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The cell wall integrity pathway (CWI) is a MAPK-mediated signaling route essential for yeast cell response to cell wall damage, regulating distinct aspects of fungal physiology. We have recently proven that the incorporation of a genetic circuit that operates as a signal amplifier into this pathway allows for the identification of novel elements involved in CWI signaling. Here, we show that the strong growth inhibition triggered by pathway hyperactivation in cells carrying the "Integrity Pathway Activation Circuit" (IPAC) also allows the easy identification of new stimuli. By using the IPAC, we have found various chemical agents that activate the CWI pathway, including the aminoglycoside neomycin. Cells lacking key components of this pathway are sensitive to this antibiotic, due to the disruption of signaling upon neomycin stimulation. Neomycin reduces both phosphatidylinositol-4,5-bisphosphate (PIP2) availability at the plasma membrane and myriocin-induced TORC2-dependent Ypk1 phosphorylation, suggesting a strong interference with plasma membrane homeostasis, specifically with PIP2. The neomycin-induced transcriptional profile involves not only genes related to stress and cell wall biogenesis, but also to amino acid metabolism, reflecting the action of this antibiotic on the yeast ribosome. [ABSTRACT FROM AUTHOR]

The cell wall integrity (CWI) MAPK pathway of budding yeast Saccharomyces cerevisiae is specialized in responding to cell wall damage, but ongoing research shows that it participates in many other stressful conditions, suggesting that it has functional diversity. The output of this pathway is mainly driven by the activity of the MAPK Slt2, which regulates important processes for yeast physiology such as fine-tuning of signaling through the CWI and other pathways, transcriptional activation in response to cell wall damage, cell cycle, or determination of the fate of some organelles. To this end, Slt2 precisely phosphorylates protein substrates, modulating their activity, stability, protein interaction, and subcellular localization. Here, after recapitulating the methods that have been employed in the discovery of proteins phosphorylated by Slt2, we review the bona fide substrates of this MAPK and the growing set of candidates still to be confirmed. In the context of the complexity of MAPK signaling regulation, we discuss how Slt2 determines yeast cell integrity through phosphorylation of these substrates. Increasing data from large-scale analyses and the available methodological approaches pave the road to early identification of new Slt2 substrates and functions. [ABSTRACT FROM AUTHOR]

Azoles are one of the most widely used drugs to treat fungal infections. To further understand the fungal response to azoles, we analyzed the MAPK circuitry of the model yeast Saccharomyces cerevisiae that operates under treatment with these antifungals. Imidazoles, and particularly clotrimazole, trigger deeper changes in MAPK phosphorylation than triazoles, involving a reduction in signaling through the mating pathway and the activation of the MAPKs Hog1 and Slt2 from the High-Osmolarity Glycerol (HOG) and the Cell Wall Integrity (CWI) pathways, respectively. Clotrimazole treatment leads to actin aggregation, mitochondrial alteration, and oxidative stress, which is essential not only for the activation of both MAPKs, but also for the appearance of a lowmobility form of Slt2 caused by additional phosphorylation to that occurring at the conserved TEY activation motif. Clotrimazole-induced ROS production and Slt2 phosphorylation are linked to Tpk3- mediated PKA activity. Resistance to clotrimazole depends on HOG and CWI-pathway-mediated stress responses. However, Pkc1 and other proteins acting upstream in the pathway are not critical for the activation of the Slt2 MAPK module, suggesting a novel rewiring of signaling through the CWI pathway. We further show that the strong impact of azole treatment on MAPK signaling is conserved in other yeast species. [ABSTRACT FROM AUTHOR]

