scholarly journals Mechanism of Mpk1 Mitogen-Activated Protein Kinase Binding to the Swi4 Transcription Factor and Its Regulation by a Novel Caffeine-Induced Phosphorylation

2009 ◽  
Vol 29 (24) ◽  
pp. 6449-6461 ◽  
Author(s):  
Andrew W. Truman ◽  
Ki-Young Kim ◽  
David E. Levin
Keyword(s):  
Cell Wall ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Binding Site ◽  
Mutational Analysis ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Activation Mechanism ◽  
Mitogen Activated Protein

ABSTRACT The Mpk1 mitogen-activated protein kinase (MAPK) of the cell wall integrity signaling pathway uses a noncatalytic mechanism to activate the SBF (Swi4/Swi6) transcription factor. Active Mpk1 forms a complex with Swi4, the DNA-binding subunit of SBF, conferring the ability to bind DNA. Because SBF activation is independent of Mpk1 catalytic activity but requires Mpk1 to be in an active conformation, we sought to understand how Mpk1 interacts with Swi4. Mutational analysis revealed that binding and activation of Swi4 by Mpk1 requires an intact D-motif-binding site, a docking surface common to MAPKs that resides distal to the phosphorylation loop but does not require the substrate-binding site, revealing a novel mechanism for MAPK target regulation. Additionally, we found that Mpk1 binds near the autoinhibitory C terminus of Swi4, suggesting an activation mechanism in which Mpk1 substitutes for Swi6 in promoting Swi4 DNA binding. Finally, we show that caffeine is an atypical activator of cell wall integrity signaling, because it induces phosphorylation of the Mpk1 C-terminal extension at Ser423 and Ser428. These phosphorylations were dependent on the DNA damage checkpoint kinases, Mec1/Tel1 and Rad53. Phosphorylation of Ser423 specifically blocked SBF activation by preventing Mpk1 association with Swi4, revealing a novel mechanism for regulating MAPK target specificity.

scholarly journals Nuclear Export and Plasma Membrane Recruitment of the Ste5 Scaffold Are Coordinated with Oligomerization and Association with Signal Transduction Components

2003 ◽  
Vol 14 (6) ◽  
pp. 2543-2558 ◽  
Author(s):  
Yunmei Wang ◽  
Elaine A. Elion
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Site ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Active Form ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Kinase Cascade ◽  
Protein Kinase Cascade

The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a Gβγ dimer (Ste4/Ste18) at the plasma membrane in a recruitment event that requires prior nuclear shuttling of Ste5. Genetic evidence suggests that Ste5 must oligomerize to function, but its impact on Ste5 function and localization is unknown. Herein, we show that oligomerization affects Ste5 activity and localization. The majority of Ste5 is monomeric, suggesting that oligomerization is tightly regulated. Increasing the pool of Ste5 oligomers increases association with Ste11. Remarkably, Ste5 oligomers are also more efficiently exported from the nucleus, retained in the cytoplasm by Ste11 and better recruited to the plasma membrane, resulting in constitutive activation of the mating mitogen-activated protein kinase cascade. Coprecipitation tests show that the RING-H2 domain is the key determinant of oligomerization. Mutational analysis suggests that the leucine-rich domain limits the accessibility of the RING-H2 domain and inhibits export and recruitment in addition to promoting Ste11 association and activation. Our results suggest that the major form of Ste5 is an inactive monomer with an inaccessible RING-H2 domain and Ste11 binding site, whereas the active form is an oligomer that is more efficiently exported and recruited and has a more accessible RING-H2 domain and Ste11 binding site.

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

2001 ◽  
Vol 21 (19) ◽  
pp. 6515-6528 ◽  
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.

