Cell Wall Stress Depolarizes Cell Growth via Hyperactivation of Rho1

Cells sense and physiologically respond to environmental stress via signaling pathways. Saccharomyces cerevisiae cells respond to cell wall stress by transiently depolarizing the actin cytoskeleton. We report that cell wall stress also induces a transient depolarized distribution of the cell wall biosynthetic enzyme glucan synthase FKS1 and its regulatory subunit RHO1, possibly as a mechanism to repair general cell wall damage. The redistribution of FKS1 is dependent on the actin cytoskeleton. Depolarization of the actin cytoskeleton and FKS1 is mediated by the plasma membrane protein WSC1, the RHO1 GTPase switch, PKC1, and a yet-to-be defined PKC1 effector branch. WSC1 behaves like a signal transducer or a stress-specific actin landmark that both controls and responds to the actin cytoskeleton, similar to the bidirectional signaling between integrin receptors and the actin cytoskeleton in mammalian cells. The PKC1-activated mitogen-activated protein kinase cascade is not required for depolarization, but rather for repolarization of the actin cytoskeleton and FKS1. Thus, activated RHO1 can mediate both polarized and depolarized cell growth via the same effector, PKC1, suggesting that RHO1 may function as a rheostat rather than as a simple on-off switch.

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Chromatin remodeling by the SWI/SNF complex is essential for transcription mediated by the yeast cell wall integrity MAPK pathway

Molecular Biology of the Cell ◽

10.1091/mbc.e12-04-0278 ◽

2012 ◽

Vol 23 (14) ◽

pp. 2805-2817 ◽

Cited By ~ 33

Author(s):

A. Belén Sanz ◽

Raúl García ◽

Jose Manuel Rodríguez-Peña ◽

Sonia Díez-Muñiz ◽

César Nombela ◽

...

Keyword(s):

Cell Wall ◽

Chromatin Remodeling ◽

Mapk Pathway ◽

Critical Role ◽

Transcriptional Response ◽

Wall Stress ◽

Cell Wall Integrity ◽

Mitogen Activated Protein Kinase ◽

Cell Wall Stress ◽

Mitogen Activated Protein

In Saccharomyces cerevisiae, the transcriptional program triggered by cell wall stress is coordinated by Slt2/Mpk1, the mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, and is mostly mediated by the transcription factor Rlm1. Here we show that the SWI/SNF chromatin-remodeling complex plays a critical role in orchestrating the transcriptional response regulated by Rlm1. swi/snf mutants show drastically reduced expression of cell wall stress–responsive genes and hypersensitivity to cell wall–interfering compounds. On stress, binding of RNA Pol II to the promoters of these genes depends on Rlm1, Slt2, and SWI/SNF. Rlm1 physically interacts with SWI/SNF to direct its association to target promoters. Finally, we observe nucleosome displacement at the CWI-responsive gene MLP1/KDX1, which relies on the SWI/SNF complex. Taken together, our results identify the SWI/SNF complex as a key element of the CWI MAPK pathway that mediates the chromatin remodeling necessary for adequate transcriptional response to cell wall stress.

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Coordination of the Cell Wall Integrity and High-Osmolarity Glycerol Pathways in Response to Ethanol Stress in Saccharomyces cerevisiae

Applied and Environmental Microbiology ◽

10.1128/aem.00551-19 ◽

2019 ◽

Vol 85 (15) ◽

Cited By ~ 6

Author(s):

Nisarut Udom ◽

Pakkanan Chansongkrow ◽

Varodom Charoensawan ◽

Choowong Auesukaree

Keyword(s):

