appears to be a critical step in signal transduction, because fusion of membrane-targeting signals to Ste5p induces activation of pheromone responses in the absence of added pheromone (384). A striking result is that Ste5p with an NH₂-terminal truncation removing the G-binding domain is nonfunctional unless fused to membrane-targeting signals (384). Thus, plasma membrane localization of Ste5p is sufficient for signaling.

Ste5p localizes to the nucleus when untethered from G (97, 283). Thus, Ste5p may be part of the signaling machinery that shuttles signals to the nucleus, perhaps released from Ste4p in pheromone-activated cells. It should, however, be pointed out that nuclear localization of Ste5p is not sufficient for signaling (97, 283). In addition, the situation may not be as simple as a single protein or protein complex shuttling signals to the nucleus: there may be multiple mechanisms acting in parallel. Deletion of the MEK gene STE7 enhances Ste5p-Ste5p interaction in the two-hybrid system, suggesting that Ste7p-Ste5p and Ste5p-Ste5p complex formation might be mutually exclusive, i.e., that Ste5p dimerization might lead to Ste7p ejection (505). Ste5p preferentially interacts with the underphosphorylated, preactivated form of Ste7p, suggesting that phosphorylation of Ste7p might induce its release from the complex with Ste5p (61). Perhaps Ste7p, like the animal cell MEK (191), also carries signals to the nucleus. The tight complex formed between Ste7p and Fus3p (19, 21) suggests that instead of individual kinases, a complex of MEKK and MAPK may be the molecular species that carries signals to the nucleus. Movement of a protein or protein complex from the cytoplasm to the nucleus will require its dissociation from other cytoplasmic proteins. This may require more than one regulatory event or cooperative changes in protein conformation, especially in the case of ternary or higher-order complexes, where a protein must dissociate from more than one binding partner before it can break free of the complex (372).

Activation of transcription. Among the many aspects of the mating pathway that have been investigated so far, its regulation of transcription is fairly well understood. Pheromone stimulation activates the transcription of many different genes. Among the products of these genes are proteins that activate (e.g., Fus3p [113]) or inhibit (e.g., Msg5p [104, 519]) signaling on the pheromone response pathway and proteins needed for cell fusion (e.g., Fus1p [303, 466]), nuclear fusion (e.g., Kar4p [228]), and other mating-related functions. What these genes have in common is that they contain in their promoter region repeats of a pheromone response element (PRE) that is necessary and sufficient for pheromone regulated transcription (156, 222). The MAPK cascade mediates pheromone induction of transcription of PRE-containing genes through phosphorylation and activation of at least three nuclear proteins: Dig1p (68) (also called Rst1p [457]), Dig2p (68) (also called Rst2p [457]), and Ste12p (441).

dig1 dig2

dig1 dig2

The MAPK pathway plays another important role in mediating cell cycle arrest in response to pheromone (494). Conjugation of two haploid mating partners is accompanied by the synchronization of the cell cycles of the two cells such that they both contribute 1N content of DNA to the zygote product of their union. Thus, mating pheromone-treated cells arrest at a position in the cell cycle prior to bud formation and initiation of DNA synthesis: they arrest as unbudded cells with a 1N DNA content. Growth of the G₁-arrested cell is not inhibited but redirected into the formation of mating projections. This pheromone-induced cell cycle arrest in G₁ involves signaling through the MAPK cascade (112, 113, 132, 469) and the cell cycle inhibitor Far1p (55, 141, 469).

To explain the mechanism of cell cycle arrest and how the MAPK pathway is involved, we first review the mechanisms that regulate cell cycle progression at the G₁/S transition in yeast (331). Formation of a bud, initiation of DNA synthesis, and duplication of the spindle pole body mark the progression of a yeast cell into S phase, past a G₁/S transition point called START. These post-START events require the activation of cyclin-cyclin-dependent kinase complexes consisting of the kinase Cdc28p and one of three G₁ cyclins: Cln1p, Cln2p, or Cln3p. An active G₁ cyclin-Cdc28p complex is needed to induce the degradation of a cyclin-dependent kinase inhibitor that is specific for B-type cyclin-Cdc28p complexes (413). This protein inhibitor, called Sic1p (344) (also called Sbd25p [106]), blocks the activity of Cdc28p in complex with the B-type cyclins Clb5p and Clb6p but not the activity of G₁ cyclin-Cdc28p complexes. The B-type cyclin-Cdc28p complex, freed of its inhibitor protein, activates DNA replication (414). The mechanism responsible for activation of bud initiation by the G₁ cyclin-Cdc28p complex is independent of Sic1p (408, 468).

