The proliferation of MAP kinase signaling pathways in yeast

The proliferation of MAP kinase signaling pathways in yeast David E Levin and Beverly Errede Johns Hopkins

University,

Baltimore,

Mitogen-activated

protein

distinct,

pathways

signaling

response

to mating

growth,

cell

osmolarity.

kinases function

include

pseudohyphal

sporulation,

kinases

of North Carolina,

in at least five,

in yeast. These

pheromone,

integrity,

These

and University

and their

and

to

activating

Hill,

USA

physiologically

pathways

development

response

upstream

Chapel

that mediate and invasive

high

extracellular

kinases

signaling modules that, in at least some cases, exist as multiprotein

comprise

complexes.

Studies during the past year have revealed that the Ste5 protein of the mating pheromone

response

among the protein

pathway

serves as a scaffold to promote

kinases in this pathway, and to prevent their with

Current

Opinion

interactions interaction

kinases of other modules. in Cell Biology

Introduction Intracellular signaling modules made up of three sequentially acting protein kinases couple a diverse array of upstream signaling events to a variety of outputs in eukaryotic cells (reviewed in [l-4]). The enzymes of the core modules are members of families known as mitogenactivated protein kinases (MAPKs, also called extracellular signal regulated kinases, ERKs), MAPK/ERK kinases (MEKs), and MEK kinases (MEKKs). The use of a series of protein kinases in such modules provides both a potential mechanism for rapid signal amplification and multiple sites for integration of signal input and divergence of signal output. This organization also provides multiple sites for signal modulation. The budding yeast Saccharomyces cerevisiae uses MAPK signaling modules in at least five physiologically distinct signaling pathways (reviewed in [5-B]). This brief review updates the expanding list of MAPK activation pathways in yeast, and summarizes the organization of these pathways. In most cases, related but separate MAPK modules have evolved to mediate each pathway. A striking recent discovery reveals, however, that in some cases different pathways use MAPK modules with shared components. This finding, combined with the multiplicity of MAPKmediated pathways in yeast, raises the issue of how the cell is able to direct a signal through the appropriate set of protein kinases to activate the required output. We also discuss the mechanisms by which fidelity among pathway components may be maintained.

Mating response pathway Haploid S. cerevisiae cells of opposite mating types can fuse with each other to form a zygote that gives rise to diploid descendants. The binding of peptide mating

1995,

7:197-202

factors from one cell type to specific receptors on the opposite cell type coordinates the requisite interactions between the two mating partners. These peptides act as negative growth factors that cause the responding cell to arrest in the G1 phase of the the cell cycle. The peptides are also differentiation factors that induce changes in cell morphology (formation of mating projections) and in the transcription of genes necessary for cytoplasmic and nuclear fusion. The intracellular signaling pathway that mediates the response to mating factor comprises a heterotrimeric G protein and several sequentially acting protein kinases [6] (Fig. la). Binding of mating factor to its receptor triggers dissociation of the G protein. The free G&r dimer stimulates a MAPK activation module by an as yet unknown mechanism that involves the Ste20 and Ste5 proteins. The Stell protein (the MEKK of this module) phosphorylates and activates Ste7 (the MEK), which in turn phosphorylates and activates Fus3 and Kssl (the MAPKs) [9*,10]. Both Fus3 and Kssl phosphorylate Ste7, Ste5, and the Ste12 transcription factor, though in none of these cases has the functional relevance of the modification been proven [ll-131. The Far1 protein, a negative regulator of Gl-cyclin/CDC28 function, and thence of cell cycle progression, is a substrate of Fus3 not shared with Kssl. There is evidence that the FuB-phosphorylated form of Far1 preferentially interacts with the cell cycle machinery [I4,15]. The existence of multiple MAPK modules in a single cell brings into focus the question of how separate pathways maintain the fidelity of signal transmission. This past year brought unexpected insights into this question. Results from two-hybrid analyses revealed that Stel 1, Ste7, Fus3, and Kssl associate independently with Ste5. Similar analyses also showed that some of the protein kinases associate with each other independently of their association

Abbreviations HOG-high

osmolarity

glycerol; MAPK-mitogen-activated

protein

kinase; MEK-MAPIVERK

0 Current Biology Ltd ISSN 0955-0674

kinase; MEKK-MEK

kinase.

