UVA Induces Ser381 Phosphorylation of
p90RSK/MAPKAP-K1 via ERK and JNK Pathways*
Yiguo
Zhang
,
Shuping
Zhong,
Ziming
Dong§,
Nanyue
Chen
,
Ann
M.
Bode
,
Wei-ya
Ma
, and
Zigang
Dong
¶
From
The Hormel Institute, University of Minnesota,
Austin, Minnesota 55912 and the § Department of
Pathophysiology, Henan Medical University,
Zhengzhou 450052, Peoples Republic of China
Received for publication, May 29, 2000, and in revised form, January 2, 2001
 |
ABSTRACT |
UVA exposure plays an important role in the
etiology of skin cancer. The family of p90-kDa ribosomal S6 kinases
(p90RSK/MAPKAP-K1) are activated via phosphorylation. In
this study, results show that UVA-induced phosphorylation of
p90RSK at Ser381 through ERKs and JNKs, but not
p38 kinase pathways. We provide evidence that UVA-induced
p90RSK phosphorylation and kinase activity were time- and
dose-dependent. Both PD98059 and a dominant negative mutant
of ERK2 blocked ERKs and p90RSK Ser381
phosphorylation, as well as p90RSK activity. A dominant
negative mutant of p38 kinase blocked UVA-induced phosphorylation of
p38 kinase, but had no effect on UVA-induced Ser381
phosphorylation of p90RSK or kinase activity. UVA-induced
p90RSK phosphorylation and kinase activity were markedly
attenuated in JnK1
/
and
JnK2
/
cells. A dominant negative
mutant of JNK1 inhibited UVA-induced JNKs and
p90RSK phosphorylation and kinase activity, but had no
effect on ERKs phosphorylation. PD169316, a novel inhibitor of JNKs and
p38 kinase, inhibited phosphorylation of p90RSK, JNKs, and
p38 kinase, but not ERKs. However, SB202190, a selective inhibitor of
p38 kinase, had no effect on p90RSK or JNKs
phosphorylation. Significantly, ERKs and JNKs, but not p38 kinase,
immunoprecipitated with p90RSK when stimulated by UVA and
p90RSK was a substrate for ERK2 and JNK2, but not p38
kinase. These data indicate clearly that p90RSK
Ser381 may be phosphorylated by activation of JNKs or ERKs,
but not p38 kinase.
 |
INTRODUCTION |
The incidence of nonmelanoma and melanoma skin cancers has been
increasing for several decades in most parts of the world (1-3), but
mainly in populations of European origin (3, 4). Approximately 90% of
nonmelanoma skin cancers are thought to be caused by ultraviolet
(UV)1 exposure (5-7). The UV
part of the solar electromagnetic spectrum is divided into UVC
(200-290 nm), UVB (290-320 nm), and UVA (320-400 nm) (5, 8), and UVA
is also subdivided into UVA2 (320-340 nm) and UVA1 (340-400 nm) (9,
10). Although UVC radiation can induce skin cancers by damaging DNA
(11), UVC is not pertinent to sunlight-induced human carcinogenesis
because UVC is completely absorbed by the earth's stratospheric ozone
layer and does not reach the surface of the earth (9). On the other
hand, although UVB is only absorbed partially by the ozone layer (5, 6, 9) and can induce skin cancer by generating DNA damage (11), the
proportion of UVB in the solar UV is small. Therefore, the risk of
UVB-induced acute and chronic damage, including skin cancer (4, 8, 11),
is diminished and can be blocked by limiting sunlight exposure during
midday hours, wearing protective clothing, and using sunscreens (4, 8).
On the other hand, UVA is not absorbed by the ozone layer and thus the
amount of UVA radiation reaching the earth's surface is ~20 times
greater than that of UVB (5, 8). Therefore, UVA may be a major
component of the solar UV radiation contributing to skin cancer.
Results from epidemiological (4, 8) and animal (5, 6, 8, 12) studies
support the concept that recreational UV exposure may play an important role in the etiology of human skin cancer. UV-induced signal
transduction pathways may be a significant component in the mechanism
of UV-induced carcinogenesis (5, 6, 9, 13). However, most recent reports focus on UVC- or UVB-induced signal transduction (13, 14) and
little is known regarding pathways induced by UVA.
Extracellular signals have been shown to activate mitogen-activated
protein kinase (MAPK) cascades including extracellular signal-regulated
kinases (ERKs), c-Jun NH2-terminal kinases
(JNKs), and p38 kinase (p38) (15). Among the first substrates of ERKs are the family of 90-kDa ribosomal S6 kinases (p90RSK, also
known as MAPKAP-K1) (16). The MAPKAP-K1 family is activated via
phosphorylation and shown to be ubiquitous and versatile mediators of
signal transduction (17, 18). These signaling molecules are the focus
of much interest due to their potent ability to be phosphorylated by
activation of ERKs (18) and their involvement in regulation of various
cellular functions (16, 19). Initially, p90RSK was isolated
from Xenopus and identified on the basis of its ability to
phosphorylate the 31-kDa protein S6 that is a component of the 40 S
ribosomal subunit in vitro (20, 21). As a signal-transducing serine/threonine kinase, p90RSK is an important member of a
growing subfamily of MAPKs-activated protein kinases (16, 18,
22) that contain two distinct kinase catalytic domains in a single
polypeptide chain. The two domains are the amino-terminal kinase domain
(NTD) and the carboxyl-terminal kinase domain (CTD) (16, 22). With
regard to primary structure, the NTD of p90RSK is most
closely related to p70 S6 kinase (p70S6K) (16, 23). It was
shown to phosphorylate exogenous substrates for p90RSK,
including the cAMP response element-binding protein (24), c-Fos (25), and the estrogen receptor (26). These
substrates suggest that p90RSK may play a role in
transcriptional regulation. The CTD of p90RSK is related to
camodulin-dependent protein kinases and is most similar to
phosphorylase kinase (16). The NTD may also be activated by
phosphorylation of the CTD (16). ERKs were shown to phosphorylate and
activate p90RSK in vivo (27, 29). To date, six
phosphorylation sites have been identified, three of which are
phosphorylated by MAPKs in vitro (28). MAPKs-catalyzed
phosphorylation of Ser364 and Thr574 is
critical for activation of the NTD and CTD, respectively, and the
phosphorylation of Ser381 catalyzed by the CTD is also
important for activation of the NTD (28, 29). The Ser381
site is located on a linker region between NTD and CTD (16, 30). Recent
studies suggest that phosphorylation of Ser381 creates a
docking site for PDK1 (31).