Caspases are a family of cysteine proteases that play an essential role in inflammation, apoptosis, cell death, and development. Here we delve into the effects caused by heterologous expression of human caspase-1 in the yeast Saccharomyces cerevisiae and compare them to those of caspase-8. Overexpression of both caspases in the heterologous model led to their activation and caused mitochondrial hyperpolarization, damage to different organelles, and cell death. All these effects were dependent on their protease activity, and caspase-8 was more aggressive than caspase-1. Growth arrest could be at least partially explained by dysfunction of the actin cytoskeleton as a consequence of the processing of the yeast Bni1 formin, which we identify here as a likely direct substrate of both caspases. Through the modulation of the GAL1 promoter by using different galactose:glucose ratios in the culture medium, we have established a scenario in which caspase-1 is sufficiently expressed to become activated while yeast growth is not impaired. Finally, we used the yeast model to explore the role of death-fold domains (DD) of both caspases in their activity. Peculiarly, the DDs of either caspase showed an opposite involvement in its intrinsic activity, as the deletion of the caspase activation and recruitment domain (CARD) of caspase-1 enhanced its activity, whereas the deletion of the death effector domain (DED) of caspase-8 diminished it. We show that caspase-1 is able to efficiently process its target gasdermin D (GSDMD) when co-expressed in yeast. In sum, we propose that S. cerevisiae provides a manageable tool to explore caspase-1 activity and structure–function relationships. [ABSTRACT FROM AUTHOR]

The cell wall integrity (CWI) pathway mediates the response of Saccharomyces cerevisiae to cell wall alterations. Stress at the cell surface is detected by mechanosensors, which transduce the signal to a protein kinase cascade that involves Pkc1, Bck1, Mkk1/Mkk2, the mitogen‐activated protein kinase (MAPK) Slt2 and the transcription factor Rlm1. We incorporated a positive feedback loop into this pathway by placing a hyperactive MKK1 allele under the control of the Rlm1‐regulated MLP1 promoter. This circuit operates as a signal amplifier and leads to a highly increased Slt2 activation under stimulating conditions. Triggering the CWI pathway in cells engineered with this circuit, which we have named the Integrity Pathway Activation Circuit (IPAC), results in strong growth inhibition. Exploitation of this hypersensitive phenotype allowed the identification of novel proteins that contribute in signalling to Rlm1 in response to cell surface stressing agents such as Congo red, zymolyase and SDS. Among these proteins, the MAPK kinase kinase Ssk2 of the osmoregulatory high‐osmolarity glycerol (HOG) pathway, but not its paralogue Ssk22, proved to be necessary for the SDS‐induced IPAC‐mediated growth inhibition. We found the existence of an Ssk1‐independent Ssk2‐Pbs2‐Hog1‐CWI pathway signalling axis that contributes to Slt2 activation in response to cell surface stress. We also demonstrated that the MAP kinase kinases Mkk1 and Pbs2 and the MAPKs Slt2 and Hog1 of the HOG and CWI pathways interact physically, forming a complex. Our results show how a simple synthetic circuit can be used as a powerful tool for a better understanding of signalling pathways. [ABSTRACT FROM AUTHOR]

