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Mutational Analysis Suggests That Activation of the Yeast Pheromone Response Mitogen-activated Protein Kinase Pathway Involves Conformational Changes in the Ste5 Scaffold Protein
Abstract:Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases G␥ (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with G␥. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5⌬ mut… Show more
“…Our interpretation contrasts the view of Sette et al (2000) who have proposed that the active form of Ste5 undergoes stronger interactions between the N and C halves of the protein, either through intramolecular contacts in a closed monomer or intermolecular contacts in an antiparallel dimer. In their study, hyperactivating mutations in either the N terminus (Ste5 P44L) or C termini (Ste5 S770K) caused better coprecipitation of the N-terminal and C-terminal halves of the protein with no effect on oligomerization.…”
Section: The Availability Of the Ring-h2 Domain Determines The Amountcontrasting
confidence: 99%
“…Two lines of evidence lead us to favor the possibility that the major form of Ste5 is an autoinhibited monomer in which contacts between the N-and C-terminal halves of the protein decrease the accessibility of the RING-H2 domain and Ste11 binding site ( Figure 8D). First, previous work indicates that the N-and C-terminal halves of Ste5 can associate (Sette et al, 2000). Second, we find that the major pool of Ste5 does not efficiently associate with Ste4 and Ste11 unless Ste5 has an increased capacity to oligomerize.…”
Section: The Major Form Of Ste5 In Cells Is An Inactive Monomermentioning
confidence: 51%
“…In light of our findings, the P44L and S770K mutations would be predicted to increase the accessibility of nearby RING-H2 and leucine-rich domains for oligomerization. This prediction is supported by findings in Sette et al (2000): 1) GST-Ste5P44L(1-518) associates more readily with Ste5P44L(1-518) than it does with wild-type Ste5(1-518); and 2) GST-Ste5 (1-518) and GST-Ste5P44L(1-518) both associate more readily with full-length Ste5P44L than with full-length Ste5. Furthermore, our model does not rule out the possibility that interactions between the N and C termini of the protein could be involved in activation of Ste5; for example, N to C interactions could occur between dimers or within a dimer (see Elion, 2001 for a discussion).…”
Section: The Availability Of the Ring-h2 Domain Determines The Amountmentioning
confidence: 52%
“…The previous results can be reconciled by postulating that the need for regulated recruitment to the plasma membrane is bypassed if the amount of Ste5 in the cytoplasm is high enough to allow interactions with Ste20 at the cell cortex. This interpretation is supported by the fact that overexpressed Ste5(C177AC180A)-GST still requires Ste20 to activate the pathway (Sette et al, 2000). It is also supported by the observation that signal transduction can be restored to a Ste5⌬49-66 mutant that fails to shuttle through the nucleus when it is overexpressed (Elion, 2002).…”
Section: Oligomerization Does Not Bypass a Requirement For Recruitmenmentioning
confidence: 83%
“…However, biochemical and two-hybrid analysis suggests that Ste5 oligomerization could occur constitutively (Feng et al, 1998). More recent analysis suggests that Ste5 undergoes an activating conformational change of interactions between the N-and C-terminal halves of the protein that regulates the associated kinases by allostery or alignment (Sette et al, 2000). Conformational changes could also regulate oligomerization and association with signaling components (Elion, 2001).…”
The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a G␥ 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.