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

2010 ◽  
Vol 21 (9) ◽  
pp. 1609-1619 ◽  
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.

scholarly journals Hog1 Mitogen-Activated Protein Kinase (MAPK) Interrupts Signal Transduction between the Kss1 MAPK and the Tec1 Transcription Factor To Maintain Pathway Specificity

2009 ◽  
Vol 8 (4) ◽  
pp. 606-616 ◽  
Author(s):  
Teresa R. Shock ◽  
James Thompson ◽  
John R. Yates ◽  
Hiten D. Madhani
Keyword(s):  
Signal Transduction ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Dna Binding ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Nuclear Accumulation ◽  
Hog Pathway ◽  
Pathway Activation ◽  
Mitogen Activated Protein

ABSTRACT In Saccharomyces cerevisiae, the mating, filamentous growth (FG), and high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) signaling pathways share components and yet mediate distinct responses to different extracellular signals. Cross talk is suppressed between the mating and FG pathways because mating signaling induces the destruction of the FG transcription factor Tec1. We show here that HOG pathway activation results in phosphorylation of the FG MAPK, Kss1, and the MAPKK, Ste7. However, FG transcription is not activated because HOG signaling prevents the activation of Tec1. In contrast to the mating pathway, we find that the mechanism involves the inhibition of DNA binding by Tec1 rather than its destruction. We also find that nuclear accumulation of Tec1 is not affected by HOG signaling. Inhibition by Hog1 is apparently indirect since it does not require any of the consensus S/TP MAPK phosphorylation sites on Tec1, its DNA-binding partner Ste12, or the associated regulators Dig1 or Dig2. It also does not require the consensus MAPK sites of the Ste11 activator Ste50, in contrast to a recent proposal for a role for negative feedback in specificity. Our results demonstrate that HOG signaling interrupts the FG pathway signal transduction between the phosphorylation of Kss1 and the activation of DNA binding by Tec1.

scholarly journals Putative Membrane Receptors Contribute to Activation and Efficient Signaling of Mitogen-Activated Protein Kinase Cascades during Adaptation of Aspergillus fumigatus to Different Stressors and Carbon Sources

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Lilian Pereira Silva ◽  
Dean Frawley ◽  
Leandro José de Assis ◽  
Ciara Tierney ◽  
Alastair B. Fleming ◽  
...  
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Aspergillus Fumigatus ◽  
Fungal Pathogen ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Hog Pathway ◽  
Content Type ◽  
High Osmolarity Glycerol ◽  
Mitogen Activated Protein

ABSTRACT The high-osmolarity glycerol (HOG) response pathway is a multifunctional signal transduction pathway that specifically transmits ambient osmotic signals. Saccharomyces cerevisiae Hog1p has two upstream signaling branches, the sensor histidine kinase Sln1p and the receptor Sho1p. The Sho1p branch includes two other proteins, the Msb2p mucin and Opy2p. Aspergillus fumigatus is the leading cause of pulmonary fungal diseases. Here, we investigated the roles played by A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p putative homologues during the activation of the mitogen-activated protein kinase (MAPK) HOG pathway. The shoA, msbA, and opyA singly and doubly null mutants are important for the cell wall integrity (CWI) pathway, oxidative stress, and virulence as assessed by a Galleria mellonella model. Genetic interactions of ShoA, MsbA, and OpyA are also important for proper activation of the SakAHog1p and MpkASlt2 cascade and the response to osmotic and cell wall stresses. Comparative label-free quantitative proteomics analysis of the singly null mutants with the wild-type strain upon caspofungin exposure indicates that the absence of ShoA, MsbA, and OpyA affects the osmotic stress response, carbohydrate metabolism, and protein degradation. The putative receptor mutants showed altered trehalose and glycogen accumulation, suggesting a role for ShoA, MsbA, and OpyA in sugar storage. Protein kinase A activity was also decreased in these mutants. We also observed genetic interactions between SlnA, ShoA, MsbA, and OpyA, suggesting that both branches are important for activation of the HOG/CWI pathways. Our results help in the understanding of the activation and modulation of the HOG and CWI pathways in this important fungal pathogen. IMPORTANCE Aspergillus fumigatus is an important human-pathogenic fungal species that is responsible for a high incidence of infections in immunocompromised individuals. A. fumigatus high-osmolarity glycerol (HOG) and cell wall integrity pathways are important for the adaptation to different forms of environmental adversity such as osmotic and oxidative stresses, nutrient limitations, high temperatures, and other chemical and mechanical stresses that may be produced by the host immune system and antifungal drugs. Little is known about how these pathways are activated in this fungal pathogen. Here, we characterize four A. fumigatus putative homologues that are important for the activation of the yeast HOG pathway. A. fumigatus SlnASln1p, ShoASho1p, MsbAMsb2p, and OpyAOpy2p are genetically interacting and are essential for the activation of the HOG and cell wall integrity pathways. Our results contribute to the understanding of A. fumigatus adaptation to the host environment.

