From the
Tristetraprolin (TTP) is a potential transcription factor that
contains three PPPPG repeats and two putative CCCH zinc fingers. TTP is
encoded by the early response gene Zfp-36, which is highly
expressed in response to growth factors and in several hematopoietic
cell lines. In the present studies, we investigated the possibility
that TTP is phosphorylated in intact cells. In NIH/3T3 cells that were
made to overexpress TTP constitutively, we found that the protein was
phosphorylated on serine residues, and that this phosphorylation was
rapidly (within 10 min) stimulated by several mitogens. In cell-free
assays, recombinant mouse TTP was a substrate for the mitogen-activated
protein (MAP) kinase. By a combination of protease digestion
experiments and site-directed mutagenesis strategies, we found that
serine 220 was phosphorylated by p42 MAP kinase in vitro.
Expression of mutant TTP in fibroblasts confirmed that serine 220 was
one of the major, mitogen-stimulated phosphorylation sites on the
protein in intact cells. These results suggest that TTP may be
phosphorylated by MAP kinases in vivo and that this
phosphorylation may regulate its function.
Mouse tristetraprolin (TTP)
Expression of the gene that encodes TTP,
Zfp-36(7) , is induced by several mitogens including
serum, insulin, platelet-derived growth factor (PDGF), fibroblast
growth factor (FGF), and phorbol 12-myristate 13-acetate
(PMA)
(1, 2, 3) . In quiescent mouse fibroblasts,
TTP mRNA is virtually undetectable, but it accumulates within 10 min of
mitogen stimulation, peaks at 30 min, and returns to basal levels
within 2 h. This is due primarily to increased transcription, and the
resulting mRNA has a half-life of 15-30
min
(1, 8) . In the mouse, TTP mRNA is highly expressed
in lung, intestine, lymph node, spleen, and thymus, with lower
expression in liver and kidney, and negligible expression in
brain
(1, 2) . In addition, the mRNA is constitutively
highly expressed in several hematopoietic cells, with very high
expression in macrophages and neutrophils, and somewhat lower
expression in B and T lymphocytes.
In the present study, we demonstrate that TTP is a
phosphoprotein, and we show that its phosphorylation on serine residues
is stimulated by several mitogens including serum, PDGF, FGF, and PMA,
but not by dibutyryl cAMP or forskolin. Furthermore, we show that
serine 220 is one of the residues that is phosphorylated by
p42
The plasmid
encoding GST/TTP, pGEX-TTP, was created by ligating the 1.1-kb
NcoI-BglII fragment of DU2 into the SmaI
site of pGEX-2T. The plasmids encoding pGEX-TTP(163-215) and
pGEX-TTP(163-235) were created by first using the polymerase
chain reaction to create the appropriate DU2 (TTP cDNA) fragments with
HindIII and XbaI sites added at the 5` and 3` ends of
each fragment, respectively. DNA primers
5`-GGCCTCTAGACCTAGCTCTCCCTGGCCAGCCC-3` and
5`-CGATAAGCTTTTACCCAGGGGGCGGTGGGGAGCT-3` were used to amplify the
region encoding amino acids 163-215; DNA primers
5`-GGCCTCTAGACCTAGCTCTCCCTGGCCAGCCC-3` and
5`-CGATAAGCTTTTATCTTCGAGTCACAGGGGTCCC-3` were used to amplify the
region encoding amino acids 163-235. Next, the amplified DNA
fragments were subcloned into the HindIII, XbaI site
of pGEX-KG
(12) . pGEX-TTP(S220A) was created by first changing
the codon that encodes S220 in DU2 from TCC to GCC by the polymerase
chain reaction primer overlap mutagenesis technique
(13) . Next,
the 1.7-kb NcoI-EcoRV fragment was cleaved from the
mutagenized TTP cDNA plasmid and ligated into the NcoI,
HindIII site of pGEX-KG. Similarly, pmMT-TTP(S220A) was
created by ligating a 1.1-kb BamHI-BglII fragment
from the mutagenized TTP plasmid into the BamHI site of
pmMT-neo.