In mammalian cells, p90RSK is activated in response to a
broad range of cellular perturbations (16, 32, 33), including oncogenic
transformation (32), insulin (17, 32), growth factors (33), phorbol
esters (16, 33), growth hormone, changes in cAMP levels, heat shock,
ionizing radiation, and T cell receptor activation (16, 17, 32, 33).
The p90RSK signal transduction pathways are involved in
cell growth, proliferation, differentiation, and apoptotic death (16).
In this study, we demonstrated that p90RSK is
phosphorylated and activated when cells are exposed to UVA irradiation.
To examine the potential dependence of UVA-induced phosphorylation of
p90RSK on MAPK pathways, we employed dominant negative
mutants of ERK2, JNK1, and p38 kinase, knockout
JnK1 and JnK2 cells, and
an inhibitor of ERKs and a novel inhibitor of JNKs and p38 kinase. From
these studies, we conclude that ERKs and JNKs, but not p38 kinase, are involved in UVA-induced p90RSK activation and
phosphorylation at Ser381.
 |
EXPERIMENTAL PROCEDURES |
Reagents and Antibodies--
Eagle's minimum essential medium
(MEM) and fetal bovine serum (FBS) were from BioWhittaker, Inc.
(Walkersville, MD); Dulbecco's modified Eagle's medium (DMEM),
L-glutamine, and G418 sulfate were from Life Technologies,
Inc. (Grand Island, NY); gentamicin sulfate was from BioWhittaker, Inc;
aprotinin, leupeptin, TPA (12-O-tetradecanoylphorbol-13-acetate), PD98059, and
SB202190 were purchased from Sigma; LY294002 was from Biomol Research
Laboratories, Inc. (Plymouth Meeting, PA); PD169316 was from
AlexisTM Biochemicals, Inc. (San Diego, CA); epidermal
growth factor (EGF) was from Collaborative Research (Madison, WI). The
specific antibodies against phosphorylated sites of ERKs
(Tyr204 of p44 and p42), JNKs
(Thr183/Tyr185), and p38 kinase
(Thr180/Tyr182), and antibodies to
nonphospho-ERKs, -JNKs, -p38 kinase, and -STAT3 were from New England
BioLabs, Inc. (Beverly, MA). Activated ERK1, ERK2, JNK1, JNK2, and p38
kinases were from Upstate Biotechnology, Inc. (Lake Placid, NY). The
phospho-specific p90RSK (Ser381) polyclonal
antibody (New England BioLabs, Inc.) detects endogenous levels of
Ser381-phosphorylated p90RSK recognizing
FRGFS*FVATG, where S* signifies phosphorylated serine (29), but this
antibody shows some cross-reactivity with RSK2 and RSK3 when
phosphorylated at the homologous serine. The specific antibody against
Thr360/Ser364 phosphorylated p90RSK
(New England BioLabs, Inc.) also shows some cross-reactivity with the
homologous phosphorylated sites of RSK3, but not with those of RSK2 or
MSK1. Therefore, analysis of RSK with these specific antibodies
indicates phosphorylated levels of total RSKs including RSK1, RSK2, and
RSK3. The antibody against nonphospho-p90RSK1 was purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
p90RSK1 (MAPKAP-K1a) antibody was from Upstate
Biotechnology, Inc.
UVA Irradiation of Cells--
The UVA source used was a Philips
TL100w/10R system from Ultraviolet Resources International (Lakewood,
OH). It consists of a Magnetek transformer number 799-XLH-TC-P, 120 volts 60 hertz, and six bulbs each six feet long. UVA irradiation
filtered through about 6 mm of plate glass, eliminating UVB and UVC
light at all wavelengths below 320 nm, was performed on cultured cells
in the UVA box with two ventilation fans installed to eliminate thermal stimulation. These adjustments were necessary because the normal UVA
lamps also produce a small amount of UVB and UVC.
Stable Transfectants and Cell Culture--
The CMV-neo vector
plasmid was constructed as previously reported (34). Mouse epidermal
JB6 promotion sensitive Cl 41 and stable transfectants with CMV-neo
mass (Cl 41) (35) or with dominant negative mutant cell lines for JNK1
(DNM-JNK1) (34), p38 kinase (DNM-p38) (36), or ERK2 (DNM-ERK2) (37)
were established as reported previously (35-37). They were cultured in
monolayers using Eagle's MEM supplemented with 5% heat-inactivated
FBS, 2 mM L-glutamine, and 25 µg/ml
gentamicin at 37 °C in humidified air with 5% CO2.
Before each experiment, these transfectants were selected with G418 and
tested with their phospho-specific MAPKs antibodies.
Phosphorylation of ERKs, JNKs, and p38--
Immunoblot analysis
for phosphorylated proteins of ERKs, JNKs, and p38 kinase was carried
out using the phospho-specific MAPK antibodies as reported previously
(34-37). STAT3 was used as an internal control to determine equal
loading of protein. The antibody-bound protein complexes were detected
by Western immunoblotting using a chemiluminescent detection system
(ECL, New England BioLabs, Inc.). Some transfer membranes were washed
with stripping buffer (7 M guanadine hydrochloride, 50 mM glycine, pH 10.8, 0.05 mM EDTA, 0.1 M KCl, and 20 mM
-mercaptoethanol) and
reprobed with other primary phospho-specific or nonphospho-specific antibodies.
Analysis of p90RSK Phosphorylation with
Phospho-specific Antibodies--
Cells (5 × 105)
were seeded into 100-mm dishes and cultured for 24 to 48 h. After
the cells reached 80-90% confluence, the Cl 41, DNM-ERK2, DNM-JNK1, or
DNM-p38 cells were starved for 24 to 48 h in MEM containing 0.1%
FBS, 2 mM L-glutamine, and 25 µg/ml gentamicin. After treatment with UVA or kinase inhibitors as
indicated (prior to UVA irradiation), the cells were washed once with
ice-cold phosphate-buffered saline and lysed in 200 µl of SDS sample
buffer (62.5 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 10% (v/v)
glycerol, 50 mM dithiothreitol, and 0.1% bromphenol blue).