Cell cycle arrest by mating pheromone involves Far1p-dependent (367) and Far1p-independent processes (468). Far1p expression is normally restricted to the G₁ phase (305) by mechanisms of cell cycle-dependent transcription and protein turnover (167, 306, 347). Results from early studies indicated that Far1p is a cyclin-dependent kinase inhibitor that inhibits the activity of G₁ cyclin-Cdc28p complexes, but not that of B-type cyclin-Cdc28p complexes (196, 368). However, a more recent study could not detect a pheromone-induced reduction in the activity of the Cln2p-associated Cdc28p kinase, even though these complexes retain Far1p (141). Nevertheless, Far1p is required for pheromone-induced inhibition of G₁ cyclin-Cdc28p-dependent responses such as the expression of CLN1 and CLN2 (472). The MAPK Fus3p (but not Kss1p) is also required for cell cycle arrest in response to mating pheromone (112, 113). The functions of Fus3p and Far1p are linked, because pheromone induces the Fus3p-catalyzed phosphorylation of Far1p (56, 119, 141, 367, 469). G₁ cyclin-Cdc28p also phosphorylates Far1p (167, 367, 469) and thereby stimulates its degradation by a ubiquitin-dependent mechanism (167). Fus3p-catalyzed phosphorylation appears to have the opposite effect of stabilizing the Far1p protein (unpublished results cited in reference 167).

Far1p is a bifunctional molecule, required not only for cell cycle arrest but also for chemotropism (107, 473). This latter function is not connected to the function of the MAPK Fus3p or to that of the rest of the MAPK cascade (410). The way in which these two functions of Far1p are coordinated is not yet clear. Interestingly, the mechanism by which Far1p mediates cell cycle arrest is also not well understood at present (141).

The effects of pheromone on the cell cycle may be more complex than altering the activity of Far1p. In the absence of Cln1p, Cln2p, and the cyclin-dependent kinase inhibitor Sic1p, pheromone induces a Far1p-independent arrest of the cell cycle (468). In cells that have reduced activity of the Cln class of cyclin, another type of cyclin-cyclin-dependent kinase complex containing the Cdc28p-related protein Pho85p becomes critical for cell cycle progression (121, 308). The mRNA level for one of the Pho85p-associated cyclins, Pcl1p, is rapidly down-regulated by pheromone treatment (309) with a time course similar to that of the pheromone-induced decrease in Cln1p and Cln2p mRNA (495). Perhaps the Far1p-independent cell cycle arrest induced by pheromone treatment in cln1 cln2 sic1 cells (468) reflects parallel regulation of the Pho85p kinase through transcriptional control of expression of the Pcl1p cyclin associated with Pho85p. The Far1p-Cdc28p paradigm also suggests that the Pho85p inhibitor Pho81p (409) might be a target of regulation by the pheromone pathway. However, Pho85p interacts with many different cyclins, and its physiological functions appear to be very complex (9). For example, although Pcl1p and Pcl9p mRNAs are decreased by pheromone treatment, other Pho85p cyclins show no change or an increase in mRNA expression after addition of pheromone (309). Finally, the role of the MAPK pathway in the Far1p-independent cell cycle arrest by pheromone has not yet been determined.

