From the Department of Microbiology and Cancer
Center, University of Virginia Health Sciences Center, Charlottesville,
Virginia 22908, the ¶ Department of Cellular and Molecular
Pharmacology, University of California, San Francisco, California
94143, and the
Cancer Research Program, Garvan Institute of
Medical Research, St. Vincent's Hospital, Darlinghurst, New South
Wales 2010, Australia
Received for publication, January 16, 2003, and in revised form, February 18, 2003
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ABSTRACT |
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The mitogen-activated protein kinases
are key regulators of cellular organization and function. To understand
the mechanisms(s) by which these ubiquitous kinases affect specific
cellular changes, it is necessary to identify their diverse and
numerous substrates in different cell contexts and compartments. As a
first step in achieving this goal, we engineered a mutant ERK2 in which
a bulky amino acid residue in the ATP binding site (glutamine 103) is changed to glycine, allowing this mutant to utilize an analog of ATP
(cyclopentyl ATP) that cannot be used by wild-type ERK2 or other
cellular kinases. The mutation did not inhibit ERK2 kinase activity or
substrate specificity in vitro or in vivo. This
method allowed us to detect only ERK2-specific phosphorylations within a mixture of proteins. Using this ERK2 mutant/analog pair to
phosphorylate ERK2-associated proteins in COS-1 cells, we identified
the ubiquitin ligase EDD (E3 identified by
differential display) and the nucleoporin Tpr
(translocated promoter region) as
two novel substrates of ERK2, in addition to the known ERK2 substrate
Rsk1. To further validate the method, we present data that confirm that
ERK2 phosphorylates EDD in vitro and in vivo.
These results not only identify two novel ERK2 substrates but also
provide a framework for the future identification of numerous cellular
targets of this important signaling cascade.
The mitogen-activated protein kinase
(MAPK)1 or extracellular
signal-regulated kinase (ERK) pathway is an evolutionarily conserved signaling pathway that regulates many cellular processes,
including proliferation, differentiation, gene transcription,
and cellular migration (1). Activation of the Ras oncogene stimulates
the membrane recruitment and activation of the Raf protein kinases (2-4), which in turn phosphorylate and activate MAPK kinase or ERK
kinases (MEK) 1 and 2. These in turn phosphorylate and activate ERK1
and ERK2 (5). When in the inactive state, ERKs are anchored in the
cytoplasm due to a basal association with the MEKs. Following activation, the ERKs migrate to various cellular locations, including the nucleus, microtubules, focal contacts, and others (6-9) where they
phosphorylate a variety of substrates. These diverse phosphorylations allow the ERKs to orchestrate a complex but coordinated response to
extracellular signals. The pattern and timing of substrate selections
determines the specific biological outcome.
The identification and characterization of the direct targets of a
particular signaling pathway is paramount to understanding the function
of that pathway within the cell. The Ras to ERK pathway is a key
regulator of many cellular processes. Although there are many
substrates of the ERKs that have been identified (10, 11), the diverse
roles of the ERKs within the cell suggest that many unknown substrates
remain to be identified. Identification of the kinase that directly
phosphorylates a particular substrate can be difficult due to the
overlap of consensus phosphorylation sequences, redundancy of kinases
that can phosphorylate a particular site, differences between in
vivo and in vitro specificity, and the diversity of
kinases that can be present in a reaction, even in "pure" protein
preparations. Several different approaches to determine novel MAPK
substrates have been used (12). These include two-hybrid analysis (13,
14), proteomic approaches under conditions of ERK activation (11),
phosphorylation of column fractions from cells (15), and a solid phase
phosphorylation assay (16).
To identify additional novel substrates of ERK2, we employed a method
developed by Shokat and coworkers (17, 18) to specifically label direct
substrates of a particular protein kinase in a mixture of cellular
proteins. Protein kinases contain similar ATP binding domains,
including conserved residues that come into close contact with the
N-6 position of ATP. Mutation of the ATP binding site at one
or both of these conserved positions to a smaller amino acid residue
can allow the mutant protein kinase, but not other cellular protein
kinases, to utilize analogs of ATP that contain a bulky substituent on
the N-6 position. When radiolabeled ATP analog is used in a
kinase reaction with the mutant kinase and a mixture of cellular
proteins, only direct substrates of the mutant kinase become
radiolabeled. This method has been successfully used to identify direct
substrates for the Src tyrosine kinase (19) and for the MAPK JNK
(20).
We used an engineered mutant of ERK2 to search for new ERK substrates
that physically associate with the MAPK. As validation of our method,
our assay resulted in the labeling and identification of Rsk1, a known
ERK2 substrate (7). We also identified two novel ERK2 substrates, the
ubiquitin ligase EDD (E3 identified by
differential display) and the nucleoporin Tpr
(translocated promoter region). EDD
is the human homolog of the Drosophila gene hyperplastic discs and was originally identified as a gene
up-regulated in response to progestin (21). EDD is believed to serve as
an E3 ubiquitin ligase due to the presence of a HECT domain
(homology to E6-AP carboxyl
terminus) in its carboxyl terminus that binds ubiquitin
through a single cysteine residue (21). Honda et al. (22)
have identified the topoisomerase II beta binding protein TopBP1 as a
ubiquitination target of EDD. Tpr is a nucleoporin that is a
component of the nuclear basket of the nuclear pore complex (23, 24).
Functional studies suggest a role for Tpr in the nuclear export of
proteins with a nuclear export signal (25).