Coxiella burnetii is a Gram-negative obligate parasitic bacterium that causes the disease Q-fever in humans. To establish its intracellular niche, it utilizes the Icm/Dot type IVB secretion system (T4BSS) to inject protein effectors into the host cell cytoplasm. The host targets of most cognate and candidate T4BSS-translocated effectors remain obscure. We used the yeast Saccharomyces cerevisiae as a model to express and study six C. burnetii effectors, namely AnkA, AnkB, AnkF, CBU0077, CaeA and CaeB, in search for clues about their role in C. burnetii virulence. When ectopically expressed in HeLa cells, these effectors displayed distinct subcellular localizations. Accordingly, GFP fusions of these proteins produced in yeast also decorated distinct compartments, and most of them altered cell growth. CaeA was ubiquitinated both in yeast and mammalian cells and, in S. cerevisiae, accumulated at juxtanuclear quality-control compartments (JUNQs) and insoluble protein deposits (IPODs), characteristic of aggregative or misfolded proteins. AnkA, which was not ubiquitinated, accumulated exclusively at the IPOD. CaeA, but not AnkA or the other effectors, caused oxidative damage in yeast. We discuss that CaeA and AnkA behavior in yeast may rather reflect misfolding than recognition of conserved targets in the heterologous system. In contrast, CBU0077 accumulated at vacuolar membranes and abnormal ER extensions, suggesting that it interferes with vesicular traffic, whereas AnkB associated with the yeast nucleolus. Both effectors shared common localization features in HeLa and yeast cells. Our results support the idea that C. burnetii T4BSS effectors manipulate multiple host cell targets, which can be conserved in higher and lower eukaryotic cells. However, the behavior of CaeA and AnkA prompt us to conclude that heterologous protein aggregation and proteostatic stress can be a limitation to be considered when using the yeast model to assess the function of bacterial effectors. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae plays a beneficial role in health because of its intrinsic nutritional value and bio-functional properties, which is why it is also used as a dietary supplement. However, the perception that S. cerevisiae is harmless has changed due to an increasing number of infections caused by this yeast. Given this scenario, we have tested whether viable strains contained in dietary supplements displayed virulence-associated phenotypic traits that could contribute to virulence in humans. We have also performed an in vivo study of the pathogenic potential of these strains using a murine model of systemic infection by intravenous inoculation. A total of 5 strains were isolated from 22 commercial products and tested. Results highlight one strain (D14) in terms of burden levels in brains and kidneys and ability to cause death, whereas the other two strains (D2 and D4) were considered of low virulence. Our results suggest a strong relationship between some of the virulence-associated phenotypic traits (ability to grow at 39°C and pseudohyphal growth) and the in vivo virulence in a mouse model of intravenous inoculation for isolates under study. The isolate displaying greatest virulence (D14) was evaluated in an experimental murine model of gastrointestinal infection with immunosuppression and disruption of mucosal integrity, which are common risk factors for developing infection in humans, and results were compared with an avirulent strain (D23). We showed that D14 was able to spread to mesenteric nodes and distant organs under these conditions. Given the widespread consumption of dietary supplements, we recommend only safe strains be used. [ABSTRACT FROM AUTHOR]

Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol 3-kinase (PI3K) pathway couples receptor-mediated signaling to essential cellular functions by generating the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate. This pathway is implicated in multiple aspects of oncogenesis. A low-cost bioassay that readily measures PI3K inhibition in vivo would serve as a valuable tool for research in this field. Using heterologous expression, we have previously reconstituted the PI3K pathway in the model organism Saccharomyces cerevisiae. On the basis of the fact that the overproduction of PI3K is toxic in yeast, we tested the ability of commercial PI3K inhibitors to rescue cell growth. All compounds tested counteracted the PI3K-induced toxicity. Among them, 15e and PI-103 were the most active. Strategies to raise the intracellular drug concentration, specifically the use of 0.003% sodium dodecyl sulfate and the elimination of the Snq2 detoxification pump, optimized the bioassay by enhancing its sensitivity. The humanized yeast-based assay was then tested on a pilot scale for high-throughput screening (HTS) purposes using a collection of natural products of microbial origin. From 9600 extracts tested, 0.6% led to a recovery of yeast growth reproducibly, selectively, and in a dose-dependent manner. Cumulatively, we show that the developed PI3K inhibition bioassay is robust and applicable to large-scale HTS. [ABSTRACT FROM PUBLISHER]

The phosphoinositide phosphatase SopB/SigD is a type III secretion system effector that plays multiple roles in Salmonella internalization and intracellular survival. We previously reported that SopB complexed with and inhibited the small GTPase Cdc42 when expressed in a yeast model system, independently of its phosphatase activity. Here we show that human Cdc42, but not Rac1, interacts with catalytically inactive SopB when coexpressed in Saccharomyces cerevisiae. This interaction occurs with both constitutively active and non-activatable Cdc42, suggesting that SopB binds Cdc42 independently of its activation state. By mutational analysis we have narrowed the Cdc42-interacting region of SopB to the first 142 amino acids, and isolated a collection of point mutations in this region, mainly affecting leucine residues conserved in the related Shigella IpgD protein. Such mutations yielded SopB unable to interact with Cdc42 but maintained phosphatase activity. SopB mutant proteins defective for binding Cdc42 were ubiquitinated upon translocation in mammalian cells, but their localization to the Salmonella-containing vacuole was reduced compared with wild-type SopB. Whereas invasion of mammalian cells by Salmonella bearing these sopB mutations was not affected, intracellular replication was less efficient, suggesting that SopB-Cdc42 interaction contributes to the adaptation of Salmonella to the intracellular environment. [ABSTRACT FROM AUTHOR]