197

198

Cell regulation

(a) Mating

(b) Pseudohyphal development

(c)

(4

(4

Cell integrity

High osmolarity glycerol response

Sporulation

Fig. 1.

8 1995 Current Opinion in Cell Biology

Comparison

pathways

with Ste5 [16**,17*,18*]. These findings suggested that Ste5 and the protein kinases form a physical complex. Support for this proposal came from biochemical studies showing that Ste5 and the kinases co-sediment in glycerol gradients [16**]. These various observations are consistent with Ste5 acting as a scaffold for the components of the MAPK module. This physical organization could facilitate both activating and attenuating interactions between the kinases. It may also minimize cross-interactions between these kinases and those of other MAPK modules. Support for the latter role has come from the behavior of a gain-of-function Ste7 variant, whose ability to function within other pathways (such as the cell integrity pathway) is restricted by the presence of Ste5 (B Yashar, B Errede, unpublished data). Another question that arises from the multiplicity of MAPK activation pathways in yeast is whether there are mechanisms for the coordination of their activities. Recent studies on the mating response documents that such coordination is important. As part of the mating process, cells undergo polarized growth to elaborate mating projections. If cells are defective in the MAPK module controlling cell integrity, they lyse as they attempt to make these projections [19]. Furthermore, treatment of cells with mating factor activates Mpkl, the MAPK of the cell integrity pathway. This activation is not an early response to mating factor, but is coincident with projection formation [19], which occurs approximately one hour after activation of the mating pathway [20]. Although the mechanism for this coordination has not been determined, the timing of events suggests the possibility that projection formation generates the signal for activation of Mpkl (see below). Interestingly, Ca2+ and the putative Mid1 Ca2+ channel are also required at this point in the mating process [21,22”]. These correlations hint that

of MAPK

activation

in S. cerevisiae.

Ca2+ could be a second messenger involved in activation of the cell integrity pathway during morphogenesis. Pseudohyphal

development

and invasive growth

pathways Diploid cells can undergo a dimorphic switch to a pseudohyphal form in response to nitrogen starvation. During this switch, cells change their shape and budding pattern to form chains resembling the hyphae of filamentous fungi [23]. Haploid cells growing on agar plates also respond to nutrient deprivation by penetrating the agar, a process termed the ‘invasive growth response’ [24*]. Induction of the pseudohyphal program and the invasive growth response depends on the function of a MAPK module that uses components from the mating response pathway (Fig. lb). The Ste20, Stell, and Ste7 proteins are required for these responses, but Ste5, Fus3, and Kssl are not [24*,25*]. Presumably at least one distinct MAPK functions as part of the module(s) mediating the responses to nutritional cues. The Ste12 transcription factor, one of the targets of the mating pathway, is also required for the starvation responses [24*,25*]. Although there is no information on Stel2-dependent transcriptional responses to starvation conditions, one possibility is that Stel2 acts cooperatively with transcription factors that are specific to the starvation pathway. Because Ste5 is not required for either nutritional response, it is tempting to speculate that analogues of Ste5 are present and that these function to put the kinases in the appropriate physical assemblies. The situation is particularly interesting in haploid cells where all the components for the mating and invasive growth pathways co-exist (some of the components of the mating

MAP kinasesignalingpathwaysin yeast Levin and Errede 199 pathway are not expressed in diploid cells). In this regard, the finding that Fus3 and Kssl are not required for, but play a regulatory role in, the invasive growth response [24 °] could be explained if they influence associations of Ste11 and Ste7 with different scaffolds.

