From the Institut für Zellbiologie, Abteilung
Molekularbiologie, Universität Tübingen, D-72076
Tübingen, Germany, the § Max-Delbrück-Center
für Molekulare Medizin, D-13122 Berlin-Buch, Germany, the
¶ Institut für Klinische Chemie, Medizinische Hochschule
Hannover, D-30623 Hannover, Germany, and the ** Innovationskolleg
Zellspezialisierung, Martin-Luther-Universität,
06120 Halle, Germany
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ABSTRACT |
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Several growth factor- and calcium-regulated
kinases such as pp90rsk or CaM kinase IV can phosphorylate the
transcription factor serum response factor (SRF) at serine 103 (Ser-103). However, it is unknown whether stress-regulated kinases can
also phosphorylate SRF. We show that treatment of cells with
anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate induces
phosphorylation of SRF at Ser-103 in both HeLa and NIH3T3 cells. This
phosphorylation is dependent on the kinase p38/SAPK2 and correlates
with the activation of MAPKAP kinase 2 (MK2). MK2 phosphorylates SRF
in vitro at Ser-103 with similar efficiency as the small
heat shock protein Hsp25 and significantly better than CREB. Comparison
of wild type murine fibroblasts with those derived from MK2-deficient
mice (Mk( The serum response element
(SRE),1 a growth factor- and
stress-regulated promoter sequence, is essential for the expression of
immediate early genes such as c-fos and egr-1 (1,
2). SREs are bound by the transcription factor serum response factor (SRF) permitting the recruitment of SRF accessory factors like the
Ets-family ternary complex factors (TCFs). SRF binds with its MADS
domain as a homodimer to the CArG box, which has the consensus sequence
C2(A/T)6G2 (1). Ternary complex
factors, namely Elk-1, Sap-1, and Net-1 can only bind to the
c-fos SRE via adjacent GGAT Ets-binding sites in the
presence of SRF. In addition, on its 3'-site the c-fos
SRF-binding site is flanked by an AP-1-like site (3). In
vivo footprinting studies suggest that all three sites of the
c-fos SRE element are continuously occupied (4).
The SRE is a converging point for several signal transduction cascades.
Particularly well documented is the role of TCFs as nuclear targets in
signal transduction. C-terminal TCF phosphorylation by MAP kinases such
as ERKs, JNKs, or p38 members results in transcriptional activation
(5-11). In contrast, the role of SRF in phosphorylation-regulated gene
expression is not well understood. Small GTP-binding proteins of the
Rho family regulate c-fos expression via SRF (12, 13). However, it is unclear which kinase cascade transmits the signal toward
SRF, and whether SRF is a direct target of phosphorylation, or instead
acts primarily as a docking element for another protein. SRF contains
several phosphorylation sites (14), although functional consequences,
particularly of C-terminal SRF phosphorylation, have not been
identified unequivocally. N-terminal phosphorylation including that of
Ser-103 immediately N-terminal to the MADS box affects the
interaction of SRF with its DNA recognition sequence (14-16).
Two groups of kinases have been shown so far to phosphorylate SRF at
Ser-103: pp90rsk, also known as MAPKAP-K1, which is activated
by serum via ERKs, as well as CaM kinases II and IV, which are
regulated by calcium levels (16, 17). We wondered whether a link
existed between activation of gene expression by Rho GTPases and SRF
phosphorylation at Ser-103. Here we investigated whether agents known
to activate SRF-dependent gene expression, possibly
involving activation of or cooperation with Rho GTPases, cause SRF
phosphorylation at Ser-103. In addition, we intended to identify
kinases phosphorylating SRF. Using in-gel kinase assays we screened for
SRF kinases induced by anisomycin, tetrafluoroaluminate, arsenite, and
stress factors, and measured the resulting SRF phosphorylation of
Ser-103. We show that these factors activate p38-dependent
SRF kinases. In particular we identify MAPKAP-K2 (MK2) as an SRF kinase
and demonstrate that MK2 contributes to SRF phosphorylation at Ser-103
in vivo. In Mk2( Cell Culture and Cell Treatment--
Cells were kept at
37 °C, 5% CO2 and 92% humidity. NIH3T3 cells were
cultured in Dulbecco's modified Eagle's medium containing 10% fetal
calf serum (FCS), 100 units/ml penicillin, and 100 mg/l streptomycin,
HeLa cells in minimal essential medium containing 10% FCS, 100 units/ml penicillin, and 100 mg/liter streptomycin. For
immortalization, mouse embryonic fibroblasts derived from Mk2(+/+) and Mk2( Cell Extracts and Immunoprecipitations--
For the preparation
of cell extracts cells were grown on 10-cm dishes. After treatment,
cell culture supernatants were discarded and cell layers were washed
with 5 ml of ice-cold phosphate-buffered saline (PBS) containing 50 mM sodium fluoride and 2 mM
Na3VO4. The washing solution was removed
completely followed by addition of 400 µl of lysis buffer (10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 30 mM sodium pyrophosphate, 5 µM
ZnCl2, 1% Triton X-100, 400 nM ocadaic acid,
10 mM 4-nitrophenyl phosphate, 20 mM
To immunoprecipitate MK2 and JNKs, cell extracts (25 µl) were
incubated with 10 µl of a 50% suspension of anti-MK2/3 (20) or
anti-JNK (Pharmingen) antibody-protein Sepharose G (3 µg of antibody)
for 2 h on ice. After centrifugation and washing twice with lysis
buffer containing 500 mM NaCl followed by two further washes with lysis buffer, the conjugate was resuspended in 20 µl of
sample buffer and subjected to an in-gel kinase assay.
EMSA Supershift Studies--
Reaction mixtures (10 µl)
contained 10 mM Tris-Cl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 5% glycerol, 0.05% low fat milk, 1 mM dithiothreitol, 0.5 g/l salmon sperm DNA, 25,000 cpm of
32P-labeled DNA probe containing the c-fos SRE
(100 fmol, 6 ng) and 30-50% (v/v) cell extract. To analyze the SRF
phosphorylation status at Ser-103, 0.5 µl of affinity purified
anti-P-SRF antibody was included in the reaction mixture. This
phospho-specific antiserum recognizes SRF when phosphorylated at
Ser-103 (16, 17). After a 30-min incubation period at room temperature
the DNA-protein complexes were separated on a 5% polyacrylamide gel
containing 0.5 × Tris borate buffer at 10 V/cm. After
electrophoresis gels were dried and exposed to PhosphorImaging
screens (Fuji).