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

2008 ◽  
Vol 28 (8) ◽  
pp. 2579-2589 ◽  
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.

scholarly journals Pkh1 and Pkh2 Differentially Phosphorylate and Activate Ypk1 and Ykr2 and Define Protein Kinase Modules Required for Maintenance of Cell Wall Integrity

2002 ◽  
Vol 13 (9) ◽  
pp. 3005-3028 ◽  
Author(s):  
Françoise M. Roelants ◽  
Pamela D. Torrance ◽  
Natalie Bezman ◽  
Jeremy Thorner
Keyword(s):  
Cell Wall ◽  
Protein Kinase ◽  
Kinase Domain ◽  
Cell Wall Integrity ◽  
Mitogen Activated Protein Kinase ◽  
Restrictive Temperature ◽  
Mitogen Activated Protein ◽  
Catalytically Active ◽  
Inducible Protein ◽  
Mammalian Protein

Saccharomyces cerevisiae Pkh1 and Pkh2 are functionally redundant homologs of mammalian protein kinase, phosphoinositide-dependent protein kinase-1. They activate two closely related, functionally redundant enzymes, Ypk1 and Ykr2 (homologs of mammalian protein kinase, serum- and glucocorticoid-inducible protein kinase). We found that Ypk1 has a more prominent role than Ykr2 in mediating their shared essential function. Considerable evidence demonstrated that Pkh1 preferentially activates Ypk1, whereas Pkh2 preferentially activates Ykr2. Loss of Pkh1 (but not Pkh2) reduced Ypk1 activity; conversely, Pkh1 overexpression increased Ypk1 activity more than Pkh2 overexpression. Loss of Pkh2 reduced Ykr2 activity; correspondingly, Pkh2 overexpression increased Ykr2 activity more than Pkh1 overexpression. When overexpressed, a catalytically active C-terminal fragment (kinase domain) of Ypk1 was growth inhibitory; loss of Pkh1 (but not Pkh2) alleviated toxicity. Loss of Pkh2 (but not Pkh1) exacerbated the slow growth phenotype of aypk1Δ strain. This Pkh1-Ypk1 and Pkh2-Ykr2 dichotomy is not absolute because all double mutants (pkh1Δ ypk1Δ, pkh2Δ ypk1Δ, pkh1Δ ykr2Δ, and pkh2Δ ykr2Δ) were viable. Compartmentation contributes to selectivity because Pkh1 and Ypk1 were located exclusively in the cytosol, whereas Pkh2 and Ykr2 entered the nucleus. At restrictive temperature,ypk1-1tsykr2Δ cells lysed rapidly, but not in medium containing osmotic support. Dosage and extragenic suppressors were selected. Overexpression of Exg1 (major exoglucanase), or loss of Kex2 (endoprotease involved in Exg1 processing), rescued growth at high temperature. Viability was also maintained by PKC1 overexpression or an activated allele of the downstream protein kinase (BCK1-20). Conversely, absence of Mpk1 (distal mitogen-activated protein kinase of thePKC1 pathway) was lethal inypk1-1tsykr2Δ cells. Thus, Pkh1-Ypk1 and Pkh2-Ykr2 function in a novel pathway for cell wall integrity that acts in parallel with the Pkc1-dependent pathway.

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