These experiments have demonstrated that TTP is a serine
phosphoprotein and that its phosphorylation in fibroblasts is
stimulated by several mitogens, including FCS, PMA, PDGF, and FGF, but
not by forskolin or dibutyryl cAMP. Phosphorylation of at least 2
serine residues on TTP is enhanced following FCS stimulation, and we
have identified serine 220 as 1 of those residues. In addition, we have
shown that serine 220 is phosphorylated in cell-free assays by one of
the MAP kinase isoforms, p42
Since no function has been ascribed to TTP, as a
transcription factor or otherwise, we cannot determine whether or not
this covalent modification is functionally significant. However, the
fact that the M
The facts that TTP is encoded by an
early response gene, that it contains two putative zinc fingers, and
that it is a nuclear protein, all suggest (but do not prove) that it is
a transcription factor. MAP kinases are known to phosphorylate several
other transcription factors, and this alters function of these proteins
in several ways
(20) . In some cases, MAP kinase phosphorylation
alters DNA binding. For example, when ATF-2 that has been isolated from
cells is treated with protein phosphatase 2A, ATF-2-binding to the DNA
cAMP response element is markedly reduced
(30) . However, in
vitro phosphorylation of ATF-2 by MAP kinase can restore DNA
binding activity
(30) . Conversely, in the case of c-Jun, the MAP
kinases p42
MAP kinase phosphorylation of several other
transcription factors is thought to alter the trans-activating
ability of these proteins. For example, serine 62 in c-Myc is a major
phosphorylation site in vivo and also a phosphorylation site
for MAP kinase in vitro(24, 25) .
Phosphorylation of c-Myc at serine 62 has been associated with
increased trans-activation of gene
expression
(24, 25) . Similarly, the
trans-activating ability of the basic-leucine zipper
transcription factor NF-IL6 is also increased by MAP kinase
phosphorylation, in this case, on threonine 235 (31). It is conceivable
that MAP kinase phosphorylation might alter the function of TTP in a
similar way; however, the putative trans-activating domains of
TTP have not yet been identified, so this possibility cannot be tested
at this point.
Other potential functional changes induced by TTP
phosphorylation could include effects on nuclear-cytoplasmic
partitioning and/or protein-protein interactions. For example, the
yeast transcription factor SWI5 is localized to the nucleus in G
To determine
exactly how phosphorylation regulates TTP function, it will be
important to determine the remaining serine phosphorylation site(s).
However, although serine 220 was clearly the dominant phosphorylation
site in our cell-free kinase assays using p42
We are very grateful to Drs. Paul Dent and Tom
Sturgill for the purified p42 MAP kinase.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a basic,
proline-rich protein of M
33,600 that contains
three PPPPG repeats
(1) . Also known as Nup475
(2) and
TIS11 (3), TTP contains two putative CCCH zinc fingers, and the
recombinant protein has been shown to bind
Zn
(2) . Recently, several other proteins have
been recognized that contain similar types of putative zinc finger
structures, including those encoded by the mammalian cDNAs TIS11b
(cMG1)
(3, 4) and TIS11d
(3) , those encoded by
the Drosophila genes unkempt(5) and
DTIS11(6) , and those encoded by the yeast genes
CTH1 and CTH2.
(
)
Although TTP
has been localized to the cell nucleus in mouse fibroblasts
(2) and may well be a transcription factor, neither TTP nor any
of the related zinc finger proteins has been shown to bind to specific
DNA target sequences.
(
)
Recently,
targeted disruption of Zfp-36 in mouse embryonic stem cells
has been used to create mice that do not express TTP.
(
)
These mice are normal at birth, but soon become runted and
cachectic, develop patchy alopecia and dermatitis, and often die within
the first few months after birth. In addition, the mice have a marked
increase in granulocytes in the bone marrow, spleen, lymph nodes, and
peripheral blood, and a decrease in B and T lymphocytes in the spleen
and peripheral blood. This phenotype suggests that TTP could play a
role in granulopoiesis or in the development of other hematopoietic
cell lineages.
in vitro and that this site is also
phosphorylated in intact cells. These experiments suggest that, in
addition to controlling the availability of TTP protein by regulating
gene transcription, certain mitogens may also control its function by
regulating its reversible phosphorylation.
Plasmid Subcloning
pmMT-neo was created by
replacing the 4.3-kb EcoRI-SalI human -actin
gene promoter fragment in pH
Apr-1-neo
(9) with the 0.7-kb
EcoRI-BglII mouse metallothionein gene fragment from
pmMT-1
(10, 11) . pmMT-TTP was then created by ligating a
1.1 HindIII-BglII mouse TTP cDNA fragment from clone
DU2
(1) , which contains the entire TTP coding region, into the
HindIII, BamHI site of pmMT-neo.
Cells and Culture
NIH/3T3 cells (ATCC CRL 1658,
American Type Culture Collection) were maintained in Dulbecco's
modified Eagle's medium (DMEM) (Hazleton Research Products, St.