Purification of Serum Response Factor--
To express
recombinant SRF, a BamHI/EcoRI fragment
containing the SRF gene with an N-terminal His-tag was cloned into
pILA503 (21). His-tagged SRF was purified from Escherichia
coli BL21-LysS/pILASRF after heat induction. Pelleted cells were
resuspended in 5 ml of buffer A (6 M guanidinium-HCl, 100 mM NaH2PO4, 10 mM Tris, 0.1% Triton X-100, pH 8.0) per g of pellet net weight and swirled for
1 h at room temperature. After addition of 1.25 ml of Ni-NTA agarose (Qiagen) the suspension was incubated under rotation for another 45 min. The suspension was transferred into a column and washed
three times with 4 column volumes of buffer C (8 M urea, 100 mM NaH2PO4, 10 mM
Tris, 0.1% Triton X-100, pH 6.3). SRF was eluted with 8 column volumes
of buffer E (8 M urea, 100 mM
NaH2PO4, 10 mM Tris, 0.1% Triton
X-100, pH 4.1). Fractions of 2 ml were collected and analyzed by
SDS-PAGE. SRF-containing fractions were combined, glycerol was added to
10%, and the solution was dialyzed against 100 fraction volumes of
dialysis buffer (100 mM NaH2PO4, 10 mM Tris, 0.1% Triton X-100, pH 8.0) containing 4 M urea and 25% glycerol at 4 °C. After every 2 h
the dialysis buffer was exchanged against fresh buffer containing a
2-fold lower concentration of urea. After final dialysis overnight at
4 °C against urea-free dialysis buffer containing 43% glycerol, SRF
aliquots were frozen in liquid nitrogen and stored at In-Gel Kinase Assay--
Cell extracts were mixed with an equal
volume of sample buffer (20% glycerol, 2% SDS, 25 mg/l bromphenol
blue, 5% 2-mercaptoethanol, 125 mM Tris-Cl, pH 6.8),
heated for 5 min at 95 °C and cooled on ice. The samples were loaded
on 10% or 12% SDS-polyacrylamide gels containing 2.5 mg/l His-SRF or
40 mg/liter of the other substrates used. After electrophoresis at 20 V/cm the gels were incubated for 30 min in 250 ml of DR buffer (25 mM HEPES pH 7.4, 10 mM dithiothreitol) containing 20% 2-propanol followed by 30 min in 250 ml of DR buffer. The proteins were denatured by incubation for 1 h in 150 ml of DR
buffer containing 6 M guanidinium thiocyanate with exchange of the buffer after 30 min. Renaturation of electrophoresed proteins was performed by incubation of the gel in 250 ml of DR buffer containing 0.04% Tween 20 at 4 °C overnight, followed by a 2-h incubation with two buffer exchanges. After incubation at room temperature for 1 h in 240 ml of kinase buffer (25 mM
HEPES pH 7.4, 10 mM dithiothreitol, 10 mM
MgCl2, 90 µM Na3VO4),
the gel was swirled in 10 ml of kinase buffer containing 5 MBq of
[ In Vitro Phosphorylation and Immunoblotting--
For in
vitro phosphorylation, constitutively active kinase MK2 Peptide Sequencing--
For the detection of MK2 phosphorylation
sites in SRF, 20 µg of either full-length SRF or core SRF (amino
acids 90-244 of SRF) were labeled as described above, with the
exception of an extended labeling time of 8 h.
32P-Labeled SRF (0.4 mg/ml) was digested with 0.4 µg of
endopeptidase Lys-C (Roche Molecular Biochemicals, Mannheim, Germany)
in 25 mM Tris-HCl buffer, pH 8.1, for 14 h at
37 °C. Digestion was terminated by addition of one-fifth volume of
10% trifluoroacetic acid. Peptides generated were fractionated by
reversed phase-HPLC applying the mixture to a µRPC C2/C18 SC 2.1/10
narrow bore column from the SMARTTM System (LKB Pharmacia, Sweden)
with peak detection. The peptides were eluted with 0.1%
trifluoroacetic acid buffer using a gradient of increasing
concentrations of acetonitrile (5-50%). Elution was for 60 min and a
flow rate of 100 µl/min at room temperature was applied. About 80 fractions were automatically collected and subjected to Cerenkov
counting. Peptide identification in the radioactive fractions was first
performed conventionally with a Procise protein sequencer (Perkin-Elmer
Applied Biosystems model 494A) operating with the routine sequencer
program. Then, another part of the fraction was covalently bound to a
Sequelon arylamine membrane (Millipore) and solid phase sequenced
collecting the anilinothiazolinones after their extraction from the
arylamine disc (methanol/water, 9:1, 1 mM phosphate) with
the fraction collector of an Applied Biosystems 477A sequencer. At each
cycle of Edman degradation 32P radioactivity released was measured.
Induction of SRF Phosphorylation at Ser-103 Correlates with the
Activation of p38/SAPK2 and MK2--
Stimulation of serum-starved
NIH3T3 fibroblasts with FCS results in a rapid and transient induction
of SRF phosphorylation at Ser-103, which, at least in part, can be
attributed to the activity of the protein kinase pp90rsk
(MAPKAP-K1) (16). However, other downstream kinases of the MAPK
cascades, such as MAPKAP-K2 (MK2) (22) may also be involved in SRF
phosphorylation. The closely related MAPKAP-K3 (MK3) (23, 24) has
recently been shown not to phosphorylate SRF (25). To examine more
specifically the role of stress-activated protein kinase (SAPK)
pathways of the JNK and p38 classes, we stimulated HeLa cells with
anisomycin and tetrafluoroaluminate which have been recently shown to
induce immediate early gene expression including c-fos by
activating or cooperating with Rho GTPases (12, 19, 13). We analyzed
kinase activities and SRF phosphorylation by EMSA supershift assays
using an
Using EMSA supershift analysis we first examined the extent of SRF
Ser-103 phosphorylation before and after treatment of cells with the
different agents. As a DNA-binding probe we used the c-fos
SRE sequence. Addition of a phospho-Ser-103-specific
To characterize potential SRF kinases induced by anisomycin, sodium
fluoride, and tetrafluoroaluminate, the HeLa cell lysates used in Fig.
1 were analyzed by in-gel kinase assays with copolymerized SRF (Fig.
2A). Several bands, particularly at 50, 55, and 60 kDa become visible after induction, with the 50-kDa band being the most
prominent one. This increase in intensity cannot be attributed to
autophosphorylation, since the corresponding region in the substrate-free control gel shows only weakly activated kinase bands
(Fig. 2C, compare indicated bands in lanes 3, 4, 7, or 10 with lane 1). Interestingly,
anisomycin (lanes 2-4) induces the corresponding kinases
within 15 min and, therefore, more rapidly than either sodium fluoride
(lanes 5-7) or tetrafluoroaluminate which required 30 min
(lanes 8-10). With these three stimulators the induction
lasts for at least 60 min. Aluminum chloride does not induce any SRF
kinase (lanes 11-13). Therefore, the activation kinetics of
these SRF kinases parallel the phosphorylation kinetics of SRF at
Ser-103 (Fig. 1).
Since the size of the 55-kDa band seen in Fig. 2A agrees
with the reported molecular weight of JNK2 (26), we examined JNK/SAPK1 activation in parallel in the same lysates using, instead of SRF, c-Jun
as in-gel substrate (Fig. 2B). In c-Jun-containing gels, a
different pattern of induced kinases compared with SRF gels is
observed. Whereas the 50-kDa kinase is absent, two bands of 46 and 55 kDa are strongly induced. Thus, these two bands very likely reflect
activated JNK1 and -2. As already seen for SRF kinases (Fig.
