Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway
2006; Springer Nature; Volume: 25; Issue: 13 Linguagem: Inglês
10.1038/sj.emboj.7601192
ISSN1460-2075
AutoresKazuo Tatebayashi, Katsuyoshi Yamamoto, Keiichiro Tanaka, Taichiro Tomida, Takashi Maruoka, Eri Kasukawa, Haruo Saito,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle15 June 2006free access Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway Kazuo Tatebayashi Kazuo Tatebayashi Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Katsuyoshi Yamamoto Katsuyoshi Yamamoto Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Keiichiro Tanaka Keiichiro Tanaka Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Taichiro Tomida Taichiro Tomida Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Takashi Maruoka Takashi Maruoka Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Eri Kasukawa Eri Kasukawa Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Haruo Saito Corresponding Author Haruo Saito Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Kazuo Tatebayashi Kazuo Tatebayashi Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Katsuyoshi Yamamoto Katsuyoshi Yamamoto Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Keiichiro Tanaka Keiichiro Tanaka Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Taichiro Tomida Taichiro Tomida Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Takashi Maruoka Takashi Maruoka Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Eri Kasukawa Eri Kasukawa Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Haruo Saito Corresponding Author Haruo Saito Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Author Information Kazuo Tatebayashi1,‡, Katsuyoshi Yamamoto1,‡, Keiichiro Tanaka1,2, Taichiro Tomida1, Takashi Maruoka1,2, Eri Kasukawa1 and Haruo Saito 1,2 1Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, Minato-ku, Tokyo, Japan 2Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Bunkyo-ku, Tokyo, Japan ‡These authors contributed equally to this work *Corresponding author. Division of Molecular Cell Signaling, Institute of Medical Sciences, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5449 5505; Fax: +81 3 5449 5701; E-mail: [email protected] The EMBO Journal (2006)25:3033-3044https://doi.org/10.1038/sj.emboj.7601192 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The yeast high osmolarity glycerol (HOG) signaling pathway can be activated by either of the two upstream pathways, termed the SHO1 and SLN1 branches. When stimulated by high osmolarity, the SHO1 branch activates an MAP kinase module composed of the Ste11 MAPKKK, the Pbs2 MAPKK, and the Hog1 MAPK. To investigate how osmostress activates this MAPK module, we isolated both gain-of-function and loss-of-function alleles in four key genes involved in the SHO1 branch, namely SHO1, CDC42, STE50, and STE11. These mutants were characterized using an HOG-dependent reporter gene, 8xCRE-lacZ. We found that Cdc42, in addition to binding and activating the PAK-like kinases Ste20 and Cla4, binds to the Ste11–Ste50 complex to bring activated Ste20/Cla4 to their substrate Ste11. Activated Ste11 and its HOG pathway-specific substrate, Pbs2, are brought together by Sho1; the Ste11–Ste50 complex binds to the cytoplasmic domain of Sho1, to which Pbs2 also binds. Thus, Cdc42, Ste50, and Sho1 act as adaptor proteins that control the flow of the osmostress signal from Ste20/Cla4 to Ste11, then to Pbs2. Introduction When exposed to hyperosmotic extracellular environments, the budding yeast (Saccharomyces cerevisiae) activates the high osmolarity glycerol (HOG) signaling pathway, which culminates in phosphorylation, activation, and nuclear translocation of the Hog1 MAP kinase (MAPK). Activated Hog1 initiates an adaptive program that includes adjustments in cell cycle progression, regulation of protein translation, induction or repression of various genes, and synthesis and intracellular retention of the compatible osmolyte glycerol (Gustin