As discussed above, the pheromone response pathway regulates the cell cycle but the converse is also true. For example, the basal level of protein kinase activity of MEK Ste7p and MAPK Fus3p fluctuates during the cell cycle, reaching a peak in early G₁ (481). The activity of the MAPK cascadehigh in early G₁ and low in late G₁correlates well with the amount of mRNA for different pheromone-dependent genes (346, 347, 517). These molecular changes in the absence of pheromone may allow the cell to be maximally responsive to pheromone in early G₁, a cell cycle position close to the pheromone arrest point in late G₁. The cell cycle regulation of the MAPK cascade and pheromone-dependent genes appears to be mediated through the G₁ cyclins Cln1p and Cln2p, cyclins that reach their peak expression level in late G₁ (392). Hence, overexpression of CLN2 represses the mating pathway (346, 481). Analyses of various mutants that either allow the Cln2p repression or block its effect suggest that the target of Cln2p repression is downstream of the G Ste4p. The MEKK Ste11p or one of the proteins involved in activating the MAPK cascade are the current candidates for the target of Cln2p repression (481). Another potential connection between Cln2p and the mating pathway is at the level of the MAPK substrates Dig1p and Dig2p, repressors of the transcription factor Ste12p (68, 457). Cln1p and Cln2p each show specific interactions with Dig1p and Dig2p in the two-hybrid system (457). While the functional significance of this interaction has not yet been determined, it is tempting to speculate that positive regulation of Dig1p and Dig2p by Cln2p-Cdc28p or Cln1p-Cdc28p might repress Ste12p and shut off the pheromone response, thereby enhancing recovery. In summary, there is a reciprocal relationship between the activities of the pheromone response pathway and the G₁ cyclin-Cdc28p complex, regulator of the G₁/S cell cycle transition, with each inhibiting the other. This situation allows the cell to make a clean switch from one function to the other, from budding to mating or vice versa.

Time is an important parameter when one considers the physiological and molecular properties of a signaling pathway like a MAPK cascade. For responding to environmental changes or a potential sexual partner, the rapidity of signaling in a pathway has tremendous selective advantage. Fus3p becomes phosphorylated and active within 1 to 2 min after -factor treatment (17, 62, 142). Other yeast MAPK pathways (discussed below) show a similar speed of response to stimuli. Another time-related factor is the relationship between the signal duration (e.g., pheromone) and the output response generated by the cell. Short-term activation of the MAPK pathway (~1 h) is sufficient to activate transcriptional responses to pheromone, while sustained activation (~3 h) is needed for cell cycle arrest (77). In a different system, the PC12 neuronal cell line, sustained activation of a MAPK cascade is required to induce differentiation and cessation of cell division. Transient activation of the cascade leads instead to increased cell proliferation (291).

Another time-related factor is the important function of turning off an activated pathway, allowing a cell to adjust to changing levels of an external stimulus. There are multiple mechanisms for down-regulating an activated mating-pheromone pathway, and attenuation of signaling on the MAPK cascade is part of the story. Following pheromone treatment, Ste7p activates the downstream MAPK Fus3p by inducing its phosphorylation (19, 114, 119, 271). However, the MAPKs Fus3p (119) and Kss1p (19) also phosphorylate the upstream MEK Ste7p; this phosphorylation appears to be part of a negative-feedback mechanism to shut off the MEK (142, 271, 526). Fus3p also phosphorylates the Ste5p scaffold protein (114), but the function of this modification is unknown. Several phosphatases act on the MAPK Fus3p: the dual-specificity phosphatase Msg5p (104, 519) and the tyrosine phosphatases Ptp2p and Ptp3p (519). The basal level of Fus3p phosphorylation is controlled mainly by the Ptp3p phosphatase (519). Pheromone treatment induces the expression of Msg5p (104), which then acts together with Ptp3p to inactivate Fus3p (519). Expression of PTP2 and PTP3 is not altered by pheromone treatment (519). Thus, deletion of these phosphatases delays the rate of recovery of pheromone-treated cells from cell cycle arrest whereas phosphatase overexpression speeds recovery. The location of these phosphatases in yeast is not known, but in animal cells similar phosphatases are localized to the nucleus (207).

Under specific culture conditions, diploid yeast will undergo a dimorphic switch and differentiate to form pseudohyphae, growing as filaments of extended and connected cells to form rough-edged colonies that invade solid medium. The physiological and genetic conditions necessary for this differentiation response have only recently been investigated. Starvation for nitrogen appears to induce the response (147), but other environmental factors may be important (260). Only a subset of commonly used laboratory strains have the right complement of genes to perform the switch (147, 258). The pseudohyphal response requires the cells to be diploid, although haploid strains can be induced to invade solid medium (395). The pseudohyphal response of diploid cells is characterized by changes in bud site selection from bipolar to unipolar, cell elongation, and invasive growth, each of which can be separated by mutation (323). This switch in cell properties from the "yeast" state to the pseudohyphal state probably involves multiple signaling pathways (31, 146, 147, 223, 264), one of which is very similar to the pheromone response pathway (257, 278, 395). The other pathways are not well defined at this writing, but at least one pathway appears to contain a G subunit encoded by GPA2 (223, 264). Gpa2p appears to act in the same pathway as a G-protein-coupled, seven-transmembrane receptor encoded by GPR1 (503), the Mep2p ammonium permease gene (262), and a downstream protein kinase encoded by SCH9 (460, 503). Although the interactions between upstream components in the two pathways are not fully resolved, here the term filamentation-invasion pathway will be used for the former pathway that contains a MAPK cascade similar to that of the pheromone response pathway.