Cell Wall ◽

Wall Stress ◽

Mitogen Activated Protein Kinase ◽

Yeast Cells ◽

Ethanol Stress ◽

Specific Cell ◽

Hog Pathway ◽

Cell Wall Stress ◽

Cell Wall Remodeling ◽

Mitogen Activated Protein

ABSTRACT During fermentation, a high ethanol concentration is a major stress that influences the vitality and viability of yeast cells, which in turn leads to a termination of the fermentation process. In this study, we show that the BCK1 and SLT2 genes encoding mitogen-activated protein kinase kinase kinase (MAPKKK) and mitogen-activated protein kinase (MAPK) of the cell wall integrity (CWI) pathway, respectively, are essential for ethanol tolerance, suggesting that the CWI pathway is involved in the response to ethanol-induced cell wall stress. Upon ethanol exposure, the CWI pathway induces the expression of specific cell wall-remodeling genes, including FKS2, CRH1, and PIR3 (encoding β-1,3-glucan synthase, chitin transglycosylase, and O-glycosylated cell wall protein, respectively), which eventually leads to the remodeling of the cell wall structure. Our results revealed that in response to ethanol stress, the high-osmolarity glycerol (HOG) pathway plays a collaborative role with the CWI pathway in inducing cell wall remodeling via the upregulation of specific cell wall biosynthesis genes such as the CRH1 gene. Furthermore, the substantial expression of CWI-responsive genes is also triggered by external hyperosmolarity, suggesting that the adaptive changes in the cell wall are crucial for protecting yeast cells against not only cell wall stress but also osmotic stress. On the other hand, the cell wall stress-inducing agent calcofluor white has no effect on promoting the expression of GPD1, a major target gene of the HOG pathway. Collectively, these findings suggest that during ethanol stress, the CWI and HOG pathways collaboratively regulate the transcription of specific cell wall biosynthesis genes, thereby leading to adaptive changes in the cell wall. IMPORTANCE The budding yeast Saccharomyces cerevisiae has been widely used in industrial fermentations, including the production of alcoholic beverages and bioethanol. During fermentation, an increased ethanol concentration is the main stress that affects yeast metabolism and inhibits ethanol production. This work presents evidence that in response to ethanol stress, both CWI and HOG pathways cooperate to control the expression of cell wall-remodeling genes in order to build the adaptive strength of the cell wall. These findings will contribute to a better understanding of the molecular mechanisms underlying adaptive responses and tolerance of yeast to ethanol stress, which is essential for successful engineering of yeast strains for improved ethanol tolerance.

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PKC1 Is Essential for Protection against both Oxidative and Nitrosative Stresses, Cell Integrity, and Normal Manifestation of Virulence Factors in the Pathogenic Fungus Cryptococcus neoformans

Eukaryotic Cell ◽

10.1128/ec.00146-08 ◽

2008 ◽

Vol 7 (10) ◽

pp. 1685-1698 ◽

Cited By ~ 83

Author(s):

Kimberly J. Gerik ◽

Sujit R. Bhimireddy ◽

Jan S. Ryerse ◽

Charles A. Specht ◽

Jennifer K. Lodge

Keyword(s):

Cell Wall ◽

Nitrosative Stress ◽

Pathogenic Fungus ◽

Fungal Growth ◽

Mitogen Activated Protein Kinase ◽

Cell Integrity ◽

Wild Type ◽

Pathway Activation ◽

Mitogen Activated Protein ◽

Kinase Cascade

ABSTRACT Cell wall integrity is crucial for fungal growth, survival, and pathogenesis. Responses to environmental stresses are mediated by the highly conserved Pkc1 protein and its downstream components. In this study, we demonstrate that both oxidative and nitrosative stresses activate the PKC1 cell integrity pathway in wild-type cells, as measured by phosphorylation of Mpk1, the terminal protein in the PKC1 phosphorylation cascade. Furthermore, deletion of PKC1 shows that this gene is essential for defense against both oxidative and nitrosative stresses; however, other genes involved directly in the PKC1 pathway are dispensable for protection against these stresses. This suggests that Pkc1 may have multiple and alternative functions other than activating the mitogen-activated protein kinase cascade from a “top-down” approach. Deletion of PKC1 also causes osmotic instability, temperature sensitivity, severe sensitivity to cell wall-inhibiting agents, and alterations in capsule and melanin. Furthermore, the vital cell wall components chitin and its deacetylated form chitosan appear to be mislocalized in a pkc1Δ strain, although this mutant contains wild-type levels of both of these polymers. These data indicate that loss of Pkc1 has pleiotropic effects because it is central to many functions either dependent on or independent of PKC1 pathway activation. Notably, this is the first time that Pkc1 has been implicated in protection against nitrosative stress in any organism.