et al, 1998; Hohmann, 2002). The current view of the HOG pathway is summarized in Figure 1A. Yeast has two putative osmosensors, Sln1 and Sho1, and perhaps a third one, Msb2, which are all structurally distinct and functionally independent of each other (Maeda et al, 1994, 1995; O'Rourke and Herskowitz, 2002). Signals emanating from these sensors converge at a common MAPK kinase (MAPKK), Pbs2, which is the specific activator of Hog1 (Brewster et al, 1993; Maeda et al, 1994, 1995). The entire signal pathway from the cell surface sensors to the Hog1 MAPK will be referred to as the HOG pathway, while the branches upstream of Pbs2 are called the SLN1 and SHO1 branches, respectively. Figure 1.Ste20 and Cla4 are redundant in the HOG pathway. (A) Schematic model of the yeast HOG signal pathway. The horizontal bar represents the plasma membrane. The role of Msb2 in HOG pathway is unclear (O'Rourke and Herskowitz, 2002; Cullen et al, 2004). (B) 8xCRE-lacZ expression accurately reflects the osmotic activation of the HOG pathway. Wild-type (TM141) and its derivatives with the indicated genotypes were transformed with the pRS414-8xCRE-lacZ reporter plasmid. β-Galactosidase activity was assayed before (−) or after (+) 30 min exposure to 0.4 M NaCl. Strains used are TM141, QG137, TM260, FP54, TM257, FP75, FP67, QG153, QG147, and QG148. (C) Degradation of Cla4-td at a nonpermissive temperature. KY461 (ssk2/22 ste20 cla4-td), KY463 (STE11-Q301P ssk2/22 ste20 cla4-td), and QG147 (ssk2/22 ste20) were grown exponentially at 25°C, transferred to 37°C, and at the indicated times, cell extracts were prepared. The HA-tagged Cla4-td protein was detected by immunoblotting using anti-HA antibody. (D) Ste20 and Cla4 are redundant in the SHO1 branch. Exponentially growing cells of the indicated genotypes carrying the pRS414-8xCRE-lacZ reporter plasmid were transferred from 25°C to 37°C 1 h before osmostress was applied. β-Galactosidase activity was assayed before (−) or after (+) 30 min exposure to 0.4 M NaCl. Strains used are TM257, QG147, and KY461. Download figure Download PowerPoint The Sho1 osmosensor contains four transmembrane segments and a cytoplasmic SH3 domain that binds a proline-rich motif in the N-terminal region of Pbs2 (see Figure 5A). Sho1 is predominantly localized to the cytoplasmic membrane at regions of polarized growth, such as the emerging bud and the bud neck (Raitt et al, 2000; Reiser et al, 2000, 2003). Sho1 is absolutely required for activation of Hog1 via the SHO1 branch. In addition, Cdc42, Ste20, Ste50, and Ste11 have also been implicated in the SHO1 pathway (Posas and Saito, 1997; O'Rourke and Herskowitz, 1998; Posas et al, 1998; Raitt et al, 2000; Reiser et al, 2000). Cdc42 is a Rho-type small G-protein, and binds and activates Ste20, a prototype of the PAK family protein kinases (Lamson et al, 2002; Ash et al, 2003). Activated Ste20 phosphorylates the MAPKK kinase (MAPKKK) Ste11 on Ser-281, Ser-285, and Thr-286, and thus dissociates the Ste11 N-terminal inhibitory domain from its C-terminal catalytic domain (van Drogen et al, 2000). Ste50, an SAM (Sterile Alpha Motif) domain-containing protein, binds Ste11, itself another SAM domain-containing protein, via a heterotypic SAM–SAM interaction (Posas et al, 1998; Ramezani Rad et al, 1998; Grimshaw et al, 2004). Many of these proteins are also involved in other MAPK pathways in yeast. Sho1, Cdc42, Ste20, Ste50, and Ste11, have been implicated in the filamentous growth (FG) pathway, and at least Cdc42, Ste20, and Ste11 are also essential in the mating pheromone responsive MAPK pathway (Dohlman and Thorner, 2001). In spite of the involvement of these molecules in multiple pathways, there is no nonphysiological crosstalk activation between the pathways. Thus, pheromone stimulation activates only the Fus3/Kss1 MAPKs, and osmotic stimulation normally activates only the Hog1 MAPK (Posas and Saito, 1997). Specific docking interactions appear to play a