Molecular Biology of the Cell
Vol. 16, 3552–3561, August 2005
Phosphorylation of Phosphoprotein Enriched in Astrocytes
(PEA-15) Regulates Extracellular Signal-regulated
Kinase-dependent Transcription and Cell Proliferation
Joseph Krueger,* Fan-Li Chou,* Angela Glading,* Erik Schaefer,†and
Mark H. Ginsberg*
*University of California–San Diego, La Jolla, CA 92093-0726; and†BioSource International, Hopkinton, MA
Submitted November 17, 2004; Revised April 20, 2005; Accepted May 17, 2005
Monitoring Editor: Martin A. Schwartz
Cell cycle progression is dependent on the nuclear localization and transcriptional effects of activated extracellular
signal-regulated kinase (ERK)1 and ERK2 mitogen-activated protein (MAP) kinases (ERK1/2). Phosphoprotein enriched
in astrocytes (PEA-15) binds ERK1/2 and inhibits their nuclear localization, thus blocking cell proliferation. Here, we
report that phosphorylation of PEA-15 blocks its interaction with ERK1/2 in vitro and in vivo and that phosphorylation
of both Ser104and Ser116is required for this effect. Using phosphomimetic and nonphosphorylatable mutants of PEA-15,
we found that PEA-15 phosphorylation abrogates its capacity to block the nuclear localization and transcriptional
activities of ERK1/2; this phosphorylation therefore enables the proliferation of cells that express high levels of PEA-15.
Additionally, we report that PEA-15 phosphorylation can modulate nontranscriptional activities of ERK1/2, such as the
modulation of the affinity of integrin adhesion receptors. Finally, we used a novel anti-phospho-specific PEA-15 antibody
to establish that PEA-15 is phosphorylated in situ in normal mammary epithelium. These results define a novel
posttranslational mechanism for controlling the subcellular localization of ERK1/2 and for specifying the output of MAP
The mitogen-activated protein (MAP) kinases extracellular
signal-regulated kinase (ERK)1 and ERK2 (hereafter referred
to as ERK1/2), play a critical role in cell cycle progression by
phosphorylating transcription factors such as Elk-1 (Gille et
al., 1995). To phosphorylate these transcription factors,
ERK1/2 must enter the nucleus. The nuclear import and
export of ERK1/2 are regulated by a variety of protein–
protein interactions that can therefore serve as important
control points in cell proliferation. Indeed, several proteins
have been identified that can bind to ERK1/2 and prevent its
nuclear accumulation, including ?-arrestin, calponin, mito-
gen-activated protein kinase phosphatase-3, dominant neg-
ative mitogen-activated protein kinase kinase (MEK), and
phosphoprotein enriched in astrocytes (PEA-15) (Menice et
al., 1997; Camps et al., 1998; Dang et al., 1998; Formstecher et
al., 2001; Robinson et al., 2002; Tohgo et al., 2002; Whitehurst
et al., 2004). Among these, PEA-15, a small death effector
domain (DED)-containing protein, sequesters ERK in the
cytoplasm, thereby inhibiting cell proliferation in a variety
of cell types and contributing to cellular senescence (Form-
stecher et al., 2001; Gaumont-Leclerc et al., 2004). Impor-
tantly, PEA-15 binding does not interfere with ERK1/2 ac-
tivation in vitro or in vivo nor does it inhibit ERK1/2’s
capacity to phosphorylate substrates, including nuclear sub-
strate in vitro (Formstecher et al., 2001; Gaumont-Leclerc et
al., 2004). PEA-15 alters ERK signaling by retaining ERK1/2
in the cytoplasm by blocking nuclear import and by promot-
ing nuclear export (Formstecher et al., 2001; Whitehurst et al.,
2004). Thus, PEA-15 is a protein that controls cell prolifera-
tion by preventing ERK1/2 accumulation in the nucleus.
Numerous biological functions have been ascribed to
PEA-15 since its discovery in astrocytes (Araujo et al., 1993).
Its amino acid sequence is completely conserved in human,
mouse, rat, and hamster and is widely expressed in tissues,
including brain, breast, lung, and prostate (Araujo et al.,
1993; Danziger et al., 1995; Estelles et al., 1996). PEA-15
expression is increased in type II diabetes and its overex-
pression in fibroblasts or transgenic mice inhibits glucose
transport (Condorelli et al., 1998). Furthermore, the presence
of a DED in PEA-15 suggests a regulatory role in apoptosis;
in some systems, it can protect cells from receptor-mediated
apoptosis (i.e., tumor necrosis factor-related apoptosis-in-
ducing ligand, Fas, and tumor necrosis factor [TNF]-?) (Con-
dorelli et al., 1999; Estelles et al., 1999; Kitsberg et al., 1999;
Zvalova et al., 2001; Renault et al., 2003; Sharif et al., 2003).
PEA-15 also binds to and regulates the expression of phospho-
lipase D1 and the activity of p90 ribosomal S6 kinase 2 (Zhang
et al., 2000; Vaidyanathan and Ramos, 2003). PEA-15 has been
ascribed a role in a variety of diseases, including squamous cell
carcinoma, glioma, breast cancer, astrogliosis, and diabetes
(Bera et al., 1994; Hwang et al., 1997; Condorelli et al., 1998;
This article was published online ahead of print in MBC in Press
on May 25, 2005.
Address correspondence to: Mark H. Ginsberg (mhginsberg@
Abbreviations used: DED, death effector domain; DISC, death in-
ducing signaling complex; ERK1/2, extracellular signal-regulated
kinase 1 and 2; MAP, mitogen-activated protein kinase; PEA-15,
phosphoprotein enriched in astrocytes.
3552© 2005 by The American Society for Cell Biology
Glienke et al., 2000; Tsukamoto et al., 2000; Dong et al., 2001;
Embury et al., 2001; Underhill et al., 2001; Sharif et al., 2004).
Thus, PEA-15 is a multifunctional protein with roles in multi-
ple physiological and pathological processes.
As noted above, there is compelling evidence that PEA-15
expression leads to inhibition of cell proliferation by binding
to ERK1/2 to prevent their transcriptional activities (Form-
stecher et al., 2001; Gaumont-Leclerc et al., 2004). Yet, PEA-15
is expressed in certain tumor cells lines that proliferate rap-
idly and cultured astrocytes continue to proliferate while
expressing the protein at high levels (Araujo et al., 1993;
Estelles et al., 1996). Furthermore, PEA-15 binds to the ki-
nase-insert domain of ERK2; this interaction blocks the abil-
ity of MEK1 to activate ERK2 (Whitehurst et al., 2004). Par-
adoxically, over expression of PEA-15 leads to MEK1- and
MEK2-dependent increases in ERK1/2 activation (Ramos et
al., 2000). The capacity of PEA-15 to stimulate ERK1/2 acti-
vation and its presence in rapidly dividing cells suggest that
the interaction of PEA-15 with ERK1/2 may be subject to
regulation by posttranslational modifications of one of the
The structure of PEA-15 suggests that phosphorylation
could regulate its binding to ERK1/2. In cultured astrocytes,
PEA-15 is phosphorylated on two Ser residues, Ser104and
Ser116. Protein kinase C (PKC) phosphorylates Ser104and
calcium/calmodulin kinase (CamKII) or Akt phosphorylate
Ser116(Araujo et al., 1993; Danziger et al., 1995; Kubes et al.,
1998). PEA-15 is composed of the N-terminal DED and a C
terminus that forms a less structured “tail” (Hill et al., 2002).
