MAPK Pathways in Budding Yeast
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MAPK Pathways in Budding Yeast
MAPK (Mitogen-activated protein kinases) are serine-threonine protein kinases that are activated by diverse stimuli ranging from cytokines, growth factors, neurotransmitters, hormones, cellular stress, and cell adherence. MAPKs are expressed in all eukaryotic cells. The basic assembly of MAPK pathways is a three-component module conserved from yeast to humans. The MAPK module includes three kinases that establish a sequential activation pathway comprising a MKKK (MAPK Kinase Kinase), MKK (MAPK Kinase), and MAPK. Yeast probably represents the experimental model where the organization and regulation of MAPK pathways are best understood. Presently, five MAPK pathways have been well characterized in the budding Yeast, Saccharomyces. cerevisiae. The four MAPKs present in vegetative cells, Fus3, Kss1, Hog1, and SLT2/Mpk1, are involved in the mating-pheromone response, filamentation-invasion pathway, high osmolarity growth, and cell integrity pathway, respectively. The fifth one, Smk1, is believed to play a role in spore wall assembly. An important concept on MAPK pathways that has emerged from yeast studies is that the kinases employed in MAPK pathways are organized into modules. This is achieved by tethering to scaffold proteins as well as by direct interaction between the different kinases of the module. Organization into modules ensures segregation of the pathways from other signaling events in the cells and also allows the use of a given component kinase in more than one MAPK module without affecting the specificity of the response mediated by the MAPK pathways (Ref.1).

The best-defined yeast MAPK pathway in S. cerevisae is involved in the mating of haploid cells. The haploid cells have two sexual phenotypes characterized by the expression of a set of genes involved in mating that are not expressed in diploids. The mating response to generate diploids is controlled by the Alpha-pheromones and A-pheromones that bind to their respective receptors that are coupled to a heterotrimeric G-protein. Pheromone binding to its receptor leads to G-protein activation and the dissociation of the Beta-Gamma subunit complex from Alpha-GTP. The S. cerevisiae genes whose disruption inhibited mating and caused sterility were designated as sterile (Ste) genes. The seven transmembrane receptors for the Alpha- and A-factors are designated as Ste2 and Ste3, respectively, and are coupled to a heterotrimeric G-protein. Pheromone activation of the G-protein induces the dissociation of the heterotrimeric G-protein subunits designated Gpa1 (Alpha-subunit), Ste4 (Beta-subunit), and Ste18 (Gamma-subunit). The released Beta-Gamma subunit complex (Ste4/Ste18) activates Ste20 and interacts with the scaffolding protein Ste5, resulting in the stimulation of the MAPK module MKKK Ste11/MKK Ste7/MAPK Fus3. The activated form of Fus3 is thought to translocate to the nucleus, where it mediates pheromone induction of transcription of PRE (Pheromone Response Element)-containing genes through phosphorylation and activation of at least three nuclear proteins: Dig1 (also called Rst1)/Dig2 (also called Rst2), Ste12 and Far1. These targets effect changes in gene expression and block cell-cycle progression. Another physiological effect of pheromone, reoriented cellular polarity, requires a different biochemical module, CDC42 (Cell Division Cycle-42), a p21 GTPase of the Ras superfamily. The membrane-tethered, activated G-protein is thought to lead to localized activation of the GEF (Guanine nucleotide Exchange Factor) for CDC42 and, thereby, to localized activation of CDC42. Exchange of GDP for GTP on CDC42 is activated by CDC24, and the hydrolysis of the CDC42-bound GTP to GDP is predicted to be regulated by the GAPs (GTPase-Activating Proteins) Bem3 and Rga1. Once activated, CDC42 can organize the actin cytoskeleton as it does in vegetative cells. CDC42 may also activate Ste20 and influence signaling through the MAP kinase cascade. Bem1, like CDC42, interacts with several proteins important for the function of the actin cytoskeleton in polarized growth. Bem1, like CDC42, interacts with several proteins important for the function of the actin cytoskeleton in polarized growth (Ref.2).

