From the University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235-9041
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ABSTRACT |
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MEK1 and MEK2 contain a proline-rich insert not present in any other known MEK (MAP (mitogen-activated protein)/ERK (extracellular signal-regulated kinase) kinase) family members. We examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells. Deletion of the insert had no effect on either the activity of MEK1 or on its ability to bind to Raf-1. Both wild type and constitutively active MEK1 coimmunoprecipitated with Raf-1 whether or not the insert was present. Deletion of the insert did not reduce activation of MEK1 by EGF or activated Raf in cells. The proline-rich insert enhanced the ability of an otherwise equally active MEK1 protein to regulate endogenous ERKs in mammalian cells. Overexpression of either constitutively active MEK1 lacking the insert or ERK2 compensates for the weaker in vivo activity of the MEK1 deletion mutant. Expression of the insert in cells reduced activation of ERKs by EGF. We conclude that the proline-rich insert is not the site of the MEK-Raf interaction and that the polyproline insert is required for its efficient activation of downstream ERKs in cells.
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INTRODUCTION |
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The MAP1 kinases ERK1 and ERK2 are the terminal enzymes in a three-protein kinase cascade committed to their activation (1, 2). The upstream components include the isoforms of the protein kinase Raf and the MAP/ERK kinases MEK1 and MEK2. The three kinase cascade is regulated by GTP ligation of Ras. GTP-bound Ras binds to Raf with high affinity and causes Raf translocation to the membrane where it is activated.
MEK activity was originally discovered by Ahn and Krebs by fractionating lysates of stimulated and unstimulated cells and mixed resulting fractions to identify ERK activators (3, 4). Subsequently, MEKs were purified by this group and others, and the two isoforms that activate ERK1 and ERK2 were cloned and expressed (5-8). Reconstitution experiments using purified MEK1 and MEK2 and ERK1 and ERK2 have demonstrated the quantitative activation of these MAP kinases requires only MEKs (9), although molecules that influence the kinetics of activation have been reported (10). Similarly, in vitro activation of MEK1 and MEK2 by Raf isoforms has been confirmed (11-13). Mansour et al. (14) and others identified mutations that created constitutively active forms of MEK1 and MEK2. When introduced into mammalian cells these activated MEK mutants increased ERK activity and induced effects associated with activation of the Ras/ERK pathway, such as transformation of fibroblasts, development of tumors in nude mice, and outgrowth of neurites in PC12 cells (14, 15).
The multienzyme MAP kinase cascade allows for amplification, because MEK1 and MEK2 are present in considerable excess of Raf. However, the MEKs and ERKs in this pathway are present at roughly equal concentrations and in some cells MEKs are in excess (16). This suggests that the MEK to ERK step exists not for amplification but for kinetic regulation (17). The presence of this step also provides additional mechanisms for sensing coincident and distinct regulatory inputs (18).
MEK1 and MEK2 contain a proline-rich insert in the C-terminal domain of the kinase catalytic core that is not present in any other known MEK family members either in higher eukaryotes or in yeast. This insert contains phosphorylation sites for proline-directed and other protein kinases and is phosphorylated in intact cells (18). In vitro, MEK1 is phosphorylated in this insert by ERKs, Cdk2, and the p21-activated kinase PAK1, among others (12, 18-20). Jelinek et al. (19) reported that a MEK1 point mutant lacking one of these phosphorylation sites no longer forms complexes with endogenous Raf. In a follow-up study they deleted the proline-rich insert from MEK1 and found that it was no longer efficiently activated in cells nor did it cause transformation (21). These data suggested that the polyproline insert of MEK1 was involved in interaction with the Ras-Raf complex, presumably through direct binding to Raf. More recently, Frost et al. (18) found that mutation of other phosphorylation sites in this proline-rich insert also reduced Raf-MEK binding, further supporting the idea that changes in the MEK1 proline-rich insert modulate coupling to Raf.
In this study we deleted the proline-rich insert of MEK1 and expressed it as a separate polypeptide with the aim of demonstrating that the insert directly binds to Raf. In addition, we examined its effects on activation of ERKs in vitro. Deletion of the proline-rich insert from a constitutively active form of MEK1, MEK1 R4F, had little effect on its in vitro protein kinase activity or its ability to coimmunoprecipitate with Raf-1, but significantly impaired its ability to activate ERKs in mammalian cells. Our findings are consistent with two conclusions. First, although phosphorylation within the proline-rich insert modulates binding of MEK to Raf, it is not the site of the interaction. Second, the polyproline insert of MEK1 is required for its efficient activation of downstream ERKs in cells.