Cell integrity pathway mediated by protein kinase C The S. cerevisiae PKC1 gene encodes a homolog of the a-, ~-, and T-subtypes of mammalian protein kinase C [26]. Although Pkcl exhibits a similar substrate specificity to that of the mammalian enzymes, its activity is not stimulated in vitro by cofactors such as phospholipids, Ca 2+, or diacylglycerol [27,28]. Because a mutation in Pkcl that is predicted to confer cofactor independence on the enzyme resulted in elevated activity in vitro, it was proposed that this enzyme is regulated by cofactors, but that appropriate in vitro conditions for activation have not yet been found [27]. A second yeast gene encoding a protein kinase C isoform was reported recently, but this claim was shown to be spurious [29]. Loss of PKC1 function results in a cell lysis defect that is due to a deficiency in cell wall construction. Addition of osmotic stabilizing agents to the growth medium prevents cell rupture and allows proliferation [30,31]. Several additional components of this signaling pathway were identified by isolating genes whose mutational activation or expression at high levels coUld suppress the cell lysis defect resulting from pathway inactivation. Among theseare four genes that encode protein kinases proposed to catalyze a protein phosphorylation cascade (Fig. lc). These include a MEKK homolog (BCK1; [32]), a redundant pair o f M E K homologs ( M K K 1 / M K K 2 ; [33]), and a MAPK homolog (MPK1; [34]; initially designated SLT2; [35]). Deletion of any of these components resuits in cell lysis when cells are grown at 37°C [32-35]. Because loss of PKC1 function results in a more severe defect (lysis at any temperature) than does loss of function of the downstream components, it was proposed that Pkcl mediates a bifurcated pathway, with the MAPK cascade functioning on one branch [6]. The function of the other branch is not yet clear. Direct activation of Bckl by Pkcl has not been demonstrated, but such an. interaction is suggested by the observation that the latter selectively phosphorylates the former in vitro [36]. The most interesting recent development regarding this pathway concerns identification of the signals that activate the MAPK branch. Studies of Mpkl protein kinase activity (or tyrosine phosphorylation of Mpkl by M k k l / 2 as an indirect measure of Mpkl activation) have revealed that this enzyme is activated by three signals. Activation results from growth at high temperature, a drop in extracellular osmolarity (3( Kamada, DE Levin, unpublished data; M C Gustin, personal communication), or exposure to or-factor (see above). The activity of Mpkl is modulated over a 170-fold range by changes in growth temperature - - the enzyme is very

weakly active in cells growing at 23°C, moderately active in cells growing at 30°C (the optimal growth temperature for this species), and highly active in cells growing at 37°C. The time course of activation of Mpkl by elevated temperature is relatively slow (peaking in 20-30 min), but its activity is sustained indefinitely. In contrast, Mpkl activation by reduced osmolarity is very rapid (peaking within I min), but transient, with activity returning to pre-activation levels within approximately 5 min. Activation of Mpkl in response to elevated temperature or reduced osmolarity requires the function of pathway components proposed to lie upstream, including Pkcl. Although these signals may represent independent inputs to the same pathway, it is also possible that they activate the pathway through a common mechanism - - stretch of the plasma membrane. Coordinate expansion of the plasma membrane and the cell wall during growth and a-factor induced morphogenesis is essential for maintenance of cell integrity, but the gross distortion of these structures during the formation of mating projections may cause an imbalance to develop that could be detected as stretch of the membrane. The observation that survival of the morphogenesis induced by et-factor requires Mpkl, and that this enzyme is activated at the onset o f projection formation, is consistent with this model. Similarly, a persistent imbalance between wall and membrane expansion could develop during cell growth at elevated temperature due to increased membrane fluidity. This model could explain both the delay in activation of Mpkl by these signals (the time required for an imbalance to develop), and the persistence of its activity in cells growing at elevated temperature. In support of this model is the observation that growth in high osmolarity medium prevents the activation of Mpkl by elevated temperature (3( Kamada, DE Levin, unpublished data), presumably by preventing the membrane from stretching. Finally, an abrupt increase in turgor pressure, such as that caused by a rapid reduction in extracellular osmolarity, would cause the plasma membrane to stretch. However, once pressure is relieved by normalizing intracellular osmolarity, the pathway could be inactivated, consistent with the observation that Mpkl activation is transient under these conditions. Yeast cells possess mechanosensitive ion channels [37] which could act as sensors for activation of this pathway. Interestingly, the isolation of a mutant in a gene encoding a candidate for such an ion channel was reported recently from a screen for mutants that die when exposed to 0t-factor [22°°]. The Pkcl protein behaves in vitro as though it is part of a complex with other proteins [27,28]. The results of twohybrid analyses with PKC1 also suggest that Pkcl is in a complex with other pathway components (G Paravicini, M Snyder, personal communications). Specifically, Pkcl interacts with both Bckl and Mkkl, and, interestingly, the interaction between Pkcl and Mkkl is disrupted in a bck I deletion mutant (G Paravicini, personal communication). Although a STE5-1ike component that functions

