Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono‐phosphorylated Pbs2 MAP 2K
2020; Springer Nature; Volume: 39; Issue: 5 Linguagem: Inglês
10.15252/embj.2019103444
ISSN1460-2075
AutoresKazuo Tatebayashi, Katsuyoshi Yamamoto, Taichiro Tomida, Akiko Nishimura, Tomomi Takayama, Masaaki Oyama, Hiroko Kozuka‐Hata, Satomi Adachi‐Akahane, Yuji Tokunaga, Haruo Saito,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoArticle3 February 2020Open Access Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-phosphorylated Pbs2 MAP2K Kazuo Tatebayashi Corresponding Author Kazuo Tatebayashi [email protected] orcid.org/0000-0002-3131-7030 Laboratory of Molecular Genetics, Frontier Research Unit, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Katsuyoshi Yamamoto Katsuyoshi Yamamoto Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Taichiro Tomida Taichiro Tomida Department of Physiology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan Search for more papers by this author Akiko Nishimura Akiko Nishimura Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tomomi Takayama Tomomi Takayama Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Masaaki Oyama Masaaki Oyama Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroko Kozuka-Hata Hiroko Kozuka-Hata Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Satomi Adachi-Akahane Satomi Adachi-Akahane Department of Physiology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan Search for more papers by this author Yuji Tokunaga Yuji Tokunaga Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan Search for more papers by this author Haruo Saito Corresponding Author Haruo Saito [email protected] orcid.org/0000-0001-7891-1689 Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Kazuo Tatebayashi Corresponding Author Kazuo Tatebayashi [email protected] orcid.org/0000-0002-3131-7030 Laboratory of Molecular Genetics, Frontier Research Unit, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Katsuyoshi Yamamoto Katsuyoshi Yamamoto Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Taichiro Tomida Taichiro Tomida Department of Physiology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan Search for more papers by this author Akiko Nishimura Akiko Nishimura Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Tomomi Takayama Tomomi Takayama Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Masaaki Oyama Masaaki Oyama Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroko Kozuka-Hata Hiroko Kozuka-Hata Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Satomi Adachi-Akahane Satomi Adachi-Akahane Department of Physiology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan Search for more papers by this author Yuji Tokunaga Yuji Tokunaga Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan Search for more papers by this author Haruo Saito Corresponding Author Haruo Saito [email protected] orcid.org/0000-0001-7891-1689 Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Search for more papers by this author Author Information Kazuo Tatebayashi *,1,2,3, Katsuyoshi Yamamoto2, Taichiro Tomida4, Akiko Nishimura2,3, Tomomi Takayama2,3, Masaaki Oyama5, Hiroko Kozuka-Hata5, Satomi Adachi-Akahane4, Yuji Tokunaga6 and Haruo Saito *,2 1Laboratory of Molecular Genetics, Frontier Research Unit, Institute of Medical Science, The University of Tokyo, Tokyo, Japan 2Division of Molecular Cell Signaling, Institute of Medical Science, The University of Tokyo, Tokyo, Japan 3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan 4Department of Physiology, School of Medicine, Faculty of Medicine, Toho University, Tokyo, Japan 5Medical Proteomics Laboratory, Institute of Medical Science, The University of Tokyo, Tokyo, Japan 6Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan *Corresponding author. Tel: +81 3 5449 5479; E-mail: [email protected] *Corresponding author. Tel: +81 3 5449 5479; E-mail: [email protected] The EMBO Journal (2020)39:e103444https://doi.org/10.15252/embj.2019103444 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The MAP kinase (MAPK) Hog1 is the central regulator of osmoadaptation in yeast. When cells are exposed to high osmolarity, the functionally redundant Sho1 and Sln1 osmosensors, respectively, activate the Ste11-Pbs2-Hog1 MAPK cascade and the Ssk2/Ssk22-Pbs2-Hog1 MAPK cascade. In a canonical MAPK cascade, a MAPK kinase kinase (MAP3K) activates a MAPK kinase (MAP2K) by phosphorylating two conserved Ser/Thr residues in the activation loop. Here, we report that the MAP3K Ste11 phosphorylates only one activating phosphorylation site (Thr-518) in Pbs2, whereas the MAP3Ks Ssk2/Ssk22 can phosphorylate both Ser-514 and Thr-518 under optimal osmostress conditions. Mono-phosphorylated Pbs2 cannot phosphorylate Hog1 unless the reaction between Pbs2 and Hog1 is enhanced by osmostress. The lack of the osmotic enhancement of the Pbs2-Hog1 reaction suppresses Hog1 activation by basal MAP3K activities and prevents pheromone-to-Hog1 crosstalk in the absence of osmostress. We also report that the rapid-and-transient Hog1 activation kinetics at mildly high osmolarities and the slow and prolonged activation kinetics at severely high osmolarities are both caused by a common feedback mechanism. Synopsis Upstream transmembrane osmosensors activate the Hog1 MAP kinase (MAPK) cascade for induction of osmoadaptive responses in yeast. Here, osmostress is shown to also directly enhance the activating phosphorylation of Hog1 by the MAPK kinase (MAP2K) Pbs2. MAP3K Ste11 phosphorylates only one of the two activating phosphorylation sites in Pbs2. Mono-phosphorylated Pbs2 phosphorylates Hog1 only when the reaction between Pbs2 and Hog1 is enhanced by osmostress. The downstream osmostress-mediated potentiation of Hog1 phosphorylation prevents cross-talk activation of Hog1 by mating pheromones. The osmotic enhancement of Hog1 phosphorylation also prevents basal activation of Hog1 in the absence of osmostress. Osmostress-induced glycerol accumulation exerts either positive or negative feedback on Hog1 activation, depending on osmostress severity. Introduction The family of mitogen-activated protein kinases (MAPKs) are major intracellular signal transducers in eukaryotic cells and are associated with many human diseases (Chen et al, 2001; Dhanasekaran & Johnson, 2007). Each MAPK is activated in a three-tiered kinase cascade composed of a MAPK kinase kinase (MAPKKK or MAP3K), a MAPK kinase (MAPKK or MAP2K), and a MAPK. In the canonical model of the MAPK cascades, an activated MAP3K activates a cognate MAP2K by phosphorylating two conserved serine (Ser) and/or threonine (Thr) residues in the flexible activation loop of the MAP2K. In turn, an activated MAP2K activates a cognate MAPK by phosphorylating the conserved Thr and tyrosine (Tyr) residues in the latter's activation loop. MAPK cascades are highly conserved from yeast to mammalian species, so much so that the mammalian MAPK p38 can functionally complement the yeast MAPK Hog1 (Han et al, 1994). A MAPK signal transduction pathway commonly comprises, in addition to the core MAPK cascade, an upstream transmembrane receptor or sensor that detects specific extracellular stimuli, and downstream MAPK substrate molecules (effectors) both in the cytoplasm and in the nucleus. Several different MAPK pathways often co-exist within a cell. In yeast, for example, four MAPKs (Slt2/Mpk1, Kss1, Fus3, and Hog1) are expressed in a cell (Gustin et al, 1998). If inappropriate crosstalk occurred between two MAPK cascades, a stimulus aimed at activation of only one of these cascades could incite irrelevant or even detrimental responses. Different MAPKs in a species are highly homologous to each other, and so are MAP2Ks. Thus, prevention of inappropriate crosstalk between MAPK cascades requires elaborate