From the Hormel Institute, University of Minnesota,
Austin, Minnesota 55912 and the § Department of
Pathophysiology, Henan Medical University,
Zhengzhou 450052, People's Republic of China
Received for publication, October 3, 2000, and in revised form, March 28, 2001
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ABSTRACT |
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Ultraviolet light A (UVA) plays an important role
in the etiology of human skin cancer, and UVA-induced signal
transduction has a critical role in UVA-induced skin carcinogenesis.
The upstream signaling pathways leading to p70S6K
phosphorylation and activation are not well understood. Here, we
observed that UVA induces phosphorylation and activation of p70S6K. Further, UVA-stimulated p70S6K activity
and phosphorylation at Thr389 were blocked by wortmannin,
rapamycin, PD98059, SB202190, and dominant negative mutants of
phosphatidylinositol (PI) 3-kinase p85 subunit (DNM- Ultraviolet light A
(UVA)1 (320-400 nm)
comprises ~95% of the total solar UV reached the earth (1),
because all the ultraviolet C (UVC) (200-290 nm) and most of the
ultraviolet B (UVB) (290-320 nm) radiation are absorbed by the
earth's stratospheric ozone layer (2). Currently, UVA, like UVB, is
considered to be a complete carcinogen (3) and to play an important
role in the etiology of human skin cancer (4). But the activation of
signaling molecules and their pathways implicated in the process
following UVA irradiation (5, 6) are not well understood. Therefore, the study of UVA-induced signal transduction will help in understanding the molecular mechanisms underlying UVA-induced carcinogenesis.
Activation of tumor cell proliferation requires an accelerated rate of
protein synthesis, which is regulated in part by intracellular activation of several signaling protein kinase cascades that interact with the translational machinery of the ribosome (7). Among them, S6 is
a component of ribosomal proteins and is located at the interface
between 40 and 60 S ribosomal proteins (8). Phosphorylation of S6 at
multiple serine sites on its C terminus was shown to be correlated with
increased translation, especially of mRNAs containing a
polypyrimidine tract in their 5'-untranslated regions (9). This family
of mRNAs constitutes as few as 100-200 genes but makes up 20-30%
of the total cellular mRNA, indicating that they are important for
cell cycle progression. The family of serine/threonine kinases that
mediate S6 phosphorylation are known as ribosomal S6 kinases, one of
which is a 70-kDa S6 kinase (p70S6K) (8). Accumulating
evidence suggests that the prominent role of p70S6K
activation in mitogenesis may be to promote translation of mRNAs necessary for cell growth and division and to generate many of the
molecules necessary for driving the cell cycle from
G0/G1 to S phase (9).
Initially, p70S6K was isolated from mitogen-stimulated
Swiss mouse 3T3 cells (10). Subsequently, two isoforms of
p70S6K (p70S6K/p85S6K, collectively
termed p70S6K or S6K1) were found in purification, cloning,
and expression studies (11). Both isoforms are encoded by the same
transcript with alternative translational start sites (12). Based on
evidence that an additional 23-amino acid extension at the N terminus
of p85S6K was shown to function as a nuclear localization
signal, p85S6K appears to be exclusively nuclear, whereas
p70S6K is largely cytoplasmic (11, 12). The
p85S6K may be responsible for phosphorylation of the free
chromatin-bound nuclear form of S6 (13, 14). Recently, deletion of the
p70S6K gene was shown to have no effect on S6
phosphorylation, 5'-untranslated region mRNA translation, or the
rate of cell growth, but it resulted in a small mouse phenotype (15).
In p70S6K Although p70S6K is known to be activated by various stimuli
including growth factors, cytokines,
12-O-tetradecanoylphorbol-13-acetate (TPA), oncogenic
products, Ca2+, and inhibitors of protein synthesis (5,
21), the signal transduction pathway mediating p70S6K is
poorly understood. An array of independently regulated protein kinases
(12, 22, 23) are known to activate 70S6K via
phosphorylation of at least eight Ser/Thr sites in its three separate
domains. Thr229 in the p70S6K activation loop
within the catalytic domain has been shown to be phosphorylated
in vivo through the phosphatidylinositol (PI)-3 kinase
pathway (24) and in vitro selectively by
3-phosphoinositide-dependent protein kinase 1 (PDK1) (25).
Thr229 phosphorylation has been shown to enable
p70S6K activity and be repressed by wortmannin, an
inhibitor of PI 3-kinase (26). Additionally, similar to
Thr229, Ser371 located in the kinase extension
domain, has been shown to influence p70S6K activity, and
its phosphorylation is also regulated by the PI 3-kinase-dependent pathway (27). Thr389 is
another site for mitogen-stimulated phosphorylation and is situated in
a conserved 65-amino acid segment located immediately C-terminal to the
catalytic domain (12, 22). It plays an especially important role in
p70S6K activation, because it influences both the
phosphorylation of Thr229 in vitro by PDK1 and
p70S6K activity (22). Although p70S6K was shown
to be phosphorylated by mTOR in vitro (28, 71), a
p70 At the same time that p70S6K is activated by mitogens, the
extracellular signal-regulated kinases (ERKs) pathway is also
stimulated (23), indicating that the activation of p70S6K
appears to coincide with that of ERKs. The proline-directed Ser/Thr (S/T-P) sites (Ser411, Ser418,
Thr421, and Ser424) within the autoinhibitory
domain of p70S6K are in a consensus motif similar to those
known to serve as recognition determinants for mitogen-activated
protein kinases (MAPKs). Indeed, ERKs were shown to phosphorylate
p70S6K in vitro, suggesting that the Ras/ERK
pathway controlled p70S6K activation (33); however, other
studies (34, 35) showed that ERKs were neither necessary nor sufficient
for p70S6K activation. But more recently, the ERK cascade,
like PI 3-kinase/Akt cascades, has been demonstrated to be a
prerequisite for p70S6K activation (23, 36). Taken
together, p70S6K activation appears to require a complex
array of separate, concurrent phosphorylations at multiple sites
catalyzed by various protein kinases, but its precise mechanisms of
activation are as yet unclear. In our work, we provide evidence that
MAPK pathways, like the PI 3-kinase/mTOR pathways, are implicated in
phosphorylation and activation of p70S6K in response to UVA irradiation.
Reagents and Antibodies--
Chemicals were of the best grades
available commercially. Eagle's minimum essential medium (MEM) and
fetal bovine serum (FBS) were from Whittaker Biosciences, Inc.