critical role in the prevention of crosstalk between different MAPK pathways. We have shown that mammalian MAPKKs have a conserved docking site termed DVD (domain for versatile docking) at or near their C-terminus. Without the DVD site, mammalian MAPKKs cannot be bound to and phosphorylated by their specific MAPKKKs (Takekawa et al, 2005). Similarly, the yeast Ssk2 and Ssk22 MAPKKKs bind Pbs2 by a direct and specific docking interaction (Tatebayashi et al, 2003). However, a direct docking interaction with a MAPKK might be unsuitable for Ste11, because Ste11 is activated by several different upstream signals, and interacts with at least two different downstream MAPKKs (Pbs2 and Ste7) depending on the context of stimulation. In this paper, we analyzed two key steps in the SHO1 branch, namely activation of Ste11 by its upstream kinases (Ste20/Cla4), and activation of a downstream kinase (Pbs2) by activated Ste11. We demonstrate how Ste11 interacts with its upstream and downstream kinases by the adaptor functions of Cdc42, Ste50, and Sho1. Results An improved reporter for HOG pathway activation We developed a new reporter gene for the HOG pathway activation, termed 8xCRE-lacZ, which contains eight tandem repeats of the ENA1-derived CRE sequence (Supplementary Figure S1A). In wild-type cells, a 30 min treatment with 0.4 M NaCl induced the reporter gene expression 50- to 100-fold (Supplementary Figure S1B). Although 8xCRE-lacZ is a simple quadruplication of the previously published 2xCRE-lacZ reporter (Proft et al, 2001), 8xCRE-lacZ has a better stimulated-to-basal signal ratio. Since the CRE-containing promoters are regulated both positively and negatively by the Sko1 transcription factor (Proft et al, 2001; Rep et al, 2001), induction of the 8xCRE-lacZ reporter by osmostress is also dependent on the activator function of Sko1 (Supplementary Figure S1C). When unstimulated, 8xCRE-lacZ is fully repressed even in the absence of Sko1, whereas the 2xCRE-lacZ reporter gene is derepressed (Supplementary Figure S1C). Disruption of a gene that is common to both the SLN1 and SHO1 branches, for example, hog1Δ or pbs2Δ, completely abolished reporter expression (Figure 1B). Inactivation of genes specific to either the SHO1 branch (e.g., ste11Δ) or to the SLN1 branch (e.g., ssk2Δ ssk22Δ; henceforth abbreviated as ssk2/22) had no discernible effect. In contrast, combinations of gene disruptions that inactivate both branches (e.g., ssk2/22 ste11, ssk2/22 ste50, or ssk2/22 sho1) completely abolished reporter expression. Thus, 8xCRE-lacZ is a faithful reporter of the HOG pathway activation. Ste20 and Cla4 can independently activate the Ste11 MAPKKK in the SHO1 branch In the pheromone MAPK pathway, the Ste20 PAK-like kinase is the activator of the Ste11 MAPKKK (Leberer et al, 1992). Analysis of HOG pathway signaling using this new reporter indicated, however, that the ssk2/22 ste20 mutant could still show a significant response to an osmotic stimulus (Figure 1B), indicating that Ste20 is not solely responsible for the SHO1 branch. Yeast has another PAK-like kinase that is very similar to Ste20, namely Cla4. These kinases share a common essential function, because their simultaneous loss is lethal (Cvrckova et al, 1995). Disruption of the CLA4 gene had a moderate effect on the 8xCRE-lacZ reporter induction (Figure 1B). To test if Ste20 and Cla4 are redundant in the SHO1 pathway, the effect of the ste20 cla4 double mutation, which is lethal, was tested using the temperature-sensitive cla4-td mutant (Holly and Blumer, 1999). The Cla4-td protein is not only inactivated rapidly at nonpermissive temperatures, but it is also degraded (Figure 1C). 