mechanism for any MAPK cascade, but its difficulty can be most clearly exemplified by the MAPK cascades in yeast, in which three different MAPK cascades with different specificities use the same MAP3K Ste11. The MAPK Hog1 is activated by hyperosmotic stress through the high-osmolarity glycerol (HOG) pathway and orchestrates an array of osmoadaptive changes in transcription, translation, cell cycle, and metabolism (Brewster et al, 1993; Saito & Posas, 2012; Hohmann, 2015). The current widely held model of the HOG pathway is as follows (Fig 1A). The upstream portion of the HOG pathway comprises the functionally redundant SHO1 and SLN1 branches. In the SHO1 branch, osmosensing complexes composed of Sho1, Opy2, Hkr1, and Msb2 activate the MAP3K Ste11 (Tanaka et al, 2014; Tatebayashi et al, 2015; Nishimura et al, 2016; Yamamoto et al, 2016). In the SLN1 branch, the Sln1-Ypd1-Ssk1 phospho-relay mechanism activates the functionally redundant MAP3Ks Ssk2 and Ssk22 (Ssk2/22) (Posas et al, 1996). Activated Ste11 and Ssk2/22 are believed to phosphorylate the MAP2K Pbs2 at Ser-514 and Thr-518 (S514 and T518). Phosphorylated Pbs2 then activates Hog1 (Maeda et al, 1995; Posas & Saito, 1997). Figure 1. Phosphorylation of Hog1 by osmostress in the absence of the upstream osmosensors A. A schematic diagram of the Hog1 MAPK signaling pathway. B. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Cells of the indicated genotypes were stimulated with the indicated concentrations of NaCl for the indicated time. Strains used are TM257, KT207, and KY594-1. C. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strain KY594-1 was stimulated with the indicated concentrations of NaCl for 5 min. The percentages of phosphorylated Hog1 (Hog1-P [%]) were calculated as explained in Materials and Methods and are shown beneath the panel. D. Analyses of Hog1 phosphorylation by immunoblotting with anti-phospho-p38 (Hog1-P) and anti-Hog1 (total Hog1) antibodies. Yeast strain KT219 was transformed with the indicated STE11 mutant gene carried by a single-copy plasmid that is expressed from the STE11 promoter: vec, vector; WT, wild-type; DDD, S281D/S285D/T286D. Cells were incubated with (+) or without (−) 1 M NaCl for 5 min. E–H. Analyses of Hog1 phosphorylation by Phos-tag band-shift assay. Yeast strains (E) KY603-3; (F) TM142; (G) TM257; and (H) FP54 were stimulated with the indicated concentrations of NaCl for 5 min. I. Comparison of the NaCl dose–responses of Hog1 activation by various strains. Phos-tag band-shift assays shown in (C and E–H) were independently repeated three times, and average values were plotted. Data information: (C and E–H) Representative results from three independent experiments. (I) Error bars are SEM (n = 3). Source data are available online for this figure. Source Data for Figure 1 [embj2019103444-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Two other yeast MAPKs Fus3/Kss1 are activated by the mating pheromones through Ste11 and the MAP2K Ste7 (Bardwell, 2005). Although the mating pheromones activate Ste11, they do not activate Hog1 (Posas & Saito, 1997). Commonly, the absence of pheromone-to-Hog1 crosstalk is explained by the pathway insulation model, which posits that a scaffold protein holds several components of one pathway close together, so that signal flows only within that pathway (Harris et al, 2001). To prevent crosstalk, however, the scaffold proteins must hold kinases for significantly longer than the half-lives of their activities, which could be several minutes or longer. Because scaffold complexes are typically not so stable (Zalatan et al, 2012), additional mechanisms other than scaffolding of signaling complexes are likely to be necessary to effectively prevent crosstalk. Here, we report a mechanism that prevents the pheromone-to-Hog1 crosstalk and also suppresses non-specific Hog1 activation by the basal activities of