Lenexa, KS) supplemented with 10% fetal bovine serum (FCS) (Life
Technologies, Inc.). The TTP-deficient fibroblasts were prepared by
first isolating primary embryonic fibroblasts from 14-17-day
C57BL/129Sv mouse embryos (14); these embryos were the result of gene
targeting experiments in mouse embryonic stem cells in which a
Neo gene had been inserted into the protein coding region
of the gene that encodes TTP.
The primary fibroblasts
cultures were then immortalized by subjecting them to the 3T3 selective
procedure of Todaro and Green
(15) . The immortalized fibroblasts
expressed neither detectable TTP mRNA as assessed by Northern blotting
nor detectable TTP protein as assessed by immunoprecipitation of
[
S]cysteine-labeled protein.
The
TTP-deficient fibroblasts were maintained in DMEM supplemented with 10%
FCS.
Transfection
To establish stable cell lines,
plasmids were introduced into NIH/3T3 cells by CaPO transfection. 24 h before transfection, cells were plated at 0.5
10
cells/100-mm plate, and 2 h before transfection,
fresh medium was added to the plates. A solution was prepared by mixing
H
O, 10 µg of plasmid DNA, and 125 µl of 1
M CaCl
in that order for a total volume of 0.5 ml,
and this solution was added dropwise to 0.5 ml of 2
HBS (28
mM NaCl, 1.5 mM Na
PO
, 50
mM HEPES, pH 6.95) through which N
was bubbling.
30 min later, 1 ml of DNA solution was added to each plate of cells.
Following a 12-16-h incubation, the cells were washed three times
with TBS (137 mM NaCl, 5 mM KCl, 25 mM Tris,
pH 7.4), and then fresh medium was added. Neo
cells were
selected with medium supplemented with 0.3 mg/ml Geneticin (G418
sulfate) (Life Technologies, Inc.) for 2-3 weeks.
In Vitro Transcription and Translation
DU2 was
linearized with XbaI and transcribed with T7 RNA polymerase
(Life Technologies, Inc.) to obtain sense mRNA. In vitro translation was performed in a rabbit reticulocyte translation
system (Promega, Madison, WI) with 100 µCi of
[S]cysteine/reaction, incubating 1 h at 30
°C. Small aliquots of the reaction mixture were phosphorylated with
p42
as described below.
In Vitro Phosphorylation and Proteolytic
Cleavage
pGEX-TTP plasmids were transformed into Escherichia
coli strain BL21/DE3, and the GST fusion proteins were purified
from these cells with glutathione-Sepharose (Pharmacia) as described
previously
(16) . Aliquots of the GST fusion protein preparations
were analyzed on 9% denaturing polyacrylamide gels that were stained
with Coomassie Blue (Diversified Biotech, Scientific Technologies,
Raleigh, NC). The GST fusion proteins were incubated with activated
p42 for 30 min at 30 °C in 10 mM
MgCl
, 0.1 mM ATP, 1 mM dithiothreitol,
0.1 mM EGTA, 0.1 mM Na
VO
, and
25 mM Tris, pH 7.2. The purified, activated recombinant
p42
was generously provided by Dr. Paul Dent and Dr. Tom
Sturgill, University of Virginia
(17) . The reaction products
were separated on a 9% denaturing polyacrylamide gel, and the gel was
transferred to nitrocellulose with a TEX50 wet transfer apparatus
(Hoeffer, San Francisco, CA) using 25 mM Tris, 480 mM
glycine, 0.1% SDS, and 50% methanol transfer buffer. Because small
amounts of less than full-length fusion proteins were present in the
GST/TTP protein preparations, only the band shown by autoradiography to
contain the full-length GST/TTP fusion protein was excised. The
nitrocellulose piece was incubated for 16 h at 37 °C in 50%
pyridine, 50% ammonium acetate, pH 8.9, to elute the
protein
(18) , and the protein was precipitated by the addition
of 20% trichloroacetic acid and 50 µg of carrier
-globulin (Sigma). The protein pellet was resuspended and
proteolytically digested with trypsin (L-(tosylamido
2-phenyl)ethyl chloromethyl ketone-treated, Worthington, Freehold, NJ)
in 0.05 M NH
HCO
, CH
CN
(95:1), with endoproteinase Arg-C (Sigma) in 100 mM Tris, pH
8.5, with endoproteinase Glu-C (Sigma) in 100 mM Tris, pH 7.8,
or with endoproteinase Asp-N (Sigma) in 100 mM Tris, pH 8.5.
The products of these reactions were separated on 20% denaturing
polyacrylamide gels.