The C-terminal tail contains both phosphorylation sites in
proximity to residues required for ERK1/2 binding (Ramos
et al., 1998, 2000; Formstecher et al., 2001; Hill et al., 2002;
Chou et al., 2003). Furthermore, phosphorylation of Ser116
regulates the antiapoptotic function of PEA-15 and modu-
lates its targeting to the death inducing signaling complex
(DISC) (Condorelli et al., 1999; Trencia et al., 2003). In this
study, we analyzed the effect of phosphorylation on ERK1/2
binding and on cell proliferation. Here, we report that phos-
phorylation of PEA-15 blocks its interaction with ERK1/2 in
vitro and in vivo and that phosphorylation of both Ser104
and Ser116is required for this effect. Using phosphomimetic
and nonphosphorylatable mutants of PEA-15, we found that
PEA-15 phosphorylation abrogates its capacity to block the
nuclear localization and transcriptional activities of ERK1/2,
thus enabling the proliferation of cells that express high
levels of PEA-15. We also report that PEA-15 phosphoryla-
tion can modulate nontranscriptional activities of ERK1/2,
such as the regulation of the affinity of integrin adhesion
receptors (Chou et al., 2003). Finally, we used a phospho-
specific anti-PEA-15 antibody to establish that PEA-15 is
phosphorylated in tumor cells and in situ in normal tissue.
Thus, these studies define a novel posttranslational mecha-
nism for controlling the subcellular localization of ERK1/2
and for specifying the biological consequences of the MAP
kinase signaling cascade.
MATERIALS AND METHODS
Chinese hamster ovary (CHO) cells, glioma CRL-1620 cells (glioma 1620),
NIH 3T3, and MB-MDA-231 breast cancer cells (MDA-231) were obtained
from American Type Culture Collection (Manassas, VA). All cells were cul-
tured in DMEM with 10% fetal bovine serum (FBS), 1% penicillin/strepto-
mycin, 1% l-glutamine, and 1% nonessential amino acids (all from Invitrogen,
Antibodies and Immunohistochemistry
Rabbit polyclonal anti-PEA-15 (3099) was raised against a synthetic peptide
containing the C-terminal 14 amino acids (EEEIIKLAPPPKKA) of PEA-15 as
described previously (Ramos et al., 2000). Anti-PEA-15 (4513) was raised
against a glutathione S-transferase-PEA-15 fusion protein (GST-PEA-15). An-
ti-PEA-15 (4513) was absorbed with GST-agarose, adjusted to pH 8.0 in 100
mM Tris, and bound to immobilized GST-PEA-15. Affinity-purified anti-
PEA-15 (4513) was eluted using 100 mM glycine (pH 3.0) and neutralized with
1 M Tris (pH 8.0). Anti-PEA-15 phospho-S116 (p-PEA-15), which recognizes
PEA-15 when it is phosphorylated at Ser116, was produced at BioSource
International (Camarillo, CA). Antiserum was generated using a chemically
synthesized phosphorylated peptide (IRQP[pS]EEEIIKL) coupled to keyhole
limpet hemocyanin and injected into specific pathogen-free rabbits. The re-
sulting phosphorylation site-specific antibody was purified using both nega-
tive and positive peptide affinity purification. The rabbit antibody against
lamins A/C was a gift from Dr. Larry Gerace (The Scripps Research Institute,
La Jolla, CA). Antibodies against Influenza hemagglutinin (HA) tag and Rho
GDI were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-
FLAG antibody was obtained from Sigma-Aldrich (St. Louis, MO). Unless
otherwise indicated, all antibodies were used at a 1:1000 dilution. Anti-rabbit
and anti-mouse horseradish peroxidase (HRP)-conjugated antibodies (Bio-
Source International) and SuperSignal Pico Chemiluminescent Substrate
(Pierce Chemical, Rockford, IL) were used for Western blot detection.
Immunohistochemistry was performed on Formalin-fixed/paraffin-embed-
ded breast tissue sections by immunoperoxidase staining. Paraffin-embedded
sections were deparaffinized and then microwaved for 10 min in citrate buffer
(10 mM sodium citrate, 0.05% Tween 20, pH 6.0). After cooling, endogenous
peroxidase was blocked with 0.3% H2O2in phosphate-buffered saline (PBS)
(1.7 mM KH2PO4, 5.2 mM Na2HPO4150 mM NaCl, pH 7.4) for 10 min.
Further blocking was done in 1% bovine serum albumin (BSA) (Sigma-
Aldrich) in PBS for 20 min. Primary antibodies were incubated on the slides
overnight at 4°C. Affinity purified anti-PEA-15 (4513) was used at 0.1 ?g/?l,
and anti-p-PEA-15 was used at a 1:25 dilution. Equivalent amounts of control
rabbit IgG and 1% BSA were used as a control. After washing in PBS, goat
anti-rabbit HRP secondary antibodies (1:500) were added to the slides for 30
min at room temperature. Staining was then developed with aminoethylcar-
bazole chromagen. Slides were counterstained with Mayer’s hematoxylin.
Dilutions and staining conditions were validated on fixed MB-MDA-231 and
CRL 1620 cell lines before performing immunohistochemistry on breast tis-
PEA-15 cDNA expression constructs used in this work have been described
previously (Chou et al., 2003). PEA-15 cDNA was expressed from two vectors,
pCDNA3.1(?) (Invitrogen) for eukaryotic expression and pGEX2T (Amer-
sham Biosciences UK, Little Chalfont, Buckinghamshire, United Kingdom) for
in vitro protein production from bacterial cells. The PEA-15 mutant L123R
was initially described in Hill et al. (2002). All PEA-15 constructs in pCDNA3
included a C-terminal HA tag. Additional PEA-15 mutants S104D, S116D,
S104A, and S116A, were generated with the QuikChange site-directed mu-
tagenesis kit (Stratagene, La Jolla, CA) by using wild-type pGEX2T-PEA-15 or
pCDNA3-PEA-15 as the template. All plasmid constructs were verified by
DNA sequencing. The pEGFP-C1 vector was obtained from BD Biosciences
Clontech (Palo Alto, CA).
In Vitro Protein Production
BL21 competent bacteria were transfected with pGEX2T-PEA-15 plasmids
(wild-type or mutant) and induced to express protein with 1 mM isopropyl
?-d-thiogalactoside for 2–3 h. The bacteria were lysed in a PBS buffer con-
taining 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 mM dithio-
threitol (DTT), 5 ?g/ml aprotinin, and 0.5 mM leupeptin. Wild-type and
mutant GST-PEA-15 were then enriched from total bacterial lysate by binding
to glutathione-Sepharose 4B beads (Amersham Biosciences UK). Bead-bound
GST-PEA-15 was washed with PBS and eluted using 20 mM glutathione.
After dialysis against PBS, purified GST-PEA-15 was stored at ?70°C.
In Vitro Phosphorylation and ERK Binding
NIH 3T3 cells were lysed in a kinase active buffer (1% NP-40, 25 mM Tris HCl,
pH 7.4, 150 mM NaCl, 1 mM NaF, 1 mM EGTA, 2 mM DTT, 3 mM MgCl2, 2
mM CaCl2, 1 mM NaVO4, EDTA-free protease inhibitor cocktail [Roche
Diagnostics, Mannheim, Germany]) to which 1 mM ATP with 0.022%
[?-32P]ATP was added. One hundred micrograms of total cell lysate in 100 ?l
was then incubated with Sepharose-immobilized GST-PEA-15 for 2 h at 37°C
in the presence or absence of lipid activator [50 ?g/ml l-?-phosphatidyl-l-
serine sodium salt (Sigma-Aldrich) and 50 ?M phorbol 12-myristate 13-
acetate (PMA; EMD Biosciences, La Jolla, CA) in 25 mM Tris, pH 7.4. In some
cases, PKC or CamKII were inhibited by addition of 1 ?M bisindoylmaleim-
ide I (Bis) or 10 ?M KN-62 (EMD Biosciences), respectively.
