Phosphorylation of Tyr-176 of the Yeast MAPK Hog1/p38 Is Not Vital for Hog1 Biological Activity
2003; Elsevier BV; Volume: 278; Issue: 17 Linguagem: Inglês
10.1074/jbc.c300006200
ISSN1083-351X
Autores Tópico(s)Melanoma and MAPK Pathways
ResumoMitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1Y176F is biologically active, capable of induction of Hog1 target genes and of rescuinghog1Δ cells from osmotic stress. Hog1Y176Fwas not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity. Mitogen-activated protein kinases are crucial components in the life of eukaryotic cells. The current dogma for MAPK activation is that dual phosphorylation of neighboring Thr and Tyr residues at the phosphorylation lip is an absolute requirement for their catalytic and biological activity. In this study we addressed the role of Tyr and Thr phosphorylation in the yeast MAPK Hog1/p38. Taking advantage of the recently isolated hyperactive mutants, whose intrinsic basal activity is independent of upstream regulation, we demonstrate that Tyr-176 is not required for basal catalytic and biological activity but is essential for the salt-induced amplification of Hog1 catalysis. We show that intact Thr-174 is absolutely essential for biology and catalysis of the mutants but is mainly required for structural reasons and not as a phosphoacceptor. The roles of Thr-174 and Tyr-176 in wild type Hog1 molecules were also tested. Unexpectedly we found that Hog1Y176F is biologically active, capable of induction of Hog1 target genes and of rescuinghog1Δ cells from osmotic stress. Hog1Y176Fwas not able, however, to mediate growth arrest induced by constitutively active MAPK kinase/Pbs2. We propose that Thr-174 is essential for stabilizing the basal active conformation, whereas Tyr-176 is not. Tyr-176 serves as a regulatory element required for stimuli-induced amplification of kinase activity. mitogen-activated protein kinase extracellular signal-regulated kinase hemagglutinin wild type glutathioneS-transferase yeast extract, peptone, dextrose yeast nitrogen base Mitogen-activated protein kinases (MAPKs1; ERK, p38, and c-Jun N-terminal kinase) play vital roles in determining the cell program (1Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3611) Google Scholar, 2Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2864) Google Scholar, 3Ono K. Han J. Cell. Signal. 2000; 12: 1-13Crossref PubMed Scopus (1385) Google Scholar, 4Pearson G. Robinson F. Beers Gibson T. Xu B.E. Karandikar M. Berman K. Cobb M.H. Endocr. Rev. 2001; 22: 153-183Crossref PubMed Scopus (3500) Google Scholar). Despite the significant progress in our understanding of MAPK activation and catalysis (5Bellon S. Fitzgibbon M.J. Fox T. Hsiao H.M. Wilson K.P. Struct. Fold. Des. 1999; 7: 1057-1065Abstract Full Text Full Text PDF Scopus (124) Google Scholar, 6Cobb M.H. Goldsmith E.J. Trends Biochem. Sci. 2000; 25: 7-9Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 7Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 8Khokhlatchev A.V. Canagarajah B. Wilsbacher J. Robinson M. Atkinson M. Goldsmith E. Cobb M.H. Cell. 1998; 93: 605-615Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar) these issues are not fully revealed. MAPKs possess a phosphorylation motif that comprises a Thr-X-Tyr sequence. Upon activation of the relevant pathway this motif is dually phosphorylated, leading to structural changes and a dramatic increase in specific activity (7Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 9Prowse C.N. Deal M.S. Lew J. J. Biol. Chem. 2001; 276: 40817-40823Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 10Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar). Current models of MAPK activation suggest that phosphorylation of both Thr and Tyr at the phosphorylation motif is an absolute requirement for activation. Substitution of any one of these phosphoacceptors diminishes the kinase activity as detected by in vitro kinase assays (9Prowse C.N. Deal M.S. Lew J. J. Biol. Chem. 2001; 276: 40817-40823Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 10Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar, 11Cobb