Phosphorylation of Tyr-176 of the yeast MAPK Hog1/p38 is not vital for Hog1 biological 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 Hog1(Y176F) is biologically active, capable of induction of Hog1 target genes and of rescuing hog1Delta cells from osmotic stress. Hog1(Y176F) was 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.

the relevant pathway this motif is dually phosphorylated, leading to structural changes and a dramatic increase in specific activity (7,9,10). 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 (9 -11).
Although both Thr and Tyr seem to be equally important for catalysis, the three-dimensional structures of phosphorylated ERK2 and p38␥ suggested that the Thr-183 residue contributes more significantly to stabilization of the active form (5,7). 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 p38␣ (12). 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 pbs2⌬ cells from high osmolarity (12). 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.

MATERIALS AND METHODS
Yeast Strains and Media-Strains used in this study were the pbs2⌬ strain MAY1 and the hog1⌬ strain JBY13 (12). Growth conditions were described previously (12).
Plasmids-T174A or Y176F mutations were inserted into plasmids pES86-HA-HOG1 (harboring an HA-tagged HOG1 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.
Preparation of Native Cell Lysates, Western Blots, in Vitro Kinase Assay, and Detection of Thr Phosphorylation-Cell lysate preparations and kinase assays were described previously (12). Detection of Thr phosphorylation was performed by immunoprecipitation of HA-Hog1 as described previously (12) 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 (12).
RNA Preparation and Analysis-Cultures were grown to A 600 ϭ 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 (13).

Tyr-176 Is Dispensable for Biological and Catalytic Activity of Hyperactive Hog1
Mutants, whereas Thr-174 Is Essential-Dual phosphorylation of both Thr and Tyr at the phosphorylation lip is considered an absolute requirement for MAPK activation (10, 14 -17). 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 (12). 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 hog1⌬ and pbs2⌬ cells from hyperosmotic shock (Ref. 12 and Fig. 1). Also attempts to directly measure dual phosphorylation of the hyperactive mutants (expressed in pbs2⌬ cells) using ␣-phospho-p38 antibodies revealed no or very low phosphorylation levels (depending on the particular mutant; see Fig. 6 in Bell et al. (12)). However, when Thr-174 was mutated to Ala, all Hog1 variants lost the capability to rescue pbs2⌬ cells and even hog1⌬ cells from osmotic stress (12), strongly suggesting that Thr-174 has a more central role in Hog1 activity.
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, Hog1 WT completely lost its catalytic activity when mutated in either Thr-174 or Tyr-176 (Fig. 2, left panel, lanes [3][4][5][6][7][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 pbs2⌬ cells, three mutants (Y176F,D170A; Y176F,F318L; and Y176F,F318S) manifested catalytic activity (see Supplemental Fig. 1S). Interestingly the activity of Hog1 Y176F,F322L that was high in hog1⌬ cells was barely measurable in pbs2⌬ cells (see Supplemental Fig. 1S). The parental enzyme Hog1 F322L was fully active in pbs2⌬ cells (see Supplemental Fig. 2S), suggesting that this particular variant became Pbs2-dependent by the Y176F mutation. However, Hog1 Y176F,F322L could rescue pbs2⌬ cells (see Fig. 7 in Bell et al. (12)) showing that also in this case Tyr-176 is not essential for biological activity.
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. (12)).