At the end of the incubation, beads were sedimented by centrifugation at
22,000 ? g and washed three times with PBS. The bead bound PEA-15 was
solubilized by digestion of the immobilized GST-PEA-15 fusion protein with
Phosphorylation of PEA-15 Regulates ERK Functions
Vol. 16, August 20053553
thrombin (100 U/ml) in a buffer containing 100 mM Tris, pH 8.0, 150 mM
NaCl, 2.5 mM CaCl2. Eluted proteins were resolved by SDS-PAGE under
reducing conditions and transferred to nitrocellulose membranes. Incorpora-
tion of32P was assayed by autoradiography. The blots were stained with
antibodies reactive with ERK1/2 (Santa Cruz Biotechnology) to detect bound
ERK1/2 and antibodies against PEA-15 to assay loading of the affinity matrix.
CHO cells were cotransfected with 0.5 ?g of pCDNA3-FLAG-ERK2 and either
1.5 ?g of pCDNA3-HA-PEA-15, pCDNA3-HA-PEA-15 mutants, or empty
vector. Transient transfections were carried out using Lipofectamine and Plus
reagents as per manufacturer’s protocol (Invitrogen). Cells were harvested
24 h posttransfection and scraped into 1 ml of lysis buffer (20 mM HEPES, pH
7.4, 2 mM EGTA, 2 mM MgCl2, 2 mM NaVO4, protease inhibitors cocktail).
Total cell lysates were homogenized by 20 serial passages through a 27-gauge
needle and then centrifuged at 22,000 ? g for 10 min. The supernatant was
precleared with 20 ?l of protein G-Sepharose beads (?50% slurry; Amersham
Biosciences UK) at 4°C for 30 min. For each condition, 2 ?g of anti-HA
antibody and 20 ?l of protein G-Sepharose beads were added to 500 ?g of
cleared lysate, and incubated for 2 h at 4°C. Immunoprecipitates were washed
three times in lysis buffer and solubilized in 5? SDS-PAGE sample buffer
[10% (wt/vol) SDS, 250 mM Tris, pH 6.8, 500 mM DTT, 50% glycerol, 1
mg/ml bromphenol blue]. Western blotting with anti-FLAG was used to
assay ERK coimmunoprecipitation and with anti-HA to detect immunopre-
cipitated PEA-15. Separately, 25 ?g of total cell lysate was immunoblotted for
FLAG-ERK and HA-PEA-15 to verify comparable expression in all samples.
CHO cells were transfected with cDNA encoding wild-type PEA-15, phos-
phomimetic mutants, or empty vector in a 3:1 ratio to HA-tagged ERK2. Cells
were allowed to recover from transfection by allowing growth in complete
media for 24 h. Next, cells were maintained in serum-free media for an
additional 24 h. Cells were stimulated for 3 h with 10% FBS and then
harvested and suspended at 5 ? 106cells/ml in fractionation buffer (20 mM
HEPES, pH 7.5, 1.5 mM MgCl2, 5 mM KCl, 1 mM DTT, 1 mM NaVO4, with
Complete EDTA Free protease inhibitor cocktail [Roche Diagnostics]). After
incubation on ice for 20 min, cells were homogenized by shearing through a
23-gauge needle. Twenty-five microliters of total cell lysate was saved, and
the remaining sample was centrifuged at 3000 ? g to sediment the nuclei. The
supernatant was then spun at 20,800 ? g for 30 min at 4°C to separate the
sedimented membrane fraction from the soluble cytosolic fraction. The nu-
clear pellet was washed two times with fractionation buffer, resuspended in
250 ?l, and loaded onto a 250-?l cushion formed by 1 M sucrose in fraction-
ation buffer, and centrifuged at 2250 ? g for 10 min at 4°C. The pellet was then
extracted into 250 ?l of fractionation buffer containing 1% NP-40. Total,
cytosolic, and nuclear fractions were resolved by SDS-PAGE and analyzed by
Serum Response Element reporter assay
CHO cells were transfected with 1.0 ?g of wild-type pCDNA3-HA-PEA-15,
0.33 ?g of pSRE-Luc (BD Biosciences Clontech), and 0.33 ?g of pRL-TK
(Promega, Madison, WI). Cells were grown in complete medium for 24 h and
then shifted to serum-free media for 24 h. Cells were then stimulated by
addition 10% FBS for 3 h, harvested, and resuspended in 250 ?l of passive
lysis buffer (supplied with dual luciferase assay kit; Promega). Twenty mi-
croliters of total lysate was placed in one well of a 96-well plate, and 100 ?l of
luciferase assay reagent (dual-luciferase reporter assay kit; Promega) was
added. Firefly luciferase activity was assayed by measuring light emission
using a 96-well plate Lucy 2 Luminometer (Anthos Labtec Instruments,
Salzburg, Austria) with 1-s integration. One hundred microliters Stop-and-glo
reagent (dual-luciferase reporter assay kit; Promega) was added to stop firefly
luciferase activity and assay Renilla luciferase activity to correct for transfec-
tion efficiency. All conditions were assayed in triplicate in each experiment,
and each experiment was performed in triplicate.
CHO cells were transfected with 1.0 ?g of pCDNA3-HA-PEA-15 and 0.33 ?g
of pEGFP-C1 (GFP) vector as a transfection reporter. Cells were grown in
complete medium for 24 h and then shifted to serum-free media for 24 h.
Bromodeoxyuridine (BrdU, 10 ?M; BD Biosciences PharMingen, San Diego,
CA) was added and 15 min later 10% FBS was added. Cells were harvested
after 45 min, resuspended in 50 ?l of ice-cold PBS, and fixed by addition of 1
ml of 1% formaldehyde in PBS for 2 min at room temperature. After washing
one time with PBS, the cells were resuspended in 100 ?l of PBS and incubated
with 1 ml of cold 70% ethanol for 5 min on ice. The cells were then washed
two times with PBS, resuspended in 50 ?l of PBS, and incubated with 20 U of
DNase I (Roche Diagnostics) for 30 min. To detect BrdU, 1 ?l of anti-BrdU
antibody (BD Biosciences PharMingen) was added for 30 min at room tem-
perature. After one wash with PBS, 1 ?l of phycoerythrin-conjugated F(ab?)2
goat anti-mouse IgG (BD Biosciences PharMingen) was added for 30 min at
room temperature. Cells were analyzed by flow cytometry for BrdU incorpo-
ration by using green fluorescent protein (GFP) expression as a marker for
transfection. The percentage of change in BrdU incorporation was calculated
by comparing the geometric mean fluorescence intensity of the BrdU staining
of each sample to serum-starved vector-transfected cells. Whole cell lysates
were analyzed for transfected protein expression by Western blotting.
Analytical two-color flow cytometry was carried out as described previously
(Chou et al., 2003). Briefly, CHO cells were transfected with 0.1 ?g of pEGFP
as transfection marker and a combination of 1.5 ?g of pCDNA1-Raf-CAAX
with 1.5 ?g of various pCDNA3-HA-PEA-15 constructs. Transient transfec-
tions were carried out using Lipofectamine and Plus reagents using the
manufacturer’s protocol. After 24 h in normal growth media, cells were
harvested and analyzed for transfection efficiency (GFP) and integrin binding
to 3Fn-(9-11). Integrin activation was quantified as an activation index (AI) as
defined in Chou et al. (2003). The percentage of reversal of suppression was
calculated as described previously (Chou et al., 2003).