The lysed samples were scraped into 1.5-ml tubes and sonicated for 5 to
10 s. Samples containing an equal amount of protein (Bio-Rad
protein assay, Bio-Rad) were loaded into each lane of an 8%
SDS-polyacrylamide gel for electrophoresis and subsequently transferred
onto Immobilon-p transfer membrane (Millipore, Danbury, CT). The
phosphorylated p90RSK protein was selectively detected
by Western immunoblotting using a chemiluminescent detection system and
a phospho-specific antibody against phosphorylation of
p90RSK at Ser381 or
Thr360/Ser364 (28, 29). However,
p90RSK phosphorylation shows the total levels of the
homologous phosphorylated sites of RSK1, RSK2, and RSK3.
Assay for p90RSK Activity--
p90RSK
activity was measured by an immune complex kinase assay using an S6
peptide AKRRRLSSLRA as a substrate according to the procedure
recommended in the S6 kinase assay kit (Upstate Biotechnology, Inc.)
(38, 39). Briefly, cell lysates were prepared from JB6 Cl 41 cells or
JB6 Cl 41 cells with DNM-JNK1, DNM-p38, or DNM-ERK2 grown in 100-mm
dishes. After starving by replacing medium with 0.1% FBS/MEM, the
cells were irradiated with UVA (160 kJ/m2). The cells were
harvested at the times indicated and lysed in 300 µl of buffer A (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 1% (v/v) Triton X-100, 2.5 mM
sodium pyrophosphate, 1 mM
-glycerol phosphate, 1 mM Na3VO4, 1 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride).
The cell lysates were centrifuged at 17,000 × g for 5 min at 4 °C. The supernatant fractions containing equal amounts of
protein were incubated with anti-p90RSK1 (MAPKAP-K1a)
antibody at 4 °C overnight, and then for an additional 4 h with
protein-A/G plus Sepharose (Santa Cruz Biotechnology, Inc.). After
washing four times with phosphate-buffered saline, the
immunoprecipitates were incubated at 30 °C for 10 min in a mixture
of the following: 20 µl of assay dilution buffer (ADB: 20 mM MOPS, pH 7.2, 25 mM
-glycerol phosphate,
5 mM EGTA, 1 mM Na3VO4,
and 1 mM dithiothreitol), 10 µl of substrate mixture (S6 peptide in ADB), 10 µl of inhibitor mixture (20 µM PKC
inhibitor peptide, 2 µM protein kinase A inhibitor
peptide, and 20 µM compound R24571 in ADB), and 10 µl
of [
-32P]ATP (1 µCi/µl; Amersham Pharmacia
Biotech, Inc.). To stop the reaction, samples were spotted onto a
numbered P81 paper square and washed three times (5 min each) with
0.75% phosphoric acid and once (3 min) with acetone. Each sample paper
was transferred into a scintillation vial and counted in a
-scintillation counter. At the same time, immunoprecipitates with
normal non-immune serum instead of p90RSK antibody were
used as background controls. After subtraction of background from each
sample, the UVA-stimulated p90RSK activity was normalized
to unstimulated controls and is shown as fold change.
Co-immunoprecipitation of Proteins--
JB6 Cl 41 cell lysates
were prepared as described above. Following exposure to UVA (160 kJ/m2), cells were harvested at 15 or 30 min and lysed for
5 min in buffer A. Supernatant fractions were incubated at 4 °C
overnight with normal rabbit serum as a non-immune IgG control or with
antibodies against ERKs, JNKs, p38 kinase, or p90RSK1
(MAPKAP-K1a) and for an additional 4 h with protein-A/G plus Sepharose (4 °C). Samples were then washed four times with
phosphate-buffered saline and the final pellet resuspended in 3 × SDS sample buffer. The immunoprecipitated proteins were analyzed by
using Western immunoblotting as recommended by New England BioLabs,
Inc. (40, 41). Immunoprecipitates of ERKs, JNKs, or p38 kinase were
incubated with anti-phospho-p90RSK (Ser381) as
the primary antibody, whereas immunoprecipitates of p90RSK1
(MAPKAP-K1a) were incubated with phospho-specific ERKs, JNKs, or p38
kinase as the primary antibody.
Preparation and Analysis JnK+/+ of JnK1
/
, and
JnK2
/
Primary Embryo Fibroblasts--
Embryo
fibroblasts from normal JnK1
/
and
JNK2
/
knockout mice were isolated and
prepared according to the procedure of Loo and Cotman (42). Cells were
established in culture in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, and
100 µg/ml streptomycin in a humidified atmosphere of 5%
CO2 at 37 °C. For analysis of protein phosphorylation
and kinase activity, the cells were starved by replacing growth medium
with serum-free DMEM for 24 h at which time they were exposed to
UVA. The cells were lysed with SDS sample buffer and protein
concentration in the cell lysates was determined (Bio-Rad assay). Equal
amounts of protein were loaded onto an 8% SDS-polyacrylamide gel and
phosphorylated and nonphosphorylated proteins were determined by
Western blotting analysis. Kinase activity of p90RSK in
these cells was performed as described above.
In Vitro Assay for p90RSK Phosphorylation and
Activation (29, 43)--
JB6 Cl 41 cell lysates were cultured in
100-mm dishes and starved for 24 h and co-immunoprecipitation
experiments with p90RSK1 (MAPKAP-K1a) were performed as
described above. Samples containing immunoprecipitated
p90RSK1 (MAPKAP-K1a) were incubated at 30 °C for 60 min
with activated ERK2, JNK2, or p38 kinase (10 ng/ml) (Upstate
Biotechnology, Inc.) in kinase buffer (50 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 5 mM ATP, and 0.01% Brij
35). At the same time, kinase activity of ERK2, JNK2, or p38 kinase was
tested by incubating with kinase substrates, Elk1, or ATF2 fusion
proteins (1 mg/ml) (New England BioLabs, Inc.). Reactions were stopped by adding SDS sample buffer. The phosphorylation of kinase protein substrates and immunoprecipitated p90RSK protein was
analyzed by using SDS-polyacrylamide gel electrophoresis, Western
blotting, and a chemiluminescent detection system (Amersham Pharmacia
Biotech, Inc.). Total lysates taken directly from Cl 41 cells that were
irradiated with UVA (160 kJ/m2) were used as an internal
control. To further analyze whether p90RSK is activated by
MAPKs in vitro, samples containing immunoprecipitated p90RSK were incubated at 30 °C for 30 min with S6
peptide plus active ERK1, ERK2, JNK1, JNK2, or p38 kinase (10 ng/ml,
Upstate Biotechnology, Inc.) and p90RSK kinase activity was
determined as described above. At the same time, incubations of S6
peptide with MAPKs were used as internal controls.