8xCRE-lacZ reporter expression is completely abolished in the ssk2/22 ste20 cla4-td mutant, thus providing evidence that Ste20 and Cla4 are functionally redundant in the SHO1 branch (Figure 1D). Ste50 and Sho1 are required in the HOG pathway downstream of Ste11 activation To study the activation mechanism of the SHO1 pathway, we isolated mutants that obviated the need for Ste20/Cla4 for 8xCRE-lacZ expression by osmostress. This was performed by screening for osmoresistant ‘revertants’ of an ssk2/22 ste20 strain on YPGal+1.5 M sorbitol plates, where the endogenous CLA4 gene is insufficient to sustain cell growth (Raitt et al, 2000). Growth characteristics of some of the mutants are shown in Figure 2A. All the mutants thus isolated had a mutation in the STE11 gene; in all, 29 different STE11 alleles were found among 54 mutants analyzed (Supplementary Figure S2). Twenty-two alleles were clustered around the Ste20 phosphorylation sites (Figure 2B), and are likely to mimic the effect of phosphorylation by activated Ste20/Cla4 (van Drogen et al, 2000). These mutations collectively define the auto-inhibitory (AI) domain to be between amino acid residues 270 and 340. Seven additional alleles were in the C-terminal kinase catalytic domain; one of these (T596I) was identical to the previously known STE11-4 (Stevenson et al, 1992). The kinase domain mutations are clustered near the ATP-binding cleft (Figure 2C), and are thus likely to escape inhibition by the AI domain (van Drogen et al, 2000). Figure 2.Characterization of constitutively active Ste11 mutants. (A) Suppression of ste20Δ by constitutively active STE11 mutations. KY218 (ssk2 ssk22 ste11 ste20) carrying pRS416-STE11 or its derivatives was spotted on the indicated plates. Ala3 is a triple mutant of Ste20 phosphorylation sites, S281A S285A T286A. (B) Distribution of the constitutively active STE11 mutations. Below is the schematic diagram of Ste11 showing the SAM, autoinhibitory (AI) and kinase domains. AI domain is expanded above the diagram. Each black vertical bar represents a constitutively active STE11 mutation. Short bars indicate one allele at the position, while longer bars indicate two different alleles. Red bars below the horizontal line indicate phosphorylation sites. (C) The predicted three-dimensional locations of the constitutively active Ste11 mutations in the kinase domain. The crystallographic structure of the human PAK1 kinase domain was used as a model (PDB ID=1F3M). (D) Constitutively active Ste11 mutant bypasses both Ste20 and Cla4. Exponentially growing cells of the indicated genotypes carrying the pRS414-8xCRE-lacZ reporter plasmid were transferred from 25°C to 37°C 1 h before osmostress was applied. β-Galactosidase activity was assayed before (−) or after (+) 30 min exposure to 0.4 M NaCl. Strains used are KT018, KT031, and KY463. (E) Constitutively active Ste11 mutants require Ste50 and Sho1 for 8xCRE-lacZ induction. Mutant strains of the indicated genotypes carrying a 8xCRE-lacZ reporter plasmid and pRS416-STE11 or its indicated derivative were grown exponentially and incubated for 30 min with (+) or without (−) 0.4 M NaCl, before preparation of cell extracts for β-galactosidase assay. Strains used are FP75, KY213, KY444, and KY452. Download figure Download PowerPoint When a constitutively active STE11 allele was expressed from the native STE11 gene locus, the HOG pathway was activated by osmostress in the absence of both Ste20 and Cla4, as we expected (Figure 2D). However, 8xCRE-lacZ expression was induced only when hyperosmotic stress was applied. This result was unexpected, because we and others have shown previously that constitutively active Ste11 mutants (e.g., Ste11ΔN or Ste11-Asp3) could activate the Hog1 MAPK in the absence of stress (Posas and Saito, 1997; van Drogen et al, 2000). We found that the expression level of Ste11 mutant protein was responsible for these different results. Thus, if Ste11-Q301P was sufficiently overexpressed, it could induce the 8xCRE-lacZ reporter without any external osmostress (Supplementary Figure S3A), even in ste50Δ or sho1Δ cells (Supplementary Figure S3B). Correspondingly, overexpression of Ste11-Q301P rescued the osmosensitivity of both ssk2/22 ste11 ste50 and ssk2/22 