RESULTS AND DISCUSSION
PEA-15 Phosphorylation Blocks ERK1/2 Binding In Vitro
PEA-15 binding to ERK1/2 is in part mediated by the tail of
PEA-15, a region that contains both major phosphorylation
sites Ser104and Ser116(Araujo et al., 1993; Kubes et al., 1998;
Trencia et al., 2003). We therefore phosphorylated GST-
PEA-15 in vitro and examined effects on ERK binding. When
GST-PEA-15 was incubated with cell lysate in the presence
of activating lipids (PMA and phosphatidyl serine), it was
phosphorylated and lost the capacity to bind to ERK1 and
ERK2 (Figure 1A). Inhibitors of either PKC or CamKII (Bis or
KN-62, respectively) reduced PEA-15 phosphorylation by 75
and 60%, and restored the ability of PEA-15 to bind ERK1/2.
This restoration of ERK1/2 binding with only partial loss in
phosphorylation suggests that phosphorylation at both sites
is required to block the interaction of ERK1/2 with PEA-15.
To confirm that PEA-15 phosphorylation was responsible
for the inhibition of ERK1/2 binding, we individually
changed each of the phosphorylated serine residues to ala-
nine. Mutation of either Ser104or Ser116reduced in vitro
phosphorylation and prevented the loss of ERK1/2 binding
(Figure 1B). This result is consistent with previous studies
that suggested cooperative phosphorylation at the two sites
(Kubes et al., 1998). These experiments demonstrate that
PEA-15 phosphorylation inhibits ERK1/2 binding and sug-
gest that phosphorylation at both Ser104and Ser116is re-
quired for this inhibition.
PEA-15 phosphorylation in vitro with purified PKC in
combination with CamKII failed to inhibit ERK1/2 binding
(our unpublished data), suggesting that PEA-15 phosphor-
ylation, although necessary, was not sufficient to inhibit
ERK1/2 binding. To address this possibility, we incubated
GST-PEA-15 with cell lysate in the presence of activating
lipids (PMA and phosphatidyl serine); the phosphorylated
GST-PEA-15 and the lysate were then separated by centrif-
ugation. Recombination of the modified lysate with the
phosphorylated PEA-15 resulted in little binding of ERK1/2
(Figure 1C). In sharp contrast, ERK1/2 bound strongly when
unmodified cell lysate was added to the phosphorylated
PEA-15. Furthermore, the modified lysate still contained
ERK1/2 capable of binding PEA-15, as evidenced by robust
binding when modified lysate was added to unmodified
GST-PEA-15 (Figure 1C). Autoradiography of the modified
lysate revealed the presence of several newly phosphory-
lated species; however, there was no increase in phospho-
ERK1/2 as judged by immunoblotting (our unpublished
data). Thus, phosphorylation of PEA-15 is required but not
sufficient for disruption of the interaction of PEA-15 with
J. Krueger et al.
Molecular Biology of the Cell3554
Phosphomimetic Mutations in PEA-15 Disrupt Its
Interaction with ERK1/2 In Vivo
The foregoing experiments established that PEA-15 phos-
phorylation can disrupt its interaction with ERK1/2 in vitro.
To assess the effect of PEA-15 phosphorylation on ERK2
interactions and functions in vivo, we introduced phospho-
mimetic Asp mutations into Ser104and Ser116. These HA-
tagged mutants were expressed in CHO cells, and their
ability to coimmunoprecipitate with FLAG-tagged ERK2
was assayed. Wild-type PEA-15 coimmunoprecipitated with
FLAG-ERK2; however, PEA-15 (S104D) or PEA-15 (S116D)
exhibited markedly reduced coimmunoprecipitation (Figure
2A). Quantification of the data revealed that there was no
detectable coprecipitation of the S116D mutant and nearly a
70% reduction of coprecipitation with the S104D mutant. For
comparison, there was 85% reduction of coprecipitation
with PEA-15 (L123R), a mutant known to block ERK1/2
binding (Hill et al., 2002). In each of these mutants, one of the
phosphorylatable Ser residues remained, suggesting that the
other Ser residue might have become phosphorylated in
vivo, leading to the inhibition of ERK binding. To directly
test this idea, we created double mutants [PEA-15 (S104D,
S116A) and PEA-15 (S104A, S116D)]. Each of these mutants
coprecipitated with FLAG-ERK to a slightly greater extent
than wild-type PEA-15 (116 and 121% of wild-type, respec-
tively) (Figure 2B). Combined with the previous experi-
ments, these results establish that phosphorylation of
PEA-15 at Ser104and Ser116lead to disruption of its interac-
tion with ERK1 and ERK2 and that preventing phosphory-
lation at either site is sufficient to maintain ERK1/2 associ-
Phosphorylation of PEA-15 Prevents Inhibition of Cell
We previously reported that PEA-15 expression inhibits cell
proliferation (Formstecher et al., 2001). Because PEA-15 is
widely expressed, we hypothesized that PEA-15 phosphor-
ylation might alter its capacity to inhibit cell proliferation. To
assess the effect of PEA-15 phosphorylation on proliferation,
cells were transfected with PEA-15 or PEA-15 (S104D) or
(S116D) mutants, and the incorporation of BrdU was mea-
sured to assess DNA synthesis. Expression of wild-type
PEA-15 led to a 50% reduction in BrdU incorporation com-
pared with vector-transfected cells (Figure 3A). This effect
Recombinant GST-PEA-15 was immobilized on glutathione-Sepha-
rose beads and incubated with lysates of NIH 3T3 cells in kinase
active buffer containing 10 ?Ci of [?-32P]ATP in the presence or
absence of 50 mg/ml phosphatidyl serine and 100 nM phorbol
myristate acetate (lipids) at 37°C for 2 h. The beads were washed,
and bound proteins were eluted in sample buffer, separated by
SDS-PAGE, and immunoblotted with anti-ERK1/2 to assess ERK
Phosphorylation of PEA-15 blocks binding to ERK1/2.
Figure 1 (cont).
tion of PEA-15 on the beads. Incorporation of32P into PEA-15 was
detected by autoradiography of the dried blots. The ERK1/2 blots
were quantified by densitometric scanning, and the ERK bound was
divided by that bound to wild-type PEA-15 to obtain the indicated
ratios. (A) Phosphorylation of wild-type PEA-15 in the presence of
lipid activators blocked the interaction of ERK1/2 with PEA-15.
Treatment with PKC (1 ?M Bis) or CamKII (10 ?M KN-62) inhibi-
tors blocked phosphorylation of wild-type PEA-15 and restored the
interaction of ERK1/2 with PEA-15. (B) Mutation of PEA-15 Ser104
or Ser116phosphorylation sites to Ala blocked phosphorylation and
preserved the ability of PEA-15 to bind ERK1/2. (C) Phosphoryla-
tion of PEA-15 is necessary but not sufficient to inhibit ERK1/2
binding. Recombinant GST-PEA-15 was incubated with lysates of
NIH 3T3 cells in the presence of absence of lipid activators, as
described in A. Lysate and PEA-15 were then separated by centrif-
ugation and subsequently recombined for binding assays. When
lipid activator-treated lysate was added to modified PEA-15
(L*?P*) ERK1/2 binding was strongly inhibited. In contrast, com-
binations in which either the lysate or the PEA-15 had been incu-
bated in the absence of activators (L*?P, L?P*) bound ERK1/2 to
the same extent as combinations in which no lipid activator was
binding and with anti-PEA-15 to assess the reten-
Phosphorylation of PEA-15 Regulates ERK Functions
Vol. 16, August 20053555
was completely reversed by the S104D or S116D mutation,
indicating that phosphorylation of PEA-15 reverses its inhi-
bition of cell proliferation. PEA-15 wild-type and mutants
were expressed at similar levels (Figure 3B). Furthermore,
transfection of cells with nonphosphorylatable PEA-15
(S104A or S116A) mutants led to an even more profound
(75–85%) suppression of DNA synthesis (Figure 3A). This
latter result suggests that endogenous kinases phosphory-
lated transfected PEA-15, thereby attenuating its effect on
proliferation; however, the fact that wild-type PEA-15 sup-
pressed proliferation indicates that under these conditions
of serum stimulation, PEA-15 is not fully phosphorylated.