Antibody Preparation
The peptide
MDLSAIYESLQSMSHDLSSDHGGC, which corresponds to the 24
NH-terminal amino acids of mouse TTP, was synthesized and
purified as described
(19) . Several rabbit antisera were raised
against this peptide coupled to keyhole limpet hemacyanin by
Immuno-Dynamics, Inc. (La Jolla, CA). Immunospecific antibody was
purified using the immunogenic peptide and a membrane affinity
chromatography capsule (no. 11016, Amicon, Beverly, MA) as described by
the manufacturer.
Cell Labeling
Confluent 60-mm plates of cells were
serum-deprived by washing three times with DMEM and then incubating
them with DMEM supplemented with 1% (w/v) bovine serum albumin (BSA)
(Life Technologies, Inc.) for 24 h. When appropriate, the
metallothionein promoter was induced by adding sterile ZnSO solution to the plates at a final concentration of 100
µM for the final 12-15 h of the serum deprivation
period. For cells labeled with [
S]cysteine, the
cells were first washed three times with cysteine-free DMEM (Biofluids,
Rockville, MD) and then incubated with 2 ml of cysteine-free DMEM
supplemented with 1% BSA and 100 µM ZnSO
for
30 min. Next, 0.5 mCi of [
S]cysteine (DuPont
NEN) was added directly to each dish and incubated for 2 h followed by
other treatments as described under ``Results.'' For cells
labeled with
P
, the cells were first washed
three times with Krebs buffer (65 mM NaCl, 4.7 mM
KCl, 1.4 mM CaCl
, 1.2 mM
MgSO
, 25 mM NaHCO
, 25 mM
glucose, and 10 mM HEPES, pH 7.4) and then incubated in 2 ml
of Krebs buffer supplemented with 0.3 mCi of
[
P]H
PO
(DuPont), 1% BSA,
and 100 µM ZnSO
, followed by other treatments
as described under ``Results.''
Immunoprecipitation
Total cellular protein lysates
were prepared by washing labeled cells three times with ice-cold Krebs
buffer and scraping them with a rubber policeman into 0.5 ml of
ice-cold Nonidet P-40 lysis buffer (1% (w/v) Nonidet P-40, 5
mM EDTA, 0.15 M NaCl, 50 mM Tris, pH 8.3).
Following sonication for 15 s in a W-380 cup horn sonicator (Heat
Systems, Farmingdale, NY), the lysates were cleared by centrifugation
in microfuge tubes in a TL100 table-top ultracentrifuge (Beckman,
Fullerton, CA) at 88,000 g for 20 min at 4 °C. The
samples were matched for trichloroacetic acid-precipitated radioactive
counts and were then incubated with normal rabbit serum (1:100
dilution) for 1 h and 6 mg of protein A-Sepharose (Pharmacia) for an
additional 1 h. Following a 2-min centrifugation at 500 revolutions/min
in a microfuge 11 (Beckman) to pellet the protein A-Sepharose, the
supernatant was transferred to a new tube and incubated for about 12 h
with 3.3 µg of TTP antibody and for an additional 1 h with 6 mg of
protein A-Sepharose. Following four washes with wash buffer (0.5%
Nonidet P-40, 1 mM EDTA, 0.15 M NaCl, 50 mM
Tris, pH 8.3), 100 µl of an SDS buffer (30% sucrose, 0.45
M dithiothreitol, 0.06 M EDTA, 6% SDS, 0.06 mg/ml
pyronin y, diluted 1:4 with phosphate-buffered saline) was added to the
resin; the suspension was mixed vigorously, boiled for 5 min, and the
resin was pelleted by centrifugation for 5 min at 1500 revolutions/min
in a microfuge. Immunoprecipitated proteins were separated by 9%
denaturing SDS-polyacrylamide gel electrophoresis.
Cloning of a Bovine TTP cDNA
The bovine TTP cDNA
was cloned(
)
by screening a bovine aortic
endothelial cell cDNA library (Stratagene, La Jolla, CA) with a human
TTP cDNA probe
(7) . Cloning and sequencing methods were as
described previously
(1) .