(Walkersville, MD); Dulbecco's modified Eagle's medium,
L-glutamine, gentamicin, and G418 sulfate were from Life
Technologies, Inc.; aprotinin, leupeptin,
12-O-tetradecanoylphorbol-13-acetate (TPA), PD98059,
SB202190, and rapamycin were purchased from Sigma; wortmannin was from
Biomol Research Laboratories, Inc. (Plymouth Meeting, PA); PD169316 was
from Alexis® Biochemicals, Inc. (San Diego, CA); and epidermal growth
factor (EGF) was from Collaborative Research (Madison, WI). The
phosphospecific antibodies against phosphorylated sites of ERKs
(Tyr204 of p44 and p42), c-Jun N-terminal kinases
(JNKs) (Thr183/Tyr185), p38 kinase
(Thr180/Tyr182), and antibodies to
nonphospho-ERKs, -JNKs, and -p38 kinase were from New England Biolabs,
Inc. (Beverly, MA). The polyclonal antibodies against
phospho-p70S6K at Ser411,
Thr421/Ser424, or Thr389 and
anti-nonphospho-p70S6K antibodies were also from New
England Biolabs. Mouse anti-phosphospecific p70S6K
(Ser411) mouse monoclonal antibody (A-6) was from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA). Active ERK1, ERK2, JNK1,
JNK2, and p38 kinase and p70S6K S6 activity assay kits were
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY).
Stable Transfectants and Cell Culture--
The CMV-neo vector
plasmid was constructed as previously reported (37). Mouse epidermal
JB6 promotion-sensitive Cl 41 and its stable transfectants with CMV-neo
mass1 (Cl 41) (38) or with dominant negative mutants of
ERK2 (DNM-ERK2) (39), JNK1 (DNM-JNK1) (38), p38 kinase (DNM-p38) (40),
or PI 3-kinase p85 subunit (DNM- Treatment of Cells with UVA, UVB, UVC, or Kinase
Inhibitors--
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 V, 60 Hz, and six bulbs
each 6 feet long. UVA irradiation filtered through about 6 mm of plate
glass, eliminating most of 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 can also
produce a small amount of UVB and UVC. The UVB irradiation was carried
out in a UVB chamber with a transluminator emitting UVB light protons
and was fitted with an Eastman Kodak Co. Kodacel K6808 filter that
eliminates all wavelengths below 290 nm. This was also necessary,
because a normal UVB lamp can generate a small number of UVC light
protons. UVC radiation performed was from germicidal lamps. To assess
the roles of different signaling pathways in p70S6K
phosphorylation, we pretreated JB6 Cl 41 cells for 1-2 h before UVA
irradiation with Me2SO or kinase inhibitors
including PD98059, SB202190, PD169316, wortmannin, or rapamycin
dissolved in Me2SO.
Phosphorylation of ERKs, JNKs, and p38 Kinase--
Immunoblot
analysis for detection of phosphorylated proteins for ERKs, JNKs, and
p38 kinase was carried out using the phosphospecific MAPK antibodies as
reported previously (38-41). The antibody-bound protein complexes were
detected by Western immunoblotting using a chemiluminescent detection
system (ECL, New England Biolabs). Some transfer membranes were washed
with stripping buffer (7 M guanidine hydrochloride, 50 mM glycine, pH 10.8, 0.05 mM EDTA, 0.1 M KCl, and 20 mM Analysis of in Vivo Phosphorylation of p70S6K with
Phosphospecific Antibodies--
Cells (40 × 104 to
80 × 104) were seeded into 100-mm dishes and cultured
for 24-48 h until the cells reached 80-90% confluence. The Cl 41, DNM-ERK2, DNM-JNK1, DNM-p38, or DNM- p70S6K Activity Assay--
p70S6K
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) (5).
Briefly, cell lysates were prepared from JB6 Cl 41 cells or Cl 41 cell
lines expressing DNM-ERK2, DNM-JNK1, DNM-p38, or DNM- Preparation of Normal, Jnk1 In Vitro Kinase Assay for p70S6K Phosphorylation and
Activation--
JB6 Cl 41 cells were cultured in 100-mm dishes,
starved for 24 h, and then lysed in buffer A alone. The cell
lysates were centrifuged, and then the supernatant fractions were
subjected to immunoprecipitation with rabbit anti-nonphosphorylated
p70S6K polyclonal antibody as described above. Samples
containing immunoprecipitated p70S6K were incubated with
active ERK1, ERK2, JNK1, JNK2, or p38 kinases (Upstate Biotechnology)
at doses as indicated in kinase buffer (50 mM Tris-HCl, pH
7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM DTT, 5 mM ATP, and 0.01% Brij 35) (New
England Biolabs) at 30 °C for 60 min. The reactions were stopped by
adding SDS sample buffer, and phosphorylation of immunoprecipitated
p70S6K protein was analyzed by using SDS-PAGE, Western
blotting, and a chemiluminescent detection system. The first antibodies
are mouse phosphospecific p70S6K (Ser411)
monoclonal antibody (Santa Cruz Biotechnology) and rabbit
phosphospecific p70S6K (Thr389,
Thr421/Ser424) polyclonal antibodies (New
England Biolabs). To further analyze whether p70S6K is
activated by MAPKs in vitro, samples containing
immunoprecipitated p70S6K were incubated at 30 °C for 30 min with S6 peptide plus active ERK1 (10 ng/µl), ERK2 (10 ng/µl),
JNK1 (25 milliunits/µl), JNK2 (25 milliunits/µl), or p38 kinases
(10 ng/µl) (Upstate Biotechnology), and p70S6K kinase
activity was determined as described above. At the same time,
incubation of immunoprecipitated p70S6K with S6 peptide
only was used as a negative control, and incubations of S6 peptide with
MAPKs were used as internal controls. The addition of bovine serum
albumin instead of MAPKs or immunoprecipitated p70S6K
proteins was used as background control.
Determination of p70S6K
Co-immunoprecipitates--
After starvation for 48 h, JB6 Cl 41 cells were or were not irradiated with UVA at 160 kJ/m2 and
then harvested at 15 or 30 min following irradiation.
Immunoprecipitated p70S6K proteins were obtained by
incubating the cell lysates with p70S6K antibody as
described above. At the same time, immunoprecipitates with normal
nonimmune IgG serum instead of p70S6K antibody were used as
internal negative controls, and immunoprecipitates with antibodies
against PI 3-kinase p85 PI 3-Kinase Assay--
PI 3-kinase activity was determined
according to reported methods (41, 44). Briefly, JB6 Cl 41 cells or JB6
cells expressing DNM- Kinase Activity Assay for Protein Phosphatase 1-treated
p70S6K Preparations--
Deactivation of the
p70S6K preparations by protein phosphatase 1 (PP1) and
reactivation by MAPKs were examined according to reported methods (72,
73). Briefly, JB6 Cl 41 cells were starved for 36 h and then lysed
in buffer A alone. The cell lysates were clarified by centrifugation at
17,000 × g at 4 °C for 5 min, and then supernatant fractions containing equal amounts of protein were subjected to immunoprecipitation with p70S6K antibody as described
above. The p70S6K preparations were incubated at 30 °C
for 30 min with 0.5 unit of PP1 or with no PP1 (as an internal
control) in 100 µl of buffer containing 50 mM
Tris-HCl (pH 7.0), 30 mM Statistical Analysis--
Significant differences between the
p70S6K kinase activity after treatment and the
corresponding control value were determined using Student's
t test.