Data Analysis--
Some data were analyzed using the
Image-QuaNTTM Microsoft System (Molecular Dynamics,
Sunnyvale, CA). This system calculates the intensity of bands in
Western blots.
Statistical Analysis--
Significant differences in
p90RSK S6 kinase activity were determined by using
Student's t test.
 |
RESULTS |
Phosphorylation of p90RSK at Ser381 Is
Induced by UVA, TPA, and EGF--
TPA is a known tumor promoter and
acts during tumor promotion and progression (44, 45). EGF is a well
described growth factor having tumor promoting action (45). TPA and EGF
were reported to activate p90RSK via phosphorylation (16,
17, 39, 40) and were used here as positive controls for comparison with
UVA for stimulation of p90RSK phosphorylation. Our results
show that like TPA and EGF, UVA induced phosphorylation of
p90RSK (Fig. 1A).
In contrast to the non-irradiated control, UVA at a higher dose (160 kJ/m2) induced a 4.7-fold increase in phosphorylation of
p90RSK, which was 2.8 times that of either TPA or EGF (Fig.
1B).

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Fig. 1.
Dose-dependent phosphorylation of
p90RSK Ser381 induced by UVA. JB6 Cl 41 cells (8 × 105) were seeded into 100-mm dishes. After
culturing for 24 h at 37 °C in humidified air with 5%
CO2, the cells were starved for 48 h by replacing
media with 0.1% FBS/MEM. The media were again replaced with fresh
0.1% FBS/MEM 4 h before exposure to UV irradiation or chemicals.
The cells were exposed to UVA, TPA, or EGF at doses indicated. TPA- or
EGF-stimulated cell samples were used as positive controls. After an
additional incubation of 30 min, the treated cells were lysed in SDS
sample buffer and p90RSK protein and its phosphorylated
protein (A) were determined as described under
"Experimental Procedures." For comparison, the lower
panel (B) shows the ratio of phosphorylation in each
stimulated sample to unstimulated control (value of 1) using the
Image-QuaNTTM Microsoft System. B, bars,
correspond directly to bands in A. B, data are
presented as the mean and standard deviation of three independent
assays and the change in UVA-, TPA-, or EGF-stimulated phosphorylation
is significantly different from the unstimulated control (*,
p < 0.05; **, p < 0.01).
|
|
UVA-induced Ser381 Phosphorylation of
p90RSK Is Dose- and Time-dependent--
As
shown in Figs. 1 and 2A,
UVA-induced phosphorylation of p90RSK was dose- and
time-dependent. Phosphorylation of p90RSK was
observed 15 min following a low dose (80 kJ/m2) of UVA
exposure and the phosphorylation decreased by 30 min and remained low
(Fig. 2, A and B). On the other hand, a higher dose (160 kJ/m2) of UVA resulted in a peak phosphorylation
of p90RSK at 30 min and decreased thereafter (Fig. 2,
A and B).

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Fig. 2.
Time-dependent phosphorylation
and activation of p90RSK induced by UVA. JB6 Cl 41 cells (6 × 105) were cultured in 100-mm dishes until
they reached 90% confluence and then starved for 48 h in 0.1%
FBS/MEM. The cells were irradiated with UVA at 80 or 160 kJ/m2. The cell samples were harvested at 15, 30, 60, 120, 360, or 720 min after irradiation and analyzed with Western
immunoblotting and an in vitro assay for p90RSK
S6 kinase activity in samples following immunoprecipitation as
described under "Experimental Procedures." This is one of three
independent similar experiments. The upper panel
(A) shows that induction of p90RSK
phosphorylation by UVA at 80 or 160 kJ/m2 follows a time
dependent course. The center panel (B) shows the
ratio of treatment with UVA (80 or 160 kJ/m2) to
non-irradiated control (value of 1) using the Image-QuaNTTM
Microsoft System. The lower panel (C) shows that
UVA at 160 kJ/m2 activates p90RSK S6 kinase in
a time-dependent manner. Data are presented as the mean and
standard deviation of three (B) or six (C) assay
samples from three independent experiments. UVA-induced phosphorylation
and activation of p90RSK at indicated time points are
significantly different from nonirradiated controls (*,
p < 0.05; **, p < 0.01).
|
|
Activation of p90RSK by UVA Is also
Time-dependent--
To study whether phosphorylation of
p90RSK reflects p90RSK S6 kinase activity, we
investigated the effect of UVA on p90RSK S6 kinase activity
specifically. As shown in Fig. 2C, p90RSK S6
kinase was activated by UVA (160 kJ/m2) in a
time-dependent manner. The activity peaked at 30 min
following UVA and was about 2 times higher than the unstimulated
control value. These data indicate that phosphorylation (Fig.
2A) of p90RSK correlated well with
p90RSK S6 kinase (Fig. 2C).
PD98059 Inhibits UVA-induced Ser381 Phosphorylation of
p90RSK Coinciding with Inhibition of ERKs
Phosphorylation--
PD98059, an inhibitor for MAP kinase kinase
(MEK1) (40, 46), was used in experiments to study the role of MEK1/ERKs
in the UVA-induced Ser381 phosphorylation of
p90RSK. PD98059 markedly inhibited both UVA-induced
phosphorylation of ERKS and p90RSK at Ser381
(Fig. 3, A and B).
At the same time, PD98059 significantly blocked p90RSK S6
kinase activation by UVA (Fig. 3E). In contrast, PD98059 was less effective in inhibiting UVA-induced phosphorylation of JNKs and
p38 kinase (Fig. 3, C and D). Additionally,
p90RSK S6 kinase activation was also blocked by LY294002
(Fig. 3E), a PI 3-kinase inhibitor, which was used here as a
positive control. These data suggest that Ser381
phosphorylation and activation of p90RSK may be dependent
on ERKs.

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Fig. 3.
PD98059 inhibits UVA-induced
Ser381 phosphorylation and activation of
p90RSK. JB6 Cl 41 cells (8 × 105)
were seeded into 100-mm dishes and cultured for 24 h in 5%
FBS/MEM. Then the cells were starved for 48 h in 0.1% FBS/MEM.