The filamentation-invasion pathway (Fig. 3) contains a MAPK cascade (257, 395) that mediates signal transduction from two small GTP binding proteins, Ras2p (147, 324) and Cdc42p (324). Signaling from Ras2p requires the 14-3-3 proteins Bmh1p and Bmh2p (145, 396). Cdc42p acts downstream of Ras2p (324) and is required for the function of the PAK Ste20p in the filamentation-invasion pathway (239, 369). Cdc42p-Ste20p then transmits signal to the MAPK cascade. Like the pheromone response pathway, this cascade contains the MEKK Ste11p and the MEK Ste7p. However, the MAPK for the filamentation-invasion pathway is Kss1p (69, 278), in place of Fus3p (278). Also, the pheromone response pathway has Ste5p as a scaffold for the MAPK cascade (61, 218, 287, 383) while a MAPK cascade scaffold protein for the filamentation-invasion pathway has yet to be uncovered.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Filamentation-invasion pathway of S. cerevisiae. Symbols are as described in the legend to Fig. 2. See the text for details of signal transduction between different proteins on the pathway.

The filamentation-invasion pathway, like the pheromone response pathway, regulates transcription. Only two promoters have so far been identified as targets of the filamentation-invasion pathway: an upstream activating sequence in the Ty1 transposon (25, 276) and the promoter of the TEC1 gene (276). The filamentation-invasion pathway-responsive, cis-acting regulatory sequences in these promoters are related to those in pheromone-regulated genes. Both types of regulatory sequences contain a PRE (156, 222), the binding site for the transcription factor Ste12p (105). Promoters regulated by the filamentation-invasion pathway have one copy of a PRE in close proximity to a binding site for a second transcription factor called Tec1p (25, 276). The regulatory DNA sequence containing both Ste12p and Tec1p binding sites has been termed a filamentation and invasion responsive element (FRE) (276). An FRE is both necessary and sufficient for transcriptional regulation by upstream activating signals in the filamentation-invasion pathway (276, 324). Both Ste12p (257, 395) and Tec1p (143, 323) are required for the pseudohyphal response. The TEC1 promoter has an FRE, providing a positive-feedback mechanism for up-regulation of Tec1p in inducing the pseudohyphal response (276).

Dig1p and Dig2p act as negative regulators of Ste12p function in not only the pheromone response pathway but also the filamentation-invasion pathway (68, 457). Thus, dig1 dig2 cells show constitutive activation of the invasive growth response normally mediated by the filamentation-invasion pathway (68, 457).

Kss1p is the MAPK for the filamentation-invasion pathway (69, 277, 278). Historically, the observation that formation of pseudohyphae is not blocked by deleting any or all of the MAPKs in yeast led to an initial hypothesis that the filamentation-invasion pathway does not use a MAPK for signaling (257). For example, cells with or without the MAPK Kss1p show diploid pseudohyphal development on low-nitrogen medium, haploid invasive growth, and expression of FRE-lacZ (69, 257, 278, 395). However, cells with an inactivated Kss1p (with STE7 deleted or expressing a nonphosphorylatable mutant Kss1p in a kss1 background) do not undergo pseudohyphal development or haploid invasive growth and have reduced FRE-lacZ expression (20, 69, 278). These findings indicate that the unactivated form of the Kss1p kinase inhibits the pseudohyphal response. The haploid invasive growth response is inhibited not only by Kss1p but also by Fus3p (69, 278).

Induction of the pseudohyphal response by the MEK Ste7p appears to involve two effects. Ste7p-catalyzed phosphorylation of Kss1p relieves inhibition of the pseudohyphal response by Kss1p. Expression of wild-type Kss1p or a catalytically inactive but phosphorylatable mutant of Kss1p allows a kss1 fus3 strain to show invasive growth and normal levels of expression of FRE-lacZ (20). In contrast, a nonphosphorylatable Kss1p does not allow these responses (20, 69, 278). The mechanism by which nonphosphorylated Kss1p inhibits invasive growth and FRE-dependent transcription appears to be mediated by binding of the unactivated MAPK to the transcription factor Ste12p. For example, a mutant of Kss1p that binds normally to Ste7p and to the Ste12p-repressors Dig1p and Dig2p but not to Ste12p was isolated. This mutant Kss1p can no longer inhibit the pseudohyphal response (20).