In this report we describe the engineering of ERK2 to allow it to
utilize an analog of ATP. Based on sequence alignment of ERK2 with
other protein kinases and the crystal structure of ERK2, we generated
alanine and glycine mutations of ERK2 at isoleucine 82 and glutamine
103, singly or in combination, to generate an ERK2 with a larger ATP
binding pocket. Mutation of these residues did not significantly
inhibit ERK2 kinase activity in vitro or signaling in
vivo. ERK2 mutants with a glutamine to glycine mutation at
position 103 were able to efficiently utilize several ATP analogs to
phosphorylate the ERK substrate Elk1 (26) in vitro. Using ERK2 Q103G and [ Cell Culture and Plasmids--
COS-1 cells were purchased from
American Type Culture Collection (ATCC, Manassas, VA). COS-1 cells were
grown in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad,
CA) and 10% fetal bovine serum (Invitrogen). FLAG-ERK2 has been
described previously (27). FLAG-ERK2 mutants were generated using the
Transformer mutagenesis kit (Clontech, Palo Alto,
CA). Transfections were performed using LipofectAMINE (Invitrogen)
according to the manufacturer's recommendations. A plasmid for
expression of amino-terminal FLAG-tagged EDD was constructed by cloning
full-length EDD from pBluescript EDD (21) into the pCMV-Tag2B vector
(Stratagene, La Jolla, CA).
Luciferase Assays--
COS-1 cells were transfected in duplicate
with 1 µg of 5× GAL4-luciferase; 50 ng of GAL4-Elk1 (28); 100 ng of
MEK1 S218D/S222D; and 5, 25, or 100 ng of ERK2 plasmid. DNA
amounts were brought up to 2 µg of total DNA with empty vector. The
cells were incubated in serum-free media overnight and harvested at
24 h. Luciferase activity was determined on a Monolight 2010 Luminometer (Analytical Luminescence, Ann Arbor, MI).
Analog Inhibition Assays and Elk1 Phosphorylation--
FLAG-ERK2
plasmids were transfected into COS-1 cells using LipofectAMINE
(Invitrogen). The cells were later serum-starved for 4 h before
stimulation with 10 ng/ml EGF (Upstate Biotechnology, Lake Placid, NY)
for 10 min. The cells were harvested in FLAG lysis buffer (27) and
FLAG-ERKs immunoprecipitated with M2 anti-FLAG-agarose (Sigma, St.
Louis, MO). The immunoprecipitates were washed three times in lysis
buffer and twice in kinase buffer (25 mM HEPES, pH 7.4, 10 mM magnesium acetate, and 1 mM dithiothreitol).
For analog inhibition assays, duplicate reactions containing 20 µg of
myelin basic protein (MBP), 10 µM ATP, 10 µCi/reaction
[ 32P-Labeling of Cyclopentyl ADP
(cpADP)--
Nucleoside diphosphate kinase (NDPK, 200 units, Sigma)
and 800 µCi of [ Immunoprecipitations, Kinase Assays, and Two-dimensional Gel
Electrophoresis--
100-mm dishes of COS-1 cells (3 × 106 cells) were transfected with 6 µg of FLAG-ERK2 or
FLAG-ERK2-QG and were allowed to recover overnight. Transfected cells
were then serum-starved for 4-5 h and stimulated with EGF (10 ng/ml)
for 10 min. Cells were washed once with cold phosphate-buffered saline
and then lysed with hypotonic lysis buffer at 4 °C (20 mM HEPES, pH 7.4, 2 mM EGTA, 2 mM
MgCl2, 200 mM sodium orthovanadate, 2 µg/ml
aprotinin, 0.4 mM microcystin, and 2 mM
phenylmethylsulfonyl fluoride). The lysate was clarified by
centrifugation at 13,000 rpm in a microcentrifuge for 20 min. ERK2 or ERK2-QG were immunoprecipitated with anti-FLAG-M2-agarose beads
(Sigma). Immunoprecipitates were washed three times with hypotonic
lysis buffer. Kinase assays were performed with
immunoprecipitated proteins in a 40-µl reaction volume containing 25 mM HEPES, pH 7.4, 20 mM magnesium acetate; 1 mM dithiothreitol, 1 mM ATP, or cpATP; and 10 µCi of [ Identification of Novel ERK2 Substrates--
Four 150-mm dishes
of COS-1 cells (6 × 106 cells/dish) transfected with
pCDNA3 or FLAG-ERK2-QG were used. Lysis and immunoprecipitation was
performed as described above. Immunoprecipitated ERK2 and associated
proteins were eluted from the M2-agarose beads with FLAG peptide and
analyzed on 8% SDS-PAGE. Labeled proteins were excised from the gel
and microsequenced using a Finnigan LCQ ion trap mass spectrometer with
a protana nanospray ion source (W. M. Keck Biomedical Mass
Spectrometry Laboratory, University of Virginia).