2A), anisomycin (Fig. 2B, lanes 2-4) activates
both JNKs with faster kinetics than tetrafluoroaluminate (lanes
5-7) and sodium fluoride (lanes 8-10). Again, no Jun
kinase is activated by addition of aluminum chloride (lanes
11-13). The observed increase in intensities is not due to
autophosphorylation, as shown by the control gel (Fig. 2C).
Therefore, anisomycin, sodium fluoride, and tetrafluoroaluminate
apparently induce the same SRF and Jun kinases, respectively, but with
different kinetics. Aluminum chloride does not induce any kinase in the
in-gel kinase assays used here.
Based on the apparent molecular mass, the 50-kDa band seen in the SRF
gel of Fig. 2A may represent human MK2 (26). To examine directly the possible activation of MK2 and its upstream activating kinase, namely p38/SAPK2, both kinases were immunoprecipitated from the
respective HeLa cell extracts followed by analysis of their activities
by in vitro phosphorylation assays (Fig. 3). In contrast to
aluminum chloride, which does not activate either MK2 (Fig.
3A) or p38/SAPK2 (Fig. 3B), anisomycin, sodium
fluoride, and tetrafluoroaluminate lead to activation of both MK2 and
p38/SAPK2. Anisomycin activates both kinases within 15 min to maximal
extent, whereas maximal activation of MK2 and p38/SAPK2 by sodium
fluoride or tetrafluoroaluminate takes more than 30 min. In addition,
p38/SAPK2 isolated from sodium fluoride- or
tetrafluoroaluminate-treated cells exhibits only 50% of the activity
caused by anisomycin treatment. Thus, despite similar extents of MK2
activation, sodium fluoride and tetrafluoroaluminate are less potent
p38 inducers than anisomycin. However, anisomycin-, sodium fluoride-,
and tetrafluoroaluminate-mediated activation of the p38/MK2 signal
transduction pathway correlates with the induction of SRF
phosphorylation at Ser-103.
MK2 Is a Potential SRF Kinase--
Since activation of MK2
parallels SRF phosphorylation at Ser-103, we suspected that MK2 might
directly phosphorylate SRF. To test whether MK2 phosphorylates SRF
in vitro, we analyzed recombinant constitutively active MK2
and anisomycin-induced HeLa extracts using in-gel kinase assays.
Different in-gel substrates were employed (Fig.
4, A-F). Hsp25 is a published
MK2 substrate (27) and served as a positive control for renaturation of
MK2 during in-gel kinase assays. The size of recombinant MK2 is 66 kDa
due to its GST tag. In this assay both the recombinant as well as
endogenous anisomycin-induced MK2 phosphorylate Hsp25 and SRF (Fig. 4,
A and B). Recombinant MK2 also phosphorylates
c-Jun, whereas endogenous MK2 is probably masked by prominent JNK/SAPK1
activity (Fig. 4C). Endogenous MK2 phosphorylates the MK2
substrate weakly, whereas with recombinant MK2 autophosphorylation is
not visible (Fig. 4E). This finding is consistent with the
autophosphorylation of the 50-kDa band observed in Fig. 2C.
Neither recombinant nor endogenous MK2 phosphorylate the TCF/Ets
transcription factor Elk-1 (Fig. 4D) or histone H1 (Fig.
4F). However, Elk-1 (Fig. 4D), as well as c-Jun
(Fig. 4C), are efficiently phosphorylated by two 46- and 55-kDa kinases
correlating with the molecular masses of JNKs. Taken together, SRF is
phosphorylated by MK2 to a similar extent as its well established
substrate Hsp25. In contrast, the SRF-interacting TCF Elk-1 is not a
substrate for MK2 under in-gel kinase assay conditions, nor in solution in MK2 enzyme reactions (data not shown).
To verify that MK2 was the SRF kinase observed in Fig. 2A,
we immunoprecipitated MK2 from the same extracts using an antiserum specific for MK2 and MK3 followed by in-gel kinase analysis of the
precipitated kinases. Since MK3 does not phosphorylate SRF in
vitro (25), only MK2 should be visible on SRF gels. For these experiments we took again the same extracts as used in Figs. 1-3. In
addition, we examined extracts from arsenite-treated HeLa cells, since
arsenite is a potent inducer of both JNKs and p38 kinases (29). Whereas
in extracts from untreated HeLa cells no SRF kinases are precipitated,
anisomycin, sodium fluoride, tetrafluoroaluminate, and arsenite induce
the same SRF kinases to similar extents (Fig. 4G, lanes 4, 7, 10, 13, and 16). Immunoprecipitations with Arsenite Induces in NIH3T3 Cells p38-dependent
Phosphorylation of Cellular SRF at Ser-103--
Activation of
SRE-mediated gene expression in HeLa cells needs additional elements,
such as TCF-binding sites, besides the CArG box (30). In contrast, in
the mouse fibroblast line NIH3T3 the CArG box seems to be sufficient to
mediate activation of gene expression by FCS or Rho GTPases (12). Since
this signal transduction may involve SRF phosphorylation, it was of
interest to investigate a possible MK2-mediated SRF phosphorylation in
NIH3T3 cells. As an inducing agent we chose arsenite because of its
efficient and relatively restricted activation of JNK/SAPK1 and
p38/SAPK2 (29).
In contrast to HeLa extracts (Fig. 1, lane 2), NIH3T3 cell
extracts show in EMSA studies a singular band which contains the binary
SRE-SRF complex. No ternary complex containing the TCF protein ELK-1 is
observed (Fig. 5A and data not
shown). In agreement with these results, mutation of the TCF-binding
site does not change the band shift pattern (data not shown). However,
the complex may include the TCF protein Net-1 which does not cause a
significant retardation compared with the binary complex containing
only an SRF dimer (31). Treatment of serum-starved NIH3T3 cells with serum (FCS) results in a rapid induction of SRF phosphorylation as
indicated by a supershift upon addition of
As already mentioned above, arsenite causes activation of both JNK and
p38 kinase family members. In addition, recent data suggest an
arsenite-dependent activation of the ERK pathway via p38/SAPK2 (32). To distinguish between different MAPK cascades potentially involved in SRF phosphorylation, we induced serum-starved cells with arsenite in the presence of either the p38-specific inhibitor SB202190, a closely related analog of SB203590 (33), or,
alternatively, with PD98059, which prevents activation of ERKs due to
inhibition of MEK-1 stimulation (34). Pretreatment of cells with 10 or
30 µM SB202190 reduces the extent of arsenite-induced SRF
phosphorylation from 80 to 40 and 30%, respectively (Fig. 5B,
lanes 2-4). In contrast, PD98059 hardly affects the
arsenite-influenced phosphorylation status of SRF at all (lanes
5 and 6). These results suggest the p38 pathway as the
primary MAPK cascade involved in arsenite-induced SRF phosphorylation
at Ser-103. Other pathways such as the JNK/SAPK1 or ERK cascades
contribute only to a minor extent, collectively amounting to not more
than 30% of SRF Ser-103 phosphorylation.