the upstream MAP3Ks. Results Osmostress can activate the Hog1 MAPK in the absence of the upstream osmosensors Several studies have reported that Hog1 can be activated at very high osmolarity (> 1 M NaCl) in strains that are defective in both the SLN1 and SHO1 branches, such as ssk1Δ ste11Δ and ssk2/22Δ sho1Δ (Van Wuytswinkel et al, 2000; O'Rourke & Herskowitz, 2004; Zhi et al, 2013; Vázquez-Ibarra et al, 2019). Since each of the strains used in those studies expressed at least one MAP3K in the HOG pathway (Ssk2, Ssk22, or Ste11), the results were interpreted as evidence for an alternative mechanism for MAP3K activation following osmostress. However, no such MAP3K activating mechanism has been identified. To examine if Hog1 could be activated by osmostress in the absence of known upstream osmosensor signaling, we constructed a mutant yeast strain that lacked all four transmembrane proteins involved in the SHO1 branch (Sho1, Opy2, Hkr1, and Msb2) as well as the two MAP3Ks essential for the SLN1 branch (Ssk2 and Ssk22). The genotype of this strain, sho1Δ opy2Δ hkr1Δ msb2Δ ssk2Δ ssk22Δ, will be abbreviated hereafter as ΔS/O/H/M ssk2/22Δ. We measured osmostress-induced activation/phosphorylation of Hog1 using the anti-phospho-p38 immunoblotting assay (Tatebayashi et al, 2003) or a Phos-tag band-shift assay (English et al, 2015). Immunoblotting assays indicated that exposure of the ΔS/O/H/M ssk2/22Δ mutant strain to stronger osmostress (1 M NaCl) indeed induced weak Hog1 phosphorylation (Fig 1B, lanes 11–12). The fact that Hog1 was not phosphorylated in a pbs2Δ ssk2/22Δ strain (Fig 1B, lanes 5–8) indicated that the MAP2K Pbs2 was necessary for Hog1 phosphorylation at 1 M NaCl in the absence of upstream osmosensor signaling. A more detailed analysis of the Hog1 NaCl dose–response using the Phos-tag band-shift assay revealed that Hog1 was weakly phosphorylated between 0.8 M and 1.6 M NaCl in the ΔS/O/H/M ssk2/22Δ mutant strain (Fig 1C). Hog1 phosphorylation in ΔS/O/H/M ssk2/22Δ was completely abolished by deletion of STE11 (Fig 1D; compare lanes 2 and 4 in the longer exposure). Conversely, it was greatly enhanced by the presence of a constitutively active Ste11 such as Ste11-Q301P, Ste11-S281D/S285D/T286D (DDD), or Ste11-T596I (van Drogen et al, 2000; Tatebayashi et al, 2006) (Fig 1D, lanes 5–10). It has been previously observed that the endogenous-level expression of constitutively active Ste11 mutant does not activate Hog1 unless osmostress is applied (Lamson et al, 2006; Tatebayashi et al, 2006). Using the ΔS/O/H/M ssk2/22Δ STE11-Q301P strain, we determined the dose–response of Hog1 phosphorylation at 5 min at various NaCl concentrations (Fig 1E). The observed dose–response clearly differed from the dose–responses of the wild-type (WT) strain (Fig 1F), a SHO1 branch-only strain (ssk2/22Δ; Fig 1G), or an SLN1 branch-only strain (ste11Δ; Fig 1H). The extents of Hog1 phosphorylation in these strains at 5 min are summarized in Fig 1I. Enhancement of the Pbs2-Hog1 reaction by osmostress involves a genuine osmosensing mechanism That the ΔS/O/H/M ssk2/22Δ STE11-Q301P mutant cell that lacks both the SHO1 and SLN1 branches could activate Hog1 in response to osmostress suggested that there may be a previously undefined sensing mechanism that is distinct from both the Sho1 and Sln1 osmosensors. To determine if phosphorylation of Hog1 in ΔS/O/H/M ssk2/22Δ STE11-Q301P was a specific reaction to NaCl or a general reaction to osmostress, we examined if Hog1 could be activated not only by NaCl but also by the non-ionic sorbitol. At very high concentrations, two solutions with the same osmolar concentrations (e.g., 1 M NaCl and 2 M sorbitol) do not necessarily have the same osmotic pressure. However, when compared at the same osmotic pressures expressed in M pascal (MPa) units (Fig EV1A), NaCl and sorbitol induced Hog1 phosphorylation almost identically in WT