Salmonella uses type III secretion systems (TTSS) to deliver pathogenic proteins into the host cells. These translocated effectors induce bacterial internalization and intracellular proliferation by targeting important cellular processes that are conserved among eukaryotes. Here, we assessed the feasibility of performing a genetic screen in yeast to identify novel Salmonella effectors, by searching for genes that produce toxicity when expressed in this model system. We identified several known TTSS-translocated effectors and found that two of them, SteC and SseF, from Salmonella enterica serovar Typhimurium, interfere with cytoskeletal dynamics as they do in mammalian cells. We also identified 11 genes of unknown function (seven from S. Typhi and four from S. Typhimurium) that display features commonly showed by effector proteins, such as a (G+C) content lower than the average for the chromosome, suggesting their acquisition by horizontal transfer processes. Five of these proteins are highly conserved only among Salmonella serovars, whereas the other six are also conserved in other pathogenic or opportunistic enterobacteria. Moreover, we identified other proteins that share specific activity domains with either translocated or bacterial-confined proteins known to be involved in pathogenesis, which might also act as virulence proteins. [ABSTRACT FROM AUTHOR]

The Mitogen-Activated Protein Kinase (MAPK) Slt2 is central to signaling through the yeast Cell Wall Integrity (CWI) pathway. MAPKs are regulated by phosphorylation at both the threonine and tyrosine of the conserved TXY motif within the activation loop (T190/Y192 in Slt2). Since phosphorylation at both sites results in the full activation of MAPKs, signaling through MAPK pathways is monitored with antibodies that detect dually phosphorylated forms. However, most of these antibodies also recognize monophosphorylated species, whose relative abundance and functionality are diverse. By using different phosphospecific antibodies and phosphate-affinity (Phos-tag) analysis on distinct Slt2 mutants, we determined that Y192- and T190-monophosphorylated species coexist with biphosphorylated Slt2, although most of the Slt2 pool remains unphosphorylated following stress. Among the monophosphorylated forms, only T190 exhibited biological activity. Upon stimulation, Slt2 is first phosphorylated at Y192, mainly by the MAPKK Mkk1, and this phosphorylation is important for the subsequent T190 phosphorylation. Similarly, dephosphorylation of Slt2 by the Dual Specificity Phosphatase (DSP) Msg5 is ordered, with dephosphorylation of T190 depending on previous Y192 dephosphorylation. Whereas Y192 phosphorylation enhances the Slt2 catalytic activity, T190 is essential for this activity. The conserved T195 residue is also critical for Slt2 functionality. Mutations that abolish the activity of Slt2 result in a high increase in inactive Y192-monophosphorylated Slt2. The coexistence of different Slt2 phosphoforms with diverse biological significance highlights the importance of the precise detection of the Slt2 phosphorylation status. [ABSTRACT FROM AUTHOR]