UVA Induces Activation of p70S6K in a
Time-dependent Manner--
Activation of
p70S6K was reported to occur through multisite
phosphorylations (12, 22, 23). We investigated the effect of UVA on
total p70S6K kinase activity and found that UVA (160 kJ/m2) stimulated p70S6K kinase activity in a
time-dependent manner (Fig.
1A; p < 0.001). The activity peaked 30 min after irradiation and then decreased by 60 min. UVA-activated p70S6K kinase activity was about 3 times that of the unstimulated control 30 min following irradiation.
Recently, UVB was shown to induce p70S6K activation (30,
45), and here, UVB (8 kJ/m2) was used as a positive control
and stimulated p70S6K activity about 2.3 times compared
with the control (Fig. 1A).
UVA Induces Phosphorylation of p70S6K--
TPA and EGF
are well known tumor promoters that act during tumor promotion and
progression (46, 47), and UVC is also an experimental carcinogen (48).
TPA, EGF, and UVC were reported to induce p70S6K
phosphorylation (5, 30, 49, 50) and were therefore used as positive
controls in this study. Our data showed that UVA (80 and 160 kJ/m2) induced phosphorylation of p70S6K at
Ser411, Thr421/Ser424, and
Thr389. The phosphorylation was equivalent to or greater
than that induced by UVC (60 kJ/m2), TPA (10 ng/ml), or EGF
(100 ng/ml) (Fig. 1B).
UVA-induced Phosphorylation of p70S6K Is Dose- and
Time-dependent--
UVA-induced phosphorylation of
p70S6K Ser411, Thr389, and
Thr421/Ser424 in a dose-dependent
(Fig. 2A) and
time-dependent (Fig. 2B) manner. Maximum
stimulation occurred at all four sites with a UVA dose of 160 kJ/m2 after 30 min followed by a decreased level by 120 min. However, phosphorylation at Ser411 and
Thr421/Ser424, but not Thr389,
showed a biphasic pattern with the first peak occurring at 30 min and a
second peak occurring at 360 min following UVA irradiation (Fig.
2B). The time course pattern of phosphorylation coincides very well with the pattern of p70S6K kinase activity (Fig.
1A), suggesting that phosphorylation of p70S6K
may indirectly reflect p70S6K kinase activity.
Wortmannin and DNM- Rapamycin Blocks UVA-induced Activation and Phosphorylation of
p70S6K at All Four Sites--
Many experiments have shown
that the mTOR signaling pathway is involved in activation and
phosphorylation of p70S6K (12, 22, 28, 31, 32). Our data
showed that UVA-induced phosphorylation of p70S6K at
Thr389 was completely abrogated by pretreatment with
rapamycin (Fig. 4A), a
selective mTOR inhibitor that abolishes mTOR function both in
vivo and in vitro (12, 53). UVA-induced phosphorylation of p70S6K at Ser411 and
Thr421/Ser424 also was partially blocked by
rapamycin pretreatment (Fig. 4A). Furthermore, our data
demonstrated that p70S6K kinase activation by exposure to
UVA irradiation was significantly (p < 0.01)
suppressed by rapamycin pretreatment (Fig. 4B). These results suggest that mTOR may preferentially mediate p70S6K
phosphorylation at Thr389, leading to its activation.
PD98059 and DNM-ERK2 Inhibit UVA-induced Activation and
Phosphorylation of p70S6K at All Four Sites--
ERKs were
reported to phosphorylate p70S6K in vitro (33),
but later reports indicated that p70S6K activation was
independent of the Ras/MAPK pathway (34, 35). However, more recently,
ERKs were shown to be required for activation of p70S6K
in vivo (22, 23). In our present experiments, we used
PD98059, an inhibitor of MEK1 (50, 54) to determine the role of ERKs in
activation and phosphorylation of p70S6K at
Thr389, Ser411, and
Thr421/Ser424. Our data showed that
pretreatment of JB6 Cl 41 cells with PD98059 markedly blocked
UVA-induced ERK phosphorylation and p70S6K phosphorylation
at Thr421/Ser424 and Thr389 and
partially inhibited phosphorylation of p70S6K at
Ser411 (Fig. 5A).
Further, UVA-stimulated p70S6K kinase activity was also
significantly suppressed (p < 0.001) by PD98059
pretreatment (Fig. 5C). However, Kamakura et al.
(55) reported that PD98059 inhibits EGF-induced ERK5 activation.
Therefore, to further study the role of ERKs in the phosphorylation and
activation of p70S6K, we used a JB6 cell line stably
expressing the protein of a dominant negative mutant of ERK2 (DNM-ERK2)
(39). As expected, UVA-induced phosphorylation and activation of ERKs
were blocked by DNM-ERK2 (Fig. 5B and data not shown). Our
data also showed that, following UVA irradiation, p70S6K
phosphorylation at Thr421/Ser424 and
Thr389 was almost completely blocked, and the
phosphorylation at Ser411 was partially inhibited in
DNM-ERK2 cells compared with control Cl 41 cells (Fig. 5B).
Moreover, p70S6K kinase activity induced by UVA was
significantly (p < 0.001) abrogated in DNM-ERK2 cells
compared with control cells (Fig. 5D). On the other hand,
PD98059 pretreatment (Fig. 5A) and DNM-ERK2 expression (Fig.
5B) had no effect on UVA-induced p38 kinase phosphorylation. Thus, these results suggest that ERKs may be involved in
p70S6K activation and phosphorylation at
Thr421/Ser424, Thr389, and
Ser411 in JB6 cells exposed to UVA irradiation.
SB202190 and DNM-p38 Inhibit UVA-induced p70S6K
Activation and Phosphorylation at Thr389 but Not
Ser411 or Thr421/Ser424--
To
determine whether p38 kinase has an effect on activation and
phosphorylation of p70S6K, we employed SB202190, a
selective inhibitor of p38 kinase (56). Our data showed that
pretreatment of JB6 cells with SB202190 prevented phosphorylation of
p38 kinase and p70S6K at Thr389 but had no
effect on ERK and p70S6K phosphorylation at
Ser411 or Thr421/Ser424 following
UVA irradiation (Fig. 6A). We
observed that SB202190 pretreatment also significantly
(p < 0.01) suppressed p70S6K activation
after cells were exposed to UVA irradiation (Fig. 6C). To
further investigate the role of p38 kinase in UVA-stimulated activation
and phosphorylation of p70S6K, we used a JB6 cell line
expressing a dominant negative mutant of p38 kinase (DNM-p38) (40). As
expected, DNM-p38 selectively inhibited phosphorylation and activation
of p38 kinase, but not ERKs, in response to UVA (Fig. 6B and
data not shown). Furthermore, DNM-p38 only blocked Thr389
phosphorylation but had no effect on phosphorylation of
p70S6K at Ser411 or
Thr421/Ser424, compared with corresponding
control Cl 41 cells (Fig. 6B). In addition, UVA-stimulated
p70S6K kinase activity was suppressed (p < 0.05) in DNM-p38 cells compared with control Cl 41 cells (Fig.