“…Our interpretation contrasts the view of Sette et al (2000) who have proposed that the active form of Ste5 undergoes stronger interactions between the N and C halves of the protein, either through intramolecular contacts in a closed monomer or intermolecular contacts in an antiparallel dimer. In their study, hyperactivating mutations in either the N terminus (Ste5 P44L) or C termini (Ste5 S770K) caused better coprecipitation of the N-terminal and C-terminal halves of the protein with no effect on oligomerization.…”
Section: The Availability Of the Ring-h2 Domain Determines The Amountcontrasting
confidence: 99%
“…Two lines of evidence lead us to favor the possibility that the major form of Ste5 is an autoinhibited monomer in which contacts between the N-and C-terminal halves of the protein decrease the accessibility of the RING-H2 domain and Ste11 binding site ( Figure 8D). First, previous work indicates that the N-and C-terminal halves of Ste5 can associate (Sette et al, 2000). Second, we find that the major pool of Ste5 does not efficiently associate with Ste4 and Ste11 unless Ste5 has an increased capacity to oligomerize.…”
Section: The Major Form Of Ste5 In Cells Is An Inactive Monomermentioning
confidence: 51%
“…In light of our findings, the P44L and S770K mutations would be predicted to increase the accessibility of nearby RING-H2 and leucine-rich domains for oligomerization. This prediction is supported by findings in Sette et al (2000): 1) GST-Ste5P44L(1-518) associates more readily with Ste5P44L(1-518) than it does with wild-type Ste5(1-518); and 2) GST-Ste5 (1-518) and GST-Ste5P44L(1-518) both associate more readily with full-length Ste5P44L than with full-length Ste5. Furthermore, our model does not rule out the possibility that interactions between the N and C termini of the protein could be involved in activation of Ste5; for example, N to C interactions could occur between dimers or within a dimer (see Elion, 2001 for a discussion).…”
Section: The Availability Of the Ring-h2 Domain Determines The Amountmentioning
confidence: 52%
“…The previous results can be reconciled by postulating that the need for regulated recruitment to the plasma membrane is bypassed if the amount of Ste5 in the cytoplasm is high enough to allow interactions with Ste20 at the cell cortex. This interpretation is supported by the fact that overexpressed Ste5(C177AC180A)-GST still requires Ste20 to activate the pathway (Sette et al, 2000). It is also supported by the observation that signal transduction can be restored to a Ste5⌬49-66 mutant that fails to shuttle through the nucleus when it is overexpressed (Elion, 2002).…”
Section: Oligomerization Does Not Bypass a Requirement For Recruitmenmentioning
confidence: 83%
“…However, biochemical and two-hybrid analysis suggests that Ste5 oligomerization could occur constitutively (Feng et al, 1998). More recent analysis suggests that Ste5 undergoes an activating conformational change of interactions between the N-and C-terminal halves of the protein that regulates the associated kinases by allostery or alignment (Sette et al, 2000). Conformational changes could also regulate oligomerization and association with signaling components (Elion, 2001).…”
The Ste5 scaffold activates an associated mitogen-activated protein kinase cascade by binding through its RING-H2 domain to a G␥ 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.
Due to the central position of scaffold proteins in numerous signaling networks, especially in MAPK pathways, considerable efforts have been made to identify new scaffolds and to characterize their function and regulation. Most of our knowledge stems from studies of yeast MAPK scaffolds, but the identification of such scaffolds in higher eukaryotes provided a new dimension to this field and led to exciting and promising new insights into the regulation of MAPK signaling. In this review, we shortly summarize the well-established basic functions of scaffolds in yeast and highlight concepts emerging from recent studies in yeast and higher eukaryotes. In particular, we discuss how scaffolds may actively influence MAPK signaling by inducing conformational changes of bound kinases or substrates, by controlling the localization of activated MAPK and the extent and output of MAPK activation, and by modulating MAPK kinetics through the recruitment of phosphatases or ubiquitin-ligases. Finally, we summarize the current knowledge of scaffold regulation, and how these events may be functionally important for MAPK signaling.
Apoptosis signal-regulating kinase 1 (ASK1) is a MAPKKK family member which activates c-Jun N-terminal kinase (JNK) and p38. In non-stressed cells, ASK1 exists as an inactive complex with the reduced form of thioredoxin. Oxidative stress such as hydrogen peroxide (H2O2) disrupts the ASK1-thioredoxin complex by oxidization of thioredoxin and thereby activates ASK1. The precise mechanism by which ASK1 is activated after its release from thioredoxin is unknown. Here we show that phosphorylation of Thr845 at the activation loop is essential for ASK1 to be activated by H2O2. ASK1 appears to form a silent homo-oligomer through its C-terminal coiled-coil region in non-stressed cells. Following H2O2 treatment, pre-existing ASK1 oligomer undergoes conformational change and creates a new interface within an oligomer, which ultimately leads to trans-autophosphorylation of Thr845. Thus, direct interaction via the coiled-coil region is required for self-scaffolding but not sufficient for activation of ASK1. Importantly, Thr845 of ASK1 can also be trans-phosphorylated by an unidentified Thr845 kinase in response to H2O2 treatment. We propose that this potential Thr845 kinase may be an ignition kinase that triggers Thr845 phosphorylation in oligomerized and activation-competent forms of ASK1.
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