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Zds1/Zds2–PP2ACdc55 complex specifies signaling output from Rho1 GTPase

The Journal of Cell Biology ◽

10.1083/jcb.201508119 ◽

2016 ◽

Vol 212 (1) ◽

pp. 51-61 ◽

Cited By ~ 9

Author(s):

Erin M. Jonasson ◽

Valentina Rossio ◽

Riko Hatakeyama ◽

Mitsuhiro Abe ◽

Yoshikazu Ohya ◽

...

Keyword(s):

Cell Wall ◽

Cell Growth ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Signaling Specificity ◽

Actin Organization ◽

Cell Wall Damage ◽

Polarized Cell Growth ◽

Mitogen Activated Protein ◽

Guanosine Triphosphatase

Budding yeast Rho1 guanosine triphosphatase (GTPase) plays an essential role in polarized cell growth by regulating cell wall glucan synthesis and actin organization. Upon cell wall damage, Rho1 blocks polarized cell growth and repairs the wounds by activating the cell wall integrity (CWI) Pkc1–mitogen-activated protein kinase (MAPK) pathway. A fundamental question is how active Rho1 promotes distinct signaling outputs under different conditions. Here we identified the Zds1/Zds2–protein phosphatase 2ACdc55 (PP2ACdc55) complex as a novel Rho1 effector that regulates Rho1 signaling specificity. Zds1/Zds2–PP2ACdc55 promotes polarized growth and cell wall synthesis by inhibiting Rho1 GTPase-activating protein (GAP) Lrg1 but inhibits CWI pathway by stabilizing another Rho1 GAP, Sac7, suggesting that active Rho1 is biased toward cell growth over stress response. Conversely, upon cell wall damage, Pkc1–Mpk1 activity inhibits cortical PP2ACdc55, ensuring that Rho1 preferentially activates the CWI pathway for cell wall repair. We propose that PP2ACdc55 specifies Rho1 signaling output and that reciprocal antagonism between Rho1–PP2ACdc55 and Rho1–Pkc1 explains how only one signaling pathway is robustly activated at a time.

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Transcriptional Coregulation by the Cell Integrity Mitogen-Activated Protein Kinase Slt2 and the Cell Cycle Regulator Swi4

Molecular and Cellular Biology ◽

10.1128/mcb.21.19.6515-6528.2001 ◽

2001 ◽

Vol 21 (19) ◽

pp. 6515-6528 ◽

Cited By ~ 84

Author(s):

Kristin Baetz ◽

Jason Moffat ◽

Jennifer Haynes ◽

Michael Chang ◽

Brenda Andrews

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Wall ◽

Transcription Factor ◽

Protein Kinase ◽

Mapk Pathway ◽

Mitogen Activated Protein Kinase ◽

Regulatory Subunit ◽

Cell Integrity ◽

Mitogen Activated Protein

ABSTRACT In Saccharomyces cerevisiae, the heterodimeric transcription factor SBF (for SCB binding factor) is composed of Swi4 and Swi6 and activates gene expression at the G1/S-phase transition of the mitotic cell cycle. Cell cycle commitment is associated not only with major alterations in gene expression but also with highly polarized cell growth; the mitogen-activated protein kinase (MAPK) Slt2 is required to maintain cell wall integrity during periods of polarized growth and cell wall stress. We describe experiments aimed at defining the regulatory pathway involving the cell cycle transcription factor SBF and Slt2-MAPK. Gene expression assays and chromatin immunoprecipitation experiments revealed Slt2-dependent recruitment of SBF to the promoters of the G1 cyclinsPCL1 and PCL2 after activation of the Slt2-MAPK pathway. We performed DNA microarray analysis and identified other genes whose expression was reduced in both SLT2and SWI4 deletion strains. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated and reveal a role for Swi4, the DNA-binding component of SBF, which is independent of the regulatory subunit Swi6. Some of the Swi4- and Slt2-dependent genes do not require Swi6 for either their expression or for Swi4 localization to their promoters. Consistent with these results, we found a direct interaction between Swi4 and Slt2. Our results establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond its prominent role in controlling cell cycle transcription.