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MATERIALS AND METHODS |
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Plasmids and Proteins--
Recombinant His6-K52R
ERK2 and active ERK2 were expressed and purified as described (9, 18).
MEK1, MEK1R4F, MEK2R4F (kindly provided by Natalie Ahn, University of
Colorado, Boulder, CO), MEKK-C, and PAK1 were expressed as described
(9, 12, 22-24). To delete the proline-rich insert, residues 265-301,
fragments encoding the N- and C-terminal sequence were created by PCR
using pRSETA MEK1 R4F as template, digested, and ligated into pGEXKG to
produce pGEXKG MEK1 R4Fins. A similar approach was used to generate
MEK1
ins. Restriction sites were incorporated during PCR to
facilitate subcloning into other vectors. The MEK1 insert, corresponding to amino acids 265-301, was amplified by PCR and subcloned into pGEXKG and pEBG. All PCR products were sequenced to
confirm correct amplification. Recombinant proteins expressed in
bacteria were purified on Ni2+-nitrilotriacetic
acid-agarose followed by Mono Q. Amounts of recombinant MEK1 proteins
were quantitated by immunoblotting with antibodies to the N and C
termini of MEK1, followed by 125I-labeled secondary
antibodies. The blots were quantitated using a phosphorimager.
Cell Culture, Transfection, and Preparation of
Extracts--
Human embryonic kidney 293 or NIH 3T3 cells were grown
in Dulbecco's modified Eagles medium containing 10% fetal bovine
serum and 1% L-glutamine (24). 293 cells at no less than
80% confluence were transfected with DNA by calcium phosphate
coprecipitation (25) and maintained for 24-48 h after transfection.
Prior to stimulation cells were deprived of fetal bovine serum for
16-18 h and then treated with EGF (50 ng/ml). Cells were homogenized in ERK lysis buffer and supernatants were prepared from them as described (26). Transfection efficiency was assessed by expression of
recombinant -galactosidase from a cotransfected plasmid (27).
Immunoprecipitation and Immunoblotting-- The monoclonal antibody to the HA epitope was purchased from Berkley Antibody Company. Anti-ERK antisera Y691, X837, and A249 were raised as described (28, 29). Antibodies to the proline-rich inserts of MEK1 (A2227) and MEK2 (A2228) were produced as described (24). Antibodies to the C termini of MEK1 (SC-219) and Raf-1 (SC-133) were purchased from Santa Cruz Biotechnology. Antibodies that recognize the activated forms of ERK1 and ERK2 (20291) were from Promega, and their selective recognition of the high activity forms of ERK1 and ERK2 was confirmed as described elsewhere (23). For immune complex kinase assays supernatants were incubated with the indicated antibodies and protein A-Sepharose with rotation for 2 h at 4 °C as described (24). The beads were washed with 0.25 M Tris-HCl (pH 7.6) plus 0.1 M NaCl followed by 0.25 M Tris-HCl and resuspended for kinase assay. Immunoblots probed with the above antisera were developed with the Amersham ECL kit.
Kinase Assays-- MEK activity was measured in 50 µl of 10 mM Hepes (pH 8.0), 10 mM MgCl2, 1 mM benzamidine, 1 mM dithiothreitol, 100 µM ATP (1-15 cpm/pmol) containing 50 µg/ml recombinant K52R ERK2 at 30 °C for 30 min using purified or immunoprecipitated kinase associated with 10 µl of protein-A-Sepharose beads. The reactions were stopped with sodium dodecyl sulfate-loading buffer. ERK activity was measured using myelin basic protein (MBP) as substrate in 30 µl of 10 mM Hepes (pH 8.0), 10 mM MgCl2, 1 mM benzamidine, 1 mM dithiothreitol, 100 µM ATP (1 cpm/fmol) at 30 °C for 30 min (24).