In Vitro Kinase Reaction and Phosphoamino Acid
Analysis--
COS-1 cells were transfected with either FLAG-ERK2,
FLAG-ERK2-QG, pCMV-Tag-EDD (FLAG-EDD), or pCMV-Tag2B. After overnight recovery, cells were serum-starved for 5 h. The cells were
stimulated with EGF (10 ng/ml) for 10 min and lysed in hypotonic lysis
buffer, and FLAG-ERKs were immunoprecipitated with anti-FLAG-M2-agarose beads. Immunoprecipitated proteins were washed three times with hypotonic lysis buffer. Immunoprecipitated FLAG-EDD or pCMV-Tag2B (control) was mixed with immunoprecipitated ERK2 or ERK2-QG, and kinase
reactions were carried out as described above with
[ Metabolic Labeling and Phosphopeptide Analysis--
100-mm
dishes of COS-1 cells (~3 × 106 cells) were
transfected with 7 µg of FLAG-EDD and 1 µg of HA-ERK2 and were
allowed to recover overnight. Cells were serum-starved for 1 h in
Dulbecco's modified Eagle's medium followed by phosphate-free RPMI
(Invitrogen) for 1 h. Cultures were then labeled in phosphate-free
RPMI containing 3 mCi/ml carrier-free 32P (PerkinElmer Life
Sciences) for 3 h followed by stimulation with EGF (10 ng/ml) for
10 min. Cultures treated with the MEK inhibitor UO126 were incubated
with 50 mM inhibitor during the 3-h labeling period and
during EGF stimulation. Cells were lysed in M2 lysis buffer (27)
followed by immunoprecipitation with anti FLAG-M2-agarose beads.
Immunoprecipitates were washed five times with M2 lysis buffer,
resolved on 8% SDS-PAGE gels, and transferred to nitrocellulose
membranes followed by autoradiography. Bands corresponding to the
labeled EDD were excised, and tryptic peptides were analyzed by
two-dimensional separation on thin-layer cellulose plates (30).
Based on the crystal structure of ERK2 bound to ATP (31) and
sequence alignment of the ATP binding site of several protein kinases
(18), we observed that both isoleucine 82 and glutamine 103 are at
conserved positions that come into close contact with the
N-6 position of adenine in ATP. We hypothesized that
mutation of one or both of these residues to either alanine or glycine would generate a larger ATP binding pocket enabling the mutant kinase
to accept and utilize ATP analogs with bulky side groups at the
N-6 position. We therefore generated five ERK2 mutants that
contained either single or double mutations of these residues to either
alanine or glycine. The five mutants generated were: ERK2 I82A; ERK2
Q103A; ERK2 Q103G; ERK2 I82A/Q103A; and ERK2 I82A/Q103G (Fig.
1A). All ERK2 constructs
encoded an amino-terminal FLAG tag.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]cyclopentyl-ATP, we were able to
radiolabel EDD, Tpr, and Rsk1 as ERK2-associated substrates in COS-1
cells. We further validated these results by demonstrating that EDD was
phosphorylated by ERK2 in vitro and in an
MEK-dependent manner in cells in response to EGF. This work
describes the generation of a molecular tool for the identification of
many more targets of this important signaling cascade.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (6000 Ci/mmol, PerkinElmer Life Sciences,
Boston, MA), and either buffer control or 100 µM ATP
analog. The reactions were incubated at 30 °C for 10 min, resolved
on a gel, and transferred to nitrocellulose. MBP bands were excised and
quantitated by Cerenkov counting. Each value was compared with the mean
of the control reactions (no analog), averaged with its duplicate, and
graphed as a percentage of the control, which was set to 100%. For
Elk1 phosphorylation, the immunoprecipitated ERKs were mixed with 1 µg of GST-Elk1 and either 100 µM ATP or 100 µM ATP analog. The reactions were incubated at 30 °C
for 10 min, resolved on a gel, and immunoblotted with anti-phospho-S383
Elk1 antibodies (New England BioLabs, Beverly, MA).
-32P]ATP were added in a 100-ml
reaction containing HBS (150 mM NaCl, 20 mM
HEPES, pH 7.4, and 5 mM MgCl2). The reaction
was allowed to equilibrate at 30 °C for 5 min.
32P-Labeled NDPK was purified from free
[
-32P]ATP by two successive rounds of Microspin G50
columns (Amersham Biosciences, Piscataway, NJ). 1000 pmol of cpADP was
added to the 32P-labeled NDPK in HBS, and the reaction was
carried out for 20 min at 30 °C. After the reaction, contents were
transferred to a Microcon-YM30 (Millipore, Bedford, MA) and centrifuged
at 13,000 rpm for 15 min. With this procedure we usually obtained
~200 µCi of purified [
-32P]cyclopentyl-ATP
([
-32P]cpATP).
-32P]ATP or [
-32P]cpATP for
15 min at 30 °C. Reactions were stopped by addition of an equal
volume of 2× SDS-sample buffer and followed by boiling for 5 min. 20 µl of these reactions was analyzed on a 10% SDS-PAGE gel. The
resolved proteins were visualized by the Vorum silver staining protocol
(29), and the gel was autoradiographed by exposing to Kodak Biomax MS
film (Eastman Kodak, Rochester, NY). Remaining samples were clarified
of M2-agarose beads by passing through wizard columns (Promega,
Madison, WI), and the proteins were precipitated with 5 volumes of cold
acetone. Precipitated proteins were resuspended in 260 ml of
two-dimensional sample buffer (7 M urea, 2 M
thiourea, 4% CHAPS, 2% dithiothreitol, and 1% IPG buffer (pH
4-7, Amersham Biosciences)) and resolved on Immobiline Dry
Strips (Amersham Biosciences, pH 4-7 linear (L), 13 cm) for 28,000 V-h. The proteins were resolved on 10% SDS-PAGE gels in the
second dimension. Silver staining and autoradiography were then
performed on the gels.
-32P]cpATP for 3 min. For time course kinase
reactions, immunoprecipitated proteins were eluted from the M2-agarose
beads with FLAG peptide prior to mixing them. Kinase reactions were
performed as mentioned above with [
-32P]cpATP. Samples
were resolved on 8% SDS-PAGE gels and transferred to polyvinylidene
difluoride membranes followed by autoradiography. Bands corresponding
to labeled EDD were excised, and phosphoamino acid analysis was
performed as described (30).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ERK2 ATP binding site mutants.