Filamentous invasive growth of S. cerevisiae also requires multiple elements of the MAPK signaling cascade that are also components of the mating pheromone response pathway. The MAPK cascade mediates signal transduction in filamentation-invasion pathway from two small GTP binding proteins, Ras2 and CDC42. Signaling from Ras2 requires the 14-3-3 proteins BMH1 (Brain Modulosignalin Homolog-1) and BMH2 (Brain Modulosignalin Homolog-2) and possibly Sho1 receptor. CDC42 acts downstream of Ras2 and is required for the function of the Ste20 in the filamentation-invasion pathway. CDC42-Ste20 then transmits signal to the MAPK cascade. This cascade contains the MEKK Ste11 and the MEK Ste7. The MAPK for the filamentation-invasion pathway is Kss1. The MAPK Kss1 has a dual role in regulating filamentous invasive growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MEK (MAPK/ERK kinase) Ste7; in contrast, the inhibitory function of Kss1 requires neither. Unphosphorylated Kss1 binds directly to the transcription factor Ste12 and forms a protein complex that also contains Tec1, and the inhibitory proteins Dig1 or Dig2. Upon phosphorylation through a MAPK cascade, Kss1 dissociates from the complex, thereby destabilizing the Ste12-Dig association. Activated Kss1 phosphorylates and activates Ste12, leading to binding of Ste12 in combination with Tec1 to genes containing a Ste12/Tec1 composite binding site, referred to as a FRE (Filamentous and invasive growth Response Element). FRE, a combination of TCS (TEA/ATTS Consensus Sequence) and PRE (Pheromone Response Element), mediates the binding of the heterodimer formed by the association of the transcriptional activators Tec1 and Ste12. Thus, the MAPK Kss1 plays a key role in the transcriptional control of genes regulated by FRE both by derepression and activation (Ref.3).

In addition to mating and filamentation-invasion, yeast responds to their environment with metabolic changes that involve MAPK pathways. For example, under conditions of high osmolarity Ste11 can lead to activation of Hog1 but does not induce mating-specific genes. The Hog pathway is activated predominantly by two independent mechanisms that lead to the activation of either the Ssk2 and Ssk22 or the Ste11 MAPKKKs, respectively. The first mechanism involves a two-component osmosensor, composed of the Sln1-Ypd1-Ssk1 proteins. The Sln1 transmembrane protein has intrinsic histidine kinase activity and is a homologue of bacterial two-component signal transducers. Using a phospho-relay mechanism involving the Ypd1 and Ssk1 proteins, Sln1 is able to control the activity of Ssk1, which in turn interacts with and regulates the Ssk2 and Ssk22 MAPKKKs and subsequent Pbs2 activation. Pbs2 activation can also be achieved by a second, independent mechanism that involves the transmembrane protein Sho1, the MAPKKK Ste11, the Ste11-binding protein Ste50, the Ste20 PAK (p21-Activated Kinase) and the small GTPase CDC42. Activation of Pbs2 by Ste11 requires the interaction of Pbs2 with Sho1 and, although this interaction is thought to be regulated, the basic activation mechanism for this remains unclear. Once activated, Pbs2 phosphorylates and activates the Hog1 MAPK. In budding yeast, Hog1 MAPK plays a key role in global gene regulation. These osmostress-regulated genes are implicated mainly in carbohydrate metabolism, general stress protection, protein production and signal transduction. This global change in transcription could account, at least in part, for the metabolic adjustments required for osmostress adaptation. In yeast, five transcription factors are known to be controlled by the Hog1 MAPK. Hot1, Smp1, Msn2 and Msn4 activate, whereas Sko1 represses or activates, different subsets of osmotic-inducible and Hog1-regulated genes. The Hog pathway also plays a role in mediating the hyperosmotic stress-induced expression of stress response genes, recovery of cell morphogenesis, and repression of the pheromone response pathway (Ref.4).