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RESULTS |
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Deletion of the Proline-rich Insert Has Little Influence on the
Activity of MEK1 in Vitro--
To examine the interactions mediated by
the proline-rich insert, it was important to demonstrate that deletion
of the insert did not influence MEK activity. Therefore, we determined
the impact of the proline-rich insert on the in vitro kinase
activity of MEK1. Because it had been reported that deletion of the
insert prevented activation of MEK1 by Raf (21), we initially studied the properties of a constitutively active form of MEK1 in the absence
of this insert. We deleted 37 amino acids (residues 265-301 of human
MEK1) from the constitutively active mutant MEK1R4F and expressed
MEK1R4F and MEK1R4Fins as histidine-tagged proteins in bacteria.
MEK1R4F
ins displays a marked increase in electrophoretic mobility
concomitant with deletion of the proline-rich domain. Recognition of
MEK1R4F
ins with antibodies to both termini, along with sequencing of
the mutant cDNA, confirms that the apparent size of the protein is
correct and due neither to inadvertent mutations nor to proteolytic
cleavage (Fig. 1A).
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Deletion of the Insert Impairs the Function of MEK1R4F in Mammalian
Cells--
Based on their in vitro activities, we expected
that both MEK1R4F and MEK1R4Fins would cause equal activation of
ERKs in cells. Their expression in transfected cells was equivalent,
based on immunoblotting lysates with antibodies to both the N terminus (not shown) and the C terminus of MEK1 (Fig.
2A). The absence of the
proline-rich domain was confirmed by blotting the transfected lysates
with an antibody that recognizes the MEK1 insert (A2227); a signal was
detected only from MEK1R4F, not from MEK1R4F
ins (Fig.
2B).
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Phosphorylation of the MEK1 Insert-- Because the MEK1 insert contains known phosphorylation sites, we tested its phosphorylation in vitro by several protein kinases (Fig. 4). ERK2 and PAK1 phosphorylated the MEK1 insert, whereas MEKK1, MEK1, and MEK2 did not. This is consistent with phosphorylation of the intact protein on S298 by PAK1 and on T292 by ERK2 and other protein kinases (12, 18, 20, 21).
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Deletion of the Proline-rich Insert Does Not Prevent Binding
of MEK1 to Raf-1--
An original aim was to determine whether
the MEK1 proline-rich insert binds to Raf. Therefore, we measured the
ability of MEK1R4F with and without the insert to coimmunoprecipitate
with endogenous Raf-1. HA-MEK1R4Fins and HA-MEK1R4F were transiently expressed in 293 cells and immunoprecipitated with an anti-HA antibody.
Antibodies against the C terminus of MEK1 were used to show that
equivalent amounts of HA-MEK1R4F and HA-MEK1 R4F
ins were
immunoprecipitated (Fig. 5A).
Endogenous Raf-1 coimmunoprecipitated with both forms of MEK1R4F, as
assessed by immunoblotting with an anti-Raf-1 antibody (Fig.
5B). To confirm that the enhanced activity of the MEK1
ins
mutant did not account for its interaction with Raf, perhaps through a
feedback phosphorylation by ERKs, we examined the ability of wild type
MEK1 and MEK1
ins to coprecipitate with Raf-1. Again we found that
Raf-1 was associated with MEK1 whether or not it contained the
proline-rich insert (Fig. 5C), although wild type MEK1 bound
to Raf less well than did MEK1R4F. These data suggest that the
proline-rich insert is not the Raf-1 binding site. To determine whether
the proline-rich insert bound to Raf in the absence of the MEK1
catalytic core, the insert-GST fusion protein was overexpressed in 293 cells. Lysates were incubated with glutathione beads. Proteins
associated with the beads were separated on gels and immunoblotted with
antibodies against Raf-1. Under these conditions, Raf-1 binding to the
insert could not be detected (not shown).
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Deletion of the Proline-rich Insert Does Not Reduce Activation of
MEK1 by EGF--
Because MEK1 lacking the insert coimmunoprecipitated
with Raf-1, we tested the ability of wild type MEK1 with and without the insert to be activated by growth factors and Raf. Previously published work suggested that without the insert MEK1 would be poorly
activated (21). Contrary to expectations, the time courses and extents
of activation of wild type MEK1 and MEK1ins by EGF were identical
(Fig. 5D). As confirmation that the cells were equally
responsive to EGF, activation of endogenous ERKs was monitored. In
cells transfected with MEK1 or MEK1
ins, ERKs were equally activated
by EGF (not shown). We also tested the ability of a constitutively
activated Raf-1 mutant S259D to activate MEK1 when they were
cotransfected. Again, we found that both wild type MEK1 and MEK1
ins
were activated to a similar extent (not shown).