Expression of TTP in NIH/3T3 Cells
NIH/3T3 cells
were serum deprived for 24 h and exposed to
[S]cysteine for 4 h and to 20% (v/v) FCS for
varying times during those 4 h. Total cellular protein lysates were
prepared, and protein was immunoprecipitated from the lysates with a
polyclonal antibody that had been raised against a peptide
corresponding to the NH
-terminal 24 amino acids of TTP
(Fig. 1). A protein species was precipitated that migrated as a
broad band with an apparent molecular mass of about 43 kDa. This
protein was not detectable in quiescent NIH/3T3 cells but accumulated
within 1 h of FCS exposure, peaked at about 2 h, and was decreased but
still present at 4 h. Although TTP has a predicted molecular mass of
33.6 kDa, several observations indicate that the observed 43-kDa
protein is TTP. First, the time course of expression of the protein
following FCS stimulation lagged slightly behind that of the TTP mRNA
which is present in these cells within 10 min of serum exposure, peaks
at 30-45 min, and returns to near basal levels within 2
h
(1) . Second, no protein of this M
was
precipitated by the preimmune serum (data not shown). Third,
precipitation of the protein could be competed completely by the
addition of the immunogenic peptide to the lysates prior to
immunoprecipitation with the TTP NH
-terminal antibody (not
shown). Fourth, when a similar experiment was performed using a
polyclonal antibody that had been raised against a peptide
corresponding to the COOH-terminal 20 amino acids of TTP, a protein was
precipitated that had the same apparent electrophoretic mobility and
the same time course of expression (not shown). In this experiment, the
corresponding immunogenic peptide also blocked precipitation of the
protein with the TTP COOH-terminal antibody (not shown). Finally, the
43 kDa species was not precipitated by the TTP NH
-terminal
antibody from lysates isolated from serum-stimulated fibroblasts
prepared from TTP-deficient mice (see below). Taken together, these
experiments indicate that the TTP NH
-terminal antibody was
able to precipitate TTP from cell lysates; the remainder of the
immunoprecipitation experiments that are described in this paper were
performed using this antibody.
Figure 1:
Time course of
FCS-stimulated TTP accumulation in NIH/3T3 cells. Confluent,
serum-deprived cells were exposed to [S]cysteine
for a total of 4 h and to 20% FCS for the indicated times. Cell lysates
were prepared and used for immunoprecipitation with the TTP
NH
-terminal antibody. The precipitated protein was
separated on a 9% SDS-polyacrylamide gel. See the text for further
details.
Because almost no TTP is detectable
in serum-deprived cells, we established cell lines that constitutively
express TTP, even in the serum-deprived state, by stably transfecting
NIH/3T3 cells with a plasmid containing the TTP cDNA linked to the
metallothionein (mMT) promoter. In this plasmid, the 3`-untranslated
region of the TTP cDNA, which contains several AU-rich sequences that
are known to destabilize the TTP mRNA, had been removed and replaced
with an SV40 polyadenylation signal. The plasmid also contained a
neo gene; consequently, neo
cell clones were
isolated following transfection, and three of these cell lines were
assayed for expression of exogenous TTP by immunoprecipitation of
[
S]cysteine-labeled protein (Fig. 2). Line
A did not express exogenous TTP, but only expressed endogenous TTP
following a 2-h exposure to FCS. However, both lines B and C expressed
TTP in the quiescent state, and interestingly, there was a shift in
electrophoretic mobility of the protein following a 2-h FCS exposure.
This suggested that FCS caused a covalent modification of TTP of some
type, possibly phosphorylation.
Figure 2:
Effect of FCS on TTP protein expression in
mMT/TTP-expressing NIH/3T3 cells. Confluent NIH/3T3 (3T3)
cells or NIH/3T3 cells that stably expressed mMT/TTP
(A-C) were serum-deprived for 24 h; 100 µM
ZnSO was added for the final 15 h. The cells were then
incubated with [
S]cysteine for 2 h followed by
an additional 2 h of incubation under control conditions or with 20%
FCS. Cell lysates were prepared, protein was precipitated with the TTP
NH
-terminal antibody, and the precipitated protein was
separated on a 9% SDS-polyacrylamide gel. See the text for further
details.
Phosphorylation of TTP
To determine if FCS
stimulated phosphorylation of TTP, mMT/TTP-expressing cells (line
B) were exposed to P
for 4 h and to FCS
for varying times during those 4 h, and an immunoprecipitation was
performed of lysates from those cells (Fig. 3). In quiescent
cells, two protein species were precipitated that were each about 43
kDa. The upper species was detectable in untransfected, serum-deprived
NIH/3T3 cells as well as in TTP-deficient fibroblasts that had been
exposed to similar conditions (Fig. 3); these results indicate
that the upper band is not TTP, but rather, a co-immunoprecipitating
protein that probably contains a shared epitope. However, the lower
species was not detectable in untransfected, serum-deprived NIH/3T3
cells, or in TTP-deficient fibroblasts, under similar conditions
(Fig. 3), suggesting that it is basally phosphorylated, exogenous
TTP. Within 10 min of FCS stimulation, the lower species was no longer
detectable, and there was a marked increase in the intensity of the
upper species; this effect was constant to at least 2 h (
Fig. 3
and data not shown). Because the effect was not seen in
immunoprecipitated protein from FCS-stimulated, untransfected NIH/3T3
cells (Fig. 3), FCS probably caused an increase in TTP
phosphorylation and a change in TTP electrophoretic mobility so that it
then comigrated with the epitope-related upper band. Therefore, it can
be concluded that although TTP is phosphorylated in quiescent cells
that constitutively express it, FCS causes a substantial increase in
TTP phosphorylation and a corresponding shift in its electrophoretic
mobility, similar to the shift seen with
[
S]cysteine-labeled TTP in NIH/3T3 cells
following FCS stimulation (Fig. 2).