M.H. Goldsmith E.J. J. Biol. Chem. 1995; 270: 14843-14846Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar). Although both Thr and Tyr seem to be equally important for catalysis, the three-dimensional structures of phosphorylated ERK2 and p38γ suggested that the Thr-183 residue contributes more significantly to stabilization of the active form (5Bellon S. Fitzgibbon M.J. Fox T. Hsiao H.M. Wilson K.P. Struct. Fold. Des. 1999; 7: 1057-1065Abstract Full Text Full Text PDF Scopus (124) Google Scholar, 7Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). Upon phosphorylation, Thr-183 forms ionic and hydrogen bonds with the N-terminal domain, thereby promoting domain closure. Tyr-185 is positioned to participate in substrate recognition. Recently we reported the isolation of MAPK kinase-independent hyperactive MAPK mutants of both the yeast Hog1 and the human p38α (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). These MAPKs were rendered intrinsically active by point mutations in either the L16 domain (mutations F318L, F318S, F322L, W320R, and W332R in Hog1) or the phosphorylation lip (D170A in Hog1) and were shown to rescue pbs2Δ cells from high osmolarity (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Although manifested very high basal activities, the catalytic activity of the mutants was further increased when cells were exposed to osmotic stress. The goal of this work was to examine the exact role of Tyr-176 or Thr-174 phosphorylation in Hog1 catalytic and biological activity. We show that in the hyperactive mutants Tyr-176 is required mainly for enhancing catalytic activity following osmostress, whereas Thr-174 is essential for biological and catalytic activity although not necessarily as a phosphoacceptor. Unexpectedly, when Tyr-176 was replaced with Phe in the wild type Hog1 enzyme, most of its catalytic activity was abolished, but its biological activity was maintained. We suggest that Thr phosphorylation stabilizes an active catalytic conformation that is independent of Tyr phosphorylation. Tyr phosphorylation serves to further amplify the basal activity in response to external signals. Strains used in this study were thepbs2Δ strain MAY1 and the hog1Δ strain JBY13 (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Growth conditions were described previously (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). T174A or Y176F mutations were inserted into plasmids pES86-HA-HOG1 (harboring an HA-taggedHOG1 coding sequence under the ADH1 promoter) and pRS1 (harboring the full-length HOG1 sequence with its native promoter and an HA tag at the N terminus). Construction details will be provided upon request. Cell lysate preparations and kinase assays were described previously (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Detection of Thr phosphorylation was performed by immunoprecipitation of HA-Hog1 as described previously (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) followed by Western blot analysis using rabbit anti-phosphothreonine antibodies (Zymed Laboratories Inc.). Hog1 protein levels in the same blots were detected by stripping the blot and reincubation with monoclonal anti-HA antibodies 12CA5 as described previously (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Cultures were grown toA600 = 0.4–0.5. Next cells were split in half, collected by centrifugation, and resuspended in the same medium or in medium containing 1 m NaCl. 20-ml samples were removed at the indicated time points for RNA isolation. RNA was analyzed by the S1 method (13Chen W. Tabor S. Struhl K. Cell. 1987; 50: 1047-1055Abstract Full Text PDF PubMed Scopus (97) Google Scholar). Dual phosphorylation of both Thr and Tyr at the phosphorylation lip is considered an absolute requirement for MAPK activation (10Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar, 14Bardwell L. Cook J.G. Voora D. Baggott D.M. Martinez A.R. Thorner J. Genes Dev. 1998; 12: 2887-2898Crossref PubMed Scopus (141) Google Scholar, 15Gartner A. Nasmyth K. Ammerer G. Genes Dev. 1992; 6: 1280-1292Crossref PubMed Scopus (219) Google Scholar, 16Schuller C. Brewster