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DISCUSSION |
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MEK1 and MEK2 contain a proline-rich insert that is not present in any of the other known MEK family members. This insert is phosphorylated in intact cells and mutation of at least two different insert phosphorylation sites diminishes the ability of MEK1 to associate with Raf-1 in cell lysates (18, 19). Furthermore, it has been reported that the insert is necessary for cell transformation (21). To understand the underlying mechanisms, we examined the effect of removing the MEK1 polyproline insert on MEK activity, its binding to Raf, and its ability to activate ERKs in cells.
We find that deletion of the insert has no effect on the activity of MEK1, either in vitro or isolated from cells, and, in contrast to earlier suggestions, removing the insert has no effect on the Raf-1 binding of MEK1. Both wild type and constitutively active MEK1 coimmunoprecipitate with Raf-1 whether or not the insert is present, indicating that the insert is not required for MEK-Raf binding. In support of this conclusion, the insert alone did not bind Raf in lysates. Apparently the phosphorylation or mutation of residues such as Thr292 and Ser298 within the insert can influence MEK1-Raf-1 binding either positively or negatively. Mutation of Thr292 to Ala inhibited the association of MEK1 with Ras-Raf complexes (19). A decrease in Raf binding was noted for MEK1 mutants with both Ser298 and Thr292 mutated to Ala (18). On the other hand, phosphorylation of Ser298 by PAK1 enhances the activation of MEK1 by Raf and growth factors, indicating that the proline insert modulates MEK1 function in cells (18).
Consistent with the lack of effect of deleting the proline-rich insert on binding of MEK1 to Raf, deletion of the insert does not reduce activation of MEK1 by EGF or activated Raf in cells. Catling et al. (21) reported that a similar MEK1 deletion protein was not activated by Raf or growth factors. Perhaps the difference in behavior of these MEK1 deletion mutants is because of the difference in the residues that were excised. The previously described MEK1 deletion mutant removed residues 270-307 (21).
We also found that the insert increases the efficiency of activation of
downstream ERKs by MEK1. Factors in addition to the enzyme, MEK1, and
the substrate, ERK2, apparently assist in the coupling of this kinase
cascade in mammalian cells. Weber and colleagues reported that
constitutively active MEK1 with the proline-rich sequence transformed
fibroblasts, whereas constitutively active MEK1 without the proline
insert did not (21). In that study, however, the activity of the
endogenous MAP kinase was not measured. Here we show directly that the
proline-rich insert unique to MEK1 and MEK2 enhances the ability of an
otherwise equally active MEK protein to regulate endogenous ERKs in
mammalian cells. The finding that overexpression of either
MEK1R4Fins or ERK2 compensates for the weaker in vivo
activity of the proline-rich deletion mutant suggests that the
intracellular process mediated by the proline-rich insert increases the
effective concentration of the substrate ERK2. The reduced activation
of ERKs in cells expressing the insert alone supports this
interpretation. Perhaps the insert binds to Ste5p-like scaffolding
proteins that affect MEK-ERK coupling (31, 32). An alternative might be
that the insert is modified or interacts with other factors in a manner
that further increases MEK activity. In either case, the reduced
ability of MEK1R4F
ins to activate ERKs is consistent with the
reduced ability of a constitutively active MEK1 mutant with a similar
but not identical deletion to transform cells (21).
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ACKNOWLEDGEMENTS |
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We thank members of the Cobb laboratory for helpful comments about the manuscript, Megan Robinson for certain reagents, Natalie Ahn for numerous MEK constructs, and Tina Arikan for secretarial assistance.
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FOOTNOTES |
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* This work was supported in part by Grant DK34128 (to M. H. C.) from the National Institutes 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.
Supported by Predoctoral Training Grant GM07062. This work was
done in partial fulfillment of the requirements for Ph.D.
1 The abbreviations used are: MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP/ERK kinase; PCR, polymerase chain reaction; EGF, epidermal growth factor; HA, hemagglutinin; MBP, myelin basic protein; GST, glutathione S-transferase.
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REFERENCES |
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