The tumour suppressor PTEN is frequently downregulated, mutated or lost in several types of tumours and congenital disorders including PHTS (PTEN Hamartoma Tumour Syndrome) and ASD (Autism Spectrum Disorder). PTEN is a lipid phosphatase whose activity over the lipid messenger PIP3 counteracts the stimulation of the oncogenic phosphatidylinositol 3-kinase (PI3K) pathway. Recently, several extended versions of PTEN produced in the cell by alternative translation initiation have been described, among which, PTEN-L and PTEN-M represent the longest isoforms. We previously developed a humanized yeast model in which the expression of PI3K in Saccharomyces cerevisiae led to growth inhibition that could be suppressed by co-expression of PTEN. Here, we show that the expression of PTEN-L and PTEN-M in yeast results in robust counteracting of PI3K-dependent growth inhibition. N-terminally tagged GFP-PTEN-L was sharply localized at the yeast plasma membrane. Point mutations of a putative membrane-binding helix located at the PTEN-L extension or its deletion shifted localization to nuclear. Also, a shift from plasma membrane to nucleus was observed in mutants at basic amino acid clusters at the PIP2-binding motif, and at the Cα2 and CBR3 loops at the C2 domain. In contrast, C-terminally tagged PTEN-L-GFP displayed mitochondrial localization in yeast, which was shifted to plasma membrane by removing the first 22 PTEN-L residues. Our results suggest an important role of the N-terminal extension of alternative PTEN isoforms on their spatial and functional regulation. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol (4,5)- bis phosphate [PtdIns(4,5)P 2 ] is essential for recognition of the plasma membrane inner leaf by protein complexes. We expressed mammalian class I phosphoinositide 3-kinase (PI3K) in Saccharomyces cerevisiae to eliminate PtdIns(4,5)P 2 by its conversion into PtdIns(3,4,5)P 3 , a lipid naturally missing in this yeast. This led to loss of actin function and endocytosis defects, causing a blockage in polarized secretion. Also, the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway was activated, triggering a typical transcriptional response. In the absence of PtdIns(4,5)P 2 at the plasma membrane, the Pkc1 protein kinase upstream the CWI MAPK module localized to post-Golgi endosomes marked by SNARE Snc1 and Rab GTPases Ypt31 and Ypt32. Other components at the head of the pathway, like the mechanosensor Wsc1, the GTPase Rho1 and its activator the GDP/GTP exchange factor Rom2, co-localized with Pkc1 in these compartments. Chemical inhibition of PI3K proved that both CWI activation and Pkc1 relocation to endosomes are reversible. These results suggest that the CWI pathway is able to respond to loss of plasma membrane identity from recycling endosomes. [ABSTRACT FROM AUTHOR]

The lack of signaling through MAPK pathways leads to a defective cellular response to the corresponding stimulus, but an improper hyperactivation of these routes results in deleterious effects as well. Protein phosphorylation is an activating modification for signal transmission through components of MAPK pathways and thus, protein phosphatases are key negative regulators of these cellular routes by limiting excessive signaling activity. However, in contrast to most of the protein kinases operating in MAPK pathways, protein phosphatases usually exhibit redundancy and promiscuity, which has limited the identification of their function. In order to identify new putative phosphatases operating in Saccharomyces cerevisiae MAPK signaling, we have taken advantage of growth inhibition promoted by overproduction of constitutively active components of the mating and cell wall integrity (CWI) pathways to perform a screen with a collection of 43 protein phosphatases or phosphatase-regulatory proteins. The phosphatases able to alleviate the induced growth inhibition when overproduced were further studied by testing their capacity to downregulate expression of mating and CWI responsive promoters and the consequences of their removal on MAPK signaling. Epistasis analysis placed the Ser/Thr protein phosphatase Ppq1 as a regulator of the mating MAPK module downstream the MAPKKK Ste11. The dual specificity phosphatase Yvh1 was found to be important for the maintenance of cell wall integrity and appropriate signaling through the CWI pathway. Moreover, we have found that Ptc2 and Ptc4 bind to the CWI MAPK Slt2. Together with known phosphatases of the mating and CWI pathway, as Msg5 or Ptp2, other putative negative regulators of both pathways that came up in the screening were Ptc2, Oca2 and Ptp1. We show that Ptp1 physically interacts with Slt2 and the mating MAPK Fus3. Elimination of Ptp1 results in increased signaling through these pathways, suggesting that this tyrosine phosphatase, like Ptp2 and Ptp3, plays a downregulatory role on both MAPKs. [ABSTRACT FROM AUTHOR]

The Saccharomyces cerevisiae cell wall integrity (CWI) pathway took this name when its role in the cell response to cell wall aggressions was clearly established. The receptors involved in sensing the damage, the relevant components operating in signaling to the MAPK Slt2, the transcription factors activated by this MAPK, as well as some key regulatory mechanisms have been identified and characterized along almost 30 years. However, other stimuli that do not alter specifically the yeast cell wall, including protein unfolding, low or high pH, or plasma membrane, oxidative and genotoxic stresses, have been also found to trigger the activation of this pathway. In this review, we compile almost forty non-cell wall-specific compounds or conditions, such as tunicamycin, hypo-osmotic shock, diamide, hydroxyurea, arsenate, and rapamycin, which induce these stresses. Relevant aspects of the CWI-mediated signaling in the response to these non-conventional pathway activators are discussed. The data presented here highlight the central and key position of the CWI pathway in the safeguard of yeast cells to a wide variety of external aggressions. [ABSTRACT FROM AUTHOR]