The cells were incubated for 1.5 h with PD98059 at the doses
indicated and then irradiated with UVA at 160 kJ/m2. The
cell samples were harvested 15 or 30 min after irradiation. The
p90RSK phosphorylation and its kinase activity as well as
phosphorylated ERKs, JNKs, and p38 kinase were analyzed as described
under "Experimental Procedures." The sample membrane was stripped
and reprobed with different primary antibodies. This figure represents
one of three independent similar experiments. The figure shows that
PD98059 blocks UVA-induced phosphorylation of p90RSK
Ser381 (A) and ERKs (B) in parallel
and has little effect on UVA-induced phosphorylation of JNKs
(C) or p38 kinase (D). E, data are
presented as the mean and standard deviation of five assay samples from
three independent experiments. The panel shows that
pretreatment of cells with PD98059 or LY294002 prior to UVA irradiation
significantly blocks UVA-stimulated p90RSK activation
compared with treatment with UVA only (*, p < 0.05;
**, p < 0.01). Here, LY294202 was used as a positive
control.
|
|
DNM-ERK2 Blocks UVA-induced Phosphorylation of Both
p90RSK at Ser381 and ERKs--
The concept
that PD98059 is a MEK1-specific inhibitor was challenged by the report
of Kamakura et al. (47), in which PD98059 was shown to
inhibit ERK5 activation in response to EGF, oxidant, and osmotic
stresses. Therefore, to further confirm the role of ERKs in UVA-induced
phosphorylation of p90RSK at Ser381, a dominant
negative ERK2 mutant (37) was used in this experiment. UVA-induced
phosphorylation of p90RSK at Ser381 (Fig.
4, A and D) and
ERKs (Fig. 4, B and D) was inhibited and p90RSK S6 kinase activity was also significantly blocked in
DNM-ERKs cells (Fig. 4E). However, phosphorylation of JNKs
was only weakly affected and no effect on p38 phosphorylation was
observed (Fig. 4C). Additionally, the basal expression level
of p90RSK in DNM-ERK2 cells was slightly decreased as
compared with control Cl 41 cells (Fig. 4, A and
B). These data confirmed that ERKs play an important role in
the activation of p90RSK via phosphorylation at
Ser381 and in the expression of the basal level of
p90RSK.

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Fig. 4.
DNM-ERK2 blocks UVA-induced
Ser381 phosphorylation and activation of
p90RSK. JB6 Cl 41 cells and Cl 41 stable
transfectants, DNM-ERK2, were cultured for 24 h until 90%
confluency was reached. The cells were starved for 48 h in 0.1%
FBS/MEM and then harvested 15, 30, or 60 min after UVA irradiation at
40, 80, or 160 kJ/m2. The phosphorylated
p90RSK, ERKs, JNKs, and p38 kinase proteins, as well as
p90RSK S6 kinase activity, were measured as described under
"Experimental Procedures." The sample membrane was stripped and
reprobed with different antibodies. Nonphosphorylated STAT3 was used as
an internal control. This is one of three independent similar
experiments. The figure shows that DNM-ERK2 blocks UVA-induced changes
in basal and phosphorylated p90RSK protein (A)
and ERKs phosphorylation (B). C, shows DNM-ERK2
weakly inhibits phosphorylation of JNKs and has no effect on p38
phosphorylation. D, shows that inhibition of
p90RSK by DNM-ERK2 parallels its inhibition of ERKs. Each
value is the relative ratio of UVA-treated sample to the unstimulated
Cl 41 control (value of 1) determined by analysis of Western blots
using the Image-QuaNTTM Microsoft System and represents the
mean from three independent experiments (D). E,
data are presented as the mean and standard deviation from three
independent experiments performed in duplicate and results show that
DNM-ERK2 significantly blocks p90RSK activation by UVA (160 kJ/m2) compared with corresponding control cells (**,
p < 0.01).
|
|
DNM-p38 Kinase Does Not Block Phosphorylation of p90RSK
at Ser381--
The JB6 cell line expressing DNM-p38
established in our laboratory (36) was used to study the role of p38
kinase in the UVA-induced phosphorylation of p90RSK
Ser381. We found that DNM-p38 did not inhibit the
phosphorylation of p90RSK Ser381 or ERKs (Fig.
5, A and B), but
inhibited phosphorylation of p38 kinase induced by UVA (Fig.
5B). DNM-p38 also had no significant inhibitory effect on
p90RSK kinase activity when the cells were stimulated by
UVA (Fig. 5D). These data suggested that the
Ser381 site in p90RSK is phosphorylated through
the ERKs pathway, but not through the p38 kinase pathway in
UVA-irradiated JB6 cells.

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Fig. 5.
DNM-p38 does not block UVA-induced
Ser381 phosphorylation and activation of
p90RSK. JB6 Cl 41 cells and Cl 41 stable
transfectants, DNM-p38, were treated as described in the legend to Fig.
4. The p90RSK and p38 proteins and their phosphorylated
proteins, as well as phosphorylated ERKs and p90RSK S6
kinase activity were analyzed as described under "Experimental
Procedures." Nonphosphorylated STAT3 was used as an internal
control. The sample membrane was stripped and reprobed with different
antibodies. This is one of three similar experiments. A,
shows that DNM-p38 did not block UVA-induced phosphorylation of
p90RSK. B, shows that DNM-p38 blocked
UVA-induced phosphorylation of p38 kinase, but not ERKs (C).
D, data are presented as the mean and standard deviation
from three independent experiments performed in duplicate and show that
DNM-p38 does not significantly inhibit p90RSK activation by
UVA at 160 kJ/m2 compared with corresponding control cells
(p > 0.10).
|
|
Basal and Phosphorylated p90RSK Is Blocked in Knockout
JNK1
/
and JNK2
/
Cells--
To study
whether the JNK pathway plays a role in the activation and basal
expression of p90RSK, we prepared primary embryo
fibroblasts from wild-type JnK (JnK+/+) and knockout JnK (JnK1
/
or
JnK2
/
) mice. Our results showed that basal
and phosphorylated levels of p90RSK at Ser381
and Thr360/Ser364 (Fig.
6A) and JNKs (Fig.
6B), as well as p90RSK S6 kinase activity (Fig.
6D), were lower in JnK1
/
and
JnK2
/
cells than those in control
JnK+/+ cells. However, basal and phosphorylated ERKs (Fig.
6C) were not significantly changed in
JnK1
/
and JnK2
/
cells. These data suggested that the JNK pathway is involved in basal
expression of p90RSK, probably having an indirect effect on
UVA-induced phosphorylation and activation of p90RSK. Thus,
whether UVA-stimulated activation of p90RSK
Ser381 requires the JNK pathway was further tested with
DNM-JNK1 and PD169316.