Besides activation of MK2 (22), the p38 pathway induces several other
downstream kinases such as MK3 (23, 24), MNKs (35, 36), MSKs (37), and
PRAK (38) (for reviews, see Refs. 18 and 28). Of all these kinases,
only MK2 is exclusively activated by p38/SAPK2 (22). Whereas MK3 does
not phosphorylate SRF in vitro (25), other kinases have not
been tested yet. Thus, we wondered whether MK2 was the major SRF kinase
in NIH3T3, and whether NIH3T3-derived JNKs also phosphorylated SRF,
thereby paralleling the results obtained with HeLa cell extracts. To
test for this, we performed in-gel kinase assays with SRF as in-gel
substrate using the identical NIH3T3 extracts investigated in Fig.
5B. Extracts from serum-starved NIH3T3 cells show a major
band at 98 kDa and minor bands at 200 kDa and around 60 kDa (Fig.
6A, lane 1). Since these bands
are also visible with similar intensities in control gels (data not
shown), they are due to autophosphorylation of unidentified protein
kinases. NIH3T3 extracts differ from HeLa extracts in the pattern of
arsenite-induced SRF kinases (Fig. 6A, lanes 2 and
3). Particularly the 50-kDa band corresponding to human MK2
is missing in NIH3T3 extracts (lane 2). Instead, arsenite
treatment of NIH3T3 results in the induction of a kinase band around 45 kDa and in increased intensities of two bands at 55 and 60 kDa
(lane 3). All three bands can be also seen weakly in the
control gels lacking substrate, but particularly the p55 and p45 bands
are significantly stronger in the SRF-containing gels (Fig. 6A,
lane 3 and data not shown). Thus, the protein kinases corresponding to 55 and 45 kDa, although capable of
autophosphorylation, also phosphorylate SRF in this experiment. The
sizes of the two bands correlate well with molecular masses published
for two murine MK2 isoforms (26) and, therefore, would correspond
precisely to the results obtained with HeLa extracts (lane
2). Interestingly, SB202190 (Fig. 6A, lanes 4-6), but
not PD98059 (lanes 7-9), completely inhibits activation of
the p45 kinase, whereas the intensity of the p55 band is only partially
reduced. It is unlikely that this band is only less susceptible than
p45 to SB202190, since increasing the inhibitor concentration to
100 µM does not cause a further reduction of this band
(lane 6). Instead, the reason for this partial reduction
might be the comigration of the p55 form of MK2 with another SRF kinase
of 55 kDa, such as JNK2 which has the same apparent molecular mass
(26). A similar band was already observed in HeLa extracts (Fig.
2A). Thus, based on these inhibitor studies, in both HeLa
and NIH3T3 cells, arsenite induces MK2 via the p38 kinase cascade. In
addition, at least JNK2 may also phosphorylate SRF whereas the Erk
pathway apparently does not contribute to arsenite-stimulated SRF
phosphorylation. Furthermore, the activation kinetics of these kinases
and their inhibition by SB202190 suggest that MK2 phosphorylates SRF at
Ser-103.
As already stated above, the sensitivities of p45 and p55 to treatment
with SB202190 suggests that they are murine MK2 isoforms (26), which
are activated by p38/SAPK2. To further characterize these observed SRF
kinase activities, we immunoprecipitated MK2/3 from the cell extracts
already used in Figs. 5B and 6A and analyzed the
MK2 activation status by in-gel kinase assays using SRF as in-gel
substrate (Fig. 6B). The MK2 Phosphorylates SRF at Ser-103 in Vitro--
To confirm the
data obtained with cell extracts and to identify the SRF site
phosphorylated by MK2, we incubated recombinant SRF protein with a
constitutively active form of MK2 in the presence of
[
The sequence flanking Ser-103 of SRF, i.e.
LKRSLS103EM, conforms to the consensus sequence
LXRXXSXX required for MK2-mediated phosphorylation. To examine if purified MK2 phosphorylates SRF in
vitro at Ser-103, SRF was phosphorylated as described in the previous paragraph followed by immunoblotting with the
To further investigate this notion, the major phosphorylation site for
MK2 within the SRF substrate was identified empirically. For this
purpose, SRF was labeled in vitro by MK2 as described above
and was eluted from the gel, digested using endopeptidase Lys-C and the
resulting peptides were fractionated by reversed phase-HPLC. In two
fractions (asterisks in Fig. 7C)
32P-radioisotope could be detected indicating the existence
of phosphopeptides in these samples. Subsequent Edman degradation of
these samples revealed two peptides with different lengths both
starting with residue Arg-100 of SRF and ending with either Lys-135 or
Lys-138, respectively. Detection of 32P release during each
Edman degradation cycle (Fig. 7D) demonstrates single
phosphorylation of SRF by MK2 at position Ser-103, supporting the data
obtained above with the phosphorylation specific antiserum. Taken
together, the above results demonstrate that MK2 phosphorylates SRF at
Ser-103, and that this is the major site of phosphorylation.
MK2 Contributes to SRF Phosphorylation at Ser-103 in Vivo--
The
results presented so far demonstrate that inside cells the kinetics and
inhibition profiles of SRF phosphorylation and MK2 activity overlap,
and that MK2 can phosphorylate SRF at Ser-103 in vitro.
However, the question remained whether MK2 is a true in vivo
SRF kinase, and if so, whether it is a major SRF kinase. Other
arsenite-induced kinases such as PRAK may also phosphorylate SRF at
Ser-103. To test for MK2-dependent SRF phosphorylation in vivo, we used embryonal fibroblasts derived from
MK2-deficient mice obtained by homologous recombination. These mice
were generated in a separate
study.3 As a control we used
fibroblasts derived from wild-type mice. Analysis of SRF
phosphorylation at Ser-103 was performed by EMSA supershift studies
using extracts of these Mk2( SRF and SRF-containing complexes are important nuclear relays
between cellular signaling and gene activity. SRF serves both as a
direct and indirect target of signaling cascades. SRF contains multiple
phosphorylation sites, of which Ser-103 has been investigated in more
detail (14, 15, 41, 42). Phosphorylation of Ser-103 has been shown
previously to be regulated by growth factors or calcium levels and to
be mediated, at least in vitro, by pp90rsk, CaMKII,
CaMKIV, and protein kinase A (16, 17, 43). We show in this report that
activation of SAPK pathways by agents such as anisomycin, arsenite,
sodium fluoride, or tetrafluoroaluminate leads to SRF phosphorylation
at Ser-103 (Figs. 1, 5, and 8). Thus, SRF is targeted by several kinase
cascades (Fig. 9) and, therefore, is a
converging point for both growth factor-regulated and stress-activated signaling cascades as is the case for its interaction partners Elk-1,
Net-1, and Sap-1a (6, 8, 10, 11, 44).