The yeast Saccharomyces cerevisiae is a model organism that has been thoroughly exploited to understand the universal mechanisms that govern signaling pathways. Due to its ease of manipulation, humanized yeast models that successfully reproduce the function of human genes permit the development of highly efficient genetic approaches for molecular studies. Of special interest are those pathways related to human disease that are conserved from yeast to mammals. However, it is also possible to engineer yeast cells to implement functions that are naturally absent in fungi. Along the years, we have reconstructed several aspects of the mammalian phosphatidylinositol 3-kinase (PI3K) pathway in S. cerevisiae. Here, we briefly review the use of S. cerevisiae as a tool to study human oncogenes and tumor suppressors, and we present an overview of the models applied to the study of the PI3K oncoproteins, the tumor suppressor PTEN, and the Akt protein kinase. We discuss the application of these models to study the basic functional properties of these signaling proteins, the functional assessment of their clinically relevant variants, and the design of feasible platforms for drug discovery. [ABSTRACT FROM AUTHOR]

The yeast cell wall integrity MAPK Slt2 mediates the transcriptional response to cell wall alterations through phosphorylation of transcription factors Rlm1 and SBF. However, the variety of cellular functions regulated by Slt2 suggests the existence of a significant number of still unknown substrates for this kinase. To identify novel Slt2 targets, we generated and characterized an analog-sensitive mutant of Slt2 (Slt2-as) that can be specifically inhibited by bulky kinase inhibitor analogs. We demonstrated that Slt2-as is able to use adenosine 5'-[')'-thio]- triphosphate analogs to thiophosphorylate its substrates in yeast cell extracts as well as when produced as recombinant proteins in Escherichia coli. Taking advantage of this chemical-genetic approach, we found that Slt2 phosphorylates the MAPK phosphatase Msg5 both in the N-terminal regulatory and C-terminal catalytic domains. Moreover, we identified the calcineurin regulator Rcn2, the 4E-BP (translation initiation factor eIF4E-binding protein) translation repressor protein Caf20, and the Golgi-associated adaptor Gga1 as novel targets for Slt2. The Slt2 phosphorylation sites on Rcn2 and Caf20 were determined. We also demonstrated that, in the absence of SLT2, the GGA1 paralog GGA2 is essential for cells to survive under cell wall stress and for proper protein sorting through the carboxypeptidase Y pathway. Therefore, Slt2-as provides a powerful tool that can expand our knowledge of the outputs of the cell wall integrity MAPK pathway. [ABSTRACT FROM AUTHOR]

The PTEN phosphoinositide 3-phosphatase is a tumor suppressor commonly targeted by pathologic missense mutations. Subject to multiple complex layers of regulation, its capital role in cancer relies on its counteracting function of class I phosphoinositide 3-kinase (PI3K), a key feature in oncogenic signaling pathways. Precise assessment of the involvement of PTEN mutations described in the clinics in loss of catalytic activity requires either tedious in vitro phosphatase assays or in vivo experiments involving transfection into mammalian cell lines. Taking advantage of the versatility of the model organism Saccharomyces cerevisiae , we have developed different functional assays by reconstitution of the mammalian PI3K–PTEN switch in this lower eukaryote. This methodology is based on the fact that regulated PI3K expression in yeast cells causes conversion of PtdIns(4,5)P 2 in PtdIns(3,4,5)P 3 and co-expression of PTEN counteracts this effect. This can be traced by monitoring growth, given that PtdIns(4,5)P 2 pools are essential for the yeast cell, or by using fluorescent reporters amenable for microscopy or flow cytometry. Here we describe the methodology and review its application to evaluate the functionality of PTEN mutations. We show that the technique is amenable to both directed and systematic structure–function relationship studies, and present an example of its use for the study of the recently discovered PTEN-L variant. [ABSTRACT FROM AUTHOR]