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Fig. 6.
Basal and phosphorylated 90RSK is
markedly attenuated in knockout JnK1 / and
JnK2 / cells. Preparation and treatment
of primary embryo fibroblasts from the mice with wild-type
(JnK+/+) and knockout JnK1 / or
JnK2 / genes, and analysis of
p90RSK S6 kinase activity and phosphorylation at
Ser381 and Thr360/Ser364, as well
as JNKs and ERKs and their phosphorylated proteins, were performed as
described under "Experimental Procedures." The basal and
phosphorylated p90RSK (A) and JNKs
(B) were almost absent in knockout
JnK1 / and JnK2 /
cells compared with wild-type JNK+/+ cells, but the basal
and UVA-induced phosphorylation of ERKs was not significantly inhibited
in the two knockout cell lines (C). D, each
bar indicates the mean and standard deviation from three
independent experiments performed in duplicate and the data show that
p90RSK activation by UVA at 80 kJ/m2 was
significantly blocked in JnK1 / and
JnK2 / cells compared with corresponding
JnK+/+ control cells (*, p < 0.05; **,
p < 0.01).
|
|
DNM-JNK1 Inhibits UVA-induced Phosphorylation of p90RSK
and JNKs--
To further confirm that UVA-induced phosphorylation of
p90RSK occurs through the JNK pathway in vivo, a
dominant negative mutant of JNK1 (34) was used
in this experiment. Our results showed that UVA-induced phosphorylation
of p90RSK at Ser381 (Fig.
7, A and D) and
JNKs (Fig. 7B), as well as p90RSK S6 kinase
activity (Fig. 7E), were inhibited in DNM-JNK1 cells, but
inhibition of ERKs phosphorylation was not observed (Fig. 7C). These data indicated that the JNK pathway may play a
role in UVA-induced phosphorylation and activation of
p90RSK.

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Fig. 7.
DNM-JNK1 blocks UVA-induced
Ser381 phosphorylation and activation of
p90RSK. JB6 Cl 41 cells and Cl 41 stable
transfectants, DNM-JNK1, were treated as described in the legend to
Fig. 4. The phosphorylated p90RSK, JNKs, and ERKs proteins,
as well as p90RSK S6 kinase activity, were determined as
described under "Experimental Procedures." The sample membrane was
stripped and reprobed with different antibodies. This is one of three
independent similar experiments. A, shows that DNM-JNK1
inhibited UVA-induced dose-dependent phosphorylation of
p90RSK Ser381 (A) and JNKs
(B), but not ERKs (C), 15 min after UVA
irradiation. D, shows that DNM-JNK1 inhibited
time-dependent phosphorylation of p90RSK
Ser381 following UVA irradiation at 160 kJ/m2.
E, data are presented as the mean and standard deviation of
six assay samples from three independent experiments and show that
DNM-JNK1 significantly suppressed UVA (160 kJ/m2)-stimulated p90RSK S6 kinase activity
compared with corresponding control cells (*, p < 0.05).
|
|
PD169316 but Not SB202190 Blocks UVA-induced Ser381
Phosphorylation and Activation of p90RSK--
The findings
above (Fig. 5) suggest that phosphorylation and activation of
p90RSK appear to occur through a p38 kinase independent
pathway. This idea was further supported by evidence showing that,
SB202190, a selective inhibitor of p38 kinase (48), only inhibited
phosphorylation of p38 kinase, but not phosphorylation of JNKs, ERKs
(Fig. 8B), or
p90RSK at Ser381 (Fig. 8A). Another
compound, PD169316, is confirmed to be a novel inhibitor of JNKs and
p38 kinase (49, 50) and it completely blocked UVA-induced
phosphorylation of JNKs, p38 kinase (Fig. 8B), and
p90RSK Ser381 (Fig. 8A), but not
that of ERKs (Fig. 8B). Our data also showed that
p90RSK S6 kinase activity was blocked markedly by PD169316,
but not by SB202190 (Fig. 8C). These data further indicate
that UVA-induced phosphorylation and activation of p90RSK
are mediated through the JNKs, but not the p38 kinase pathway.

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Fig. 8.
PD169316 but not SB202190 completely blocks
UVA-induced phosphorylation and activation of p90RSK.
JB6 Cl 41 cells were treated as described in the legend to Fig. 3. The
cell samples were harvested 30 min after UVA irradiation.
Phosphorylated p90RSK and its S6 kinase activity, as well
as phosphorylated JNKs, p38 kinase, and ERKs, were analyzed as
described under "Experimental Procedures." The sample membrane was
stripped and reprobed with different antibodies. This is one of three
independent similar experiments. The figure shows that PD169316, a
novel inhibitor of JNKs and p38 kinase (49, 50), completely blocks
UVA-induced phosphorylation of p90RSK Ser381
(A), JNKs and p38 kinase, but not ERKs (B).
SB202190 at 0.5 µM only inhibited phosphorylation of p38
kinase, but did not inhibit phosphorylation of p90RSK
Ser381 (A), JNKs or ERKs (B).
C, data are presented as the mean and standard deviation of
six assay samples from three independent experiments and the results
show that UVA-stimulated p90RSK activation was
significantly attenuated by pretreatment with PD169316 (**,
p < 0.01), but not with SB202190 (p > 0.10) prior to UVA irradiation compared with corresponding treatment
with UVA only.
|
|
p90RSK Co-immunoprecipitates with ERKs or JNKs, but Not
p38 Kinase--
The non-phosphorylated-p90RSK
protein-antibody complex was co-immunoprecipitated strongly with
anti-phospho-ERKs and weakly with anti-phospho-JNKs, but not at all
with anti-phospho-p38 kinase as determined by Western immunoblotting
analysis (Fig. 9A). Inversely, a UVA-induced phosphorylated p90RSK was
co-immunoprecipitated weakly with the immunoprecipitates containing
nonphospho-ERKs (Fig. 9B) and -JNKs (Fig. 9C)
antibodies, but not with those containing nonphospho-p38 kinase
antibody (Fig. 9D). At the same time, immunoprecipitates
with normal non-immune IgG serum, a background control, did not react
with the above mentioned antibodies (Fig. 9, A-D). These
data indicated that a possible interaction occurs between
p90RSK and ERKs or JNKs, but not p38 kinase. Whereas the
ERK-docking site in p90RSK has been identified by Smith
et al. (51), the JNK-docking site on p90RSK
remains to be determined.