A, ERK2 ATP binding site mutants containing an
amino-terminal FLAG tag and mutations at isoleucine 82 and glutamine
103. B, COS-1 cells were transfected with ERK2 or an ERK2
mutant. The cells were serum-starved for 5 h before stimulation
with 10 ng/ml EGF for 10 min. FLAG-ERKs were immunoprecipitated and
tested for their kinase activity toward myelin basic protein
(MBP). C, ERK2 mutants can phosphorylate and
activate Elk1 in vivo. COS-1 cells were transfected with 5X
Gal4-luciferase, Gal4-Elk1, MEK1 S218D/S222D, and ERK2 or an ERK2
mutant. ERK2 plasmids were transfected at 5, 25, and 100 ng. The cells
were serum-starved after the transfection and harvested for luciferase
assays 24 h post transfection.
We first examined whether mutation of these residues affected the
ability of ERK2 to phosphorylate its substrates in vitro and
in vivo. ERK2 or an ERK2 mutant was transfected into COS-1 cells, and the cells were stimulated with EGF. In vitro
kinase assays were performed with immunoprecipitated ERK2 and the ERK2 mutants using myelin basic protein (MBP) and [-32P]ATP
as substrates. ERK2 and the mutants had roughly equivalent levels of
kinase activity (Fig. 1B), although alanine mutations at
isoleucine 82 somewhat inhibited ERK2 kinase activity toward MBP.
Similar experiments using serum stimulation or co-transfected mutationally activated MEK1 (MEK1 S218D/S222D), demonstrated that these
mutants could be phosphorylated and activated by activated MEK1 (data
not shown).
To determine if the ERK2 mutants could signal to a physiological ERK2 substrate in vivo, GAL4-Elk1 luciferase assays were performed. This system utilizes a plasmid containing a luciferase gene with five GAL4 binding sites in the promoter and a plasmid encoding a fusion of the DNA-binding domain of GAL4 and the ERK-responsive transactivation domain of Elk1 (28). MEK1 S218D/S222D was co-transfected to activate both endogenous and transfected ERK. Co-transfection of ERK2 or an ERK2 mutant stimulated luciferase expression in a dose-dependent manner (Fig. 1C). These data demonstrate that mutation of one or both residues in the ATP binding site of ERK2 did not substantially affect the localization, substrate recognition, or kinase activity of ERK2.
We next needed to identify the optimal pairing between an ATP binding
site mutant and an ATP analog. To do this, we screened each of our ERK
mutants with a panel of seven ATP analogs. We first determined the
ability of each analog to inhibit the capacity of ERK2 or an ERK2
mutant to phosphorylate MBP with [-32P]ATP. If an ATP
analog was able to compete with [
-32P]ATP for an ERK2
mutant, incorporation of radiolabeled phosphate into MBP would be
inhibited. ERK2 or an ERK2 mutant was immunoprecipitated from
transfected COS-1 cells stimulated for 10 min with EGF. The FLAG
immunoprecipitates were aliquoted equally and used in a kinase reaction
containing MBP, 10 µM ATP, and 10 µCi of
[
-32P]ATP, with or without 100 µM ATP
analog. The reactions were resolved on a gel and transferred, and
incorporation of 32P into MBP was quantified by Cerenkov
counting. Comparison of reactions containing an ATP analog (Fig.
2, lanes 2-8 in each panel)
to control reactions without analog (Fig. 2, lane 1 in each
panel) demonstrated that none of the analogs inhibited the ability of
wild-type ERK2 to utilize ATP. By contrast, many of the ATP analogs had
varied abilities to compete with [
-32P]ATP for
phosphate incorporation into MBP, suggesting that some of the ERK2
mutants were able to utilize or at least bind to the ATP analogs.
Mutation of isoleucine 82 alone had little effect on the susceptibility
of ERK2 to inhibition by an analog. Mutation of glutamine 103 to
alanine increased the susceptibility of ERK2 to inhibition by an
analog, whereas mutation of this residue to glycine had the greatest
effect on the ability of an analog to inhibit 32P
incorporation into MBP. The Q103G mutation (either by itself or in
conjunction with the I82A mutation) demonstrated a significant reduction of 32P incorporation into MBP when kinase
reactions were performed in the presence of analog. These results
suggested that residue 103 of ERK2 was the critical residue in
controlling the capacity of ERK2 to accept an ATP analog with a bulky
N-6 substituent. In particular, a substantial inhibition of
32P incorporation into MBP by ERK2 Q103G was observed with
addition of either N-6-benzyl-ATP (98.8% inhibition,
lane 2, middle panel, right side),
N-6-cyclopentyl-ATP (95.2% inhibition, lane 3,
middle panel, right side),
N-6-(3,3-dimethyl)butyl-ATP (98.4% inhibition, lane
6, middle panel, right side),
N-6-(2-phenethyl)-ATP (97.8% inhibition, lane 7,
middle panel, right side), and
N-6-(1-methyl)butyl-ATP (94.1% inhibition, lane
8, middle panel, right side).