J.L. Alexander M.R. Gustin M.C. Ruis H. EMBO J. 1994; 13: 4382-4389Crossref PubMed Scopus (442) Google Scholar, 17Madhani H.D. Styles C.A. Fink G.R. Cell. 1997; 91: 673-684Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). The recently isolated hyperactive Hog1 mutants do not require the MAPK kinase Pbs2 for their activity and therefore seem to have escaped the requirement of phosphorylation (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). This conclusion is supported by the fact that when Tyr-176 was mutated to Phe the Hog1 mutants remained biologically active, i.e. they rescued hog1Δ and pbs2Δ cells from hyperosmotic shock (Ref. 12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar and Fig.1). Also attempts to directly measure dual phosphorylation of the hyperactive mutants (expressed inpbs2Δ cells) using α-phospho-p38 antibodies revealed no or very low phosphorylation levels (depending on the particular mutant; see Fig. 6 in Bell et al. (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar)). However, when Thr-174 was mutated to Ala, all Hog1 variants lost the capability to rescuepbs2Δ cells and even hog1Δ cells from osmotic stress (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar), strongly suggesting that Thr-174 has a more central role in Hog1 activity.Figure 6Hog1Y176F is capable of inducing Hog1 target genes in hog1Δ cells.Hog1WT or Hog1Y176F was subcloned into the pRS426 plasmid and expressed under the native HOG1 promoter in hog1Δ cells. Cells were exposed or not to 1m NaCl. At the indicated time points, RNA levels were monitored by S1 analysis. HAL3 was used as a loading control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To test whether differences in biological activity of the mutants reflect differences in catalytic activity, we immunoprecipitated the various proteins and analyzed their catalytic activity in vitro. As expected, Hog1WT completely lost its catalytic activity when mutated in either Thr-174 or Tyr-176 (Fig.2, left panel, lanes 3–8). In contrast, the catalytic activity of Hog1 hyperactive mutants that also harbored a Y176F mutation was readily observed (with the exception of Y68H,Y176F that manifested low activity; Fig. 2). Notably active mutants harboring the T174A mutation lost activity altogether (see clones T174A,F318L and T174A,F318S in Fig. 2). When tested in pbs2Δ cells, three mutants (Y176F,D170A; Y176F,F318L; and Y176F,F318S) manifested catalytic activity (see Supplemental Fig. 1S). Interestingly the activity of Hog1Y176F,F322L that was high in hog1Δ cells was barely measurable in pbs2Δ cells (see Supplemental Fig. 1S). The parental enzyme Hog1F322L was fully active inpbs2Δ cells (see Supplemental Fig. 2S), suggesting that this particular variant became Pbs2-dependent by the Y176F mutation. However, Hog1Y176F,F322L could rescuepbs2Δ cells (see Fig. 7 in Bell et al. (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar)) showing that also in this case Tyr-176 is not essential for biological activity.Figure 7Hog1Y176F is phosphorylated on Thr-174 in hog1Δ cells.Hog1WT, Hog1T174A, or Hog1Y176F was expressed in hog1Δ cells that were exposed or not to 1m NaCl for 10 min. Proteins were extracted and separated by SDS-PAGE. Western blot analysis was performed with α-phospho-p38 antibodies (upper panel) followed by stripping and reincubation with α-HA antibodies. Proteins were expressed under theADH1 promoter. P-Hog1, phosphorylated Hog1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Thus, Tyr-176 phosphoacceptor is dispensable for catalytic activity of the hyperactive Hog1 mutants, but Thr-174 is essential. These results fully correlate with the biological assay (Fig. 1 of this study and Fig. 7 in Bell et al. (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar)). Most of the hyperactive Hog1 mutants acquired very high catalytic activity that is independent of salt induction. Yet this activity was further enhanced when cells were exposed to salt (see Fig. 4 in Ref. 12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). The results shown in Fig. 2 suggest that when mutated in Tyr-176 the basal