ACCELERATED PUBLICATION
Phosphorylation of Tyr-176 of the Yeast MAPK Hog1/p38 Is Not Vital for Hog1 Biological Activity*,

Michal Bell and David EngelbergDagger

From the Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel

Received for publication, January 8, 2003, and in revised form, March 9, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 rescuing hog1Delta cells from osmotic stress. Hog1Y176F 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mitogen-activated protein kinases (MAPKs1; ERK, p38, and c-Jun N-terminal kinase) play vital roles in determining the cell program (1-4). Despite the significant progress in our understanding of MAPK activation and catalysis (5-8) 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 (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 p38gamma 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 p38alpha (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 pbs2Delta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Yeast Strains and Media-- Strains used in this study were the pbs2Delta strain MAY1 and the hog1Delta 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 A600 = 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).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 hog1Delta and pbs2Delta cells from hyperosmotic shock (Ref. 12 and Fig. 1). Also attempts to directly measure dual phosphorylation of the hyperactive mutants (expressed in pbs2Delta cells) using alpha -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 pbs2Delta cells and even hog1Delta cells from osmotic stress (12), strongly suggesting that Thr-174 has a more central role in Hog1 activity.


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Fig. 1.   Hog1 hyperactive mutants harboring the mutation Y176F rescue pbs2Delta and hog1Delta cells from osmotic shock. Cultures were grown in liquid medium to A600 = 0.4 and serially diluted to obtain the indicated number of cells in 6 µl. 6 µl of each dilution were plated on a YNB-URA plate (upper panel) and on a YPD + 1.1 M NaCl plate (lower panel). Panels on the left show hog1Delta cells harboring the indicated plasmids, and panels on the right show pbs2Delta cells harboring the same plasmids. HOG1 alleles were overexpressed using the ADH1 promoter.

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 pbs2Delta 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 hog1Delta cells was barely measurable in pbs2Delta cells (see Supplemental Fig. 1S). The parental enzyme Hog1F322L was fully active in pbs2Delta cells (see Supplemental Fig. 2S), suggesting that this particular variant became Pbs2-dependent by the Y176F mutation. However, Hog1Y176F,F322L could rescue pbs2Delta cells (see Fig. 7 in Bell et al. (12)) showing that also in this case Tyr-176 is not essential for biological activity.


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Fig. 2.   Active Hog1 alleles mutated in Y176F are catalytically active. Hog1 alleles were immunoprecipitated from hog1Delta or pbs2Delta (Supplemental Figs. 1S and 2S) cells and assayed in vitro using GST-ATF2 as a substrate. Cells were either exposed or not to 1 M NaCl for 10 min. The upper panel of each image shows the autoradiogram of the in vitro kinase assay. The lower panel of each image shows Western blots of the immunoprecipitated Hog1 protein (this figure and Supplemental Fig. 1S) or Hog1 in the lysates (Supplemental Fig. 2S). HOG1 alleles were overexpressed using the ADH1 promoter.

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 Hog1D170A and Hog1F318L molecules side by side with the Hog1D170A,Y176F and Hog1F318L,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).


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Fig. 3.   Tyr-176 is important for enhancing catalytic activity of the hyperactive mutants in response to salt induction. A, activity of Hog1 alleles extracted from hog1Delta cells. The upper two panels show different exposures of the same gel on which kinase assay mixtures were separated. The lower panel shows a Western blot of the immunoprecipitated Hog1 proteins. B, activity of Hog1 alleles extracted from pbs2Delta cells. HOG1 alleles were expressed under the control of the ADH1 promoter.

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 alpha -phospho-Thr antibodies (Fig. 4). The results show that when expressed in pbs2Delta cells the active mutants manifested either barely or no detectable Thr phosphorylation (Fig. 4, right panel). When expressed in hog1Delta 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 pbs2Delta 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.


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Fig. 4.   Hog1 hyperactive alleles are not phosphorylated on Thr residues in pbs2Delta cells. Hog1 alleles were immunoprecipitated from hog1Delta cells (left panels) or pbs2Delta cells (right panels) and analyzed for Thr phosphorylation by Western blot using alpha -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 alpha -HA antibodies. Proteins were overexpressed using the ADH1 promoter.

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, Hog1Y176F was able to rescue hog1Delta 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 hog1Delta cells (Fig. 5 and Supplemental Fig. 3S).


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Fig. 5.   Hog1 mutated at Tyr-176 is biologically active. Hog1WT, Hog1T174A, or Hog1Y176F were expressed in hog1Delta cells. Cells were grown to A600 = 0.4 when each culture was diluted, and the indicated number of cells was plated on a YNB-URA plate (left) and a YPD + 1.1 M NaCl plate (right). In Supplemental Fig. 3S, hog1Delta cells harboring the plasmid pES86+, HOG1WT, or HOG1T174E were plated on a YNB-URA plate (left) and on a YPD + 0.9 M NaCl plate (right). Proteins were overexpressed using the ADH1 promoter.

Our inability to detect any catalytic activity of Hog1Y176F on 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, and STL1. In hog1Delta 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 allele PBS2DD. PBS2DD was previously shown to induce growth arrest, depending on the presence of intact Hog1 (23). 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 Hog1Y176F to 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 alpha -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. (14) who showed that alpha -phospho-ERK antibodies react with Thr-183 phosphorylated Kss1Y185F. We found (lane 6 in Fig. 7) that alpha -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 of hog1Delta cells on hyperosmotic medium (Fig. 5).