/
)) reveals MK2 as the major SRF kinase
induced by arsenite. These results demonstrate that SRF is targeted by
several signal transduction pathways within cells and establishes SRF
as a nuclear target for MAPKAP kinase 2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
) fibroblasts
arsenite-induced SRF phosphorylation at Ser-103 is greatly reduced. Our
data identify SRF as a direct intracellular target of signal
transduction cascades activated by tumor-promoting and stress-inducing
stimuli (18).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
/
)
mice2 were transfected with
pSV40Tag, coding for the SV40 large T antigen, and pREP8 (Invitrogen)
in a 10:1 mixture. Clones were selected with 3 mM
histidinol. Prior to stimulation cells were incubated for 24-48 h in
serum-free medium. Cells were stimulated with either 20% FCS, 10 mM sodium fluoride, 100 µM AlCl3,
AlF4
(100 µM AlCl3 and
10 mM sodium fluoride), 250 µM
AsNaO2 or 10 µg/ml anisomycin.
-glycerophosphate, 50 mM sodium fluoride, 2 mM Na3VO4, 500 µM
benzamidin, 1 unit/ml
-macroglobulin, 2 mg/l aprotinin, 500 µg/l
leupeptin, 500 µg/pepstatin, and 500 µM
phenylmethylsulfonyl fluoride) (19). Lysed cells were collected by
scraping, transferred into a 1.5-ml reaction tube, and vortexed.
Lysates were obtained by centrifugation for 15 min at 4 °C at
16,000 × g. Aliquots were frozen in liquid nitrogen and stored at
80 °C.
80 °C.
-32P]ATP for 1 h, followed by incubation in 250 ml of washing buffer (5% trichloroacetic acid, 10 mM
sodium pyrophosphate) for 2 days with frequent buffer exchanges.
Finally, gels were dried and exposed to PhosphorImaging screens.
3B
PC
was incubated in kinase assay buffer (50 mM sodium
-glycerophosphate, pH 7.4, 100 µM EDTA, 100 µM ATP (22 kBq/µl), 4 mM magnesium) acetate with 80 mg/liter substrate protein for 10 min at 30 °C. His-tagged SRF, His-tagged CREB and murine Hsp25 were used as substrates. For
immunoblotting analysis, 140 mg/l SRF were incubated for 30 min at
30 °C. The reaction was stopped by addition of sample buffer. After
denaturation at 65 °C for 5 min, the samples were separated by
SDS-PAGE followed by either PhosphorImaging analysis or blotting on a
PVDF membrane. Immunoblotting was essentially performed as described
(17). Blots were blocked with 4% bovine serum albumin in PBST (1.4 mM KH2PO4, 4.3 mM
Na2HPO4, 2.7 mM KCl, 137 mM NaCl, 0.1% Tween 20) for 2 h followed by overnight
incubation with affinity purified anti-P-SRF antibody (1:500 diluted in
PBST containing 4% bovine serum albumin). Phospho-SRF was visualized
by enhanced chemiluminescence (ECL, Amersham). After detection of
phospho-SRF, the blots were rinsed with methanol and shaken for 30 min
at 50 °C in stripping buffer (62.5 mM Tris-Cl, pH 6.8, 2% SDS, 100 mM 2-mercaptoethanol). After blocking with 4%
bovine serum albumin in PBST the blots were incubated with an anti-SRF
antiserum (1:200 diluted), and SRF was detected by enhanced chemiluminescence.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-phospho-Ser-103-SRF antiserum (Fig.
1), by in-gel kinase assays (Fig.
2) and by directly measuring kinase
activities in all cell extracts (Fig. 3).
Since tetrafluoroaluminate is formed in solution by mixing aluminum
chloride and sodium fluoride, we included these two compounds as
controls in our study. In all assays shown in Figs. 1-3, we analyzed
the identical extracts to allow a direct comparison of the results.
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Fig. 1.
Stress-induced SRF phosphorylation in
HeLa cells. Extracts from HeLa cells stimulated for the indicated
times with anisomycin (Aniso), sodium fluoride
(NaF), aluminum chloride (AlCl3), or
tetrafluoroaluminate (AlF4 ) were
subjected to EMSA supershift analysis as described under "Materials
and Methods. In lanes 2-14,
-phospho-Ser-103-SRF
antiserum was included in the binding mixtures. SRF+Elk,
control with recombinant SRF and Elk-1 proteins; untreated,
control HeLa extracts. Bands corresponding to binary complexes
(SRF), ternary complexes (SRF+TCF), or the
supershifted phospho-SRF-containing complexes (Phospho-SRF
or PhosphoSRF+TCF, respectively) are indicated on the
left.
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Fig. 2.
Induction of SRF kinases and c-Jun kinases in
HeLa cells. Identical HeLa cell lysates as used in Fig. 1 were
examined by in-gel kinase analysis. The length standards are shown on
the left of each gel, the copolymerized substrate (SRF,
c-Jun, or no substrate) is indicated on the right side. All
three gels were run using the same extracts, whereby the SRF gel was
run on a different day than the c-Jun and the control gels. Panel
A, in-gel kinase assay with SRF as copolymerized substrate. The
arrow indicates the major induced SRF kinase of 50 kDa. As
shown, this kinase activity corresponds to MK2. Panel B,
in-gel kinase assay with c-Jun as copolymerized substrate. The
arrows indicate induced c-Jun kinases of 46 and 55 kDa,
corresponding to JNK1 and -2, respectively. Panel C, in-gel
kinase assay without any copolymerized substrate. This gel serves as a
control for autophosphorylation. The arrow indicates an
autophosphorylating kinase of 50 kDa comigrating with MK2.
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Fig. 3.
Induction of MK2 and p38/SAPK2 in HeLa
cells. MK2 (Panel A) or p38/SAPK2 (Panel B)
were immunoprecipitated from the identical HeLa extracts used for Figs.
1 and 2 and examined for their activity using either GST-Hsp25
(Panel A) or GST-MK2 (Panel B) as substrates for
phosphorylation. The reaction mixtures were separated on
SDS-polyacrylamide gels, and the intensities of bands corresponding to
32P-labeled substrates were quantified by PhosphorImaging
analysis. Inducers were anisomycin, sodium fluoride,
tetrafluoroaluminate, or aluminum chloride. The relative inductions
were measured 15 (black bars), 30 (hatched bars),
and 60 (gray bars) min after addition of the corresponding
inducer.