catalytic activity of the mutants was not lost, but their ability to further enhance activity in response to salt induction was compromised. To verify this point we measured kinase activity of the Hog1D170A and Hog1F318L molecules side by side with the Hog1D170A,Y176F and Hog1F318L,Y176Fderivatives. The results clearly show that the basal catalytic activity of the hyperactive mutants harboring Phe at position 176 was similar to that of the active mutants carrying the native Tyr-176 (Fig.3). However, whereas the activity of the Hog1 hyperactive molecules increased upon exposure to salt, molecules mutated in Tyr-176 were not as responsive to salt (Fig. 3). These results suggest that the catalytic activity of the hyperactive Hog1 alleles could be divided to two levels: 1) an intrinsic activity, acquired through the activating mutations, that is Pbs2-independent, salt-independent, and Tyr-176-independent; and 2) an enhanced activity that is salt-dependent. In most mutants an intact Tyr-176 is important for the enhanced activity and is dispensable for the intrinsic activity. Intact Thr-174 is essential for all levels of activity of the wild type and the hyperactive Hog1 mutants (Fig. 2). It seems that Thr-174 is essential for catalytic and biological activity of the hyperactive mutants. The question remains whether this residue is required as a phosphoacceptor or is essential due to conformational reasons. To address this question we analyzed the phosphorylation state of Thr-174 in some of the active mutants using α-phospho-Thr antibodies (Fig. 4). The results show that when expressed in pbs2Δ cells the active mutants manifested either barely or no detectable Thr phosphorylation (Fig. 4, right panel). When expressed in hog1Δ cells Thr(P) was clearly detected in all hyperactive Hog1 molecules (Fig. 4, left panel). In fact, Thr-174 in the hyperactive Hog1 alleles seemed to have elevated basal phosphorylation levels when Tyr-176 was mutated. Since the active mutants manifested clear catalytic activity in pbs2Δ cells (Fig.3B) but were not significantly phosphorylated on any Thr residue in this strain (Fig. 4), we conclude that Thr-174 is not required for kinase activity as a phosphoacceptor but rather as an essential structural component. As expected (9Prowse C.N. Deal M.S. Lew J. J. Biol. Chem. 2001; 276: 40817-40823Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 10Robbins D.J. Zhen E. Owaki H. Vanderbilt C.A. Ebert D. Geppert T.D. Cobb M.H. J. Biol. Chem. 1993; 268: 5097-5106Abstract Full Text PDF PubMed Google Scholar), when we mutated each of the phosphoacceptors in wild type Hog1 we could not measure any kinase activity (Fig. 2, lanes 5–8). We expected that these mutants would not show any biological activity either (16Schuller C. Brewster J.L. Alexander M.R. Gustin M.C. Ruis H. EMBO J. 1994; 13: 4382-4389Crossref PubMed Scopus (442) Google Scholar). Surprisingly, when overexpressed, Hog1Y176F was able to rescue hog1Δ cells from osmotic shock (Fig.5). In contrast, Thr-174 was found to be essential for biological activity as Hog1T174A or even Hog1T174E did not rescue hog1Δ cells (Fig. 5 and Supplemental Fig. 3S). Our inability to detect any catalytic activity of Hog1Y176Fon one hand (Fig. 2) and the fact that Hog1Y176F is biologically active on the other hand (Fig. 5) led us to test whether Hog1Y176F supports growth on salt by activating the authentic downstream targets of the Hog1 pathway. To this end we analyzed RNA levels of GPD1, GPP2, andSTL1. In hog1Δ cells these genes did not show significant increase in RNA levels following exposure to salt (Fig.6, lanes 1–7). In contrast, when Hog1 or Hog1Y176F were expressed, a significant elevation in the transcription of these genes was detected following salt induction (Fig. 6, lanes 12–14 and 19–21). It appears that Hog1Y176F is capable of inducing expression of these genes to nearly wild type levels, explaining its ability to support growth on salt. To test whether Hog1Y176F is capable of imposing the most extreme Hog1-dependent phenotype (i.e. growth arrest) we expressed this variant in cells that also expressed the