cells (Fig EV1B). Hog1 phosphorylation in the ΔS/O/H/M ssk2/22Δ STE11-Q301P mutant cells was also similar in response to NaCl and sorbitol (Fig EV1C and D), indicating that the Hog1 phosphorylation in the absence of the upstream osmosensors involved a genuine osmosensing mechanism. These results indicated that in the HOG signaling pathway, osmostress acts not only at the level of the upstream osmosensors (Sho1 and Sln1), but also at a point downstream of MAP3Ks. Therefore, we conclude that there is a downstream osmosensor distinct from the upstream osmosensors. Click here to expand this figure. Figure EV1. The "downstream osmosensor" responds to osmostress regardless of the type of osmostressor Conversion of NaCl and sorbitol solute concentration (molarity, M) to osmotic pressure Π (mega pascal, MPa). For more detail, see the Appendix Fig S1. Comparison of Hog1 phosphorylation induced by NaCl and sorbitol. The yeast strain TM142 (WT) was stimulated with various concentrations of NaCl or sorbitol for 5 min, and the percentage of Hog1-P was determined using a Phos-tag band-shift assay. Solution molar concentrations were converted to the corresponding osmotic pressures (MPa) using the table in (A). Phos-tag band-shift analyses of sorbitol-induced Hog1 phosphorylation. The yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was stimulated with the indicated concentrations of sorbitol for 5 min. Same as in (B), except that the yeast strain KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) was used. Source data are available online for this figure. Download figure Download PowerPoint The downstream osmosensor acts at the step of Hog1 phosphorylation by Pbs2 Next, we identified the signaling step in the HOG pathway at which the predicted downstream osmosensor functions. It has been reported that Hog1 can, when strongly overexpressed, auto-phosphorylate in the absence of Pbs2 (Maayan et al, 2012). However, Hog1 auto-phosphorylation could not account for our observations, because in our experiments Hog1 was not overexpressed, and the catalytically inactive Hog1-K52S/K53N (Alepuz et al, 2001) could be phosphorylated by osmostress in the absence of upstream osmosensors (Fig 2A, lanes 4–6). On the contrary, Pbs2 was required to phosphorylate Hog1 by the downstream osmosensor (Fig 2B, lanes 1–4). Furthermore, neither expression of Pbs2-S514A/T518A, which lacked activating phosphorylation sites, nor that of catalytically inactive Pbs2-K389M supported Hog1 phosphorylation by osmostress (Fig 2B, lanes 5–8), further indicating that active Pbs2 is necessary for Hog1 phosphorylation induced by the downstream osmosensor. In contrast, MAP3Ks were not required to phosphorylate Hog1 by the downstream osmosensor if the constitutively active Pbs2-S514D/T518D (Pbs2-DD) was present (Fig 2C, compare lanes 6 and 9). Phosphorylation of Hog1 in the presence of Pbs2-DD had a very similar dose–response to that in the presence of Ste11-Q301P (Fig 2D and E), indicating that Pbs2-DD phosphorylated Hog1 by the same mechanism. We thus concluded that osmostress enhances the Hog1 phosphorylation at the step of the Pbs2-Hog1 reaction (Fig 2F). Figure 2. Osmostress enhances the phosphorylation of Hog1 by Pbs2 A–C. Immunoblot analyses of Hog1 phosphorylation. Yeast strains (A) KT235; (B) KT209; and (C) KT234 were stimulated with the indicated concentrations of NaCl for 10 min, and phosphorylated Hog1 (Hog1-P) and total Hog1 in cell lysates were detected by immunoblotting. The relevant genotypes of the strains are indicated in the top row of each panel and are schematically shown in the diagrams at left. The second row from the top indicates the genes carried by a single-copy plasmid that are expressed from their own promoters. vec, vector; WT, wild-type. D. Phos-tag band-shift analyses of Hog1 phosphorylation. The yeast strain KT234 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was