Ste7p-catalyzed phosphorylation of Kss1p not only removes a repressor (unphosphorylated Kss1p) but also appears to generate an activator (phosphorylated Kss1p). This dual role of Kss1p can be appreciated by comparing wild-type and kss1 strains. Although cells lacking (the repressor) Kss1p show some invasiveness and expression of FRE-lacZ, the levels of each are significantly lower than that observed for KSS1⁺ cells (69, 278). Expression of hyperactive forms of either the MEKK Ste11p or the MEK Ste7p induces a strong pseudohyphal response and greatly increased FRE-lacZ expression (257, 276, 278). Cells with KSS1 deleted show no response to expression of these hypermorphic mutants (278), providing further support for the idea that Kss1p in its phosphorylated, active state is a positive regulator of the pseudohyphal response.

Kss1p has also been proposed to be part of the pheromone response pathway MAPK cascade. Kss1p, in the absence of Fus3p, allows near-wild-type levels of mating (112, 142), suggesting that Kss1p may also play a part in signaling by the pheromone response pathway. A fus3 kss1 strain is thus completely sterile. Further support for Kss1p as a mediator of mating pheromone responses is that pheromone treatment increases Kss1p kinase activity (19), although the fold increase is much lower than that for pheromone stimulation of Fus3p kinase activity (114). Furthermore, Kss1p interacts in the two-hybrid system with the pheromone pathway scaffold protein Ste5p (61), although whether this interaction is mediated through the MEK Ste7p was not tested.

However, it has been recently argued that Kss1p is not normally part of the pheromone response pathway and fills that role only when Fus3p has been deleted. Rather, it was proposed on the basis of several observations that Fus3p is the MAPK for the pheromone response pathway (277, 278), just as Kss1p is the MAPK for the filamentation-invasion pathway (69, 278). Kss1p cannot fully cover for the loss of Fus3p. For example, pheromone-induced cell cycle arrest requires Fus3p and Kss1p cannot mediate this response (112). As mentioned above, pheromone does increase Kss1p kinase activity but the increase is much lower than that for the Fus3p kinase. Pheromone effects on Kss1p kinase activity were also tested under conditions of Kss1p overexpression (19), in which Kss1p could artifactually compete with Fus3p.

Deletion of FUS3 may thus allow Kss1p to perform new functions; e.g., pheromone induces a Kss1p-dependent increase in FRE-lacZ expression but only in fus3 cells (278). A fus3 strain shows increased haploid invasive growth; kss1 or ste4 suppresses this phenotype. Haploid invasive growth of a wild-type FUS3 strain is not inhibited by ste4 (278). These observations suggest that in the absence of Fus3p, Ste4p inappropriately signals to Kss1p and therefore activates FRE-dependent transcription of invasive growth genes. One mechanism to explain the fus3 phenotype is that the absence of Fus3p allows Kss1p to bind to the MAPK binding site on Ste5p and receive signals from pheromone. The observation that a strain expressing a catalytically inactive mutant Fus3p in a fus3 strain is more sterile than a fus3 strain (278) is consistent with this possibility. The inactive Fus3p mutant had no effect when expressed in a wild-type FUS3 strain (278), showing that the mutant is not acting as a dominant negative mutant to Fus3p and, by extension, to Kss1p. Although many of these data support a model in which Fus3p is the MAPK for the pheromone response pathway, additional experimental tests are needed to fully resolve this point. For example, it is important to know whether addition of mating pheromone induces the activation of Kss1p phosphorylation or kinase activity with similar kinetics to the observed activation of Fus3p, particularly under conditions where both proteins are present at wild-type expression levels. In addition, it is important to know whether Kss1p is physically associated with Ste5p in cells under conditions of normal expression levels for both proteins.

