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⌬ mutation, yet supports mating even in ste4⌬ ste5⌬ cells when artificially dimerized by fusion to glutathione S-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5⌬ cells, but does not allow mating of ste4⌬ ste5⌬ cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa ste4⌬ ste5⌬ cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when G␤␥ is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of G␤␥ to the RING-H2 domain may induce a conformational change that promotes association of the N-and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20. INTRODUCTIONThe pheromone response pathway of the yeast Saccharomyces cerevisiae has provided a system for elucidating mechanisms that convert an extracellular signal into both a morphological response and a change in the pattern of gene expression (reviewed in Bardwell et al., 1994;Leberer et al., 1997a). Mating of haploid cells (MATa and MAT␣) requires the action of peptide pheromones: MATa cells secrete a-factor, and MAT␣ cells secrete ␣-factor. The cell surface receptors for these peptides (Ste2 in MATa cells binds ␣-factor, and Ste3 in MAT␣ cells binds a-factor) a...

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

“…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.…”

“…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.…”

“…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).…”

“…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).…”

“…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).…”