ste11 sho1 mutants (Supplementary Figure S4A and B). These results give the misleading impression that neither Ste50 nor Sho1 is needed once Ste11 is activated. In contrast, when hyperactive STE11 mutant genes (STE11-Q301P and STE11-S540P) were expressed from the native STE11 promoter, induction of the 8xCRE-lacZ reporter depended absolutely on the presence of both Ste50 and Sho1, and stress (Figure 2E). Consistently, none of the hyperactive Ste11 mutations could rescue the osmosensitive growth defect of ssk2/22 ste11 ste50 and ssk2/22 ste11 sho1 mutants (Supplementary Figure S4C, and data not shown). Thus, only by expression of the constitutively active Ste11 mutant proteins at physiologically relevant levels, a more subtle regulation of HOG pathway activation become discernible. To further analyze the role of Ste50 and Sho1 in Ste11 function, we tested the necessity for Ste50 and Sho1 for expression of the 8xCRE-lacZ reporter following activation of Ste11 by Ste20/Cla4. Activation of Ste20 and Cla4 by expression of the constitutively active Cdc42-G12V, which binds to their CRIB domains (Lamson et al, 2002), induced the 8xCRE-lacZ reporter (Figure 3A). This induction was inhibited significantly by ste20Δ and moderately by cla4Δ. More important, both Ste50 and Sho1 were needed for the 8xCRE reporter expression by constitutively activated Cdc42 (Figure 3A). Figure 3.Interaction between Cdc42 and Ste50. (A) Induction of the HOG reporter by the constitutively active Cdc42-G12V. Strains of the indicated genotypes were transformed with an 8xCRE-lacZ reporter plasmid and pYES2 (a multicopy plasmid with the GAL1 promoter) encoding either the wild-type Cdc42 (WT) or Cdc42-G12V. Expression of the Cdc42 protein was induced for 0, 2, or 4 h by 2% galactose, before preparation of cell extracts for β-galactosidase assay. Strains used are TM257, QG147, QG148, FP67, and QG153. (B) Schematic diagram of Ste50. Boxes indicate the SAM and RA domains and a central conserved region. Deletion (Δ1) and membrane-targeting (Δ1-Cpr) mutants are also shown. Cpr, C-terminal prenylation signal of Ras2. (C) The mut-153 (ste50-P318L) strain is defective in Hog1 phosphorylation. Exponentially growing cells were stimulated with 0.4 M NaCl for 5 min before cell extracts were prepared. Phosphorylated Hog1 was probed by immunoblotting using anti-phospho p38 antibody. Control cells on the left panel are TM252 and FP53. mut-95 and mut-159 are irrelevant control mutants isolated in the same screening. (D) Suppression of the osmosensitivity of ste50-P318L mutant cells by CDC42-L4P. FP67 (ssk2 ssk22 ste50) carrying pRS416-ste50-P318L was transformed with YCpIF16 (a single-copy vector with the GAL1 promoter) encoding the indicated gene, and streaked on YPGal plates with or without 0.7 M NaCl. (E, F) A membrane-targeting signal functionally substitutes for the Ste50 RA domain. Host strains of the indicated genotypes (FP67 and KT049) carrying pRS416-8xCRE-lacZ were transformed with YCpIF16-STE50 or its mutant derivatives. Cells were grown exponentially in CARaf, induced for Ste50 expression for 1.5 h by adding galactose to 2%, and incubated for an additional 30 min with (+) or without (−) 0.4 M NaCl in the media. Download figure Download PowerPoint These results demonstrate that both Ste50 and Sho1 are required even after Ste11 has been activated by Ste20/Cla4. We call such functions their post-Ste11 functions. Both Ste50 and Sho1, however, also have functions upstream of Ste11 activation, which we call their pre-Ste11 functions. In order to clearly understand how these molecules work in the HOG pathway, it is necessary to distinguish their two functional aspects. In the next section, we will define the pre-Ste11 function of Ste50. The pre-Ste11 function of Sho1, namely its osmosensor function, will be described elsewhere. Interaction between Cdc42 and Ste50 Ste50 comprises three conserved domains (Ramezani-Rad, 2003): the N-terminal SAM