MAPK phosphatases (MKPs) are negative regulators of signaling pathways with distinct MAPK substrate specificities. For example, the yeast dual specificity phosphatase Msg5 dephosphorylates the Fus3 and Slt2 MAPKs operating in the mating and cell wall integrity pathways, respectively. Like other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains. These include D-motifs, which contain basic residues that interact with acidic residues in the common docking (CD) domain of MAPKs. Here we show that Msg5 interacts not only with Fus3, Kss1, and Slt2 but also with the pseudokinase Slt2 paralog Mlp1. Using yeast two-hybrid and in vitro interaction assays, we have identified distinct regions within the N-terminal domain of Msg5 that differentially bind either the MAPKs Fus3 and Kss1 or Slt2 and Mlp1. Whereas a canonical D-site within Msg5 mediates interaction with the CD domains of Fus3 and Kss1, a novel motif (102IYT104) within Msg5 is involved in binding to Slt2 and Mlp1. Furthermore, mutation of this site prevents the phosphorylation of Msg5 by Slt2. This motif is conserved in Sdp1, another MKP that dephosphorylates Slt2, as well as in Msg5 orthologs from other yeast species. A region spanning amino acids 274-373 within Slt2 and Mlp1 mediates binding to this Msg5 motif in a CD domain-independent manner. In contrast, Slt2 uses its CD domain to bind to its upstream activator Mkk1. This binding flexibility may allow MAPK pathways to exploit additional regulatory controls in order to provide fine modulation of both pathway activity and specificity. [ABSTRACT FROM AUTHOR]

Dual-specificity protein phosphatases (DSPs) are involved in the negative regulation of mitogen-activated protein kinases (MAPKs) by dephosphorylating both threonine- and tyrosine-conserved residues located at the activation loop. Here we show that Msg5 DSP activity is essential for maintaining a low level of signaling through the cell integrity pathway in Saccharomyces cerevisiae. Consistent with a role of this phosphatase on cell wall physiology, cells lacking Msg5 displayed an increased sensitivity to the cell wall-interfering compound Congo Red. We have observed that the N-terminal non-catalytic region of this phosphatase was responsible for binding to the kinase domain of Slt2, the MAPK that operates in this pathway. In vivo and in vitro experiments revealed that both proteins act on each other. Msg5 bound and dephosphorylated activated Slt2. Reciprocally, Slt2 phosphorylated Msg5 as a consequence of the activation of the cell integrity pathway. In addition, alternative use of translation initiation sites at MSG5 resulted in two protein forms that are functional on Slt2 and became equally phosphorylated following activation of this MAPK. Under activating conditions, a decrease in the affinity between Msg5 and Slt2 was observed, leading us to suggest that the mechanism by which Slt2 controls the action of Msg5 was via the modulation of protein-protein interactions. Our results indicate the existence of posttranscriptional mechanisms of regulation of DSPs in yeast and provide new insights into the negative control of the cell integrity pathway. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae MATa haploid cells, the a-specific genes are expressed, whereas in the MATα haploid and MATa/α diploid cell types their transcription is repressed. It is shown in this report that Itc1p, a component of the ATP-dependent Isw2p-Itc1p chromatin remodelling complex, is required for the repression of a-specific genes. It has previously been reported that disruption of the ITC 1 gene leads, in MATα cells, to an aberrant cell morphology resembling the polarized mating projection of cells responding to pheromone. The activation of the pheromone signalling pathway in Itc1 mutants of both mating types was examined and found to be constitutively active in MATα itc1 but not in MATa itc1 cells. Furthermore, unlike the wild-type, MATα itc1 and MATa/α itc1/itc1 cells secrete a-factor and express significant levels of other a-specific genes. The results indicate that the inappropriate a-factor production in a MATα context, due to the derepression of the a-specific genes, produces an autocrine signalling loop that leads to the aberrant morphology displayed by MATα Itc1 cells, it is suggested that the Isw2p-Itc1p complex contributes to maintain the repressive chromatin structure described for the asg operator present in the promoters of a-specific genes. [ABSTRACT FROM AUTHOR]