constitutively active PBS2 allelePBS2DD. PBS2DD was previously shown to induce growth arrest, depending on the presence of intact Hog1 (23Bilsland-Marchesan E. Arino J. Saito H. Sunnerhag P. Posas F. Mol. Cell. Biol. 2000; 20: 3887-3895Crossref PubMed Scopus (119) Google Scholar). As shown in Supplemental Fig. 4S, Pbs2DD induced growth arrest of cells expressing Hog1WT as expected. Cells expressing Pbs2DD and Hog1Y176F were able to grow (Supplemental Fig. 4S) on galactose. Thus, Hog1Y176F is capable of executing important functions of Hog1 (Figs. 5 and 6) but is probably not maximally activated (Supplemental Fig. 4S). The ability of Hog1Y176Fto efficiently induce gene expression suggests that although the catalytic activity of Hog1Y176F was below the threshold of our in vitro assay the enzyme was activated in the cell. We could not obtain an indication that this is the case because α-phospho-Thr antibodies did not react with Hog1Y176F(Fig. 4, lanes 5 and 6). Although this result may suggest that Hog1Y176F is not phosphorylated on Thr-174, we decided to further explore the issue through the use of antibodies against the dually phosphorylated p38. We speculated that these antibodies might recognize determinants of the active conformation of the phosphorylation motif and not merely the phosphorylated residues. This idea was based on the results of Bardwell et al. (14Bardwell L. Cook J.G. Voora D. Baggott D.M. Martinez A.R. Thorner J. Genes Dev. 1998; 12: 2887-2898Crossref PubMed Scopus (141) Google Scholar) who showed that α-phospho-ERK antibodies react with Thr-183 phosphorylated Kss1Y185F. We found (lane 6 in Fig. 7) that α-phospho-p38 reacted with Hog1Y176F after salt induction. This result supports the notion that Hog1Y176F was activated in vivo to some level. We believe that this low activity was responsible for the induction of gene expression shown in Fig. 6, which enabled growth ofhog1Δ cells on hyperosmotic medium (Fig. 5). Many enzymes, receptors, and transcription factors are regulated through phosphorylation (18Hunter T. Karin M. Cell. 1992; 70: 375-387Abstract Full Text PDF PubMed Scopus (1118) Google Scholar, 19Cohen P. Eur. J. Biochem. 2001; 268: 5001-5010Crossref PubMed Scopus (493) Google Scholar, 20Cohen P. Nat. Cell Biol. 2002; 4: E127-E130Crossref PubMed Scopus (764) Google Scholar, 21Blume-Jensen P. Hunter T. Nature. 2001; 411: 355-365Crossref PubMed Scopus (3109) Google Scholar). MAPKs are considered unusual as their activation requires concomitant dual phosphorylation of neighboring Thr and Tyr residues. This report provides evidence that at least for the yeast MAPK Hog1 this dogma only partially holds. With respect to biological activity Tyr phosphorylation plays a partial role. Mutating Tyr-176 to Phe in the hyperactive Hog1 alleles revealed that this residue functions in enhancing catalytic activity of these molecules by Pbs2 but has no role in the elevated intrinsic activity of those alleles (Fig. 3). Furthermore it appears that Tyr-176 might have an inhibitory effect on the basal activity of the hyperactive mutants as Hog1F318L,Y176F shows a higher catalytic activity in comparison with Hog1F318L in pbs2Δ cells (Fig.3B, right panel, lanes 7–10). It must be noted that these roles of Tyr-176 may be specific to the hyperactive mutants. However, the results obtained with Hog1WT mutated in Tyr-176 suggested that these roles might be relevant to the native protein as well. In wild type Hog1, mutating Tyr-176 resulted in a dramatic decrease of catalytic activity below our detection level (Fig. 2). However, Hog1Y176F, unlike Hog1T174A or even Hog1T174E, was probably catalytically active at a low levelin vivo, a level sufficient for induction of target genes (Fig. 6) and for rescuing hog1Δ cells from hyperosmotic shock (Fig. 5). It was not sufficient, however, to mediate Pbs2DD-induced growth arrest (Supplemental Fig. 4S) suggesting that Tyr-176 phosphorylation is required to obtain some further increase in activity, a case similar to that observed in the hyperactive mutants (Fig. 3). The unexpected capabilities of Hog1Y176F led us to carefully inspect