domain (amino acids 32–102), which interacts constitutively with the SAM domain in Ste11; a central conserved domain of previously unknown function (amino acids 119–149); and the C-terminal Ras-association (RA) domain (amino acids 235–327) (Figure 3B). For the FG pathway, it was recently demonstrated that binding of Cdc42 to the Ste50 RA domain was essential for signaling (Truckses et al, 2006). Either the GTP or the GDP form of Cdc42 can bind the RA domain. As Cdc42 is membrane-anchored by prenylation (Johnson, 1999), binding of Cdc42 to the Ste50 RA domain will translocate the otherwise cytoplasmic Ste11–Ste50 complex to the plasma membrane, where the Cdc42-bound Ste20 is also localized. Co-localization of activated Ste20 and the Ste11–Ste50 complex activates Ste11. We therefore examined whether the Cdc42–Ste50 interaction is also involved in the HOG pathway. In a genetic screening designed to isolate mutants defective in the SHO1 branch, we found a ste50 mutant in which Pro-318 in the RA domain was altered to Leu. The ssk2/22 ste50-P318L strain was defective in Hog1 phosphorylation when stimulated by osmotic stress (Figure 3C). Although it was less osmosensitive than the null ssk2/22 ste50Δ mutant, the ssk2/22 ste50-P318L mutant failed to grow on relatively stringent osmotic conditions, such as YPGal+0.7 M NaCl (Figure 3D). Overexpression of the wild-type CDC42 gene did not suppress the osmosensitive phenotype of ssk2/22 ste50-P318L. However, we could isolate CDC42 mutants that suppressed the osmosensitivity of ssk2/22 ste50-P318L. Fourteen independently isolated CDC42 mutants all had in common a Leu-4 to Pro (L4P) mutation, suggesting that Cdc42-L4P restores the lost Ste50–Cdc42 interaction caused by Ste50-P318L. Interestingly, Leu-4 lies very close to Ile-46 on the predicted three-dimensional structure of Cdc42 based on the homologous human Cdc42Hs (Supplementary Figure S5). The Ile-46 to Met (I46M) substitution in Cdc42 inhibits the binding between Cdc42 and the Ste50 RA domain, and cdc42-I46M mutant cells are defective in the FG response (Mösch et al, 2001; Truckses et al, 2006). The positions of Leu-4 and Ile-46 are far from the GTP binding/hydrolysis domains and the effector binding regions called Switch I (residues 26–40) and Switch II (residues 58–76) (Johnson, 1999). It is therefore likely that Cdc42 binds to the Ste50 RA domain and to the Ste20/Cla4 CRIB domain via different regions of the molecule. To test if a membrane-targeting signal could functionally substitute for the Ste50 RA–Cdc42 interaction, we made Ste50 RA deletion mutants without (Ste50Δ1) or with (Ste50Δ1-Cpr) the Ras2 C-terminal prenylation site (Bhattacharya et al, 1995) (see Figure 3B). Ste50Δ1-Cpr transduces the SHO1 branch signal efficiently when stimulated by 0.4 M NaCl (Figure 3E). In contrast, the Ste50Δ1 mutant is totally defective in this respect. However, in cells that express constitutively active Ste11 (such as Q301P), the Ste50Δ1 mutant can transduce the HOG signal, because co-localization of the Ste11–Ste50 complex with Ste20 (or Cla4) is unnecessary for Ste11 activation (Figure 3F). Thus, one role of Ste50 seems to be to localize the Ste11–Ste50 complex on the membrane by binding to the membrane-linked Cdc42. As Cdc42-bound Ste20 and Cla4 are also localized on the membrane, the upstream enzyme (Ste20 or Cla4) and downstream substrate (the Ste11–Ste50 complex) will be both concentrated on the membrane. Alternatively, a single molecule of Cdc42 might bind both Ste20 (or Cla4) and the Ste11–Ste50 complex, bringing them very close together. Constitutively active Ste50 mutants Expression of Ste50Δ1 in the STE11-Q301P mutant cells still requires osmostress stimulation to induce the reporter gene (Figure 3F), whereas expression of Ste50Δ1-Cpr in the same cells spontaneously induces 8xCRE-lacZ expression without osmostress, indicating that membrane-targeting of the Ste11–Ste50 complex mimics another essential event