Tyr-176 Is Important for Increasing the Catalytic Activity of Hog1 Hyperactive Mutants in Response to Salt Induction-
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. 12). 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 Hog1 D170A and Hog1 F318L molecules side by side with the Hog1 D170A,Y176F and Hog1 F318L,Y176F derivatives. 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).
Thr-174 Is Not Required as a Phosphoacceptor for Active Hog1 Activity-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 ␣-phospho-Thr antibodies (Fig. 4). The results show that when expressed in pbs2⌬ cells the active mutants manifested either barely or no detectable Thr phosphorylation (Fig. 4, right panel). When expressed in hog1⌬ 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 pbs2⌬ 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  Fig. 2S). HOG1 alleles were overexpressed using the ADH1 promoter.
Tyr-176 Phosphorylation Is Not Essential for Hog1 Activity 14604 kinase activity as a phosphoacceptor but rather as an essential structural component.
Tyr-176 Is Not Essential for Wild Type Hog1 Biological Activity-As expected (9, 10), 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 (16). Surprisingly, when overexpressed, Hog1 Y176F was able to rescue hog1⌬ cells from osmotic shock (Fig. 5). In contrast, Thr-174 was found to be essential for biological activity as Hog1 T174A or even Hog1 T174E did not rescue hog1⌬ cells (Fig. 5 and Supplemental Fig. 3S).
Our inability to detect any catalytic activity of Hog1 Y176F on one hand (Fig. 2) and the fact that Hog1 Y176F is biologically active on the other hand (Fig. 5) led us to test whether Hog1 Y176F supports growth on salt by activating the authentic downstream targets of the Hog1 pathway. To this end we analyzed RNA levels of GPD1, GPP2, and STL1. In hog1⌬ 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 Hog1 Y176F 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 Hog1 Y176F is capable of inducing expression of these genes to nearly wild type levels, explaining its ability to support growth on salt. To test whether Hog1 Y176F 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 allele PBS2 DD . PBS2 DD was previously shown to induce growth arrest, depending on the presence of FIG. 4. Hog1 hyperactive alleles are not phosphorylated on Thr residues in pbs2⌬ cells. Hog1 alleles were immunoprecipitated from hog1⌬ cells (left panels) or pbs2⌬ cells (right panels) and analyzed for Thr phosphorylation by Western blot using ␣-phospho-Thr antibodies (P-Thr) (upper panels). Cultures were exposed or not to 1 M NaCl. Hog1 protein levels were detected by stripping the blots and reincubation with ␣-HA antibodies. Proteins were overexpressed using the ADH1 promoter.
FIG. 5. Hog1 mutated at Tyr-176 is biologically active. Hog1 WT , Hog1 T174A , or Hog1 Y176F were expressed in hog1⌬ cells. Cells were grown to A 600 ϭ 0.4 when each culture was diluted, and the indicated number of cells was plated on a YNB-URA plate (left) and a YPD ϩ 1.1 M NaCl plate (right). In Supplemental Fig. 3S, hog1⌬ cells harboring the plasmid pES86 ϩ , HOG1 WT , or HOG1 T174E were plated on a YNB-URA plate (left) and on a YPD ϩ 0.9 M NaCl plate (right). Proteins were overexpressed using the ADH1 promoter.
FIG. 6. Hog1 Y176F is capable of inducing Hog1 target genes in hog1⌬ cells. Hog1 WT or Hog1 Y176F was subcloned into the pRS426 plasmid and expressed under the native HOG1 promoter in hog1⌬ cells. Cells were exposed or not to 1 M NaCl. At the indicated time points, RNA levels were monitored by S1 analysis. HAL3 was used as a loading control.

FIG. 7. Hog1 Y176F is phosphorylated on Thr-174 in hog1⌬ cells.
Hog1 WT , Hog1 T174A , or Hog1 Y176F was expressed in hog1⌬ cells that were exposed or not to 1 M NaCl for 10 min. Proteins were extracted and separated by SDS-PAGE. Western blot analysis was performed with ␣-phospho-p38 antibodies (upper panel) followed by stripping and reincubation with ␣-HA antibodies. Proteins were expressed under the ADH1 promoter. P-Hog1, phosphorylated Hog1.
Tyr-176 Phosphorylation Is Not Essential for Hog1 Activity 14605 intact Hog1 (23). As shown in Supplemental Fig. 4S, Pbs2 DD induced growth arrest of cells expressing Hog1 WT as expected. Cells expressing Pbs2 DD and Hog1 Y176F were able to grow (Supplemental Fig. 4S) on galactose. Thus, Hog1 Y176F is capable of executing important functions of Hog1 (Figs. 5 and 6) but is probably not maximally activated (Supplemental Fig. 4S). The ability of Hog1 Y176F to efficiently induce gene expression suggests that although the catalytic activity of Hog1 Y176F 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 ␣-phospho-Thr antibodies did not react with Hog1 Y176F (Fig. 4, lanes 5 and 6). Although this result may suggest that Hog1 Y176F 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. (14) who showed that ␣-phospho-ERK antibodies react with Thr-183 phosphorylated Kss1 Y185F . We found (lane 6 in Fig. 7) that ␣-phospho-p38 reacted with Hog1 Y176F after salt induction. This result supports the notion that Hog1 Y176F 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 of hog1⌬ cells on hyperosmotic medium (Fig. 5).

DISCUSSION
Many enzymes, receptors, and transcription factors are regulated through phosphorylation (18 -21). 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 Hog1 F318L,Y176F shows a higher catalytic activity in comparison with Hog1 F318L in pbs2⌬ 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 Hog1 WT 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, Hog1 Y176F , unlike Hog1 T174A or even Hog1 T174E , was probably catalytically active at a low level in vivo, a level sufficient for induction of target genes (Fig. 6) and for rescuing hog1⌬ cells from hyperosmotic shock (Fig. 5). It was not sufficient, however, to mediate Pbs2 DD -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 Hog1 Y176F led us to carefully inspect previous studies in which MAPKs carrying similar mutations were used. Schü ller et al. (16) suggested that Hog1 T174A and Hog1 Y176F cannot support growth of hog1⌬ cells on hyperosmotic media. However, careful inspection of their data reveals that Hog1 Y176F -expressing cells (but not cells expressing Hog1 T174A ) did grow on 0.4 M KCl but grew very poorly on 0.9 M KCl (16). Tyr phosphorylation may be required for extreme conditions. The notion that Hog1 Y176F is biologically active was also raised by Warkma et al. (22).
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 Kss1 Y185F were capable of inducing invasive growth to some extent (14). Gartner et al. (15) 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 (5,7). This information coincides with our results, which show that Thr-174 is essential for both biological and catalytic activity (Bell et al. (12) 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 ϩ 1) recognition site (7,9) but may affect catalysis as well (9). 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.