6D). Therefore, these results indicated that p38 kinase may
be implicated in UVA-induced activation and phosphorylation of
p70S6K at Thr389 but not at Ser411
or Thr421/Ser424.
JNKs May be Involved in UVA-induced Activation and Phosphorylation
of p70S6K at Thr389 and Ser411 but
Not at Thr421/Ser424--
We found that
UVA-stimulated activation of p70S6K was significantly
abolished by pretreatment of JB6 cells with PD169316 (Fig. 6C), a novel inhibitor of p38 kinase and JNKs (57, 58),
suggesting that JNKs, like ERKs and p38 kinase, may be implicated in
UVA-induced activation and phosphorylation of p70S6K.
Further, we used cell lines with a dominant negative JNK1 mutant (DNM-JNK1) (38) and embryo Jnk1 Immunoprecipitated p70S6K Proteins Are Phosphorylated
by Active MAPKs in Vitro--
ERKs were previously shown to
phosphorylate p70S6K in vitro and to mediate
p70S6K activation (33). More recently, activation of the
ERK cascade, like the PI 3-kinase/mTOR cascade, was reported to be a
prerequisite for p70S6K activation (23). In our in
vitro study, immunoprecipitated p70S6K proteins from
unstimulated cell lysates were phosphorylated at Ser411
when incubated with active ERK1, ERK2, JNK1, or JNK2 but not with p38
kinase (Fig. 8A).
Phosphorylation at Thr421/Ser424 was induced
only by ERK1 and not by the other above mentioned MAPKs (Fig.
8A). However, the immunoprecipitated p70S6K
proteins were not phosphorylated at Thr389 by in
vitro incubation with active ERKs and JNKs or p38 kinase (data not
shown). Together with the results of the in vivo
phosphorylation assays, these data suggest that activation of
p70S6K by UVA might require prior phosphorylation at
Ser411 by JNKs and ERKs, at
Thr421/Ser424 by ERKs, or at other S/T-P
sites possibly by p38 kinase and a second phosphorylation at
Thr389 mediated by mTOR/PI 3-kinase or another unidentified
kinase.
p70S6K Proteins Co-immunoprecipitated with PI 3-Kinase
or Possibly PDK1 Are Activated by MAPKs in Vitro--
Prior
phosphorylation of p70S6K at S/T-P sites by MAPKs was shown
to be necessary for p70S6K activation through PDK1-mediated
regulation, and the phosphorylation at Thr229 by PDK1 and
at Thr389 by PI 3-kinase/mTOR was reported to engender
partial activation of p70S6K, but total activation of
p70S6K was not induced by MAPKs alone (12, 22, 23, 28, 59). However, our present data showed that immunoprecipitated
p70S6K proteins are activated by incubation with active
MAPKs in vitro (Fig. 8B), whereas the S6 peptide,
a substrate of p70S6K, is not phosphorylated by active
MAPKs in vitro (Fig. 8C). These results suggest
that the p70S6K proteins may co-immunoprecipitate with
other kinases that can phosphorylate the S6 peptide or with additional
p70S6K-activating kinases such as PDK1. Our studies showed
further that in the UVA-treated cells, the p70S6K proteins
co-immunoprecipitated strongly with PI 3-kinase (Fig. 8D)
and very weakly with PDK1 (Fig. 8E) but not with Akt (Fig. 8F), as compared with corresponding controls. MAPK
downstream kinases, including RSK1, RSK2, RSK3, or mitogen- and
stress-activated protein kinase, can also phosphorylate the S6 peptide.
However, the p70S6K immunoprecipitates were not
contaminated with any phosphorylated or nonphosphorylated MAPK
downstream kinases (data not shown). In addition, immunoprecipitates
from normal nonimmune serum did not contain any of the above mentioned
kinases (data not shown). Therefore, our results suggest that the
activation and phosphorylation of p70S6K may be mediated by
MAPKs in cooperation with PI 3-kinase and possibly PDK1 or an
additional unidentified p70S6K-activating kinase.
Partial Reactivation of PP1-treated p70S6K Preparations
by ERKs--
PP1-treated p70S6K preparations were employed
here to determine whether the immunoprecipitate (IP)-p70S6K
used above was partially phosphorylated at non-MAPK sites and, thus,
would be "primed" for its phosphorylation and activation by MAPKs
in vitro. Our data showed the S6 kinase activity decreased by about 40% following treatment of the IP-p70S6K with PP1
compared with that with no PP1 treatment (Fig.
9A), indicating that a partial
phosphorylation of IP-p70S6K exists in the unstimulated
state. The partial phosphorylation appeared to facilitate the induction
of p70S6K activation by MAPKs (Figs. 8B and
9A). Furthermore, partial reactivation of PP1-treated
IP-p70S6K was observed following incubation with ERKs
and p38 kinase but not JNKs (Fig. 9A). Recently, a
complex between ERKs and p70S6K was documented by
immunoprecipitation (43). Therefore, these results suggest that
p70S6K may be a potential substrate for ERKs or a kinase
dependent upon ERKs, but full activation of p70S6K may
occur via cooperation of MAPKs with phosphatidylinositol 3-kinase/PDK1
or an unidentified kinase.
Activation of p70S6K is known to occur though a
hierarchical multisite phosphorylation process directed at three
separate domains: 1) a cluster of S/T-P sites (Ser411,
Ser418, Thr421, Ser424) in an
autoinhibitory domain in the noncatalytic C-terminal tail; 2)
Thr229 in the activation loop of the catalytic domain; and
3) Thr389 and another two S/T-P sites (Ser371
and possibly Thr367) in the kinase extension domain
immediately C-terminal to the catalytic domain (12, 22). Here, we
employed phosphospecific anti-peptide antibodies to determine the
changes in phosphorylation of p70S6K at Thr389,
Ser411, and Thr421/Ser424 in
response to UVA exposure. We found that exposure of JB6 cells to UVA
irradiation stimulated p70S6K kinase activity in a
time-dependent manner and simultaneously induced a dose-
and time-dependent phosphorylation of p70S6K at
the four sites, suggesting that phosphorylation at these sites may
indirectly reflect p70S6K kinase activity. However,
upstream kinase pathways leading to phosphorylation and activation of
p70S6K are not well understood (12, 22, 25). Here, we used
selective kinase inhibitors and specific dominant negative mutants of
putative p70S6K upstream kinases to further analyze the
roles of PI 3-kinase, mTOR, and MAPKs in the regulation of
UVA-stimulated p70S6K activation and phosphorylation.