in the SHO1 branch that occurs after Ste11 has been activated. Since the Ste50Δ1 mutant protein lacks the RA domain, this second membrane-targeting event is independent of the RA domain. To identify the nature of this post-Ste11 event, we screened for STE50 mutants that had a phenotype similar to STE50Δ1-Cpr; that is, a capacity to spontaneously induce the 8xCRE-lacZ reporter when co-expressed with a constitutively active Ste11. Seven such STE50 mutants were isolated, all of which had a mutation at Asp-146 in the conserved central domain: five were D146V, one was D146A, and another was D146Y. The equivalent position of D146 in the Ste50 orthologs of other yeasts is often occupied by the conserved Glu residue (Ramezani-Rad, 2003). Co-expression of STE50-D146V and constitutively active STE11-Q301P induced the 8xCRE-lacZ reporter in the absence of any osmostress stimulus (Figure 4A). Site-directed mutagenesis of Ste50 Asp-146 to Cys, Leu, or Phe also created mutants of a similar phenotype, whereas mutations to Ser or Asn had little or no effect (Figure 4A and data not shown). We chose the strongest allele, STE50-D146F (STE50-D/F), for further characterization. Figure 4.Constitutively active Ste50 mutant. (A) Constitutively active Ste50 mutants. KT018 (STE11-Q301P ssk2 ssk22) and TM257 (ssk2 ssk22) carrying the pRS416-8xCRE-lacZ reporter plasmid were transformed with YCpIF16-STE50 or its derivative as indicated. Ste50 expression was induced by 2% galactose for 0, 3, and 5 h before cell extracts were prepared for β-galactosidase assay. (B) Schematic diagrams of the Ste50 deletion constructs used in this work. (C) Induction of the 8xCRE-lacZ reporter by the constitutively active Ste50-D146F mutant. FP67 (ssk2 ssk22 ste50) and KT049 (STE11-Q301P ssk2 ssk22 ste50) carrying the pRS416-8xCRE-lacZ reporter plasmid were transformed with YCpIF16-STE50 or one of its derivatives containing the indicated mutations. Cells were grown exponentially in CARaf, induced for Ste50 expression for 1.5 h by 2% galactose, and incubated for additional 30 min with (+) or without (−) 0.4 M NaCl in the media, before cell extracts were prepared for β-galactosidase assay. D/F, D146F. (D) Intragenic suppression of the Ste50 RA domain deletion mutants by D146F. FP67 (ssk2 ssk22 ste50) was transformed with YCpIF16 (a single-copy vector with the GAL1 promoter) encoding the indicated gene, and streaked on CAD plates with or without 1.5 M sorbitol. (E) Constitutively active Ste50-D146F requires Sho1 for 8xCRE-lacZ reporter expression. Cells of the indicated genotypes (KT018, KT031, and KT028) carrying pRS416-8xCRE-lacZ were transformed with either YCpIF16-Ste50 (WT) or YCpIF16-Ste50-D146F (D/F). Cell extracts for β-galactosidase assay were prepared as in (C). Download figure Download PowerPoint Figure 5.Constitutively active Sho1 mutants. (A) Schematic diagrams of the Sho1 mutants used in this work. TM, transmembrane segment. (B) The predicted three-dimensional locations of the constitutively active Sho1 mutations R342G and G346S in the SH3 domain. The location of W338F that abrogates Pbs2 binding is also shown. The crystallographic structure of the human Fyn SH3 domain (green) complexed with a ten-residue proline-rich peptide (yellow) was used as a homologous model (PDB ID=1FYN). (C) Constitutively active Sho1 mutants induce 8xCRE-lacZ reporter expression in the presence of Ste11-Q301P. TM257 (ssk2 ssk22) and KT018 (STE11-Q301P ssk2 ssk22) carrying the pRS414-8xCRE-lacZ reporter plasmid were transformed with pRS416-GAL1-SHO1 or one of its derivatives containing the indicated mutations. The Sho1 proteins were induced by 2% galactose for 2 h before cell extracts were prepared for β-galactosidase assay. W/F, W338F. (D) Binding of Sho1 to Ste50 and Ste11. KT045 (sho1 ste11 ste50 pbs2) was co-transformed with a YCpIF16-based plasmid (GAL1 promoter) for expression of HA-tagged protein and a p
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