200

Cell regulation

within the PKCl-mediated pathway has not been identified, the existence of such a component could explain these interactions.

High osmolarity glycerol response pathway When yeast cells are exposed to conditions of high extracellular osmolarity, they respond by producing high intracellular concentrations of glycerol and reducing membrane permeability to this solute in an effort to re-establish osmotic equilibrium with the environment. The signaling pathway that mediates this response uses the MAPK homolog Hog1 [38] and the MEK homolog Pbs2 [39] (Fig. Id), which are required for cell growth in high-osmolarity medium. PBS2-dependent tyrosine phosphorylation of Hog1 is induced rapidly upon exposure of cells to increased extracellular osmolarity [38]. In this regard, the high osmolarity glycerol (HOG) response pathway is activated in opposite fashion to the Pkcl-mediated pathway, which responds to reductions in osmolarity. Recently, elegant work described by Meada et al. [40**] indicated that signaling via the HOG pathway is mediated by a putative transmembrane receptor, designated Slnl . The SL.Nl gene is essential for growth and encodes a protein that contains a region related to the sensor histidine kinases of bacterial two-component signaling systems, and another region related to the response regulator proteins of the same systems [41]. A search for mutations that suppressed the phenotype of an sZn7 mutation yielded recessive mutations in PBS2, HOGl, and a novel gene, designated SSKl (suppressor of sensor kinase) [40**]. These observations suggest that Slnl negatively regulates the HOG pathway and that sin 1 mutants are inviable because they allow constitutive activation of this pathway The Sskl protein is closely related to bacterial response regulator proteins (targets of sensor histidine kinases), suggesting that it is coupled to Slnl. Overproduction of Sskl results in tyrosine phosphorylation of Hogl, indicating that the putative response regulator positively regulates the MAPK. Meada et al. [40**] have proposed a model in which Slnl inhibits Sskl activity, and increased extracellular osmolarity inhibits the presumptive Slnl histidine kinase, thereby allowing Sskl to activate the downstream components. Unlike PBS2 and HOGl, however, SSKl is not required for growth in high-osmolarity medium, suggesting that a redundant function exists at this level. A pair of genes encoding MEKK homologs proposed to act upstream of Pbs2 have been isolated recently (T Meada, H Saito, personal communication). Mutation of one of these genes, designated SSK2, suppresses loss of SLNl function, suggesting that it functions on the pathway activated by Slnl and Sskl. The other, designated SSK22, was identified by its high degree of sequence similarity to SSK2, but this putative kinase does not appear to be under the control of Slnl and Sskl. It seems that multiple inputs to this pathway converge on the Pbs2 MEK homolog.