Activation of the PI 3-kinase pathways was reported to be required for
phosphorylation and activation of p70S6K (12, 23, 59).
PDK1, a downstream kinase of PI 3-kinase, was clearly shown to be a
markedly selective kinase for Thr229 phosphorylation
contributing to p70S6K activation (12, 22, 25, 60). In
addition, Thr389 phosphorylation was also shown to be
mediated by PI 3-kinase/PDK1 and contributed to the activation of
p70S6K (12, 22, 60). More recent studies indicated that
Thr389 phosphorylation might create a PDK1 docking site in
p70S6K that recruits and activates PDK1, leading to
Thr229 phosphorylation and its activation (61). These
previous studies suggest that phosphorylation at Thr229 and
Thr389 occurs through the PI 3-kinase pathway and reflects
p70S6K activation in vivo. We observed that the
changes in Thr389 phosphorylation correlate with those of
p70S6K kinase activity when JB6 cells were exposed to UVA
irradiation. Moreover, UVA-stimulated p70S6K activity and
phosphorylation of Thr389, but not Ser411 or
Thr421/Ser424, were prevented by wortmannin, a
selective inhibitor of the PI 3-kinase p110 subunit, and also were
abolished by a dominant negative mutant of the PI 3-kinase p85 subunit
(DNM- However, a kinase specific for Thr389 phosphorylation
regulated by PI 3-kinase has not yet been identified. mTOR was shown to be a critical kinase in regulation of p70S6K activation.
Immunoprecipitation studies showed that p70S6K at
Thr389 was phosphorylated in vitro by mTOR (28,
71). But other studies showed that regulation of p70S6K
activation and Thr389 phosphorylation by mTOR occurs
through inhibition of protein phosphatase 2A-mediated dephosphorylation
(12, 22, 29, 31, 32). Here, UVA-stimulated p70S6K activity
and phosphorylation at Thr389 were completely abrogated by
rapamycin, an mTOR inhibitor (20, 53), suggesting that mTOR is also
required for p70S6K activation and phosphorylation at
Thr389 induced by UVA exposure. On the other hand,
UVA-induced phosphorylation at Ser411 and
Thr421/Ser424 of p70S6K were only
partially blocked by rapamycin. These data suggest that inhibition of
phosphorylation of p70S6K at the four sites leading to its
inactivation may occur via rapamycin-induced protein phosphatase 2A
activation (12, 30, 51), inasmuch as insulin-induced inhibition of
protein phosphatase 2A activity is blocked by rapamycin (62), and these
rapamycin-sensitive S/T-P sites in the autoinhibitory domain do not
serve as a substrate for mTOR in vitro (28). However, the
sites in eukaryotic initiation factor 4E-binding protein 1 phosphorylated by mTOR all contain an S/T-P motif (12), characteristic
of sites phosphorylated by proline-directed kinases (e.g.
MAPK). Therefore, mTOR might phosphorylate similar S/T-P motifs on
p70S6K in the presence of an unidentified cofactor in
vivo.
Although the role of MAPKs in the activation of the p70S6K
signaling pathway has been controversial (12), phosphorylation of Thr229 by PDK1 and of Thr389 by mTOR in
vitro results in only partial activation of p70S6K
(12, 22, 25, 28), suggesting that complete activation of
p70S6K may require additional pathways (e.g.
MAPKs). Earlier studies employing dominant interfering mutants of
Ras/Raf (35) as well as SH2 docking site mutants of platelet-derived
growth factor (34) demonstrated that MAPKs are not necessary for
p70S6K activation (35). However, later studies favor a role
for autoinhibitory domain phosphorylation by MAPKs at the S/T-P sites
for regulating p70S6K activation (23, 36).
Thr229 phosphorylation required for p70S6K
activation depends on prior phosphorylation of other sites that include
the autoinhibitory domain S/T-P sites as well as Thr389
(28, 29, 63, 64). Recently, Weng et al. (22) hypothesized that phosphorylation of the S/T-P sites recognized by MAPKs is the
first step in the hierarchical multisite phosphorylation of p70S6K that leads to its activation. In our studies,
UVA-stimulated p70S6K activity and phosphorylation at
Thr389 were inhibited by the MEK1 inhibitor,
PD98059, and also markedly blocked by a dominant negative mutant of
ERK2 (DNM-ERK2), suggesting that ERKs may be involved in
p70S6K activation and Thr389 phosphorylation.
Furthermore, UVA-induced phosphorylation at Thr421/Ser424 and Ser411 was
blocked by PD98059 and DNM-ERK2, consistent with other published reports (22, 23, 33, 36, 72). Therefore, our data and previous studies
(22, 23, 25, 33, 36, 61, 64, 65) suggest that prior phosphorylation at
Thr421/Ser424 and Ser411 by ERKs
may induce a conformational change of p70S6K that
facilitates a second phosphorylation at Thr389 contributing
to Thr229 phosphorylation and p70S6K activation
in response to UVA irradiation.
The p38 kinase and JNKs are putative upstream kinases that may be
important in the regulation of p70S6K activation (22). Wang
et al. (66) reported that p38 kinase but not JNKs is
involved in activation of p70S6K by arsenite. A p38 kinase
inhibitor, SB203580, prevented the activation of p70S6K by
mitogens (67). But recent experiments using the p38 kinase inhibitor,
SB203580, and dominant mutants of p38 kinase and JNK pathways indicated
that p38 kinase and JNKs are probably not implicated in the regulation
of p70S6K activity in response to osmotic stress (30).
However, our studies suggest that p38 kinase may also be required for
UVA-stimulated p70S6K activation and phosphorylation at
Thr389, inasmuch as UVA-stimulated p70S6K
activation and phosphorylation at Thr389 were blocked by a
p38 kinase inhibitor, SB202190, and also inhibited by a dominant
negative mutant of p38 kinase (DNM-p38). On the other hand, SB202190
and DNM-p38 had no effect on UVA-induced phosphorylation at
Ser411 or Thr421/Ser424. These
results suggest that prior phosphorylation of p70S6K at
S/T-P sites other than Ser411 and
Thr421/Ser424 in the autoinhibitory domain may
occur via activation of p38 kinase and facilitate a second
phosphorylation at Thr389 and subsequent activation of
p70S6K. But another possibility that cannot be ruled out is
that p38 kinase may be an activator of
Akt,2,3
which might have an effect on p70S6K activation.