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Fig. 6.   Hog1Y176F is capable of inducing Hog1 target genes in hog1Delta cells. Hog1WT or Hog1Y176F was subcloned into the pRS426 plasmid and expressed under the native HOG1 promoter in hog1Delta 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.


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Fig. 7.   Hog1Y176F is phosphorylated on Thr-174 in hog1Delta cells. Hog1WT, Hog1T174A, or Hog1Y176F was expressed in hog1Delta 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 alpha -phospho-p38 antibodies (upper panel) followed by stripping and reincubation with alpha -HA antibodies. Proteins were expressed under the ADH1 promoter. P-Hog1, phosphorylated Hog1.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Hog1F318L,Y176F shows a higher catalytic activity in comparison with Hog1F318L in pbs2Delta 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 level in vivo, a level sufficient for induction of target genes (Fig. 6) and for rescuing hog1Delta 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. (16) suggested that Hog1T174A and Hog1Y176F cannot support growth of hog1Delta 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 (16). Tyr phosphorylation may be required for extreme conditions. The notion that Hog1Y176F 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 Kss1Y185F 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 + 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.

    ACKNOWLEDGEMENTS

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.

    FOOTNOTES

* This study was supported by grants from the Israel Science Foundation, The Israel Cancer Research Fund, and the chief scientist of the Israeli ministry of health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1S-4S.

Dagger To whom correspondence should be addressed. Tel.: 972-2-6584718; Fax: 972-2-6584910; E-mail: engelber@mail.ls.huji.ac.il.

Published, JBC Papers in Press, March 10, 2003, DOI 10.1074/jbc.C300006200

    ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; WT, wild type; GST, glutathione S-transferase; YPD, yeast extract, peptone, dextrose; YNB, yeast nitrogen base.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Davis, R. J. (2000) Cell 103, 239-252[Medline] [Order article via Infotrieve]
2. Kyriakis, J. M., and Avruch, J. (2001) Physiol. Rev. 81, 807-869[Abstract/Free Full Text]
3. Ono, K., and Han, J. (2000) Cell. Signal. 12, 1-13[CrossRef][Medline] [Order article via Infotrieve]
4. Pearson, G., Robinson, F., Beers Gibson, T., Xu, B. E., Karandikar, M., Berman, K., and Cobb, M. H. (2001) Endocr. Rev. 22, 153-183[Abstract/Free Full Text]
5. Bellon, S., Fitzgibbon, M. J., Fox, T., Hsiao, H. M., and Wilson, K. P. (1999) Struct. Fold. Des. 7, 1057-1065[Medline] [Order article via Infotrieve]
6. Cobb, M. H., and Goldsmith, E. J. (2000) Trends Biochem. Sci. 25, 7-9[CrossRef][Medline] [Order article via Infotrieve]
7. Canagarajah, B. J., Khokhlatchev, A., Cobb, M. H., and Goldsmith, E. J. (1997) Cell 90, 859-869[Medline] [Order article via Infotrieve]
8. Khokhlatchev, A. V., Canagarajah, B., Wilsbacher, J., Robinson, M., Atkinson, M., Goldsmith, E., and Cobb, M. H. (1998) Cell 93, 605-615[Medline] [Order article via Infotrieve]
9. Prowse, C. N., Deal, M. S., and Lew, J. (2001) J. Biol. Chem. 276, 40817-40823[Abstract/Free Full Text]
10. Robbins, D. J., Zhen, E., Owaki, H., Vanderbilt, C. A., Ebert, D., Geppert, T. D., and Cobb, M. H. (1993) J. Biol. Chem. 268, 5097-5106[Abstract/Free Full Text]
11. Cobb, M. H., and Goldsmith, E. J. (1995) J. Biol. Chem. 270, 14843-14846[Free Full Text]
12. Bell, M., Capone, R., Pashtan, I., Levitzki, A., and Engelberg, D. (2001) J. Biol. Chem. 276, 25351-25358[Abstract/Free Full Text]
13. Chen, W., Tabor, S., and Struhl, K. (1987) Cell 50, 1047-1055[Medline] [Order article via Infotrieve]
14. Bardwell, L., Cook, J. G., Voora, D., Baggott, D. M., Martinez, A. R., and Thorner, J. (1998) Genes Dev. 12, 2887-2898[Abstract/Free Full Text]
15. Gartner, A., Nasmyth, K., and Ammerer, G. (1992) Genes Dev. 6, 1280-1292[Abstract]
16. Schuller, C., Brewster, J. L., Alexander, M. R., Gustin, M. C., and Ruis, H. (1994) EMBO J. 13, 4382-4389[Abstract]
17. Madhani, H. D., Styles, C. A., and Fink, G. R. (1997) Cell 91, 673-684[Medline] [Order article via Infotrieve]
18. Hunter, T., and Karin, M. (1992) Cell 70, 375-387[Medline] [Order article via Infotrieve]
19. Cohen, P. (2001) Eur. J. Biochem. 268, 5001-5010[Abstract/Free Full Text]
20. Cohen, P. (2002) Nat. Cell Biol. 4 (5), E127-E130[CrossRef][Medline] [Order article via Infotrieve]
21. Blume-Jensen, P., and Hunter, T. (2001) Nature 411, 355-365[CrossRef][Medline] [Order article via Infotrieve]
22. Warmka, J., Hanneman, J., Lee, J., Amin, D., and Ota, I. (2001) Mol. Cell. Biol. 21, 51-60[Abstract/Free Full Text]
23. Bilsland-Marchesan, E., Arino, J., Saito, H., Sunnerhag, P., and Posas, F. (2000) Mol. Cell. Biol. 20, 3887-3895[Abstract/Free Full Text]


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