Thus, the capacity of PEA-15 to inhibit cell proliferation is
regulated by its phosphorylation.
PEA-15 Phosphorylation Reverses the Biological
Consequences of Its Interaction with ERK1/2
The foregoing experiments established that PEA-15 phos-
phorylation blocks its capacity to interact with ERK1/2 and
to inhibit cell proliferation. Cell proliferation, in part, de-
pends on ERK1/2 entering the nucleus where they can
phosphorylate transcription such as Elk-1, c-myc, c-fos, and
c-jun, resulting in cell cycle progression (Whitmarsh and
Davis, 1996). PEA-15 expression blocks nuclear accumula-
tion of activated ERK1/2, promoting its retention in the
cytosol; this effect is dependent on its ability to bind ERK1/2
(Formstecher et al., 2001; Whitehurst et al., 2004). We there-
fore examined the effect of the phosphomimetic mutations
on PEA-15’s capacity to inhibit ERK2 nuclear translocation
and ERK-dependent transcription. We transfected cells with
PEA-15 or phosphomimetic mutants and examined serum-
stimulated ERK2 nuclear translocation. Serum stimulation
induced nuclear accumulation of ERK2, which was almost
completely blocked in cells transfected with PEA-15 (Figure
4A). In contrast, PEA-15 (S104D) or (S116D) mutants did not
with ERK2 in cells. CHO cells were cotransfected with cDNAs
encoding FLAG-tagged ERK2, and HA-tagged wild-type PEA-15 or
the indicated PEA-15 mutants. Lysates of these cells were immuno-
precipitated with anti-HA antibody, and the immunoprecipitates
were fractionated by SDS-PAGE and immunoblotted with anti-
FLAG antibody to detect coprecipitated ERK2. These immunopre-
cipitates also were blotted with anti-PEA-15 to verify consistent
immunoprecipitation. The whole cell lysates were blotted with anti-
FLAG to verify expression of the recombinant ERK2. The blots were
quantified by densitometric scanning and the quantity of coprecipi-
tated ERK2 was divided by ERK2 precipitated with wild-type
PEA-15 to obtain the indicated ratios. (A) Phosphomimetic muta-
tions (S104D and S116D) in PEA-15 reduce interaction with ERK2.
Note that the S116D mutant blocked coprecipitation of ERK2 with
PEA-15 to a greater extent than the L123R mutant, previously re-
ported to disrupt the PEA-15-ERK interaction (Hill et al., 2002). The
S104D mutant also inhibited the PEA-15–ERK2 interaction but to a
lesser extent than either of the other two mutants. (B) Phosphory-
lation of both Ser104and Ser116is required to block interaction with
ERK1/2. The free Ser on each Asp mutant was mutated to a
nonphosphorylatable Ala, creating double mutants PEA-15(S104A,
S116D) and PEA-15(S104D, S116A). Each of these double mutants
restored the interaction of PEA-15 with ERK1/2.
Phosphomimetic PEA-15 mutations block its association
proliferation. (A) Nonphosphorylatable PEA-15 mutants suppress
cell proliferation more than wild type; phosphomimetic mutants fail
to suppress. CHO cells were transfected with cDNAs encoding
HA-PEA-15 or the indicated PEA-15 mutants. After 24 h in growth
media, the cells were placed in 0.5% serum for an additional 24 h.
BrdU (10 ?M) was added to the medium, followed by 10% serum to
stimulate cell proliferation. After 1 h, the cells were harvested, and
BrdU incorporation was assayed by staining with anti-BrdU and
quantified using flow cytometry. Data were acquired as geometric
mean fluorescence intensity of BrdU staining and are expressed as
percentage of BrdU incorporation, where 100% is basal BrdU incor-
poration of vector-transfected serum-starved cells. Transfection
with PEA-15 blocked serum stimulation of proliferation. In contrast,
both phosphomimetic mutants (S104D, S116D) did not block cell
proliferation. Both nonphosphorylatable mutants (S104A, S116A)
reduced proliferation less than that seen in the absence of serum. (B)
Expression of transfected PEA-15 variants. Lysates of the cells de-
scribed in A were fractionated by SDS-PAGE and immunoblotted
with PEA-15 antibody to detect transfected PEA-15.
PEA-15 phosphorylation prevents its inhibition of cell
J. Krueger et al.
Molecular Biology of the Cell 3556
block nuclear accumulation of ERK (Figure 4A). In each case,
similar levels of HA-ERK2 and HA-PEA-15 expression were
observed (Figure 4A, bottom). Nuclear fractions were free
from the cytosolic marker Rho GDI, and cytosolic fractions
were free from nuclear markers (lamins A/C) (Figure 4B).
Thus, PEA-15 phosphorylation inhibits its capacity to block
nuclear accumulation of ERK2.
After translocation to the nucleus, activation of the c-fos
transcription factor by ERK1/2 is required for transcription
of genes that contain the c-fos serum response element (SRE)
(Gille et al., 1995). Using an SRE-luciferase reporter assay, we
compared the effects of PEA-15 and phosphomimetic mu-
tants on ERK1/2-dependent transcription. PEA-15 dramati-
cally reduced serum-induced SRE reporter activity (Figure
5A). This is consistent with other studies that report a de-
crease in Elk-1 reporter activity induced by PEA-15 (Form-
stecher et al., 2001). In contrast, PEA-15 (S104D) and (S116D)
failed to block SRE activity (Figure 5A). As before, the dou-
ble mutants (S014D, S116A) and (S104A, S116D) maintained
the capacity to block SRE reporter activity (Figure 5A).
PEA-15 wild-type and mutants were expressed at similar
levels (Figure 5B). Thus, as with blockade of ERK1/2 bind-
ing, phosphorylation of both Ser residues is required to
abolish the capacity of PEA-15 to block ERK2 nuclear trans-
location and ERK1/2-dependent transcription.
In addition to its transcriptional activities, ERK1 and
ERK2 have a variety of transcription-independent effects on
cells (Cobb et al., 1994; Johnson and Lapadat, 2002; Reddy et
al., 2003; Roux and Blenis, 2004). Among these are the ca-
pacity of activated ERK1/2 to markedly reduce the affinity
of integrin adhesion receptors (Hughes et al., 1997). PEA-15
blocks ERK-dependent suppression of integrin activation by
binding to ERK1/2 (Chou et al., 2003). Therefore, we sought
to investigate the effect of phosphomimetic mutations of
PEA-15 on integrin suppression. CHO cells were transfected
with Raf-CAAX to activate ERK1/2 and suppress integrin
activation and were cotransfected with PEA-15 or PEA-15
mutants. After 24 h, the cells were detached and the binding
S116D) in PEA-15 do not block nuclear import of ERK2. CHO cells were cotransfected with HA tagged PEA-15 wild-type or mutants and
HA-ERK2, grown in 10% serum for 24 h, followed by serum-free media for 24 h. The cells were then stimulated with 10% serum for 3 h,
disrupted by osmotic shock, and fractionated into nuclear and cytoplasmic fractions. Each fraction or the total cell lysate was separated by
SDS-PAGE and immunoblotted with anti-HA to detect ERK2 and PEA-15. Wild-type PEA-15 blocked nuclear accumulation of ERK2, whereas
phosphomimetic PEA-15 (S104D and S116D) did not (top). Whole cell lysates also were assayed for HA-ERK and PEA-15 expression (middle
and bottom). (B) Validation of subcellular fractionation. Whole cell lysates or nuclear and cytosolic fraction from A were separated by
SDS-PAGE and immunoblotted with antibodies reactive for the cytosolic marker RhoGDI (bottom left) and the nuclear marker lamin A/C
PEA-15 phosphorylation prevents inhibition of nuclear translocation of ERK1/2. (A) Phosphomimetic mutations (S104D, and
Phosphorylation of PEA-15 Regulates ERK Functions
Vol. 16, August 2005 3557
of soluble cell binding domain of fibronectin [3Fn(9-11)] was
measured to assess the activation of integrin ?5?1. Trans-
fection with Raf-CAAX markedly suppressed the binding of
3Fn(9-11), and this suppression was reversed by transfection
with wild-type PEA-15. In sharp contrast, the phosphomi-
metic mutations in PEA-15 (S104D or S116D) abolished this
effect (Figure 6A). PEA-15 wild-type and mutants were ex-
pressed at similar levels (Figure 6B). Thus, PEA-15 phos-
phorylation also prevents its effects on the nontranscrip-
tional activities of ERK 1/2.