Interestingly, PD169316, a novel inhibitor of p38 kinase and JNKs (57,
58), significantly blocked UVA-stimulated p70S6K activity,
consistent with the hypothesis that JNKs appear to be involved in
p70S6K activation (22, 68). Furthermore, UVA-induced
p70S6K activity and phosphorylation at Thr389
were significantly suppressed in JB6 cells expressing a dominant negative mutant of JNK1 (DNM-JNK1) and almost completely abrogated in
Jnk1 To further examine the effects of MAPKs on phosphorylation and
activation of p70S6K, we performed in vitro
experiments. Our data showed that the p70S6K proteins
immunoprecipitated from serum-free starved JB6 cell lysates were
phosphorylated at Ser411 by active ERKs and JNKs but not
p38 kinase and were phosphorylated at
Thr421/Ser424 only by ERK1, agreeing at least
partially with the results of Mukhopadhyay et al. (33).
However, Thr389 phosphorylation was induced by MAPKs
in vitro (data not shown). Together with the in
vivo experiments, these data suggest that p70S6K
activation may be triggered through a prior phosphorylation at Ser411 by JNKs and ERKs, at
Thr421/Ser421 by ERKs, or at other S/T-P sites
possibly by p38 kinase and a second phosphorylation at
Thr389 by a downstream unidentified kinase of PI
3-kinase/mTOR. Indeed, phosphorylation at the S/T-P sites in the
autoinhibitory domain is confirmed to participate in phosphorylation of
the p70S6K activation loop, leading to its activation (25,
64, 65, 69). Additionally, we found that the p70S6K
proteins precipitated with PI 3-kinase and possibly PDK1 but not with
Akt or MAPK downstream kinases including RSK1, RSK2, RSK3, and mitogen-
and stress-activated protein kinase (Fig. 8 and data not shown).
Furthermore, our data showed that the p70S6K complex
possessed a partial basal level of phosphorylation and, thus, was
"primed" for its activation by MAPKs in vitro, although p70S6K activity was shown not to be induced by MAPKs alone
in vitro (12, 22, 23, 28, 59). In fact, active PI 3-kinase
(p110) has been shown to induce p70S6K activation and
phosphorylation at Thr229, Thr389,
Ser411, and Thr421/Ser424 in
vivo (22). Also, p70S6K forms a complex with PDK1 in
co-transfected cells (70), but the binding site has not been
identified. Recently, Gu et al. (43) reported that
interrelationships between ERKs and p70S6K were
characterized and a complex between both kinases was documented by
immunoprecipitation, suggesting that p70S6K may be a
potential substrate of ERKs or be dependent upon ERKs. This idea was
supported by our experiments showing that PP1-deactivated p70S6K preparations were partially reactivated by ERKs
in vitro, but the possibility of the existence of an
unidentified kinase or a coactivator in our reaction system cannot be
ruled out. Thus, these results suggest that full activation of
p70S6K may be induced by cooperation of MAPKs with PI
3-kinase/PDK1 or an unidentified p70S6K-activating kinase.
Another possibility remaining to be determined is that prior
autoinhibitory domain phosphorylation by MAPKs may induce a
conformational change in p70S6K that allows for a second
phosphorylation at the S/T-P sites (Ser371 and possibly
Thr367) in the kinase extension domain by MAPKs,
contributing to p70S6K activation (22, 26, 27). Overall,
MAPKs, like PI 3-kinase/mTOR, may be required for phosphorylation and
activation of p70S6K by UVA.
p85), ERK2
(DNM-ERK2), p38 kinase (DNM-p38), and JNK1 (DNM-JNK1) and were absent
in Jnk1
/
or Jnk2
/
knockout cells. The
p70S6K phosphorylation at Ser411 and
Thr421/Ser424 was inhibited by rapamycin,
PD98059, or DNM-ERK2 but not by wortmannin, SB202190, DNM-
p85, or
DNM-p38. However, Ser411, but not
Thr421/Ser424 phosphorylation, was suppressed
in DNM-JNK1 and abrogated in Jnk1
/
or
Jnk2
/
cells. In vitro assays indicated that
Ser411 on immunoprecipitated p70S6K proteins is
phosphorylated by active JNKs and ERKs, but not p38 kinase, and
Thr421/Ser424 is phosphorylated by ERK1, but
not ERK2, JNKs, or p38 kinase. Moreover, p70S6K
co-immunoprecipitated with PI 3-kinase and possibly PDK1. The complex
possibly possessed a partial basal level of phosphorylation, but not at
MAPK sites, which was available for its activation by MAPKs in
vitro. Thus, these results suggest that activation of MAPKs, like
PI 3-kinase/mTOR, may be involved in UVA-induced phosphorylation and
activation of p70S6K.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice,
another S6 kinase (S6K2) with a 70% overall amino acid homology with
p70S6K and a potential nuclear localization signal at the C
terminus was found to partially compensate for loss of
p70S6K function (15). Recently, another nuclear S6
kinase-related kinase was cloned and identified as a novel nuclear
target of Akt (16). Generally, the p70S6K family plays a
key role in the control of cell size, growth, and proliferation.
Consistent with this concept, inhibition of p70S6K
activation by microinjection of neutralizing antibodies (17) or
treatment of cells with rapamycin, an inhibitor of mammalian target of
rapamycin (mTOR)-p70S6K (18-20), severely impeded cell
cycle progression.
2-46/
CT104 construct was shown to still be activated and phosphorylated at Thr389 in vivo in the presence
of rapamycin, an mTOR inhibitor (12, 22, 29). Other studies indicated
that mTOR activity probably suppresses protein phosphatase 2A-mediated
dephosphorylation of p70S6K (30-32).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85) (41) were established as
previously reported and 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, the
transfectants were selected with G418 and tested with their phosphospecific MAPK antibodies.
-mercaptoethanol) and
reprobed with other primary phosphospecific or nonphosphospecific antibodies.
p85 cells were starved for
24-48 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 irradiation), the
cells were washed once with ice-cold phosphate-buffered saline (PBS)
and lysed in 200 µl of SDS sample lysis buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% (v/v) glycerol,
50 mM dithiothreitol (DTT), and 0.1% bromphenol blue. The
lysed samples were scraped into 1.5-ml tubes and sonicated for 5-10 s.
Samples containing equal amounts of protein (Bio-Rad protein assay)
were loaded into each lane of an 8% SDS-polyacrylamide gel for
electrophoresis (SDS-PAGE) and subsequently transferred onto an
Immobilon P transfer membrane. The phosphorylated p70S6K
protein was selectively detected by Western immunoblotting using a
chemiluminescent detection system and phosphospecific antibodies against p70S6K phosphorylation at Ser411,
Thr421/Ser424, or Thr389.
