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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
hog1 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
p38 Recently we reported the isolation of MAPK kinase-independent
hyperactive MAPK mutants of both the yeast Hog1 and the human p38 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.
Yeast Strains and Media--
Strains used in this study were the
pbs2 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).
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
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 pbs2
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).
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
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 hog1
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 hog1 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 pbs2 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 hog1 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 hog1 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.
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
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.
(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.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
strain MAY1 and the hog1
strain JBY13
(12). Growth conditions were described previously (12).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
View larger version (56K):
[in a new window]
Fig. 1.
Hog1 hyperactive mutants harboring the
mutation Y176F rescue pbs2 and
hog1
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 hog1
cells
harboring the indicated plasmids, and panels on the
right show pbs2
cells harboring the same
plasmids. HOG1 alleles were overexpressed using the
ADH1 promoter.
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 hog1
cells
was barely measurable in pbs2
cells (see Supplemental
Fig. 1S). The parental enzyme Hog1F322L was fully active in
pbs2
cells (see Supplemental Fig. 2S), suggesting that
this particular variant became Pbs2-dependent by the Y176F mutation. However, Hog1Y176F,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.
View larger version (24K):
[in a new window]
Fig. 2.
Active Hog1 alleles mutated in Y176F are
catalytically active. Hog1 alleles were immunoprecipitated from
hog1 or pbs2
(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.
View larger version (39K):
[in a new window]
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
hog1 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 pbs2
cells. HOG1
alleles were expressed under the control of the ADH1
promoter.
-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 kinase activity as a phosphoacceptor but rather as an
essential structural component.
View larger version (27K):
[in a new window]
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.
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 hog1
cells (Fig. 5
and Supplemental Fig. 3S).
View larger version (26K):
[in a new window]
Fig. 5.
Hog1 mutated at Tyr-176 is biologically
active. Hog1WT, Hog1T174A, or
Hog1Y176F were expressed in hog1 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, hog1
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.
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
-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
-phospho-ERK antibodies react with Thr-183
phosphorylated Kss1Y185F. We found (lane 6 in
Fig. 7) that
-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
hog1
cells on hyperosmotic medium (Fig. 5).
View larger version (51K):
[in a new window]
Fig. 6.
Hog1Y176F is capable of inducing
Hog1 target genes in hog1 cells.
Hog1WT or Hog1Y176F 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.
View larger version (40K):
[in a new window]
Fig. 7.
Hog1Y176F is phosphorylated on
Thr-174 in hog1 cells.
Hog1WT, Hog1T174A, or Hog1Y176F 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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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.
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).
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).
![]() |
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.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
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 |
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 |
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 |
11. |
Cobb, M. H.,
and Goldsmith, E. J.
(1995)
J. Biol. Chem.
270,
14843-14846 |
12. |
Bell, M.,
Capone, R.,
Pashtan, I.,
Levitzki, A.,
and Engelberg, D.
(2001)
J. Biol. Chem.
276,
25351-25358 |
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 |
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 |
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 |
23. |
Bilsland-Marchesan, E.,
Arino, J.,
Saito, H.,
Sunnerhag, P.,
and Posas, F.
(2000)
Mol. Cell. Biol.
20,
3887-3895 |