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Saccharomyces cerevisiae Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of thePKC1-MPK1 Cell Integrity Pathway

Journal of Bacteriology ◽

10.1128/jb.181.11.3330-3340.1999 ◽

1999 ◽

Vol 181 (11) ◽

pp. 3330-3340 ◽

Cited By ~ 194

Author(s):

Troy Ketela ◽

Robin Green ◽

Howard Bussey

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Wall ◽

Wall Stress ◽

Structural Features ◽

Mitogen Activated Protein Kinase ◽

Cell Integrity ◽

Chitin Synthesis ◽

Calcofluor White ◽

Reading Frame ◽

Cell Wall Stress

ABSTRACT The MID2 gene of Saccharomyces cerevisiaeencodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion ofMID2 causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression ofMID2 causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. α-Factor hypersensitivity ofmid2Δ mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, includingPKC1, RHO1, WSC1, andWSC2. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since mid2Δ wsc1Δ mutants are inviable on medium that does not contain osmotic support. A role for MID2 in the cell integrity pathway is further supported by the finding that MID2 is required for induction of Mpk1p tyrosine phosphorylation during exposure to α-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame, MTL1, which encodes a protein with both structural and functional similarity to Mid2p.

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Yeast Mpk1 Cell Wall Integrity Mitogen-activated Protein Kinase Regulates Nucleocytoplasmic Shuttling of the Swi6 Transcriptional Regulator

Molecular Biology of the Cell ◽

10.1091/mbc.e09-11-0923 ◽

2010 ◽

Vol 21 (9) ◽

pp. 1609-1619 ◽

Cited By ~ 30

Author(s):

Ki-Young Kim ◽

Andrew W. Truman ◽

Stefanie Caesar ◽

Gabriel Schlenstedt ◽

David E. Levin

Keyword(s):

Cell Wall ◽

Protein Kinase ◽

Transcriptional Activation ◽

Transcriptional Response ◽

Wall Stress ◽

Cell Wall Integrity ◽

Mitogen Activated Protein Kinase ◽

Nucleocytoplasmic Shuttling ◽

Nuclear Entry ◽

Mitogen Activated Protein

The yeast SBF transcription factor is a heterodimer comprised of Swi4 and Swi6 that has a well defined role in cell cycle-specific transcription. SBF serves a second function in the transcriptional response to cell wall stress in which activated Mpk1 mitogen-activated protein kinase of the cell wall integrity signaling pathway forms a complex with Swi4, the DNA binding subunit of SBF, conferring upon Swi4 the ability to bind DNA and activate transcription of FKS2. Although Mpk1–Swi4 complex formation and transcriptional activation of FKS2 does not require Mpk1 catalytic activity, Swi6 is phosphorylated by Mpk1 and must be present in the Mpk1-Swi4 complex for transcriptional activation of FKS2. Here, we find that Mpk1 regulates Swi6 nucleocytoplasmic shuttling in a biphasic manner. First, formation of the Mpk1-Swi4 complex recruits Swi6 to the nucleus for transcriptional activation. Second, Mpk1 negatively regulates Swi6 by phosphorylation on Ser238, which inhibits nuclear entry. Ser238 neighbors a nuclear localization signal (NLS) whose function is blocked by phosphorylation at Ser238 in a manner similar to the regulation by Cdc28 of another Swi6 NLS, revealing a mechanism for the integration of multiple signals to a single endpoint. Finally, the Kap120 β-importin binds the Mpk1-regulated Swi6 NLS but not the Cdc28-regulated NLS.