Sporuiation pathway When deprived of both a fermentable carbon source and a nitrogen source, diploid yeast cells undergo meiosis and package the haploid nuclei as spores. Spore formation is a complex developmental pathway in which many events must be carried out in a specific sequence (reviewed in [42]). More than 25 genes involved in this process have been grouped on the basis of their timing of expression during sporulation (early, middle, or late). Several genes expressed early in sporulation have been shown to be important for meiosis, whereas those expressed late in sporulation tend to be involved in spore maturation. How these events are coordinated is not yet understood. Two sporulation-specific genes that encode protein kinases were described recently: the SMKl gene encodes a MAPK homolog [43*], and the SPSl gene encodes a Ste20 homolog (a MEKK kinase; [44*]) (Fig. le). Circumstantial evidence suggests that these putative protein kinases function in a common pathway First, deletion mutants in SMKl or SPSl display similar defects: both mutants complete meiosis normally, forming four haploid nuclei, but fail to assemble proper spore walls. These mutants also display altered expression of late sporulation genes, further suggesting that spore maturation is controlled by this putative MAPK pathway Second, a double spsl smkl mutant displays a defect in spore formation that is no more severe than that of the single mutants, consistent with the notion that the function of one component is under the control of the other (E Winter, personal communication). Interestingly, both SPSl and SMKl are expressed specifically during sporulation, with mRNA accumulation peaking in the middle of the sporulation program (as meiosis is being completed) [43*,44*]. Expression of pathway components exclusively during the brief period in which they are required may assist in maintenance of signal fidelity by reducing the number of signaling modules present in a cell at one time. Of course, such a mechanism would not be feasible for a signaling pathway that is required to sense an extracellular signal, but is practical for a pathway that is activated as a delayed event in a developmental program.

Conclusions The diverse array of processes in which MAPK-mediated pathways function in yeast includes responses to mating pheromones, stresses and nutritional signals. Some of these pathways need not respond directly to extracellular signals, but may be activated secondarily in response to activation of other signaling pathways. Indeed, in at least one case (sporulation), the expression of pathway components is part of a developmental program that is initiated by an external signal. Additional MAPK pathways are likely to be identified in the next two years as the sequencing of the yeast genome nears completion.

MAP kinase signaling pathways in yeast Levin and Errede

The core MAPK activation module was defined initially as including three protein kinases. The emerging role of Ste5 as a scaffold for complex formation of the kinases in the mating pathway, together with evidence that components of the Pkcl-mediated pathway may also form a complex, suggests that the core module ultimately may be extended to include SteS-like proteins. Complex formation between MAPKs and their activating kinases raises some new issues. For example, what is the stoichiometry of components within the complex? If the complex is stable, it is not clear how multiple protein -kinases would function to amplifjl a signal. Perhaps component kinases associate with and dissociate from the complex depending on their activation state. For the pathways examined (the mating pathway and the Pkcl pathway), the number of MAPK molecules in a cell is much greater than that of MEK or MEKK molecules, consistent with a model in which the complex provides a site for transient association and subsequent activation of MAPKs. Additionally, Ste5 appears to promote fidelity among components of the mating pathway, perhaps by sequestering them horn components of other pathways. Is the only function of Ste5 to serve as a scaffold for protein kinases, or does it play an active role in signal transmission? Whatever the answers to these and other pertinent questions, continued study of the multiple MAPK pathways in yeast promises to reveal those features that are fundamental to MAPK signaling.

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DE Levin, sity School

Torres L, Martin H, Garcia-Saez Ml, Arroyo J, Molina M, Sanchez M, Nombela C: A protein kinase gene complements

B Errede, of North

35.

in Sac&a-

Department of Biochemistry, of Public Health, Baltimore, Department ofBiochemistry Carolina, Chapel Hill, NC

Johns Hopkins UniverMD 31305, USA.

and Biophysics, 27599, USA.

University