PEA-15 Is Phosphorylated In Vivo
PEA-15 is widely expressed in a variety of tissues and is
phosphorylated in lysates of astrocytes (Araujo et al., 1993).
We used an antibody directed against a peptide derived
from the human PEA-15 sequence corresponding to amino
acids 112–123 which Ser116was phosphorylated to examine
in vivo PEA-15 phosphorylation (p-PEA-15). This antibody
was PEA-15-specific; it recognized a single band with mobility
of authentic PEA-15 in PEA-15-transfected CHO cells. In con-
trast the anti-p-PEA-15 failed to react with the PEA-15 (S116A)
(Figure 7A), but it still reacted with the PEA-15 (S105A and
S105D) mutants (our unpublished data). The anti-p-PEA-15
failed to react with purified recombinant PEA-15; however,
phosphorylation with purified CamKII resulted in strong reac-
tivity with this antibody. Reactivity was not observed when
PEA-15 (S116A) was treated with CamKII under identical con-
ditions (Figure 7B). Thus, the anti-p-PEA-15 is both PEA-15
specific and phosphorylation specific.
Having established the specificity of the antibody, we
examined the phosphorylation status of PEA-15 in selected
tumor cell lines. The 1620 glioma cell line and MB-MDA-231
breast cancer cell lines both expressed PEA-15 that was
reactive with anti-p-PEA-15 (Figure 7C). We next sought to
assess the potential presence and phosphorylation state of
PEA-15 in normal epithelial cells. Previous studies have
shown that PEA-15 protein is expressed in normal brain and
mammary tissue (Bera et al., 1994; Danziger et al., 1995;
Estelles et al., 1996; Hwang et al., 1997; Kubes et al., 1998;
Ramos et al., 2000; Tsukamoto et al., 2000; Sharif et al., 2004).
Immunohistochemical staining of normal breast tissue re-
vealed the presence of PEA-15 in mammary epithelium and
to a much lesser extent in the mammary stroma (Figure 7D).
These mammary epithelial cells were strongly reactive with
anti-p-PEA-15, establishing that PEA-15 is phosphorylated
in situ in normal mammary epithelium. Thus, PEA-15 is
phosphorylated in vivo in cell lines and tissues.
Our studies establish that PEA-15 phosphorylation blocks
its binding to ERK1/2. The ERK2 binding surface of PEA-15
includes part of the N-terminal DED and of the C-terminal
“tail” (Hill et al., 2002). Both phosphorylation sites are within
the C-terminal tail, suggesting that phosphorylation might
sterically hinder ERK1/2 binding. The unphosphorylated
dependent transcription. (A) Phosphomimetic mutations (S104D
and S116D) in PEA-15 do not block SRE luciferase reporter activity.
CHO cells were cotransfected with PEA-15 or the indicated mu-
tants, a SRE-dependent luciferase reporter construct (pSRE-Luc),
and a constitutive Renilla reporter (pRL-TK). Cells were grown in
10% serum for 24 h, serum-free media for 24 h, and then stimulated
with 10% serum for 3 h. ERK nuclear activity was assayed as a
function of SRE luciferase reporter activity. SRE reporter activity
was corrected for transfection efficiency by the cotransfected Renilla
reporter activity. Results are reported as the percentage of increase
in SRE reporter activity compared with vector-transfected serum-
starved cells. Wild-type PEA-15 and both nonphosphorylatable mu-
tants (S0104A, S116A) markedly suppressed SRE-dependent tran-
scription. In contrast, the phosphomimetic PEA-15 mutants (S104D,
S116D) did not inhibit SRE reporter activity. Data depicted are the
mean ? SE of four determinations. (B) Expression of transfected
PEA-15. Lysates of cells described in A were fractionated by SDS-
PAGE and immunoblotted with anti-PEA-15 antibody to detect
PEA-15 phosphomimetic mutants do not block ERK-
grin suppression. (A) Effect of PEA-15 wild-type and mutants on
Raf-CAAX–induced integrin suppression. CHO cells were cotrans-
fected with cDNA encoding a constitutively active Raf kinase (Raf-
CAAX) in combination with PEA-15 or the indicated mutants. After
24 h, the cells were detached, and the binding of soluble cell binding
domain of Fn [3Fn(9-11)] binding was measured to assess the affin-
ity state of integrin ?5?1. This was quantified as an AI, where AI ?
100 * (F –Fo)/(Fm –Fo). F represents the geometric mean fluores-
cence (GMF) of 3Fn-(9-11) binding alone, Fo is the GMF of 3Fn-(9-
11) binding in the presence of 10 mM EDTA, and Fm is the GMF of
3Fn-(9-11) binding in the presence of 9EG7. The percentage of
suppression was calculated as 100 * (AIM –AIT)/AIM, where AIM
is the activation index of the vector transfected cells, and AIT is the
activation index in the presence of a Raf-CAAX or Raf-CAXX ?
PEA-15 WT or mutants. The percentage of reversal of suppression
was calculated as 100 * (AI –AIR)/AI, in which AI is the activation
index of the control cells, and AIR is the activation index in the
presence of a transfected PEA-15 WT or mutants. Wild-type PEA-15
reversed Raf-CAAX–induced integrin suppression ?50%, whereas
PEA-15 phosphomimetic mutants (S104D, S116D) failed to do so at
all. (B) Expression of transfected PEA-15 variants. Lysates of cells
described in A were fractionated by SDS-PAGE and immunoblotted
with PEA-15 antibody to detect transfected PEA-15.
Phosphomimetic PEA-15 mutants do not reverse inte-
J. Krueger et al.
Molecular Biology of the Cell3558
tail of PEA-15 is unstructured and phosphorylation may
alter its conformation to block ERK1/2 binding (Hill et al.,
2002). Interestingly, phosphorylation of PEA-15 stimulates
its recruitment to the DISC, an event presumably mediated
by the interaction of its DED with DISC components such as
FADD (Gille et al., 1995; Camps et al., 1998; Dang et al., 1998).
Because ERK1/2 also interacts with the DED of PEA-15, our
results raise the possibility that ERK1/2 binding blocks
FADD binding to PEA-15. PEA-15 phosphorylation, by dis-
placing ERK1/2, may make the DED available for binding to
FADD, enabling PEA-15 recruitment to the DISC and thus
blocking apoptosis (Menice et al., 1997; Formstecher et al.,
2001; Robinson et al., 2002).