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Signaling Domains of Mucin Msb2 in Candida albicans

Eukaryotic Cell ◽

10.1128/ec.00264-14 ◽

2015 ◽

Vol 14 (4) ◽

pp. 359-370 ◽

Cited By ~ 14

Author(s):

Marc Swidergall ◽

Lasse van Wijlick ◽

Joachim F. Ernst

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Map Kinase ◽

Cytoplasmic Tail ◽

Wall Stress ◽

Mitogen Activated Protein Kinase ◽

Basal Resistance ◽

Interacting Protein ◽

Content Type ◽

Cell Wall Stress

ABSTRACTCandida albicansadapts to the human host by environmental sensing using the Msb2 signal mucin, which regulates fungal morphogenesis and resistance characteristics. Msb2 is anchored within the cytoplasmic membrane by a single transmembrane (TM) region dividing it into a large N-terminal exodomain, which is shed, and a small cytoplasmic domain. Analyses of strains carrying deleted Msb2 variants revealed an exodomain segment required for cleavage, shedding, and all functions of Msb2. Phosphorylation of the mitogen-activated protein kinase (MAP kinase) Cek1 was regulated by three distinct regions in Msb2: in unstressed cells, N-terminal sequences repressed phosphorylation, while its induction under cell wall stress required the cytoplasmic tail (C-tail) and sequences N-terminally flanking the TM region, downstream of the proposed cleavage site. Within the latter Msb2 region, overlapping but not identical sequences were also required for hyphal morphogenesis, basal resistance to antifungals, and, in unstressed cells, downregulation of thePMT1transcript, encoding proteinO-mannosyltransferase-1. Deletion of two-thirds of the exodomain generated a truncated Msb2 variant with a striking ability to induce hyperfilamentous growth, which depended on the presence of the Msb2-interacting protein Sho1, the MAP kinase Cek1, and the Efg1 transcription factor. Under cell wall stress, the cytoplasmic tail relocalized partially to the nucleus and contributed to regulation of 117 genes, as revealed by transcriptomic analyses. Genes regulated by the C-tail contained binding sites for the Ace2 and Azf1 transcription factors and included theALScell wall genes. We concluded that Msb2 fulfills its numerous functions by employing functional domains that are distributed over its entire length.

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The Aspergillus fumigatus Phosphoproteome Reveals Roles of High-Osmolarity Glycerol Mitogen-Activated Protein Kinases in Promoting Cell Wall Damage and Caspofungin Tolerance

mBio ◽

10.1128/mbio.02962-19 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 5

Author(s):

Eliciane Cevolani Mattos ◽

Lilian Pereira Silva ◽

Clara Valero ◽

Patrícia Alves de Castro ◽

Thaila Fernanda dos Reis ◽

...

Keyword(s):