previous studies in which MAPKs carrying similar mutations were used. Schüller et al. (16Schuller C. Brewster J.L. Alexander M.R. Gustin M.C. Ruis H. EMBO J. 1994; 13: 4382-4389Crossref PubMed Scopus (442) Google Scholar) suggested that Hog1T174A and Hog1Y176F cannot support growth of hog1Δ cells on hyperosmotic media. However, careful inspection of their data reveals that Hog1Y176F-expressing cells (but not cells expressing Hog1T174A) did grow on 0.4 m KCl but grew very poorly on 0.9 m KCl (16Schuller C. Brewster J.L. Alexander M.R. Gustin M.C. Ruis H. EMBO J. 1994; 13: 4382-4389Crossref PubMed Scopus (442) Google Scholar). Tyr phosphorylation may be required for extreme conditions. The notion that Hog1Y176Fis biologically active was also raised by Warkma et al.(22Warmka J. Hanneman J. Lee J. Amin D. Ota I. Mol. Cell. Biol. 2001; 21: 51-60Crossref PubMed Scopus (146) Google Scholar). How crucial is tyrosine phosphorylation for the biological activity of MAPKs other than Hog1? In the case of Kss1, mutating Tyr-185 to Phe did not abolish biological activity completely as cells expressing Kss1Y185F were capable of inducing invasive growth to some extent (14Bardwell L. Cook J.G. Voora D. Baggott D.M. Martinez A.R. Thorner J. Genes Dev. 1998; 12: 2887-2898Crossref PubMed Scopus (141) Google Scholar). Gartner et al. (15Gartner A. Nasmyth K. Ammerer G. Genes Dev. 1992; 6: 1280-1292Crossref PubMed Scopus (219) Google Scholar) reported that in Fus3 dual phosphorylation is essential for biological activity. Importantly none of these studies provided sufficient quantitative information regarding the catalytic and biological activities of the mutated MAPK. Based on the available data, we believe that the case of Hog1 analyzed here reflects a general situation in MAPK activation. Namely for many MAPK molecules Tyr phosphorylation may not be as vital as Thr phosphorylation for biological activity. Structural studies revealed that in both ERK2 and p38 phospho-Thr forms important networks of interactions that appear to be critical for stabilizing the active conformation of the enzyme (5Bellon S. Fitzgibbon M.J. Fox T. Hsiao H.M. Wilson K.P. Struct. Fold. Des. 1999; 7: 1057-1065Abstract Full Text Full Text PDF Scopus (124) Google Scholar, 7Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). This information coincides with our results, which show that Thr-174 is essential for both biological and catalytic activity (Bell et al. (12Bell M. Capone R. Pashtan I. Levitzki A. Engelberg D. J. Biol. Chem. 2001; 276: 25351-25358Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and Fig. 2). However, it appears that in the hyperactive alleles Thr-174 is important mainly for structural reasons and not as a phosphoacceptor (Fig. 4). One may speculate that the activating mutations maneuver Thr-174 toward the L16 domain and stabilize an active conformation that is not phosphorylated. Upon phosphorylation Thr-174 forms stronger interactions with residues in L16 resulting in a more active conformation. Phospho-Tyr appears to be involved in changing the conformation of the substrate (P + 1) recognition site (7Canagarajah B.J. Khokhlatchev A. Cobb M.H. Goldsmith E.J. Cell. 1997; 90: 859-869Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 9Prowse C.N. Deal M.S. Lew J. J. Biol. Chem. 2001; 276: 40817-40823Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar) but may affect catalysis as well (9Prowse C.N. Deal M.S. Lew J. J. Biol. Chem. 2001; 276: 40817-40823Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Taking advantage of the hyperactive alleles, it was possible to obtain a more detailed insight into the role of Tyr-176 in Hog1 catalysis and to reveal that it is not essential as a stabilizer of the active conformation but is more important as an amplifier of enzyme activity. We thank Eran Blachinsky, Melanie Grably, Riki Perlman, Ella Sklan, Ariel Stanhill, and Gilad Yaakov for useful comments on the manuscript and Gustav Ammerer, Michael C. Gustin, and Francesc Posas for strains and plasmids. Download .pdf (.06 MB) Help with pdf files
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