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There are two likely explanations for the inhibition of 32P
incorporation into MBP by the addition of excess unlabeled ATP analog. First, the ATP analog could be utilized as an ATP source by the mutant
ERK2 kinase to phosphorylate MBP, thus effectively competing with the
[-32P]ATP for usage by the mutant ERK2. Alternatively,
the kinase could bind the analog but not be able to utilize it, thus
effectively blocking [
-32P]ATP from the ATP binding
site of the kinase. To differentiate between these two possibilities,
we screened ERK2, ERK2-Q103G, and ERK2-I82A/Q103G for their ability to
phosphorylate GST-Elk1 with either ATP or an ATP analog. The reactions
were resolved on a gel and immunoblotted with an antibody that
specifically recognizes phospho-S383 Elk1, the major site of
phosphorylation by ERK2. Incubation of GST-Elk1 in the absence of ERK2
did not result in S383 phosphorylation (lane 1). All three
ERK2 proteins were able to phosphorylate GST-Elk1 with normal ATP (Fig.
3, lane 2 in all p-Elk1
panels). However, wild-type ERK2 could not use any of the ATP
analogs to phosphorylate GST-Elk1 (Fig. 3, top panel,
lanes 3-9). These data support the previous result that none of the analogs could inhibit wild-type ERK2 from using
[
-32P]ATP to phosphorylate MBP.
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Conversely, both ERK2-Q103G and ERK2-I82A/Q103G were able to effectively use several ATP analogs to phosphorylate GST-Elk1. Overall, ERK2-Q103G appeared to use the analogs more effectively than ERK2-I82A/Q103G, correlating with the inhibition assay in Fig. 2. ERK2-Q103G effectively used all of the ATP analogs tested with the exception of N-6-(p-methyl)benzyl-ATP. Although this analog appeared somewhat effective in the inhibition assay, it was the least efficient of the seven and was most likely an example of an analog that simply blocks the ERK2 ATP binding site. Based on the inhibition assay and the Elk1 phosphorylation assay, we decided to use ERK2-Q103G (henceforth designated ERK2-QG) along with cyclopentyl ATP (henceforth designated cpATP) (Fig. 3, middle panel, lane 4) in our attempts to identify novel ERK2 substrates.
Detection of Direct Substrates of ERK2 with
[-32P]cpATP--
To identify relevant substrates of
ERK2, one would like to introduce labeled ATP analog into the live
cells. However, cells are impermeable to ATP, and addition of labeled
ATP analog to digitonin-permeabilized cells results in hydrolysis of
the analog ATP within 1 min (32). Furthermore, the intracellular
concentration of ATP is 3 mM (33), making it difficult to
achieve analog concentrations sufficient to compete with ATP for
kinases. Therefore, it was necessary to apply this technique to cell
lysates or fractions. We chose to express the ERK2 and ERK2-QG (mutant
ERK2) in COS-1 cells and then immunoprecipitate ERKs under
non-stringent conditions where we would expect many ERK-binding
proteins to remain associated. The immunoprecipitates were then used in
a kinase reaction with either [
-32P]ATP or
[
-32P]cpATP. In addition to ERK2 and ERK2-QG, other
co-immunoprecipitated kinases would be capable of utilizing
[
-32P]ATP to phosphorylate co-immunoprecipitated
substrates. However, reactions with ERK2-QG and
[
-32P]cpATP should result in the phosphorylation of
only those proteins that are direct ERK substrates. Therefore, we
should detect fewer radiolabeled substrates in the reactions with
[
-32P]cpATP as compared with reactions with
[
-32P]ATP.
ERK2 or ERK2-QG from unstimulated or EGF-stimulated cells were
immunoprecipitated, and associated proteins were labeled in an in
vitro kinase reaction using either [-32P]ATP or
[
-32P]cpATP. The reactions were resolved on SDS-PAGE,
silver-stained, and subjected to autoradiography (Fig.
4A). ERK2 and ERK2-QG proteins were expressed at similar levels and were activated efficiently after
EGF stimulation (Fig. 4A, silver stain). Both ERK2 and
ERK2-QG used [
-32P]ATP very efficiently, generating
similar patterns of phosphorylated substrates on SDS-PAGE (Fig.
4A, autoradiogram). Phosphorylation of substrates in the
absence of EGF stimulation was most likely due to the basal activity of
ERK2. There were noticeable differences in the substrates recognized by
ERK2 prior to and after EGF stimulation (Fig. 4A,
autoradiogram; compare lane 1 with 2, lane
3 with 4, and lane 7 with 8).
These differences are probably due to (i) differences in the
association of substrates with ERK2 depending on the phosphorylation
status of ERK2; (ii) dissociation of phosphorylated substrates from
ERK2 after EGF stimulation in vivo; or (iii) prior phosphorylation of the substrate in vivo during the 10-min
EGF stimulation.
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When the kinase reactions were performed with
[-32P]cpATP, there was a marked difference in the
pattern of labeled substrates between reactions with ERK2 and ERK2-QG.
Neither wild-type ERK2 nor its associated proteins could efficiently
utilize [
-32P]cpATP. The background phosphorylation
with wild-type ERK2 that was detected with [
-32P]cpATP
was most likely due to trace contamination with
[
-32P]ATP (~0.1%, data not shown) that occurred
when generating [
-32P]cpATP. At this level of
electrophoretic resolution, few differences were detected in the
substrates recognized by ERK2-QG in reactions with
[
-32P]ATP versus
[
-32P]cpATP (Fig. 4A, autoradiogram;
compare lanes 3 and 4 with lanes 7 and
8). However, when the resolution of the gel systems was increased by using two-dimensional gels (Fig. 4, B and
C), the advantages of the analog approach became evident.
ERK2 in vitro kinase reactions with
[-32P]ATP or [
-32P]cpATP resolved by
two-dimensional gel electrophoresis are shown in Fig. 4B.