Cell Wall ◽

Aspergillus Fumigatus ◽

Protein Kinases ◽

Wall Stress ◽

Mapk Signaling ◽

Fungal Cell ◽

Content Type ◽

Cell Wall Stress ◽

Cell Wall Damage ◽

Mitogen Activated Protein

ABSTRACT The filamentous fungus Aspergillus fumigatus can cause a distinct set of clinical disorders in humans. Invasive aspergillosis (IA) is the most common life-threatening fungal disease of immunocompromised humans. The mitogen-activated protein kinase (MAPK) signaling pathways are essential to the adaptation to the human host. Fungal cell survival is highly dependent on the organization, composition, and function of the cell wall. Here, an evaluation of the global A. fumigatus phosphoproteome under cell wall stress caused by the cell wall-damaging agent Congo red (CR) revealed 485 proteins potentially involved in the cell wall damage response. Comparative phosphoproteome analyses with the ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutant strains from the osmotic stress MAPK cascades identify their additional roles during the cell wall stress response. Our phosphoproteomics allowed the identification of novel kinases and transcription factors (TFs) involved in osmotic stress and in the cell wall integrity (CWI) pathway. Our global phosphoproteome network analysis showed an enrichment for protein kinases, RNA recognition motif domains, and the MAPK signaling pathway. In contrast to the wild-type strain, there is an overall decrease of differentially phosphorylated kinases and phosphatases in ΔsakA, ΔmpkC, and ΔsakA ΔmpkC mutants. We constructed phosphomutants for the phosphorylation sites of several proteins differentially phosphorylated in the wild-type and mutant strains. For all the phosphomutants, there is an increase in the sensitivity to cell wall-damaging agents and a reduction in the MpkA phosphorylation upon CR stress, suggesting these phosphosites could be important for the MpkA modulation and CWI pathway regulation. IMPORTANCE Aspergillus fumigatus is an opportunistic human pathogen causing allergic reactions or systemic infections, such as invasive pulmonary aspergillosis in immunocompromised patients. The mitogen-activated protein kinase (MAPK) signaling pathways are essential for fungal adaptation to the human host. Fungal cell survival, fungicide tolerance, and virulence are highly dependent on the organization, composition, and function of the cell wall. Upon cell wall stress, MAPKs phosphorylate multiple target proteins involved in the remodeling of the cell wall. Here, we investigate the global phosphoproteome of the ΔsakA and ΔmpkC A. fumigatus and high-osmolarity glycerol (HOG) pathway MAPK mutants upon cell wall damage. This showed the involvement of the HOG pathway and identified novel protein kinases and transcription factors, which were confirmed by fungal genetics to be involved in promoting tolerance of cell wall damage. Our results provide understanding of how fungal signal transduction networks modulate the cell wall. This may also lead to the discovery of new fungicide drug targets to impact fungal cell wall function, fungicide tolerance, and virulence.

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Yeast Mpk1 Mitogen-Activated Protein Kinase Activates Transcription through Swi4/Swi6 by a Noncatalytic Mechanism That Requires Upstream Signal

Molecular and Cellular Biology ◽

10.1128/mcb.01795-07 ◽

2008 ◽

Vol 28 (8) ◽

pp. 2579-2589 ◽

Cited By ~ 80

Author(s):

Ki-Young Kim ◽

Andrew W. Truman ◽

David E. Levin

Keyword(s):

Cell Wall ◽

Protein Kinase ◽

Dna Binding ◽

Transcriptional Activation ◽

Kinase Activity ◽

Wall Stress ◽

Mitogen Activated Protein Kinase ◽

Protein Kinase Activity ◽

Mitogen Activated Protein

ABSTRACT The cell wall integrity mitogen-activated protein kinase (MAPK) cascade of Saccharomyces cerevisiae drives changes in gene expression in response to cell wall stress. We show that the MAPK of this pathway (Mpk1) and its pseudokinase paralog (Mlp1) use a noncatalytic mechanism to activate transcription of the FKS2 gene. Transcriptional activation of FKS2 was dependent on the Swi4/Swi6 (SBF) transcription factor and on an activating signal to Mpk1 but not on protein kinase activity. Activated (phosphorylated) Mpk1 and Mlp1 were detected in a complex with Swi4 and Swi6 at the FKS2 promoter. Mpk1 association with Swi4 in vivo required phosphorylation of Mpk1. Promoter association of Mpk1 and the Swi4 DNA-binding subunit of SBF were codependent but did not require Swi6, indicating that the MAPK confers DNA-binding ability to Swi4. Based on these data, we propose a model in which phosphorylated Mpk1 or Mlp1 forms a dimeric complex with Swi4 that is competent to associate with the FKS2 promoter. This complex then recruits Swi6 to activate transcription. Finally, we show that human ERK5, a functional ortholog of Mpk1, is similarly capable of driving FKS2 expression in the absence of protein kinase activity, suggesting that this mammalian MAPK may also have a noncatalytic function in vivo.

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