Figure 3:
FCS-induced TTP phosphorylation in NIH/3T3
cells, TTP-deficient fibroblasts (TTP(-/-)), and
mMT/TTP-expressing cells. Confluent cells were serum-deprived for 24 h;
100 µM ZnSO was added for the final 15 h. The
cells were then incubated with
P
for 2 h
followed by an additional 15 min of incubation with 20% FCS. Cell
lysates were prepared, protein was precipitated with the TTP
NH
-terminal antibody, and the protein was separated on a 9%
SDS-polyacrylamide gel. Other details are as described in the
text.
To determine if other
mitogens could cause a similar increase in TTP phosphorylation,
mMT/TTP-expressing cells were labeled with P
and exposed to several mitogens for 15 min, and TTP from these
cells was immunoprecipitated (Fig. 4). FCS, PMA, PDGF, and FGF
all caused a substantial increase in TTP phosphorylation, but dibutyryl
cAMP and forskolin had no detectable effect; these studies suggested
that the cyclic AMP-dependent protein kinase probably did not mediate
TTP phosphorylation in cells under these conditions.
Figure 4:
Effect
of mitogens on TTP phosphorylation in mMT/TTP-expressing NIH/3T3 cells.
Confluent cells were treated as described in the legend to Fig. 3.
Following a 2-h exposure to P
, the cells were
exposed to control conditions, 1% (v/v) dimethyl sulfoxide
(DMSO), 20% (v/v) FCS, 1.6 µM PMA, 10 ng/ml PDGF,
10 nM FGF, 1 mM dibutyryl cAMP, or 100
µM forskolin for 15 min. Immunoprecipitation was then
carried out as described in the legend to Fig.
3.
To determine
which amino acids on TTP are phosphorylated in vivo, the
intensely phosphorylated upper band from FCS-stimulated
mMT/TTP-expressing cells, and both the upper and lower bands from
quiescent mMT/TTP-expressing cells, were each isolated and subjected to
phosphoamino acid analysis. In each case, only phosphoserine was
detected (data not shown).
TTP Phosphorylation by p42
Many signaling cascades that are initiated by
extracellular signals at cell surface receptors converge on a common
signaling pathway that results in the activation of mitogen-activated
protein (MAP) kinases (also known as extracellular signal-regulated
kinases, or ERKs) (see Refs. 20 and 21 for reviews). Among the many
substrates that have been identified for the MAP kinases are several
transcription factors including c-Jun
(22) , c-Fos
(23) ,
c-Myc
(24, 25) , and p62in
Vitro
(26) . To
determine whether TTP was also a MAP kinase substrate, TTP was
synthesized in a rabbit reticulocyte lysate system, and the
S-labeled TTP was incubated with one of the activated MAP
kinase isoforms, p42
(17) (Fig. 5). The
electrophoretic mobility of TTP following incubation with p42
was shifted relative to protein that had been incubated under
control conditions (Fig. 5), indicating that p42
can phosphorylate TTP in vitro. This electrophoretic
shift was similar to that seen with TTP isolated from FCS-stimulated
NIH/3T3 cells (Fig. 2). To identify the site(s) in TTP that were
phosphorylated by p42
, a GST/TTP fusion protein was
created and used as a p42
substrate. Following
incubation of GST/TTP with p42
in the presence of
[
P]ATP, GST/TTP-
P was isolated
and exhaustively digested with several proteases (Fig. 6). One of
these proteases, endoproteinase Asp-N, cleaved the protein to produce
one major phosphopeptide of about 6 kDa. Among the TTP fragments that
are predicted to be created following endoproteinase Asp-N digestion
are two fragments that are about 6 kDa (Fig. 7). Consequently,
two other fusion proteins were created that fused GST with these two
TTP fragments, amino acids 163-215 and amino acids 163-235,
and each fusion protein was incubated with p42
(Fig. 8). Although GST/TTP(163-235) was a substrate
for p42
under these conditions, GST/TTP(163-215)
was not, indicating that the phosphorylation site(s) was (were) between
amino acids 216 and 235. In this region of TTP, there are 3
serines
(1) , but only serine 220 falls within the MAP kinase
phosphorylation consensus sequence of P-X-S/T-P
(20) .