Nonphosphorylated p70S6K was used as a control to verify
equal protein loading.
p85 grown in
100-mm dishes. After starving by replacing medium with 0.1%
FBS-MEM, the cells were or were not pretreated with inhibitors as
described above and then irradiated with UVA (160 kJ/m2).
The cells were harvested at the times indicated and lysed in 300 µl
of buffer A containing 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 clarified by centrifugation at 17,000 × g
for 5 min at 4 °C. The supernatant fractions containing equal
amounts of proteins were incubated with p70S6K antibody at
4 °C overnight and then for an additional 4 h with protein
A/G-Sepharose beads (Santa Cruz Biotechnology). After washing four
times with PBS, the immunoprecipitates were incubated at 30 °C for
10 min in a mixture of the following: 20 µl of assay dilution buffer
(20 mM MOPS, pH 7.2, 25 mM
-glycerol
phosphate, 5 mM EGTA, 1 mM
Na3VO4, and 1 mM DTT), 10 µl of
substrate mixture (S6 peptide in assay dilution buffer), 10 µl of
inhibitor mixture (20 µM PKC inhibitor peptide, 2 µM protein kinase A inhibitor peptide, and 20 µM compound R24571 in assay dilution buffer), and 10 µl
of [
-32P]ATP (1 µCi/µl; Amersham Pharmacia
Biotech). To stop the reaction, each sample was spotted onto a numbered
P81 paper square and washed three times (5 min each) with 0.75%
phosphoric acid and once (5 min) with acetone. Each sample paper was
transferred into a scintillation vial containing 5 ml of scintillation
fluid and then counted in a
-scintillation counter. At the same
time, immunoprecipitates isolated by nonimmune IgG serum instead
of the p70S6K antibody were used as background controls.
After subtraction of background from each of the samples, the
UVA-stimulated p70S6K kinase activity was normalized to
unstimulated controls and expressed as -fold change.
/
, and Jnk2
/
Primary Embryo
Fibroblasts--
Embryonic 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 Dulbecco's modified
Eagle's medium supplemented with 10% FBS, 2 mM
L-glutamine, 100 units/ml of penicillin, and 100 µg/ml of streptomycin in a humidified atmosphere of 5% CO2 at
37 °C. For analysis of protein phosphorylation, the cells were
starved by replacing growth medium with serum-free Dulbecco's modified
Eagle's medium for 12 h, at which time cells were exposed to UVA.
The cells were lysed with SDS sample buffer, and the protein
concentration in the supernatant fraction of the cell lysates was
determined (Bio-Rad assay). Equal amounts of protein were resolved by
8% SDS-PAGE, and phosphorylated and nonphosphorylated
p70S6K proteins were determined by Western blotting
analysis. Additionally, p70S6K kinase activity in these
cell lines was measured following immunoprecipitation procedures as
described above.
(Z-8), p90 ribosomal S6 kinase (RSK)
3 (C-20), phosphotyrosine (PY99 from Santa Cruz Biotechnology), PDK1,
Akt, RSK1, RSK2, and mitogen- and stress-activated protein
kinase (Upstate Biotechnology) were used as positive controls. To examine whether p70S6K co-immunoprecipitates with PI
3-kinase, immunoprecipitated p70S6K proteins and
corresponding PI 3-kinase controls were incubated with
phosphatidylinositol 4,5-diphosphate (Sigma), a preferential substrate
of PI 3-kinase, and then PI 3-kinase activity was determined by running
thin layer chromatography plates as described below. To further test
whether p70S6K co-immunoprecipitates with the other above
mentioned kinases, immunoprecipitated p70S6K proteins and
positive controls were subjected to SDS-PAGE and Western immunoblotting
with corresponding nonphosphorylated or phosphospecific antibodies
according to the procedure as described above.
p85 were cultured in monolayers in 100-mm
dishes. Then cells were starved for 24 h in serum-free MEM. At
1.5 h following pretreatment with or without wortmannin at the
doses indicated, the cells were irradiated with UVA at 160 kJ/m2. After an additional incubation for 15 min at
37 °C, the cells were washed once with ice-cold PBS and lysed in
buffer B (20 mM Tris-HCl, pH 7.4, 137 mM NaCl,
1 mM MgCl2, 10% glycerol, 1% Nonidet P-40, 1 mM DTT, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin,
leupeptin, and pepstatin). The lysates were centrifuged, and the
supernatant fractions containing equal amounts of protein were
incubated overnight at 4 °C with 20 µl of agarose beads previously
conjugated with mouse monoclonal phosphotyrosine antibody (PY99) or
with rabbit polyclonal PI 3-kinase p85
antibody (Z-8). The
immunocomplex-bound beads were washed twice with each of the following
buffers: 1) PBS containing 1% Nonidet P-40, 1 mM DTT, and
0.1 mM Na3VO4; 2) 100 mM Tris-HCl, pH 7.6, 0.5 M LiCl, 1 mM DTT, and 0.1 mM
Na3VO4; 3) 10 mM Tris-HCl, pH 7.6, 100 mM NaCl, 1 mM DTT, and 0.1 mM
Na3VO4. The beads were incubated for 5 min on
ice in 20 µl of buffer 3, and then 20 µl of 0.5 mg/ml phosphatidylinositol 4,5-diphosphate previously sonicated in substrate buffer (50 mM HEPES, pH 7.6, 1 mM EGTA, 1 mM NaH2PO4) was added. After
incubation for 5 min at room temperature, 10 µl of the reaction buffer (10 mM Tris-HCl, pH 7.6, 60 mM
MgCl2, 250 µM ATP containing 10 µCi of
[
-32P]ATP) was added, and then the beads were
incubated for an additional 10 min at 30 °C. The reaction was
stopped by the addition of 15 µl of 4 N HCl and
subsequently 130 µl of chloroform/methanol (1:1, v/v). After
vortexing for 30 s, 30 µl of lower phospholipid-containing chloroform phase was spotted onto thin layer chromatography plates coated with Silica Gel H containing 1% potassium oxalate (Analtech Inc., Newark, DE) that were baked at 110 °C for at least 1 h
before use. The plates were developed in tanks containing
chloroform/methanol/NH4OH/H2O (60:47:2:11.3,
v/v/v/v) until the solvent reached the top of the plates. The plates
were dried at room temperature and autoradiographed. The
phosphatidylinositol 3,4,5-trisphosphate phosphate blotting was
quantified by the Image Quant software (Molecular Dynamics, Inc., Sunnyvale, CA).
-mercaptoethanl, 0.01% Brij-35
(Sigma). Then PP1 activity was inhibited by the addition of NaF (10 mM). Deactivated p70S6K preparations were saved
by centrifugation and then incubated at 30 °C for 30 min with ERK1
(10 ng/µl), ERK2 (10 ng/µl) (New England Biolabs), JNK1 (50 milliunits/µl), JNK2 (50 milliunits/µl), p38 kinase (10 ng/µl)
(Upstate Biotechnology), and subsequent p70S6K S6 kinase
activity assay was performed as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation and phosphorylation of
p70S6K at Ser411, Thr389, and
Thr421/Ser424 induced by UVA.