-SRF antiserum
to the DNA binding reaction causes a mobility supershift of
phospho-SRF-containing protein-DNA complexes (16, 17). In these assays,
HeLa cell extracts show two bands (Fig. 1). According to control lanes
with recombinant protein (Fig. 1, lane 1), the faster
migrating band represents the binary complex containing only SRF
homodimer bound to the SRE whereas the slower band represents the
ternary complex containing SRE, SRF homodimer, and an additional TCF
protein. In extracts of uninduced cells, addition of
-phospho-Ser-103-SRF antiserum causes only a very weak supershift
(Fig. 1, lane 2). Therefore, only a minor fraction of SRF is
phosphorylated at Ser-103 in uninduced HeLa cells. Treatment of these
cells for 15, 30, or 60 min with anisomycin induces SRF phosphorylation
at Ser-103, leading to a complete supershift of both SRF containing
bands (Fig. 1, lanes 3-5). No obvious decrease in the
degree of SRF phosphorylation is observed during 60 min of anisomycin
treatment. The tyrosine phosphatase inhibitor sodium fluoride also
induces SRF phosphorylation, albeit with slower kinetics compared with anisomycin (Fig. 1, lanes 6-8). No significant SRF
phosphorylation can be detected after 15 min, whereas after 30 and 60 min both the binary and the ternary complex bands are completely
supershifted by
-phospho-Ser-103-SRF antiserum. Aluminum chloride
does not induce SRF phosphorylation (Fig. 1, lanes 9-11).
However, tetrafluoroaluminate induces SRF phosphorylation with similar
kinetics as sodium fluoride (Fig. 1, lanes 12-14). Indeed,
we observed no difference between sodium fluoride and
tetrafluoroaluminate in any assay used in this report.
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Fig. 4.
MK2 is an SRF kinase in HeLa extracts.
Panels A-F, characterization of anisomycin-stimulated
kinases by in-gel kinase assays. Constitutively active MK2
(GST-MK2 PC) or cell extracts from either untreated or
anisomycin-stimulated (Aniso) HeLa cells were analyzed by
in-gel kinase assays. The copolymerized substrates were Hsp25
(Panel A), SRF (Panel B), c-Jun (Panel
C), Elk-1 (Panel D), MK2 (Panel E), or
histone H1 (Panel F), as indicated at the bottom.
Bands corresponding to recombinant MK2 are indicated on the
left. Those bands corresponding to endogenous MK2 or
JNK/SAPK1 are indicated at the right. The size of
recombinant MK2 is 66 kDa due to its GST tag. Panel G.
In-gel kinase assay of immunoprecipitates. JNK/SAPK1s (lanes 3, 6, 9, 12, and 15) and MK2/3 (lanes 4, 7, 10, 13, and 16) were immunoprecipitated from HeLa lysates
and analyzed for their activation status by in-gel kinase assays with
SRF as copolymerized substrate. The treatment of the cells prior to
lysis is indicated at the top of the gels. The same lysates
as in Figs. 1-3 were examined. In addition to Fig. 2, lysates of
arsenite-treated cells are also included. Cells were lysed 30 min after
addition of anisomycin (Aniso) or arsenite, or 60 min after
adding sodium fluoride (NaF) or tetrafluoroaluminate
(AlF4
).
-JNK IP,
immunoprecipitates obtained with an
-JNK antibody;
-MK
IP, immunoprecipitates obtained with an
-MK2/3 antibody. The
length standard is shown on the left. The black
arrows indicate MK2 bands, the white arrows JNK/SAPK1
bands. In lane 15, some spillover of lane 16 is
noticeable.
-MK2/MK3 antiserum yields two inducible bands of different intensities. The more
intense band of 50 kDa agrees with the published size of human MK2
(26). The slower migrating band may represent a second isoform of human
MK2 similar to what has also been described in rat and rabbit (26).
Therefore, MK2 phosphorylates SRF in vitro. Since the sizes
of the 46- and 55-kDa SRF kinases observed in Fig. 2A agree
with the molecular masses of JNK1 and -2, we also immunoprecipitated
JNKs using
-JNK antibody to examine whether JNKs can phosphorylate
SRF. The
-JNK antibody precipitates two SRF kinase bands of 46 and
55 kDa (Fig. 4G) corresponding to JNK1 and JNK2 (26). This
finding suggests JNKs as potential SRF kinases and points to an
involvement of the JNK/SAPK1 cascade in signal transmission onto
SRF.
-phospho-Ser-103-SRF antiserum. After 10 min the phosphorylation reaches its maximum, but decreases after 20 min to background levels (Fig. 5A, lanes 1-6). Thus, in agreement with previously published results (16), FCS induces a transient SRF phosphorylation at Ser-103. Addition of
arsenite to serum-starved cells also leads to SRF phosphorylation at
Ser-103, but different phosphorylation kinetics are observed. Instead
of a rapid and transient phosphorylation, a slower but steady increase
of SRF phosphorylation to 50% within 45 min is seen (Fig. 5A,
lanes 7-12). In conclusion, arsenite treatment of NIH3T3 cells
causes phosphorylation of SRF at Ser-103 with different kinetics than
serum treatment. This difference is congruent with the slow and long
lasting activation of SAPKs by arsenite due to the inhibition of a SAPK
specific phosphatase activity (29).
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Fig. 5.
Arsenite-induced SRF phosphorylation at
Ser-103 is dependent on p38/SAPK2.
Panel A, EMSA supershift analysis with extracts from serum-
or arsenite-stimulated NIH3T3 cells. Serum-starved cells were treated
with either 20% FCS or 250 µM arsenite
(AsNaO2) followed by lysis at the indicated time points.
The extracts were subjected to EMSA supershift analysis with the
c-fos SRE as DNA probe. The bottom band reflects
SRF-containing DNA-protein complex, the top band contains
phospho-SRF supershifted with -phospho-Ser-103-SRF antiserum.
Panel B, EMSA supershift analysis of the effects of kinase
inhibiting drugs. Serum-starved cells were treated for 1 h with
the indicated concentrations of kinase inhibitors prior to incubation
with 250 µM arsenite for 45 min.
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Fig. 6.
Arsenite-induced SRF kinase in NIH3T3
identified as MK2. Panel A, in-gel kinase assays with
NIH3T3 cell lysates. Identical cell extracts as in Fig. 5B
were analyzed by an in-gel kinase analysis with SRF as copolymerized
substrate. Concentrations of the used kinase inhibitors (SB202190 and
PD98059) are indicated. The arrows indicate the induced MK2 bands at 45 and 55 kDa. The length standard is shown on the left.
Arsenite-stimulated HeLa extract (HeLa) served as a positive control.
Panel B, identification of murine MK2 as a SRF kinase.
Identical lysates as used in Fig. 5B and A of
this figure were immunoprecipitated with antisera against MK2 and
analyzed by in-gel kinase assays as described in Panel A.
MK2 IP, immunoprecipitates obtained with -MK2/3 antibody;
SB, cell extracts pretreated for 1 h with 30 µM SB202190; PD, cell extracts pretreated for
1 h with 30 µM PD98059. The MK2 isoforms are
indicated by arrows, the protein standards are shown at the
left.
-MK2/3 antiserum precipitates both the 45- and 55-kDa bands (lane 4). Cell extracts from
SB202190-treated cells do not contain active MK2 (lane 6),
whereas PD98059 hardly affects their activation (lane 8).
Thus, in NIH3T3 as well as in HeLa cells, arsenite activates MK2 via
p38/SAPK2 which in turn phosphorylates SRF, suggesting this signal
transmission as a common intracellular pathway.