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Fig. 9.
p90RSK co-immunoprecipitates with
ERKs or JNKs, but not p38 kinase. JB6 Cl 41 cells were cultured
for 24 h in 100-mm dishes and then starved for 48 h. Fifteen
or 30 min following UVA irradiation (160 kJ/m2), the cells
were harvested and lysed in 300 µl of immunoprecipitation buffer A. Nonirradiated cells were used as a negative control.
Co-immunoprecipitation and subsequent Western immunoblotting analysis
were performed as described under "Experimental Procedures." The
upper three panels (A) show that
nonphosphorylated p90RSK proteins co-immunoprecipitate with
phosphorylated ERKs and JNKs, but not p38 kinases. The lower
three panels show that nonphosphorylated ERKs (B) and
JNKs (C), but not p38 kinase (D) proteins
co-immunoprecipitate weakly with phosphorylated p90RSK.
Additionally, non-immune IgG controls (IP: serum)
were negative. P/C indicates total cell lysates
as an internal positive control. IP, immunoprecipitation;
WB, Western blotting.
|
|
Phosphorylation of p90RSK Ser381 by
Activated ERK2 and JNK2, but Not p38 Kinase in Vitro--
To further
study the role of MAP kinases in the phosphorylation of
p90RSK, we performed kinase assays by incubating
p90RSK protein with activated ERK2, JNK2, or p38 kinases
in vitro. As shown in Fig.
10, Elk1, a substrate was for ERK2,
phosphorylated by ERK2 (Fig. 10A) and ATF2, a substrate of
JNK and p38 kinase, was phosphorylated by JNK2 and p38 kinase (Fig.
10B). The p90RSK Ser381 was
phosphorylated by activated ERK2 (Fig. 10C) and JNK2 (Fig. 10, C and D), but not by p38 kinase (Fig.
10D). Furthermore, immunoprecipitated p90RSK was
activated in vitro by active ERK1, ERK2, JNK1, and JNK2, but
not p38 kinase (Fig. 10E). At the same time, the S6 peptide was not phosphorylated by MAPKs (Fig. 10F). These data
further suggested that UVA-induced phosphorylation of
p90RSK Ser381 occurs through activation of ERKs
and JNKs, but not p38 kinase.

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Fig. 10.
Activated ERK2 and JNK2, but not p38 kinase
phosphorylate and activate p90RSK in
vitro. Cell lysates were prepared from Cl 41 cells
cultured in 100-mm dishes and starved for 24 h.
Immunoprecipitation with anti-p90RSK1 antibody, in
vitro assay for p90RSK phosphorylation and subsequent
Western immunoblotting analysis, as well as, in vitro assay
for activation of immunoprecipitated p90RSK1 by MAPKs, were
performed as described under "Experimental Procedures." Activity of
ERKs, JNKs, and p38 kinase was tested by incubation with their
substrates, fusion protein Elk1 (for ERK2) or ATF2 (for JNK2 and p38
kinase). Total lysates from SDS lysis of Cl 41 cells were used as
internal controls in the in vitro p90RSK
phosphorylation assays. Incubation of immunoprecipitated
p90RSK with no MAPKs was used as a negative control in the
in vitro p90RSK activation assays. At the same
time, incubations of S6 peptide with MAPKs, but without
immunoprecipitated p90RSK, were performed as internal
control experiments. The figure shows that ERK2 phosphorylates Elk1
(A), JNK and p38 kinase both phosphorylate ATF2
(B), and ERK2 and JNK2 (C), but not p38 kinase
(D), phosphorylate immunoprecipitated p90RSK
proteins at Ser381. The lower panel
(E) shows that the S6 kinase activity found in
immunoprecipitates of p90RSK that were incubated with
active ERK1, ERK2, JNK1, or JNK2 in vitro was significantly
greater (*, p < 0.05; **, p < 0.01)
than the S6 kinase activity of p90RSK in the absence of
MAPKs, but the p90RSK activity induced by incubating with
p38 kinase is not different from that by incubating with no p38 kinase
(p > 0.10). F, shows that in the absence of
p90RSK, incubation of the S6 peptide with each of the MAPKs
in vitro did not induce phosphorylation that was
significantly different from the control level (p > 0.10). Data are presented as the mean and standard deviation of five
assay samples from three independent experiments (E and
F).
|
|
 |
DISCUSSION |
Two major signaling pathways regulating cell function that are
activated by various stimuli (16, 52, 53, 54) include: 1) the
phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B/Akt
pathway which lead to cell survival (55) and 2) the Ras-ERK pathway,
which is important in cell division and differentiation (56). The
p90RSK is known to be a downstream kinase of the
Raf-MEK-ERK protein kinase cascade and it contains two kinase catalytic
domains, the NTD and CTD (16, 22). The CTD of p90RSK kinase
is activated by ERKs, which leads to activation of NTD kinase (16).
Thus, p90RSK represents a continuation of the ERKs cascade
with two additional protein kinase activities (16). However, purified
p90RSK that had been deactivated by treatment with
phosphatase in vitro could only be activated partially by
incubation with active ERKs (18, 28, 57). This suggests that besides
ERKs-dependent pathways, p90RSK may also be
activated by ERKs-independent pathways.
Recently, full activation of the NTD kinase of p90RSK was
shown to require cooperation of three kinases: ERKs (58, 59), the CTD
kinase of p90RSK (16), and PDK1
(3-phosphoinositide-dependent protein kinase-1, a newly
identified downstream kinase of PI 3-kinase). Ser221 in the
activation loop of the NTD of p90RSK is known to be
phosphorylated by PDK1 and this phosphorylation is proven to be
essential for the activation of all p90RSK isoforms (29,
60-62). Furthermore, Ser221 phosphorylation leading to
activation of the NTD kinase was shown to require Ser381
phosphorylation in the linker region of p90RSK (28, 29).
This notion is further supported by the results of Frödin
et al. (31) who reported that Ser381
phosphorylation may create a docking site that recruits and activates PDK1 and induce a conformational change of p90RSK that
facilitates PDK1-mediated Ser221 phosphorylation
contributing to p90RSK activation (31). These previous
studies suggest that Ser381 phosphorylation is an important
rate-limiting step of p90RSK activation. In our study,
Ser381 phosphorylation is shown to be induced by UVA
irradiation and correlates with UVA-stimulated p90RSK S6
kinase activity. However, little is known regarding whether ERK-independent pathways are involved in Ser381
phosphorylation of p90RSK. Like Ser221,
Ser381 phosphorylation may also be regulated through the PI
3-kinase pathway, inasmuch as UVA-stimulated p90RSK
activity and Ser381 phosphorylation are blocked by a PI
3-kinase inhibitor LY294002 (Fig. 3E and data not shown).