stimulated with the indicated concentrations of NaCl for 5 min. E. Comparison of the NaCl dose–responses of Hog1 activation by constitutively active Ste11-Q301P and constitutively active Pbs2-DD. Phos-tag band-shift assays shown in (D) and Fig 1E were independently repeated three times, and the average values were plotted. F. A scheme illustrating the step in the Hog1 MAPK cascade at which osmostress acts to enhance Hog1 phosphorylation. Data information: (E) Error bars are SEM (n = 3). Source data are available online for this figure. Source Data for Figure 2 [embj2019103444-sup-0004-SDataFig2.pdf] Download figure Download PowerPoint Generally speaking, two mechanisms are possible at the step of the Pbs2-Hog1 reaction to increase the Hog1 phosphorylation: (i) enhancement of Hog1 phosphorylation by Pbs2, and (ii) inhibition of Hog1 dephosphorylation by phosphatases. An excellent example of the latter mechanism is the activation of Hog1 by arsenite, which inhibits the major Hog1 phosphatases Ptp2 and Ptp3 (Lee & Levin, 2018). We thus examined if the osmotic enhancement of Hog1 phosphorylation at the step of the Pbs2-Hog1 reaction also involves the inhibition of these phosphatases. In a ΔS/O/H/M ssk2/22Δ STE11-Q301P strain, deletion of either PTP2 or PTP3 alone had essentially no effect (Fig EV2). As expected, deletion of both PTP2 and PTP3 together increased the Hog1 phosphorylation even without osmotic stress. More important, 5-min treatment of the ptp2Δ ptp3Δ strain further increased the extent of Hog1 phosphorylation. It should be noted that about 30% of the unphosphorylated Hog1 present before NaCl addition was phosphorylated both in the PTP2 PTP3 and ptp2 ptp3 strains. Thus, osmotic enhancement of Hog1 phosphorylation must be attained by promotion of Hog1 phosphorylation by Pbs2, but not by inhibition of Hog1 dephosphorylation. Click here to expand this figure. Figure EV2. Protein tyrosine phosphatases Ptp2/Ptp3 are not involved in the osmotic enhancement of the Pbs2-Hog1 reaction Yeast strains of the indicated genotypes (shown below the graph) were grown exponentially and were either untreated or exposed to 1.0 M NaCl for 5 min. Hog1 phosphorylation was determined using the Phos-tag band-shift assay. Strains used are KY603-3 (ΔS/O/H/M ssk2/22Δ STE11-Q301P) and its derivatives, KT303 (ptp2Δ), KT307 (ptp3Δ), and KT305 (ptp2Δ ptp3Δ). Data information: Error bars are SEM (n = 3). Download figure Download PowerPoint The Hog1 L16 domain is necessary for the osmotic enhancement of the Pbs2-Hog1 reaction To investigate the physiological role as well as the molecular mechanism of the osmotic enhancement of the Pbs2-Hog1 reaction, we tried to isolate Hog1 mutants that are unable to be enhanced. We based our screen for Hog1 mutants that cannot be osmotically enhanced on the expectation that such Hog1 mutants would not be phosphorylated at 1.0 M NaCl in the ΔS/O/H/M ssk2/22Δ STE11-Q301P strain. Furthermore, we thought that the C-terminal non-catalytic region of Hog1 was especially promising for the search of Hog1 mutants that cannot be osmotically enhanced, as this domain contains the highly conserved common docking (CD) domain that binds Pbs2 (Murakami et al, 2008) and the moderately conserved L16 domain (Fig 3A) that are known to modulate Hog1 activation (Maayan et al, 2012). Figure 3. The Hog1 L16 domain is required for the osmotic enhancement of the Pbs2-Hog1 reaction A. Alignment of the amino acid sequences of the CD (green) and L16 (pink) domains of yeast Hog1 and mammalian p38α. The alpha helix αL16 forms the core of the L16 domain (Wang et al, 1997). B. Schematic diagrams of Hog1-WT and its deletion constructs used in this study. C. Immunoblot analyses of Hog1 phosphorylation. The yeast strain KT235 was transformed with pRS416-FLAG-Hog1 (WT) or its indicated deletion derivatives. FLAG-Hog1 was immunoprecipitated (IP), and