To determine if serine 220 was phosphorylated by p42
,
another fusion protein was created that fused GST with a full-length
TTP in which serine 220 had been mutated to alanine. This fusion
protein, GST/TTP(S220A), was phosphorylated by p42
to a
much lesser extent than was GST/TTP under identical conditions
(Fig. 8). Consistent with this result, a synthetic peptide
corresponding to TTP amino acids 211-230 was a good substrate for
p42
in vitro, whereas an otherwise identical
peptide in which alanine was substituted for serine 220 was a much
poorer substrate (Fig. 9). Preliminary kinetic analysis indicated
that phosphorylation of the peptide that corresponded to amino acids
211-230 was half-maximal at about 250 µM substrate
(not shown). These experiments demonstrate that serine 220 is a major
phosphorylation site for p42
in vitro.
Figure 5:
Effect of
p42 on in vitro synthesized TTP. TTP was
synthesized in a rabbit reticulocyte lysate system and then incubated
in a kinase buffer containing ATP and no kinase (C) or
p42
(MAP) for 30 min. The incubated protein was
separated on a 9% SDS-polyacrylamide gel. The experiment was carried
out in duplicate with five times more protein in the second set of
samples.
Figure 6:
Proteolytic digestion of GST/TTP that had
been phosphorylated by p42. GST/TTP was phosphorylated
by p42
in vitro as described in the text, and
the reaction mixture was separated on a 9% denaturing polyacrylamide
gel. Full-length GST/TTP-
P was excised from the dried gel,
and the protein was eluted from the gel slice. The protein was then
incubated with nothing (control), trypsin, endoproteinase Arg-C,
endoproteinase Asp-N, or endoproteinase Glu-C. The digested protein was
separated on a 20% SDS-polyacrylamide gel. Other details are as
described in the text.
Figure 7:
Predicted TTP endoproteinase Asp-N
proteolytic fragments. The major TTP proteolytic fragments following
digestion with endoproteinase Asp-N are displayed. Diagrams of two GST
fusion proteins that were constructed based on the size of the digested
fragments are also shown.
Figure 8:
Phosphorylation of GST/TTP fusion proteins
by p42in vitro. A, the indicated
fusion proteins were incubated with p42
in the presence
of [
-
P]ATP for 30 min and then separated on
a 9% SDS-polyacrylamide gel. An autoradiograph of this gel is shown.
B, equal amounts of the total protein from the indicated GST
fusion protein preparations were separated on a 9% SDS-polyacrylamide
gel that was then stained with Coomassie Blue. This gel is identical to
that from which the autoradiograph in A was
prepared.
Figure 9:
Peptide phosphorylation by
p42. Synthetic peptides corresponding to the indicated
amino acids of the mouse TTP protein were incubated with p42
for 30 min under the conditions described in the text. Following
incubation, the peptides were separated from unincorporated
[
-
P]ATP on a 20% SDS-polyacrylamide gel; an
autoradiograph of the gel is shown. Exposure time was 1.3
h.
Phosphorylation of Serine 220 in Vivo
To
determine if serine 220 was phosphorylated in intact cells in response
to mitogen stimulation, two stable NIH/3T3 cell lines were created that
each constitutively expressed TTP(S220A) under control of the mouse
metallothionein promoter. Phosphorylation of TTP(S220A) in these cells
was compared to that of wild-type TTP in mMT/TTP-expressing cells
(Fig. 10). Following FCS stimulation, an increase in
phosphorylation of TTP(S220A) was seen, as was a shift in the
electrophoretic mobility of the mutant protein. However, the mobility
shift of TTP(S220A) was significantly decreased compared to the
mobility shift of wild-type TTP. The same effect on electrophoretic
mobility was seen in protein precipitated from both
[S]cysteine and
P
-labeled cells (Fig. 10). The fact that
the mobility shift of TTP(S220A) was decreased indicates that serine
220 is a site of FCS-stimulated TTP phosphorylation, but the fact that
some of the TTP(S220A) mobility shift remained suggests that there are
other phosphorylation site(s) that remain to be identified. The extent
of the electrophoretic shift seen with the mutant protein appeared to
be about 50% of that seen in the wild-type protein, suggesting the
possibility that only one FCS-stimulated phosphorylation site remains
to be identified in TTP(S220A).
Figure 10:
The
effect of FCS on TTP and TTP(S220A) phosphorylation in NIH/3T3 cells.