These results further demonstrated that ERK2 and its associated proteins could not utilize [
-32P]cpATP. In
vitro kinase reactions using ERK2-QG were also analyzed on
two-dimensional gels (Fig. 4C). Although the phosphorylation patterns between the [
-32P]ATP and the
[
-32P]cpATP reactions were somewhat similar, we
detected fewer substrates with [
-32P]cpATP. The use of
[
-32P]cpATP decreased the overall level of
phospho-proteins while enhancing ERK2-QG-specific phosphorylations.
These results clearly demonstrate the advantage of using
[
-32P]cpATP for specific detection of the direct
substrates of ERK2.
Identification of Novel ERK2 Substrates--
After optimizing the
conditions for the specific detection of direct substrates of ERK2-QG
with [-32P]cpATP, we scaled up the procedure to detect
co-immunoprecipitating substrates by mass spectrometry. Proteins
co-immunoprecipitating with ERK2-QG were eluted along with ERK2 from M2
beads with FLAG peptide. Kinase reactions were then performed with
[
-32P]cpATP, and the reactions were resolved on an 8%
SDS-PAGE (Fig. 5A). Two
radiolabeled silver-stained bands, one at ~80 kDa and the other at
~250 kDa, were excised from the lane with a non-EGF-stimulated (serum-free) sample (Fig. 5A, lane 2) and were
sequenced by mass spectrometry. Eleven peptides (16.3% coverage)
corresponding to the ERK2 substrate Rsk1 (34) were detected in the
80-kDa band. The isolation of a known ERK2 substrate validated our
methodology for detecting ERK2 substrates. Mass spectrometry analysis
of the 250-kDa band revealed 14 peptides (7% total coverage)
corresponding to the E3 ubiquitin ligase EDD (E3 identified
by differential display). EDD is the human
homolog of the Drosophila gene hyperplastic discs
and was originally identified as a gene up-regulated in response to
progestin (21). EDD is a 300-kDa protein and contains a region that has
extensive homology to HECT domain ubiquitin ligases (21).
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In a similar experiment we detected a radiolabeled substrate (~250 kDa) that specifically associated with activated ERK2-QG (Fig. 5B). Mass spectrometric analysis detected 23 peptides (12.4% protein coverage) corresponding to the nucleoporin Tpr (translocated promoter region) (35, 24). Tpr is a nucleoporin that is localized within the nuclear pore complex and potentially has a role in nuclear protein export (25).
Identical samples were also analyzed on pH 3-10 linear (L) two-dimensional gels and silver-stained. Most of the radiolabeled protein spots that were obtained in kinase reactions were below the detection level, and hence we could not obtain any sequence data (data not shown). In addition to phosphorylated proteins, spots corresponding to proteins that were specifically associated with ERK2, but not phosphorylated, were cored and sequenced by mass spectrometry. The substrates and ERK2-associated proteins identified are shown in Table I. Although in vitro phosphorylation of some of the associated proteins was not detected, they are still potential substrates in cells. To further validate our method of labeling ERK2-associated substrates, we attempted to confirm our results with one of the novel substrates we discovered.
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EDD Associates Specifically with ERK2 and ERK1 but Not
MEK1--
We chose to reconfirm our results with the novel
substrate EDD. For this, ERK2 and ERK2-QG were immunoprecipitated from
transiently transfected COS-1 cells, resolved on SDS-PAGE, transferred
to nitrocellulose membrane, and probed with anti-FLAG and anti-EDD antibodies. Similar to our results in Fig. 5A, EDD
associated more strongly with ERK2 and ERK2-QG in unstimulated
versus EGF-stimulated cells (Fig.
6A, lane 3 versus 4 and lane 5 versus
6). To ensure that the interaction of EDD with ERK2 was
specific, we immunoprecipitated ERK2, ERK2-QG, ERK1, and MEK1 from
transiently transfected COS-1 cells and probed the blots with anti-FLAG
and anti-EDD antibodies. ERK2, ERK2-QG, and ERK1 interacted with
endogenous EDD (Fig. 6B); however, we could not detect any
interaction between MEK1 and EDD. These results suggest that ERK2 and
ERK1, but not MEK1, specifically associate with EDD.
|
ERK2-QG Phosphorylates EDD in Vitro--
To determine if ERK2-QG
would phosphorylate EDD in vitro, FLAG-ERK2, FLAG-ERK2-QG,
FLAG-EDD, and the vector control were immunoprecipitated from
transiently transfected COS-1 cells. Immunoprecipitated ERK2 or ERK2-QG
was mixed with immunoprecipitated EDD or vector control and in
vitro kinase reactions were carried out with
[-32P]cpATP. Because ERK2 does not utilize
[
-32P]cpATP, we did not detect any phosphorylation
with vector control or EDD (Fig.
7A; lanes 1 and
2). ERK2-QG was able to efficiently phosphorylate EDD
in vitro using [
-32P]cpATP (Fig.
7A, compare lane 4 versus
3). We then performed a time course of the in
vitro kinase reaction with ERK2-QG and EDD. Labeling of EDD in the
in vitro kinase reaction reached its peak by 15 min (Fig.
7B, lane 4) with a slight decrease at 30 min
(Fig. 7B, lane 5). EDD has a total of ten SP and
ten TP sites present in 2799 amino acids, and any of these sites can be
potentially phosphorylated by ERK2. To narrow down the target sites,
phosphoamino acid analysis of in vitro phosphorylated
EDD was performed. Our results indicated that the bulk of the
phosphorylation was on serine (Fig. 7C), although we did
observe very faint phosphorylation of threonine.
|
ERK2 Phosphorylates EDD in Vivo--
It was apparent that ERK2
phosphorylated EDD in vitro at one or more serine residues.