Yeast cells use the same signaling proteins (Ste20p, Ste11p, Ste7p, and Ste12p) in two different pathways that receive different input signals and generate different outputs. Pheromone induces mating, and nitrogen starvation induces filamentation and invasion. Three factors are important in matching input signal to output response by using the same signaling proteins for the central part of two different pathways. Cell-type-specific gene expression is one such factor. To respond to mating pheromone, cells need receptors for the pheromone (Ste2p and Ste3p) plus a G protein (Gpa1p-Ste4p-Ste18p), a MAPK cascade scaffold protein (Ste5p) to transmit the signal from the receptors to the MAPK cascade, and a MAPK (Fus3p) to induce cell cycle arrest. Diploid cells do not express these components and therefore cannot respond to mating pheromone (113, 163, 235, 326, 366, 442, 486). However, haploid cells can activate either a pheromone response pathway or a filamentation-invasion pathway (395). A second factor important for determining pathway specificity is a protein complex that allows specific input signals to the MAPK cascade and then to the transcription factor. One pathway-specific protein complex has been identified for the pheromone response pathway (e.g., the Ste4p-Ste5p-MAPK cascade) but the corresponding complex for the filamentation-invasion pathway is unknown. How pathway specificity is generated at the steps involving the PAK Ste20p and the transcription factor Ste12p, respectively, has not yet been determined. The final factor important for generating specificity is input from one or more additional pathways, a critical factor for the filamentation-invasion pathway (146, 223, 259, 264). Although the molecular details of transcriptional regulation during the pseudohyphal response are sketchy at present, it seems reasonable to expect that the combination of signals from different pathways dictates which genes to turn on and which to keep off.

At first glance, a pathway with a MAPK cascade appears to be selected for speed, responding rapidly to an environmental stimulus. Proteins are complexed so that there are few steps at which a protein must diffuse randomly through the cell to find the next signaling protein in the pathway. The pheromone response pathway (142), the cell integrity pathway (89), and the HOG pathway (44) all can activate their MAPKs in minutes after initial stimulus. The large-scale cellular responses, e.g., cell adhesion and fusion during mating, to pathway activation are of course very much slower. Nevertheless, the cellular responses to these pathways still occur within one cell generation, approximately 1.5 to 3 h.

The filamentation-invasion pathway and the responses it mediates seem much slower by comparison. Growth in medium that contains limiting amounts of nitrogen activates this pathway (324) and elicits the pseudohyphal response in diploid cells (147). Depletion of cellular nitrogen is likely to present a rather slow, graded stimulus rather than a rapid, step-like stimulus like a decrease in osmolarity or addition of mating pheromone. Typical responses such as filamentous growth and invasion of agar reflect the concerted activity of many cells (147, 221, 395). Expression of the FRE-lacZ reporter for the filamentation-invasion pathway is usually assayed after growing yeast strains for many generations (324). Thus, one could view the filamentation-invasion pathway as a potentially fast pathway mediating slow responses to a slow signal. Whether this pathway can react quickly, or even needs to do so, remains to be determined. It could be that the filamentation-invasion pathway allows for a slow increase in signaling, integrating many different inputs (e.g., nitrogen starvation, carbon starvation, or a change in the surrounding physical environment) until some threshold is reached and a switch is activated.

One real gap in our understanding of the filamentation-invasion pathway is the nature of the true activating physiological signal(s) for this pathway. More genetic and physiological analysis of the nitrogen limitation condition of the cell is needed to determine what aspect of nitrogen metabolism more directly activates the MAPK cascade and its downstream gene targets in diploids. Formation of cellular filaments by haploid cells does not require nitrogen limitation per se but appears to be triggered instead by nutrient limitation (395). The physical nature of the growth medium could also play a role in activating the pseudohyphal response (260). Dimorphic switching of bacteria to a hyperflagellated, swarming-motility cell type is induced by changes in the agar support, i.e., the physical properties of the growth medium (162). Certain fungi appear to be capable of sensing external mechanical stimuli (175). How direct is the effect of nutrient limitation on the filamentation-invasion pathway? Are new proteins expressed that activate the pathway, or do preexisting proteins mediate pathway activation? Although Ste20p, Ste11p, Ste7p, and Kss1p appear to be constitutively expressed in haploid and diploid cells, other, as-yet-uncharacterized activators of the MAPK pathway might be expressed in response to nutrient deprivation. There is precedent for this type of mechanism. For example, components of the spore wall assembly pathway (see below) are expressed during the time preceding the events they regulate (131, 220).