Moxham et al. (43) indicated that JNKs may be involved in
activation and phosphorylation of RSK3, a p90RSK isoform.
We, therefore, examined whether the ERKs and JNKs pathways are involved
in UVA-stimulated Ser381 phosphorylation and activation of
p90RSK.
ERKs are known to be upstream kinases of p90RSK (15, 16,
18, 28, 51, 57). But whether Ser381 phosphorylation occurs
via activation of ERKs is not clear. Data from our present study showed
that ERKs phosphorylation (Fig. 5) and activation (data not shown) were
induced by UVA irradiation and involved in UVA-stimulated
Ser381 phosphorylation and activation of
p90RSK. Consistent with results of the studies showing that
p90RSK is phosphorylated by ERKs in response to a variety
of stimuli (16, 18, 28, 63), we provide evidence that UVA-induced phosphorylation of p90RSK Ser381, as well as
its kinase activity, is completely blocked by MEK1 inhibitor, PD98059,
and a DNM-ERK2, and the blockage was paralleled with inhibition of ERKs
by PD98059 and DNM-ERK2. In addition, PD98059 was less effective in
inhibiting UVA-induced phosphorylation of JNKs and p38 kinase, which
appears to agree with the suggestion of Smith et al. (51)
and Cheng and Feldman (64) that JNKs and p38 kinase are implicated in
p90RSK regulation. However, DNM-ERK2 also suppressed
phosphorylation of JNKs, but not p38 kinase (Fig. 4C),
suggesting that JNKs, like ERKs, but not p38 kinase, may be involved in
activation and phosphorylation of p90RSK in
vivo.
Recently, p38 kinase was identified as an upstream kinase of
p90RSK-related kinases including PLPK (65), RSK-B (66), and
MSK1 (40). Another p90RSK family member, MAPKAP kinase-2,
was also shown to be an in vivo substrate of p38 kinase and
to be mediated by the mammalian target of rapamycin pathway (68).
However, our data showed that p38 kinase may not be involved in
UVA-stimulated p90RSK activity and Ser381
phosphorylation. We provide evidence that a selective p38 kinase inhibitor, SB202190, and DNM-p38 had no effect on p90RSK
Ser381 phosphorylation and its kinase activity induced by
UVA. Although the studies of Lian et al. (46) and Horstmann
et al. (67) suggested that the p38 kinase
inhibitor-sensitive pathway was involved in activation of S6 kinases,
our data suggest that UVA-induced Ser381 phosphorylation
may not be dependent on p38 kinase. The discrepancy may be related to
cell type and the kind of stimuli.
Recently, RSK3 was reported to be activated by JNKs both in
vivo and in vitro (43). Here, we also found that
activation of JNKs may be involved in UVA-stimulated p90RSK
Ser381 phosphorylation and kinase activity. We provide
evidence that the basal expression and possibly UVA-stimulated
phosphorylation of p90RSK, as well as its kinase activity,
were abrogated in JnK1
/
and JnK2
/
cells compared with wild-type
JnK+/+ cells. Furthermore, a DNM-JNK1 almost completely
blocked UVA-induced Ser381 phosphorylation and activation of
p90RSK and the same blockage also occurred following
pretreatment with PD169316, a novel inhibitor of JNKs and p38 kinase
(49, 50), whereas SB202190 had no effect. No change in ERKs
phosphorylation was observed in these experiments. Importantly, the
observed effects of PD169316 and SB202190 were not due to their
absorption by UV (data not shown). These data indicated that
Ser381 phosphorylation and activation of p90RSK
may occur via activation of the JNKs pathway in vivo.
Moreover, ERKs, but not JNKs or p38 kinase, is shown to interact with
the COOH-terminal tails of three p90RSK isoenzymes (RSK1,
RSK2, and RSK3) (51), indicating that a docking site for ERKs, but not
for JNKs, is located in the COOH-terminal tail of p90RSK.
However, we observed that JNKs, like ERKs, but not p38 kinase, co-immunoprecipitated with p90RSK following UVA exposure,
suggesting that a binding site of JNKs is most likely located with
another region of p90RSK. This notion was supported further
by the evidence from our in vitro studies showing that
Ser381 was phosphorylated by active JNK2 and ERK2, but not
p38 kinase. Furthermore, S6 kinase activity in the immunoprecipitated
p90RSK was activated by JNKs and ERKs, but not p38 kinase
in vitro (Fig. 10E), consistent with the
suggestions of Moxham et al. (43) that JNKs are required for
p90RSK activation. Taken together, our results strongly
support the hypothesis that activation and phosphorylation of
p90RSK Ser381 by UVA is mediated ERKs and JNKs and not by
p38 kinase.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Masaaki Nomura for help on the
assays for the p90RSK activity and Andria Hansen for
secretarial assistance.
 |
FOOTNOTES |
*
This work was supported by The Hormel Foundation and
National Institutes of Health Grants CA77646 and CA81064.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.
¶
To whom correspondence should be addressed. Tel.:
507-437-9640; Fax: 507-437-9606; E-mail: zgdong@smig.net.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M004615200
 |
ABBREVIATIONS |
The abbreviations used are:
UV, ultraviolet;
MAPKs, mitogen-activated protein kinases;
ERKs, extracellular
signal-regulated kinases;
JNKs, c-Jun NH2-terminal kinases;
p38, p38 MAPK or p38 kinases;
MAPKAP-K1, mitogen-activated protein
kinase-activated protein kinase 1;
p90RSK or RSK, 90-kDa
ribosomal S6 kinases;
NTD, the amino-terminal kinase domain of
p90RSK;
CTD, the carboxyl-terminal kinase domain of
p90RSK;
PI 3-kinase, phosphatidylinositol 3-kinases;
PDK1, 3-phosphoinositide-dependent protein kinase 1;
DNM, dominant negative mutant;
MEM, Eagle's minimum essential medium;
FBS, fetal bovine serum;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
EGF, epidermal growth
factor;
UVA, ultraviolet light A;
MEK1, MAP kinase kinase;
MOPS, 4-morpholinepropanesulfonic acid.
 |
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