-32P]ATP. This reaction yields a
32P-labeled band of an apparent molecular mass of 67 kDa
(Fig. 7A, lane 2). Therefore,
MK2 also phosphorylates SRF in vitro. As positive controls,
we included again Hsp25 as a well characterized and efficient substrate
for MK2 (lane 1), and CREB (lane 3), which has
been shown to be accepted by MK2 as a substrate (39). The intensity of
the SRF signal compares very favorably with Hsp25 (lanes 1 and 2) suggesting once more that SRF is a very efficient substrate for MK2. In contrast, CREB is only very weakly phosphorylated by MK2 (lane 3). This result agrees with recent findings
that MK2 is not a CREB kinase in vivo (37).
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Fig. 7.
Identification of phosphorylation sites for
MK2 in SRF. Panel A, substrate specificity of MK2.
Recombinant Hsp25, SRF, or CREB were incubated with
constitutively active MK2 (MK2 3B
PC) in the presence of
[
-32P]ATP. Reaction mixtures were separated by
SDS-PAGE followed by PhosphorImaging analysis. The length marker is
shown on the right, the substrates are indicated at the
top of the gel. Panel B, immunoblot detection of
in vitro phosphorylated SRF. Recombinant SRF was subjected
to immunoblotting, either directly (SRF) or after incubation with a
constitutively active form of MK2 (SRF+MK2). On the top, the
blot was developed with
-phospho-Ser-103-SRF antiserum. On the
bottom, the blot was stripped and total SRF was detected
with an
-SRF antibody. Panel C, elution profile of Lys-C
peptides from SRF by reversed-phase HPLC. Two phosphorylated fractions
are marked with asterisks. Both fractions contain a peptide
with identical N-terminal sequence but different length (see
panel D). Panel D, identification of the amino
acid serine 103 in SRF phosphorylated by MK2. Peptides labeled with an
asterisks were either sequenced conventionally on an Applied
Biosystems 494 A sequencer to identify amino acid residues or on an
Applied Biosystems 477 A sequencer by solid phase sequencing after
linking the peptides covalently to a Sequelon arylamine membrane via
their terminal carboxylate group. The figure show amounts of
32P radioactivity released after each cycle of Edman
degradation and the amino acid residues identified by conventional
Edman sequencing.
-phospho-Ser-103-SRF antiserum. In the absence of ATP and MK2, only
a very weak SRF band can be seen (Fig. 7B) due to the low
affinity of this antibody for unphosphorylated SRF (16). However,
incubation of SRF with ATP and MK2 results in an intense band of 67 kDa
indicating that SRF is phosphorylated at Ser-103. Stripping and
reprobing of the membrane with an
-SRF antiserum assured equal
loading with SRF. Thus, MK2 phosphorylates SRF at Ser-103 in
vitro and, given the above MK2 activity profiles in extracts of
treated cells, MK2 is likely to be responsible for arsenite-induced SRF
phosphorylation at the same site in vivo.
/
) fibroblasts. Arsenite
treatments were performed for 60 min. Generally, the EMSA supershift
patterns obtained with extracts from Mk2(
/
) (Fig.
8A, lanes 1-4) and wild-type
(lanes 5-8) fibroblasts were similar to those observed with
NIH3T3, with no obvious ternary complex band (Fig. 5). However, the
extent of SRF phosphorylation at Ser-103 after arsenite induction
differed significantly between knockout and wild-type cells. Whereas
with wild-type cell extracts 73% of the SRF-DNA complex was
supershifted by the
-phospho SRF antibody (Fig. 8, A, lane
8, and B), only 23% was supershifted with extracts
from MK2-deficient cells (Fig. 8, A, lane 4, and B). Therefore, inside living cells, MK2 mediates
arsenite-induced SRF phosphorylation at Ser-103 to a major extent in at
least this cell system. Kinases other than MK2 contribute only to a
minor extent to SRF phosphorylation after arsenite induction.
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Fig. 8.
SRF phosphorylation in arsenite-treated
Mk2( /
) fibroblasts. Extracts of
untreated or arsenite-treated embryonic fibroblasts were analyzed by
EMSA supershift analysis as described in Fig. 1. MK2
/
,
extracts from Mk2(
/
) fibroblasts; MK2+/+,
extracts from wild type mouse fibroblasts. Panel A, EMSA
gel. Phospho-SRF-containing complexes were detected with
-phospho-Ser-103-SRF antiserum and are indicated at the right.
Panel B, graphical representation of the percentage of
phosphorylated SRF after quantifying the relative band intensities from
A. Gray bars, untreated (lanes 2 and
6 of Panel A); hatched bars,
arsenite-treated (lanes 4 and 8 of Panel
A).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Signal transduction targeting the SRE.
The scheme shows protein kinase cascades targeting the SRE. All three
MAP kinase cascades target TCF as indicated in the bottom.
SRF is targeted by at least the ERK pathway via MK1/pp90rsk and
by the p38 pathway via MK2, but not via MK3 (25). In addition,
calcium-induced CaMKIV also phosphorylates SRF at Ser-103 (17). The
potential phosphorylation of SRF by JNKs, as suggested by our data, is
indicated by a dashed arrow. Also shown is the activation of
JNKs and p38 members by arsenite and the inhibition of p38/SAPK2 by
SB202190.
Induction of SRF phosphorylation at Ser-103 by anisomycin, arsenite, sodium fluoride, or tetrafluoroaluminate correlates kinetically, and with regard to immunoprecipitation and inhibitor studies, with the activation profiles of MK2 (see Figs. 1-6). Indeed, MK2 phosphorylates SRF at Ser-103 (Fig. 7). A comparison of the substrate properties of several putative MK2 substrates shows that CREB is a poorer substrate than Hsp25 or SRF (Fig. 7A). This result agrees with recent findings that MK2 phosphorylates another small heat shock protein, Hsp27, significantly better than CREB (45, 37). Because of this result, in combination with inhibition studies, the authors of that study concluded that not MK2 but rather Msk1 mediates the growth factor- and stress-mediated CREB activation (37). In contrast to CREB, SRF is as efficiently phosphorylated by MK2 as Hsp25 (Fig. 7A), which agrees with our observation that MK2 is the major SRF kinase in arsenite- or anisomycin-treated fibroblasts (Fig. 8 and data not shown). Interestingly, MK3 does not phosphorylate SRF in vitro (25) demonstrating for the first time different substrate preferences of these two closely related kinases. Thus, MK2 is the second example of a MAP kinase-activated SRF kinase, the other being pp90rsk (16). It remains to be examined whether other kinases downstream of MAP kinases, such as PRAK, Msk1, or Mnks, are also able to phosphorylate SRF.