immunoblotted (IB) with anti-phospho-p38 (for Hog1-P; upper panel) or anti-FLAG (for total FLAG-Hog1; lower panel) D–G. Phos-tag band-shift assay of Hog1 phosphorylation. Yeast strain (D and E) KT235 or (F and G) KT290 carrying the single-copy expression plasmid YCplac22I'-Pbs2 S514D/T518D was transformed with either pRS416-Hog1 (WT) or its indicated mutant derivatives and was treated with the indicated concentrations of NaCl for 5 min. (D) and (F) show typical results, and (E) and (G) summarize the averages of three independent experiments. H. Phos-tag band-shift assay of Hog1 phosphorylation. The yeast strain FP4 was transformed with the single-copy expression plasmid pRS416-Hog1 (WT) or pRS416-Hog1-ΔL16 and was treated with the indicated concentrations of NaCl for 5 min. The averages of three independent experiments are shown. Data information: (E, G, and H) Error bars are SEM (n = 3). Source data are available online for this figure. Source Data for Figure 3 [embj2019103444-sup-0005-SDataFig3.pdf] Download figure Download PowerPoint We constructed Hog1 expression plasmids that lacked various parts of the C-terminal non-catalytic region (Fig 3B). These constructs were individually introduced into the ΔS/O/H/M ssk2/22Δ STE11-Q301P hog1Δ strain, and their phosphorylation was assayed in the absence or presence of osmostress. There was no phosphorylation of the Hog1 deletion mutant that lacked most of the non-catalytic C-terminal domain (Δ[320–431]; Fig 3C, lanes 7–9). In contrast, the Hog1 deletion mutant that retained the L16 domain (Hog1Δ[355–431]) was phosphorylated in the presence of osmostress (Fig 3C, lanes 4–6). Finally, deletion of the L16 region alone (ΔL16 = Δ[320–350]) was sufficient to inhibit Hog1 phosphorylation, whereas HOG1 mutation D307A/D310A that abrogated the binding capacity of the CD domain (Murakami et al, 2008) had little effect (Fig 3D and E). The ΔL16 mutation also suppressed Hog1 phosphorylation driven by Pbs2-DD (Fig 3F and G). We concluded from these results that the Hog1 L16 domain was required for osmotic enhancing of Hog1 phosphorylation. Although Hog1-ΔL16 could not be phosphorylated by Pbs2-DD, it was phosphorylated in WT cells in which both the SLN1 and SHO1 branches are intact (Fig 3H), suggesting that normally activated Pbs2 (by the upstream MAP3Ks) can phosphorylate Hog1 without the osmotic enhancement of the Pbs2-Hog1 reaction. If so, these results suggest that Pbs2-DD, though mimicking the phosphorylated Pbs2, has a significantly weaker activity than the normally phosphorylated Pbs2. Inhibition of the osmotic enhancement of the Pbs2-Hog1 reaction affects the SLN1 and SHO1 branches differently Next, we examined how inhibition of the osmotic enhancement by ΔL16 affected the Hog1 phosphorylation mediated by individual upstream osmosensing branches. For this purpose, we expressed Hog1-ΔL16 in a WT strain (with both the SLN1 and SHO1 branches), an ste11Δ strain (the SLN1 branch-only), or an ssk2/22Δ strain (the SHO1 branch-only) and measured Hog1-ΔL16 phosphorylation at various NaCl concentrations. In the WT strain (Fig 4A and B) and the SLN1 branch-only strain (Fig 4C and D), phosphorylation of Hog1-ΔL16 was strongly reduced at higher NaCl concentrations (> 1.0 M) compared to that of Hog1-WT, whereas it was comparable to that of Hog1-WT at lower NaCl concentrations (< 0.4 M). In contrast, in the SHO1 branch-only strain, phosphorylation of Hog1-ΔL16 was strongly reduced at both lower and higher NaCl concentrations compared to that of Hog1-WT (Fig 4E and F). This observation was quite puzzling, as the extent of Hog1-WT phosphorylation at the lower NaCl concentration range (0.2–0.6 M NaCl) is about the same for the three strains examined. To explain this difference between the SLN1 and SHO1 branches, we hypothesized that there might be a qualitativ
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