PEA-15 is a downstream effector of multiple kinases in-
cluding PKC, Akt, and CamKII. As reported here, PEA-15
phosphorylation can regulate the proliferation of the cells
that express it. When PEA-15 is unphosphorylated, its ex-
pression leads to Ras activation and therefore ERK1/2 acti-
vation (Ramos et al., 2000). As shown here, only unphos-
phorylated PEA-15 binds ERK1/2 and blocks its nuclear
translocation and therefore cell proliferation. We found that
when PEA-15 is phosphorylated, it loses the capacity to bind
ERK1/2 and block proliferation; simultaneously, it gains the
capacity to enter the DISC and to inhibit apoptosis (Kitsberg
et al., 1999; Hao et al., 2001; Trencia et al., 2003). Protein
phosphorylation can be dynamic and reversible; hence,
PEA-15 phosphorylation may serve to regulate the shuttling
of PEA-15 among its many binding partners (Figure 8),
thereby controlling cell proliferation and survival.
Araujo, H., Danziger, N., Cordier, J., Glowinski, J., and Chneiweiss, H. (1993).
Characterization of PEA-15, a major substrate for PKC in astrocytes. J. Biol.
Chem. 268, 5911–5920.
Bera, T. K., Guzman, R. C., Miyamoto, S., Panda, D. K., Sasaki, M., Hanyu, K.,
Enami, J., and Nandi, S. (1994). Identification of a mammary transforming
gene (MAT1) associated with mouse mammary carcinogenesis. Proc. Natl.
Acad. Sci. USA 91, 9789–9793.
Camps, M., Nichols, A., Gillieron, C., Antonsson, B., Muda, M., Chabert, C.,
Boschert, U., and Arkinstall, S. (1998). Catalytic activation of the phosphatase
MKP-3 by ERK2 mitogen-activated protein kinase. Science 280, 1262–1265.
Chou, F. L., Hill, J. M., Hsieh, J. C., Pouyssegur, J., Brunet, A., Glading, A.,
Uberall, F., Ramos, J. W., Werner, M. H., and Ginsberg, M. H. (2003). PEA-15
binding to ERK1/2 MAP kinases is required for its modulation of integrin
activation. J. Biol. Chem.
Cobb, M. H., Hepler, J. E., Cheng, M., and Robbins, D. (1994). The mitogen-
activated protein kinases, ERK1 and ERK2. Semin. Cancer Biol. 5, 261–268.
Condorelli, G., Vigliotta, G., Cafieri, A., Trencia, A., Andalo, P., Oriente, F.,
Miele, C., Caruso, M., Formisano, P., and Beguinot, F. (1999). PED/PEA-15, an
anti-apoptotic molecule that regulates FAS/TNFR1-induced apoptosis. On-
cogene 18, 4409–4415.
were transfected with cDNAs encoding PEA-15 or the indicated mutants. After 48 h, the cells were lysed, fractionated by SDS-PAGE, and
analyzed by immunoblotting with either anti-phospho-PEA-15 (p-PEA-15) or an anti-PEA-15 antibody (PEA-15) that reacts with both
phosphorylated and unphosphorylated PEA-15. Note that both antibodies reacted with a polypeptide of molecular weight of ?15 k and no
other cellular polypeptides. This polypeptide was absent from immunoblots of untransfected CHO cells (our unpublished data). Mutation
of Ser116to Ala abolished reactivity of the p-PEA-15 antibody. (B) Anti-p-PEA-15 is phospho-specific. Purified recombinant PEA-15 or the
indicated mutants were phosphorylated in vitro with purified CamKII, fractionated by SDS-PAGE, and immunoblotted with anti-p-PEA-15
or anti-PEA-15. Anti-p-PEA-15 reacted with phosphorylated but not unphosphorylated PEA-15. Reactivity was abolished by mutating the
CamKII phosphorylation site, Ser116, to Ala. (C) Phosphorylation of PEA-15 in cultured cells. The indicated tumor cell lines were lysed in
sample buffer, fractionated by SDS-PAGE, and immunoblotted with either anti-PEA-15 or anti-p-PEA-15. Note that PEA-15 is expressed and
phosphorylated in both cell lines. (D) PEA-15 is phosphorylated in situ in normal breast epithelium. Sections of normal mammary tissue were
immuno peroxidase stained for PEA-15 (left) or phospho-PEA-15 (middle) and counterstained with Mayer’s hematoxylin. Preimmune rabbit
IgG was used as a staining control (right).
PEA-15 is phosphorylated in cells and tissues. (A) Phospho-specific anti-PEA-15 (anti-p-PEA-15) is PEA-15 specific. CHO cells
Phosphorylation of PEA-15 Regulates ERK Functions
Vol. 16, August 2005 3559
Condorelli, G., et al. (1998). PED/PEA-15 gene controls glucose transport and
is overexpressed in type 2 diabetes mellitus. EMBO J 17, 3858–3866.
Dang, A., Frost, J. A., and Cobb, M. H. (1998). The MEK1 proline-rich insert
is required for efficient activation of the mitogen-activated protein kinases
ERK1 and ERK2 in mammalian cells. J. Biol. Chem. 273, 19909–19913.
Danziger, N., Yokoyama, M., Jay, T., Cordier, J., Glowinski, J., and Chnei-
weiss, H. (1995). Cellular expression, developmental regulation, and phylo-
genic conservation of PEA-15, the astrocytic major phosphoprotein and PKC
substrate. J. Neurochem. 64, 1016–1025.
Dong, G., Loukinova, E., Chen, Z., Gangi, L., Chanturita, T. I., Liu, E. T., and
Van Waes, C. (2001). Molecular profiling of transformed and metastatic
murine squamous carcinoma cells by differential display and cDNA microar-
ray reveals altered expression of multiple genes related to growth, apoptosis,
angiogenesis, and the NF-?B signal pathway. Cancer Res. 61, 4797–4808.
Embury, J., Klein, D., Pileggi, A., Ribeiro, M., Jayaraman, S., Molano, R. D.,
Fraker, C., Kenyon, N., Ricordi, C., Inverardi, L., and Pastori, R. L. (2001).
Proteins linked to a protein transduction domain efficiently transduce pan-
creatic islets. Diabetes 50, 1706–1713.
Estelles, A., Charlton, C. A., and Blau, H. M. (1999). The phosphoprotein
protein PEA-15 inhibits Fas- but increases TNF-R1-mediated caspase-8 activ-
ity and apoptosis. Dev. Biol. 216, 16–28.
Estelles, A., Yokoyama, M., Nothias, F., Vincent, J. D., Glowinski, J., Vernier,
P., and Chneiweiss, H. (1996). The major astrocytic phosphoprotein PEA-15 is
encoded by two mRNAs conserved on their full length in mouse and human.
J. Biol. Chem. 271, 14800–14806.
Formstecher, E., et al. (2001). PEA-15 mediates cytoplasmic sequestration of
ERK MAP kinase. Dev. Cell 1, 239–250.
Gaumont-Leclerc, M. F., Mukhopadhyay, U. K., Goumard, S., and Ferbeyre,
G. (2004). PEA-15 is inhibited by adenovirus E1A and plays a role in ERK
nuclear export and Ras-induced senescence. J. Biol. Chem. 279, 46802–46809.
Gille, H., Kortenjann, M., Thomae, O., Moomaw, C., Slaughter, C., Cobb,
M. H., and Shaw, P. E. (1995). ERK phosphorylation potentiates Elk-1-medi-
ated ternary complex formation and transactivation. EMBO J. 14, 951–962.
Glienke, J., Schmitt, A. O., Pilarsky, C., Hinzmann, B., Weiss, B., Rosenthal, A.,
and Thierauch, K. H. (2000). Differential gene expression by endothelial cells
in distinct angiogenic states. Eur. J. Biochem. 267, 2820–2830.