A second MAPK cascade is found in budding yeast as part of the cell integrity pathway (Fig. 4). This pathway mediates cell cycle-regulated cell wall synthesis and responds to different signals including cell cycle regulation, growth temperature, changes in external osmolarity, and mating pheromone. Signaling proteins on the pathway include the GTP binding protein Rho1p (202, 340), the protein kinase C homologue Pkc1p (250), the MEKK Bck1p (242) (also called Slk1p [73]), the redundant pair of MEKs Mkk1p and Mkk2p (190), the MAPK Slt2p (465) (also called Mpk1p [241]), and the transcription factor targets Rlm1p (103, 483, 484) and SBF (275), the latter being composed of the polypeptides Swi4p and Swi6p (101, 211). There are probably many branches onto and off this pathway. For example, Rho1p interacts with and regulates more proteins than Pkc1p does (109, 170, 185, 214, 272, 300, 386) and Pkc1p regulates more than just the MAPK cascade (177, 242). Membrane proteins that potentially provide input signals to the cell integrity pathway include Wsc1p (476) (also called Hsc77p [151]), Wsc2p (476), and Wsc3p (476). Of all the MAPK cascades in yeast, this pathway is the most similar to the classical mitogen-activated ERK1-ERK2 MAPK cascade in animal cells in functional tests of the pathway components (37, 241, 256, 507). Also, both the mammalian MAPK cascade and the yeast cell integrity pathway have the same general function in their respective systems: to positively regulate growth and cell proliferation (151, 275, 288, 291, 518).

View larger version (45K): [in this window] [in a new window]   FIG. 4.   Cell integrity pathway of S. cerevisiae. This pathway appears to be regulated by several different signals listed at the top: nutrients, temperature, osmolarity, pheromone, and cyclin-dependent kinase (CDK). Where these different upstream signals feed in to the pathway is currently unknown. Whether Rlm1p and SBF mediate pathway regulation of separate (as shown) or overlapping sets of genes is not known.

Cell cycle regulation. Formation of a new bud occurs at the G₁/S transition, after START (251, 252). This process requires the activity of the cyclin-dependent kinase Cdc28p in complexes with the G₁ cyclins encoded by CLN2, CLN2, and CLN3. Among the periodic events of the cell cycle that peak at the G₁/S transition is the transcription of cell wall genes (181). Several observations suggest that Cdc28p regulates the cell integrity pathway, which in turn induces cell wall gene expression. Increasing or decreasing the activity of Cdc28p in cells induces corresponding increases or decreases in the tyrosine phosphorylation and kinase activity of the MAPK Slt2p (288, 518). Mutations in the cell integrity pathway kinases show synthetic lethality with cdc28 mutations (288, 302). Measurements of the amount of tyrosine phosphorylated Slt2p at different points in the cell cycle show a peak of phosphorylated Slt2p at the G₁/S transition (518). The time of maximum Slt2p phosphorylation thus correlates with the time of polarization of growth toward the bud tip (251). This peak of Slt2p phosphorylation correlates approximately in time with the peak in G₁ cyclin-Cdc28p activity and an increase in cell wall gene expression (181). PKC1 is required for cell cycle-dependent expression of a subset of cell wall genes (FKS1, MNN1, and CSD1) (181). A mechanism by which the cell integrity pathway regulates the expression of the cell wall genes is discussed below.

Heat stress activation. The cell integrity pathway is required both for growth of yeast at elevated temperatures (242, 250) (see below) and also for induced thermotolerance (201), i.e., the ability of cells to better survive severe heat shock if they are first exposed to mild heat shock. Not only is the MAPK cascade of this pathway required for growth and viability under heat stress conditions, but also the pathway is activated by heat stress. Increasing the growth temperature from 23 to 39°C induces the tyrosine phosphorylation of the MAPK Slt2p (201, 518) and increases its kinase activity (201). The change in Slt2p kinase activity is large (~100-fold), requires Pkc1p and the MEKK Bck1p, develops ~20 min after heat shock to its maximum at 30 min, and does not require new protein synthesis (201). The slowness of the response suggests that heat stress is not the primary signal for activation of the cell integrity pathway but instead reflects a physiological property of the cell affected by heat stress. Analysis of Slt2p tyrosine phosphorylation shows that raising the temperature increases the overall activity of the cell integrity pathway but has little effect on its periodicity during the cell cycle (518). After heat stress, Slt2p phosphorylation still peaks in late G₁ and early S phase, although the S-phase Slt2p phosphorylation appears to be increased relative to that observed at lower temperature (518).