MK2 has both nuclear and cytosolic substrates. SRF belongs to the first group, whereas the small heat-shock proteins belong to the second group. Of interest, MK2 is a nuclear kinase, which translocates to the cytoplasm upon phosphorylation within the nucleus by p38/SAPK2 (27, 46). After some 40 min of anisomycin stimulation of fibroblasts, the majority of MK2 has left the nucleus. These export kinetics correlate with the arsenite-induced phosphorylation kinetics of SRF (Fig. 5A). Thus, MK2 may phosphorylate first its nuclear targets, such as SRF, before being exported to the cytoplasm and phosphorylating cytosolic substrates, such as Hsp25. In contrast, regulation of the intracellular localization of SRF by MK2 seems to be rather unlikely, since SRF localization is not affected by mutating Ser-103 to either alanine or aspartate (43).
Besides MK2-mediated SRF phosphorylation at Ser-103 JNKs may also
phosphorylate SRF, albeit at different sites. This is not surprising,
since Ser-103 is not neighbored by a proline and therefore does not
represent a phosphorylation site for JNKs. We have not identified these
sites yet, but initial kinase assays using p54 MAP kinase- (47) and
SRF indicate that JNKs do phosphorylate SRF in vitro
4 thereby agreeing with
the immunoprecipitation results and in-gel kinase assays shown here
(Fig. 4G). Interestingly, in NIH3T3 cells only p55/JNK2 is
visible by in-gel kinase assays upon arsenite stimulation (Fig. 6).
This finding points to differences between the two cell lines HeLa and
NIH3T3 regarding the regulation of the JNK/SAPK2 pathway. Future
experiments will address the role of this signal transduction pathway
in SRF-mediated gene expression.
In NIH3T3 cells FCS induces c-fos expression via SRF by a TCF-independent mechanism. This pathway has been suggested to include heterotrimeric G proteins and Rho GTPases (12). In this context, tetrafluoroaluminate as an inducer of heterotrimeric G proteins has been shown to efficiently activate c-fos expression in transient transfection assays. In contrast, sodium fluoride, a well known phosphatase inhibitor, hardly activated c-fos expression (12). To identify possible downstream kinases the authors also tested for the activation of MAPKs, including JNKs and p38, by these agents. In their hands, neither tetrafluoroaluminate nor sodium fluoride activated these SAPKs significantly (12). This result is surprising, since at least CDC42 and Rac1 (48, 49), as well as the RhoA-specific guanosine nucleotide exchange factor mNET1 (50), are known to activate SAPKs. In our hands, both tetrafluoroaluminate and sodium fluoride efficiently activate JNKs as well as p38 and MK2 in HeLa cells. For several reasons, both compounds are likely to activate these kinases by the same mechanism. First, to obtain tetrafluoroaluminate, sodium fluoride is added to the cell culture medium in combination with aluminum chloride. Second, aluminum chloride does not activate SAPKs, and third, both sodium fluoride and tetrafluoroaluminate induce the same kinase pattern with the same kinetics. We do not know the exact mechanism, but, in analogy to the SAPK activation by arsenite, sodium fluoride may inhibit SAPK-specific phosphatases leading to an increase in SAPK activity. This model is supported by the slower kinetics of kinase induction by sodium fluoride, which is reminiscent to the SAPK activation kinetics of arsenite.
Thus, SAPKs may possibly be involved in SRF activation controlled by
Rho GTPases. In transient transfection assays, Rho GTPases can
efficiently activate SRF reporter genes (12), whereas chromosomal SRF
reporter genes are only activated by CDC42 and Rac1, but not by RhoA
(13). In contrast to RhoA, CDC42 and Rac1 are potent activators of both
JNKs and p38 members (48). In agreement with theses findings, RhoA can
activate chromosomal SRF reporters in the presence of either anisomycin
or constitutively active MKK3 (13). It was suggested that MKK3 induces
histone acetylation thereby complementing the RhoA signaling onto SRF.
However, it could not be excluded that MKK3 may also affect SRF
directly. MKK3 is known to activate p38 and p38
(51, 52, 53),
which, in contrast to p38
and p38
, are efficient MK2 activators.
Using MK2 as a phosphorylation substrate we show that anisomycin
induces at least these two p38 forms efficiently (Fig. 3B).
Since both MKK3 as well as anisomycin activate MK2, SRF phosphorylation
by MK2 may be important for SRF-dependent gene expression.
However, addition of the p38 inhibitor SB203580 did not interfere with SRF activation by a combination of RhoA and anisomycin (13). These
conflicting results, on the one hand cooperativity of RhoA with MKK3,
on the other no SB203580 effect, remain to be resolved (13).
Clearly, the in vivo significance of SRF phosphorylation at
Ser-103 remains an open question. Ser-103 lies in close proximity to
the MADS box of SRF. This conserved region is involved in
homodimerization and DNA binding. Therefore, phosphorylation of Ser-103
may have functional consequences for the formation of the
SRF2-DNA complex. In fact, SRF mutants containing an
alanine residue at position 103 display a reduced affinity toward the
CArG box whereas phosphorylation of Ser-103 increases the binding rate
in band shift assays (42, 16). In contrast, the phosphorylation status
of Ser-103 does not affect the interaction of SRF with TCFs (42).
Possible effects on the interaction with other SRF partners such as
ATF-6 (54), C/EBP (55), or CBP (56) have not yet been examined. In
addition, since SRF has been shown to be essential for mesoderm
formation during mouse embryogenesis (57) as well as for skeletal
muscle differentiation (40, 58), we are now examining the role of Ser-103 phosphorylation in these processes. Future experiments will
address the question of the functional consequences of SRF phosphorylation at Ser-103.
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ACKNOWLEDGEMENTS |
---|
We thank Bob Hipskind and Michael Kracht for
discussions. The skilled technical assistance of Kathrin Rollwage and
of Gabriele Schwedersky is acknowledged. We greatly appreciate the
generous gifts of -phospho-SRF antisera from M. Greenberg.
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FOOTNOTES |
---|
* This work was supported in part by Deutsche Forschungsgemeinschaft Grant No. 120/7-3 and the Fonds der Chemischen Industrie (to A. N.), and Deutsche Forschungsgemeinschaft Grant SFB344 (to M. G.).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.
Present address: Abbott GmbH, Max-Planck-Ring 2, D-65205
Wiesbaden, Germany.
Supported by the European TMR Network "Signaling Networks in
Development and Disease."
§§ To whom correspondence should be sent: Abt. Molekularbiologie, Institut für Zellbiologie, Auf der Morgenstelle 15, 72076 Tübingen, Germany. Fax: 49-7071-295359; E-mail: alfred.nordheim{at}unituebingen.de.
2 A. Kotlyarov et al., submitted for publication.
3 A. Kotlyarov, A. Neininger, C. Schubert, R. Eckert, C. Birchmeier, H.-D. Volk, and M. Gaestel, submitted for publication.
4 M. Kracht, R. Zinck, M. Gaestel, and A. Nordheim, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: SRE, serum response element; SRF, serum response factor; TCF, ternary complex factor; MK2, MAPKAP-K2; FCS, fetal calf serum; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; CREB, cAMP response element-binding protein; HPLC, high performance liquid chromatography; SAPK, stress-activated protein kinase; l, liter.
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REFERENCES |
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