However, it was important to know if ERK2 phosphorylated EDD in
vivo. COS-1 cells transfected with FLAG-EDD and HA-ERK2 were
metabolically labeled with or without EGF stimulation. In addition,
these labeling reactions were carried out with or without pretreatment
of the cells with the MEK1 inhibitor UO126. Two-dimensional tryptic
phosphopeptide maps were analyzed to determine if there were any
differences in the pattern of tryptic peptides with EGF treatment as
compared with non-stimulated cells and if these changes were reversible
with UO126 pretreatment. Tryptic peptide maps obtained under various
conditions are shown in Fig.
8A. These data demonstrate
that EDD was phosphorylated on many sites in unstimulated COS-1 cells
(serum-free, Fig. 8A). We detected the appearance of a new
tryptic phospho-peptide with EGF stimulation (Fig. 8A, shown
by an arrow). Pretreatment of cells with the MEK1 inhibitor
UO126 abolished the emergence of this tryptic phospho-peptide,
indicating that the phosphorylation was either by ERK2 or a downstream
kinase. To determine which of these peptides were due to direct
phosphorylation by ERK2, we compared the tryptic peptide maps of
in vivo and in vitro labeled EDD (Fig.
8B). Our results show that only a few tryptic peptides were
present in both in vivo and in vitro tryptic
peptide maps. These were most likely phosphorylated directly by ERK2,
whereas tryptic peptides that were present only in labeled EDD in
vivo were probably a result of phosphorylation by other cellular
kinases. Interestingly, the tryptic phospho-peptide that appeared after EGF stimulation was also detected with in vitro labeling
(Fig. 8C, spot 1). Because these tryptic peptides
were analyzed on different TLC plates, to be absolutely sure that the
in vitro and in vivo labeled tryptic peptides,
which are migrating at the same positions, were in fact the same
peptides, we eluted them from the plates and re-analyzed them on the
same TLC plate. Peptides 1, 2, and 3 (Fig. 8B) were eluted
from in vivo and in vitro labeled EDD peptide
maps and were spotted on the same TLC plate individually and in
concert. Results obtained for the three different peptides are shown in
Fig. 8C. It is evident from our results that peptides eluted
from in vivo and in vitro labeled EDD migrate at
the same position on the TLC plate, indicating that these peptides were identical. These results demonstrate that tryptic peptides 1, 2, and 3 are directly phosphorylated by ERK2, not a kinase downstream of ERK2 or
any other cellular kinase. Phosphoamino acid analysis of in
vitro labeled EDD demonstrated that the bulk of phosphorylation was on serine. The tryptic phosphopeptide, which appears following EGF
stimulation (Fig. 8A, indicated by arrow, and
Fig. 8B, peptide 1) also was phosphorylated on
serine (Fig. 8D), which is in agreement with the
phosphoamino acid analysis of in vitro labeled EDD
(Fig. 7C).
|
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DISCUSSION |
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To identify novel ERK2 substrates, we adopted the approach developed by Shokat and coworkers (18) that involves mutating the ATP binding site of a kinase of interest in a way that allows it to utilize modified analogs of ATP. Based on this strategy, we generated mutations in ERK2 at positions 82 and 103 in the ATP binding site. The mutations did not significantly inhibit ERK activation in response to growth factors or mutationally activated MEK. In addition, ERK2 substrate recognition and signaling were not noticeably affected by the mutations in vitro or in vivo. Our results demonstrate that glycine mutations at glutamine 103 were very effective at generating a larger ATP binding pocket, enabling this mutant, but not wild-type ERK2, to utilize several ATP analogs to phosphorylate Elk1.
To identify biologically relevant substrates, one would ideally
introduce labeled ATP analogs into live or permeabilized cells. However, because cells are impermeable to ATP analogs and labeled ATP
analogs are quickly hydrolyzed upon addition to digitonin-permeabilized cells (32) and because of the formidably high intracellular concentrations of ATP (33), the analog approach requires the use of
lysed or fractionated cells. Heterogeneous nuclear
ribonucleoprotein K was identified as a substrate of JNK using
the ATP analog method in a cell lysate after the addition of
recombinant modified JNK and a [-32P]ATP analog (20).
Addition of large quantities of a kinase to a cell lysate could result
in nonspecific or aberrant phosphorylation of substrates. Therefore, we
chose to target only those substrates that were directly associated
with ERK2. In our work, we expressed the mutant and wild-type ERK2
in vivo and then immunoprecipitated ERK2 with associated
proteins, with the rationale that this subset of the proteome
would more likely contain biologically relevant ERK2 substrates. These
experiments allowed us to identify ERK substrates in both unstimulated
and mitogen-stimulated cells. In vitro kinase reactions with
wild-type ERK2 and ERK2-QG immunoprecipitates from serum-deprived and
EGF-stimulated cells resulted in similar phosphorylation patterns when
[
-32P]ATP was used, demonstrating that the proteins
had similar binding partners and substrate specificity. The use of
[
-32P]cpATP with wild-type ERK2 resulted in minimal
incorporation of 32P into proteins, demonstrating that
wild-type ERK2 and its associated proteins could not utilize
[
-32P]cpATP. However, the use of
[
-32P]cpATP with ERK2-QG resulted in robust
phosphorylation of associated proteins. Interestingly, the labeling
pattern obtained from ERK2-QG revealed some spots that co-migrated with
spots when [
-32P]ATP was used, as well as the
disappearance of some spots and the appearance of others. The
disappearance of some labeled spots suggests that these
phosphorylations were carried out by associated kinases in the presence
of [
-32P]ATP. The appearance of new putative
substrates in the presence of [
-32P]cpATP demonstrates
that by using an ATP analog that can only be utilized by ERK2-QG, there
was an enhancement of the "signal to noise" ratio, presumably
because the ERK2-QG no longer competes with co-immunoprecipitating
kinases for ATP. The increased intensity of these minor spots strongly
supports the advantage of using the ATP analog system to identify substrates.