Another MAPK cascade is found in budding yeast as part of the cell integrity pathway. 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. The pathway is under the control of PKC. Signals activating the pathway are detected by sensors located at the cell surface, such as Slg1/WSC1 (also called Hsc77), WSC2, WSC3 and Mid2. Information is then transduced via the GDP/GTP exchange factor Rom2 to the small GTPase Rho1. The latter, like all small GTPases, is considered active in its GTP-bound and inactive in its GDP-bound state. Sac7 and Lrg1 act as GAPs (GTPase-Activating Protein) for Rho1 and thus function as negative regulators. Further GAP functions have been assigned to Bag7 and Bem2 but seem to be less crucial for Rho1 function. Similar to many other small GTPases, Rho1 has a set of different target proteins. In its GTP-bound state it binds to and thereby activates the Beta-1, 3-glucan-synthase complex. It is also involved in regulation of the actin cytoskeleton by interacting with Bni1. In addition, interaction with Skn7, a regulator of oxidative stress response, has been reported. With respect to signals ensuring cellular integrity, the main effector of Rho1 is PKC1. This kinase then activates a MAP-kinase cascade consisting of the MAPKKK Bck1, the MAPKKs MKK2 and the MAPK SLT2, also referred to as Mpk1. Rlm1 and the SBF complex (consisting of Swi4 and Swi6) have been reported as targets of the MAP kinase SLT2. Rlm1 regulates transcription of a specific set of genes. Swi4 is the DNA binding subunit and transcriptional activator of SBF and is required for normal expression of the G1 cyclin genes Cln1, Cln2, Pcl1, and Pcl2 at the G1/S transition. Swi6 is more of a regulatory subunit, because loss of Swi6 leads to constitutive intermediate levels of Cln1 and Cln2 expression. Cln1 and Cln2 are G1 cyclins that complex with the cyclin-dependent kinase CDC28 and thereby activate the G1/S transition (Ref.5).

Another yeast MAPK, Smk1, is recently discovered, which is believed to be involved in sporulation. Upon starvation for carbon and nitrogen sources, diploid yeast cells enter meiosis to generate four haploid spores. Upon completion of meiosis, the four-haploid nuclei, which still remain within a single nuclear membrane, are enveloped by the double membranous prospore wall. The spore wall is then deposited from the space between the layers of the prospore wall. The final differentiated spore wall consists of four layers. The two inner layers appear indistinguishable from the vegetative cell wall, whereas the third layer is a spore-specific structure composed primarily of chitosan and chitin. A putative MAPK module, which employs MAPK Smk1 regulate the sporulation response. Smk1 is one of the middle sporulation genes and is therefore expressed during the latter stages of meiosis and during the time when spore wall formation occurs. The Smk1-containing spore wall assembly pathway is thus required for completion of a developmental pathway that has previously been induced. Other signaling proteins that may be part of the Smk1 pathway are two protein kinases, Sps1 and Cak1. Sps1, A MKKKK homolog, is similar in sequence to members of the PAK subfamily of protein kinases. Like Smk1, Sps1 is a middle sporulation gene and is required for proper spore wall assembly. Whether the Smk1 pathway contains a MEKK or MEK has not yet been determined. Cak1 is an essential protein kinase that is required during vegetative growth for progression through the cell cycle. The cell cycle function of Cak1 is probably related to the ability of Cak1 to activate the cyclin dependent protein kinase CDC28 by phosphorylation. Cak1 also appears to regulate the Smk1 pathway. Cak1 is expressed to a higher level during the same time that Smk1 is expressed. Whether Cak1 plays a supporting or instructive role on the Smk1 pathway has not yet been determined. The mechanism by which Smk1, Sps1, and Cak1 coordinate the assembly of the spore wall is unknown. Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system. Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades. The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades (Ref.6).