Hao, C., Beguinot, F., Condorelli, G., Trencia, A., Van Meir, E. G., Yong, V. W.,
Parney, I. F., Roa, W. H., and Petruk, K. C. (2001). Induction and intracellular
regulation of tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) mediated apoptosis in human malignant glioma cells. Cancer Res. 61,
Hill, J. M., Vaidyanathan, H., Ramos, J. W., Ginsberg, M. H., and Werner,
M. H. (2002). Recognition of ERK MAP kinase by PEA-15 reveals a common
docking site within the death domain and death effector domain. EMBO J. 21,
Hughes, P. E., Renshaw, M. W., Pfaff, M., Forsyth, J., Keivens, V. M.,
Schwartz, M. A., and Ginsberg, M. H. (1997). Suppression of integrin activa-
tion: a novel function of a Ras/Raf-initiated MAP kinase pathway. Cell 88,
Hwang, S., Kuo, W. L., Cochran, J. F., Guzman, R. C., Tsukamoto, T., Ban-
dyopadhyay, G., Myambo, K., and Collins, C. C. (1997). Assignment of
HMAT1, the human homolog of the murine mammary transforming gene
(MAT1) associated with tumorigenesis, to 1q21.1, a region frequently gained
in human breast cancers. Genomics 42, 540–542.
Johnson, G. L., and Lapadat, R. (2002). Mitogen-activated protein kinase
pathways mediated by ERK, JNK, and p38 protein kinases. Science 298,
Kitsberg, D., Formstecher, E., Fauquet, M., Kubes, M., Cordier, J., Canton, B.,
Pan, G., Rolli, M., Glowinski, J., and Chneiweiss, H. (1999). Knock-out of the
neural death effector domain protein PEA-15 demonstrates that its expression
protects astrocytes from TNF?-induced apoptosis. J. Neurosci. 19, 8244–8251.
Kubes, M., Cordier, J., Glowinski, J., Girault, J. A., and Chneiweiss, H. (1998).
Endothelin induces a calcium-dependent phosphorylation of PEA-15 in intact
astrocytes: identification of Ser104 and Ser116 phosphorylated, respectively,
by PKC and calcium/calmodulin kinase II in vitro. J. Neurochem. 71, 1307–
Menice, C. B., Hulvershorn, J., Adam, L. P., Wang, C. A., and Morgan, K. G.
(1997). Calponin and mitogen-activated protein kinase signaling in differen-
tiated vascular smooth muscle. J. Biol. Chem. 272, 25157–25161.
Ramos, J. W., Hughes, P. E., Renshaw, M. W., Schwartz, M. A., Formstecher,
E., Chneiweiss, H., and Ginsberg, M. H. (2000). Death effector domain protein
PEA-15 potentiates Ras activation of extracellular signal receptor-activated
kinase by an adhesion-independent mechanism. Mol. Biol. Cell 11, 2863–2872.
Ramos, J. W., Kojima, T. K., Hughes, P. E., Fenczik, C. A., and Ginsberg, M. H.
(1998). The death effector domain of PEA-15 is involved in its regulation of
integrin activation. J. Biol. Chem. 273, 33897–33900.
Reddy, K. B., Nabha, S. M., and Atanaskova, N. (2003). Role of MAP kinase in
tumor progression and invasion. Cancer Metastasis Rev. 22, 395–403.
Renault, F., Formstecher, E., Callebaut, I., Junier, M. P., and Chneiweiss, H.
(2003). The multifunctional protein PEA-15 is involved in the control of
apoptosis and cell cycle in astrocytes. Biochem. Pharmacol. 66, 1581–1588.
Robinson, F. L., Whitehurst, A. W., Raman, M., and Cobb, M. H. (2002).
Identification of novel point mutations in ERK2 that selectively disrupt bind-
ing to MEK1. J. Biol. Chem. 277, 14844–14852.
Roux, P. P., and Blenis, J. (2004). ERK and p38 MAPK-activated protein
kinases: a family of protein kinases with diverse biological functions. Micro-
biol. Mol. Biol. Rev. 68, 320–344.
Sharif, A., Canton, B., Junier, M. P., and Chneiweiss, H. (2003). PEA-15
modulates TNF? intracellular signaling in astrocytes. Ann. N.Y. Acad. Sci.
Sharif, A., Renault, F., Beuvon, F., Castellanos, R., Canton, B., Barbeito, L.,
Junier, M. P., and Chneiweiss, H. (2004). The expression of PEA-15 (phospho-
protein enriched in astrocytes of 15 kDa) defines subpopulations of astrocytes
and neurons throughout the adult mouse brain. Neuroscience 126, 263–275.
Tohgo, A., Pierce, K. L., Choy, E. W., Lefkowitz, R. J., and Luttrell, L. M.
(2002). ?-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK
activity but inhibits ERK-mediated transcription following angiotensin AT1a
receptor stimulation. J. Biol. Chem. 277, 9429–9436.
Trencia, A., et al. (2003). Protein kinase B/Akt binds and phosphorylates
PED/PEA-15, stabilizing its antiapoptotic action. Mol. Cell Biol. 23, 4511–
Tsukamoto, T., et al. (2000). Expression of MAT1/PEA-15 mRNA isoforms
during physiological and neoplastic changes in the mouse mammary gland.
Cancer Lett. 149, 105–113.
Underhill, D. A., Vogan, K. J., Underhill, T. M., and Gros, P. (2001). Identifi-
cation of a novel, alternatively spliced isoform and single nucleotide poly-
morphisms in the murine Pea-15 gene. Mamm. Genome 12, 172–174.
(A) Unphosphorylated PEA-15 binds activated ERK1/2 and blocks
cell proliferation and reverses integrin suppression. Unphosphory-
lated PEA-15 binds activated ERK1/2 and prevents their nuclear
accumulation, thereby preventing cell proliferation. Additionally,
PEA-15 reverses activated ERK1/2-mediated integrin suppression.
(B) Phosphorylated PEA-15 does not block cell proliferation or
reverse integrin suppression, but it is recruited to the DISC to block
apoptosis. Kinases such as PKC, CamKII, or Akt can phosphorylate
PEA-15. Phosphorylated PEA-15 does not bind activated ERK1/2 and
therefore has no effect on cell proliferation or integrin activation. In
contrast, phosphorylated PEA-15 is recruited to the DISC, where it can
block TNF receptor- or Fas receptor-mediated apoptosis.
Effects of PEA-15 phosphorylation on cellular functions.
J. Krueger et al.
Molecular Biology of the Cell3560
Vaidyanathan, H., and Ramos, J. W. (2003). RSK2 activity is regulated by its Download full-text
interaction with PEA-15. J. Biol. Chem. 278, 32367–32372.
Whitehurst, A. W., Robinson, F. L., Moore, M. S., and Cobb, M. H. (2004). The
death effector domain protein PEA-15 prevents nuclear entry of ERK2 by
inhibiting required interactions. J. Biol. Chem. 279, 12840–12847.
Whitmarsh, A. J., and Davis, R. J. (1996). Transcription factor AP-1 regulation
by mitogen-activated protein kinase signal transduction pathways. J. Mol.
Med. 74, 589–607.
Zhang, Y., Redina, O., Altshuller, Y. M., Yamazaki, M., Ramos, J., Chneiweiss,
H., Kanaho, Y., and Frohman, M. A. (2000). Regulation of expression of
phospholipase D1 and D2 by PEA-15, a novel protein that interacts with them.
J. Biol. Chem. 275, 35224–35232.
Zvalova, D., Formstecher, E., Fauquet, M., Canton, B., and Chneiweiss, H.
(2001). Keeping TNF-induced apoptosis under control in astrocytes: PEA-15
as a ‘double key’ on caspase-dependent and MAP-kinase-dependent path-
ways. Prog. Brain Res. 132, 455–467.
Phosphorylation of PEA-15 Regulates ERK Functions
Vol. 16, August 2005 3561