Cells that stably expressed mMT/TTP or mMT/TTP(S220A) were
serum-deprived for 24 h; 100 µM ZnSO was added
for the final 15 h. The cells were then incubated (A) with
[
S]cysteine for 2.5 h or (B) with
P
for 2 h, followed by an additional 15 min
incubation under control conditions or with 20% FCS.
Immunoprecipitation, electrophoresis, and autoradiography were carried
out as described in the legend to Fig. 3. Note that mMT/TTP(S220A) A
cells expressed roughly comparable amounts of
S-labeled
TTP compared to wild-type mMT/TTP cells.
. These results suggest that
TTP function might be regulated by mitogens at the level of reversible
phosphorylation.
of the protein is shifted
significantly by mitogen stimulation in cells suggests that this
modification occurs in vivo with significant stoichiometry; in
fact, the lower TTP species present in the basal or serum-deprived
cells completely disappears in favor of the more highly phosphorylated
species after only 10 min of mitogen treatment. In cell-free kinase
assays, the GST-TTP fusion protein can be phosphorylated by
p42
. Finally, serine 220 and its surrounding minimal MAP
kinase consensus sequences are conserved in the
mouse
(1, 2, 3) , human
(7) , and
bovine
sequences; all of these species contain the sequence
L-S-P at this site (Fig. 11). These data suggest that there are
at least two physiologically significant phosphorylation sites on TTP,
one being serine 220, and that phosphorylation on one or more sites
occurs with a stoichiometry to make it physiologically important.
Figure 11:
Alignment of the mouse (mttp),
rat (rttp), bovine (bttp), and human (http)
TTP protein sequences. Alignments were performed by the program
Pileup (Genetics Computer Group, Madison, WI); periods in the sequences indicate spaces introduced by the program to
optimize alignments. Sequence identities are indicated by asterisks (*). The putative CCCH zinc finger regions have been placed in
brackets, and the C and H residues that may coordinate
Zn
have been highlighted. The identified MAP
kinase site in the mouse protein (Ser
) is noted with a
large arrow, and the remaining seven minimal MAP kinase
consensus sequences (S-P) that are conserved in all four proteins are
noted with small arrows.
Although our work shows that p42 can phosphorylate
TTP in cell-free assays and suggests that p42
might also
phosphorylate TTP in intact cells, it is possible that other kinases
phosphorylate TTP in intact cells rather than, or in addition to,
p42
. Because serine 220 in TTP is followed by a proline,
it seems likely that these additional kinase(s) are proline-directed
kinases. In addition to p42
, other proline-directed
kinases include two other MAP kinases, p44
(20, 21) and p54
(27) , and a group of
MAPK-related kinases, the c-Jun amino-terminal kinases
(JNKs)
(28) . Each of these kinases has a similar substrate
specificity
(20, 28) and could conceivably phosphorylate
TTP serine 220, as well as other serines in TTP. Another
proline-directed kinase is the cdc2 kinase (29). However, none of the
serines in TTP fall within the cdc2 phosphorylation consensus sequence,
S-P-X-R/K
(29) . Finally, most of the mitogens that
increase TTP phosphorylation in cells can activate protein kinase C;
however, the GST-TTP fusion protein was not a substrate for the
constitutively active form of mixed brain protein kinase C isozymes in
cell-free assays.
and p44
can phosphorylate a
site within the c-Jun COOH-terminal domain, and phosphorylation at this
site inhibits DNA binding
(22) . It is not obvious how
phosphorylation of TTP at serine 220 would alter DNA binding, since
this residue lies well outside of the putative zinc finger region,
which is between amino acids 95 to 159 in the mouse protein
(Fig. 11).
where it activates transcription of the HO endonuclease; during
other phases of the cell cycle, SWI5 translocates to the
cytoplasm
(32) . Phosphorylation of SWI5 is catalyzed by the
Cdc28 kinase in conjunction with an activating cyclin subunit and
appears to be responsible for the nuclear to cytoplasmic
translocation
(33) . Phosphorylation of TTP could also affect its
association with putative regulatory proteins, as in the
NF-
B/I-
B interaction
(34, 35) .
,
phosphorylation on other serine(s) was relatively minor in this system;
hence, the other phosphorylation sites may be difficult to identify
using the same approach. (
)In addition, efforts to identify
the other sites will be complicated by the fact that the mouse TTP
protein contains 55 serine residues; 8 of these fall into the minimal
MAP kinase recognition sequence of S-P
(1, 20, 21) and are conserved among the mouse, rat, human, and bovine
TTP proteins (Fig. 11). Efforts are currently underway to
identify these remaining site(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.