Comparison of the labeling pattern of substrates by ERK2-QG in the unstimulated and stimulated states revealed differences in labeling of associated proteins. Although some proteins were labeled under both conditions, we often observed an enhanced labeling of substrates by ERK2 immunoprecipitated from unstimulated cells. Although at first counterintuitive, these results could be due either to an EGF-stimulated decrease in association of the ERK and the substrate (e.g. note the decreased association with Rsk1 in Fig. 5A, left panel) or an increased intracellular phosphorylation of the substrate after EGF stimulation.
Several ERK binding partners in unstimulated cells, including MEK1 and Rsk1, are also ERK substrates. The main ERK2 phosphorylation site on MEK1, Thr-292, is constitutively phosphorylated in cells, and phosphorylation is not stimulated by growth factor treatment (36). Moreover, ERK leaves its MEK anchor following growth factor stimulation. For both of these reasons, we are unlikely to detect a significant phosphorylation of MEK1 in our assay. However, the most prominent phosphorylated protein band from unstimulated cells was a ~90-kDa protein that was identified as Rsk1 (34). This served to validate the assay, demonstrating that the assay allowed identification of a known ERK substrate.
The nucleoporin Tpr was identified as an ERK2 substrate in EGF-stimulated cells, but not in unstimulated cells. Tpr localizes to the nuclear pore complex. Although the function of Tpr in this complex is unclear, it may play a role in the nuclear export of proteins (25). ERK proteins are known to dimerize (6) and translocate to the nucleus (7, 8) by transport in an active, Ran-dependent manner, as well as by passive diffusion (37), although the exact mechanisms remain unknown. The interaction with and phosphorylation of Tpr by ERK following growth factor stimulation raises intriguing possibilities for both Tpr function and ERK localization. ERK phosphorylation of Tpr may play a role in Tpr-regulated transport of proteins out of the nucleus. Alternatively, ERK association with Tpr may regulate the nuclear import or export of ERK. The association of ERK2 with Tpr after EGF stimulation, but not in unstimulated cells, suggests that ERK may interact with Tpr as ERK traverses the nuclear pore.
Although the ubiquitin ligase EDD was found associated with ERK2 under both conditions, it was primarily labeled in kinase reactions from unstimulated cells. EDD was exclusively found in this phosphorylated band. The absence of phospho-labeled EDD in reactions from EGF-stimulated cells suggests that this protein was rapidly phosphorylated by ERK2 in the cells during the 10 min of EGF stimulation. Indeed, in vivo orthophosphate labeling experiments revealed the appearance of a MEK-dependent phospho-peptide in EDD 10 min after EGF stimulation. In vitro phosphorylation of EDD with ERK2-QG generated several phospho-peptides that co-migrated with those from the in vivo labeling. In particular, in vitro phosphorylation of EDD generated a phospho-peptide that co-migrated with the MEK-dependent in vivo labeled peptide. These in vivo data confirm the conclusion that EDD is a substrate for ERK2.
Both the presence of a HECT domain in the carboxyl terminus of EDD and the ability of a cysteine residue in this region to bind ubiquitin suggest that EDD acts as a ubiquitin ligase and ERK2 phosphorylation of EDD may affect this activity of EDD. Another possibility is that EDD affects the ubiquitination of ERK2. ERK2 was recently shown to be ubiquitinated and degraded in response to osmotic shock induced by sorbitol treatment (38). Although the ubiquitin ligase that induces ERK ubiquitination nation is unidentified, the association of EDD with ERK2 in the basal state may provide a mechanism by which ubiquitin-mediated degradation of ERK occurs.
In conclusion, we have created a mutant ERK2 kinase that can utilize an
analog of ATP to specifically label ERK2 substrates. These results not
only identify two novel ERK substrates, the nucleoporin Tpr and the
ubiquitin ligase EDD, but also provide a framework by which many other
ERK substrates can be identified.
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ACKNOWLEDGEMENTS |
---|
We thank members of the Parsons-Weber- Parsons group for helpful discussions, and Nicholas E. Sherman in the W. M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia for peptide sequencing. We thank Tomas Vomastek for the FLAG-MEK1 construct.
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FOOTNOTES |
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* This work was supported in part by Grants CA39076 and CA40042 (to M. J. W.) 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.
§ Both authors contributed equally to this work.
** Supported by the United States Army Medical Research and Material Command Breast Cancer Research Program Grant DAMD17-00-1-253 and the Association for International Cancer Research.
To whom correspondence should be addressed. Tel.:
434-924-5052; Fax: 434-982-0689; E-mail: mjw@virginia.edu.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M300485200
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ABBREVIATIONS |
---|
The abbreviations used are: MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; JNK, c-Jun amino-terminal kinase; EDD, E3 identified by differential display; Tpr, translocated promoter region; HECT, homology to E6-AP carboxyl terminus; E3, ubiquitin-protein isopeptide ligase; cp, cyclopentyl; EGF, epidermal growth factor; CMV, cytomegalovirus; MBP, myelin basic protein; NDPK, nucleoside diphosphate kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, glutathione S-transferase.
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