A, 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 medium with 0.1% FBS MEM. The medium was again
replaced with fresh 0.1% FBS MEM and allowed to equilibrate for 4 h before treatment. Cells were untreated or exposed to UVA (160 kJ/m2) or UVB (4 kJ/m2). UVB-stimulated cell
samples were used as a positive control, and untreated samples were
used as negative controls. After an additional incubation for 15, 30, or 60 min following UV treatment, the cells were lysed in buffer A for
immunoprecipitation. The p70S6K kinase activity was
determined as described under "Experimental Procedures." Each
bar indicates the mean and S.D. from four independent assays
performed in duplicate. UVA-stimulated activity of p70S6K
was significantly higher than unstimulated control activity (*,
p < 0.001). B, JB6 Cl 41 cells were
cultured as described above and then exposed to UVA, UVC, TPA, or EGF
at the doses indicated. UVC-, TPA-, and EGF-stimulated cell samples
were used as positive controls, and an untreated sample was used as a
negative control. After an additional incubation for 30 min following
the treatment, the cells were lysed in SDS sample buffer for
immunoblotting. Phosphorylation of p70S6K was determined as
described under "Experimental Procedures." The figure
represents one of three similar independent experiments.
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Fig. 2.
Dose- and time-dependent
phosphorylation of p70S6K at Ser411,
Thr389, and Thr421/Ser424 induced
by UVA. JB6 Cl 41 cells (6 × 105) were cultured
in 100-mm dishes until they reached 90% confluence and then were
starved for 48 h in 0.1% FBS MEM. A, the cells were
irradiated with UVA at the doses indicated and harvested after 30 min
and analyzed as described under "Experimental Procedures."
B, cells were exposed to UVA (160 kJ/m2) and
harvested at 15, 30, 120, 360, and 720 min following irradiation and
analyzed as described under "Experimental Procedures." This is one
of three similar independent experiments, and the images
show that UVA-induced phosphorylation of 70S6K at Ser411,
Thr389, and Thr421/Ser424 is dose- and
time-dependent.
p85 Inhibit UVA-induced Activation and
Phosphorylation of p70S6K at Thr389 but Not at
Ser411 or
Thr421/Ser424--
Studies have shown that PI
3-kinase and its downstream kinases, protein kinase B/Akt and PDK1, are
required for activation and phosphorylation of p70S6K (12).
Here, we assess the role of the PI 3-kinase pathway in UVA-stimulated
activation and phosphorylation of p70S6K at
Thr389, Ser411, and
Thr421/Ser424. Our data showed that UVA-induced
phosphorylation of p70S6K at Thr389, but not at
Ser411 or Thr421/Ser424, was
inhibited by pretreatment of JB6 cells with wortmannin (Fig. 3A), a selective inhibitor of
the PI 3-kinase p110 subunit (51). We also observed that wortmannin
markedly suppressed activation of PI 3-kinase (Fig. 3B) and
p70S6K (Fig. 3C; p < 0.001) in
response to UVA irradiation. Further, we used a JB6 cell line
expressing a dominant negative mutant of PI 3-kinase p85 subunit
(DNM-
p85) (41) to study the role of the PI 3-kinase pathway in
UVA-stimulated phosphorylation and activation of p70S6K. As
expected, PI 3-kinase activity induced by UVA was significantly lower
in DNM-
p85 cells (Fig. 3E), agreeing with the results of Sajan et al. (52), who showed that DNM-
p85 blocks p85
subunit-dependent PI 3-kinase and related pathway
activation. Following UVA irradiation, phosphorylation of
p70S6K at Thr389, but not at Ser411
or Thr421/Ser424 (Fig. 3D), and
p70S6K kinase activity were almost completely
attenuated (p < 0.001) in DNM-
p85 cells compared
with control Cl 41 cells expressing only CMV-neo vector (Fig.
3F). Therefore, these results indicate that the PI 3-kinase
pathway may be required for UVA-induced p70S6K activation
and phosphorylation at Thr389 but not for phosphorylation
at Ser411 or Thr421/Ser424. This
suggests that phosphorylation of p70S6K at
Ser411 and Thr421/Ser424 appears to
occur through activation of PI 3-kinase-independent pathways.
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Fig. 3.
Inhibition of activation and phosphorylation
of p70S6K at Thr389 but not at
Ser411 or Thr421/Ser424 by
wortmannin and DNM- p85. JB6 Cl 41 or
DNM-
p85 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 Cl 41 cells were or were
not preincubated for 1.5 h with wortmannin at the doses indicated
and then irradiated with UVA (160 kJ/m2), whereas
DNM-
p85 cells were treated only with UVA at the doses indicated. The
cell samples were harvested 15 or 30 min after irradiation.
Phosphorylation of p70S6K and its kinase activity as well
as PI 3-kinase activity were analyzed as described under
"Experimental Procedures." Some of the sample membranes were
stripped and reprobed with different primary antibodies. This
figure represents one of three independent similar
experiments. The images show that UVA-induced
p70S6K phosphorylation at Thr389, but not at
Ser411 or Thr421/Ser424, is blocked
by wortmannin (A) and DNM-
p85 (D). In
addition, UVA-stimulated PI 3-kinase activity was abolished by
wortmannin (B) and DNM-
p85 (E). Cells were
harvested 15 min (B and E) or 30 min
(A and D) or at the time indicated (C
and F) after UVA irradiation. Each bar represents
the mean and S.D. from three independent assays performed in duplicate.
UVA (160 kJ/m2)-induced p70S6K activity was
significantly inhibited (**, p < 0.01) by wortmannin
(0.2 µM) (C) or DNM-
p85 (F)
compared with corresponding positive controls. PIP3,
phosphatidylinositol 3,4,5-trisphosphate.
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Fig. 4.
Inhibition of UVA-induced activation and
phosphorylation of p70S6K at Thr389,
Ser411, and Thr421/Ser424 with
rapamycin. Cl 41 cells (8 × 105) were cultured
in monolayers for 24 h in 100-mm dishes and subsequently starved
for 48 h in 0.1% FBS MEM. The cells were pretreated for 1.5 h with rapamycin at the doses indicated. Then the cells were harvested
15 or 30 min after irradiation with UVA (160 kJ/m2). The
phosphorylation of p70S6K proteins and S6 kinase activity
were determined as described under "Experimental Procedures." The
sample membrane was stripped and reprobed with different antibodies.
A shows that rapamycin inhibits phosphorylation of
p70S6K at Thr389, Ser411, and
Thr421/Ser424 (30 min). This is one of three
similar independent experiments. B, each bar
indicates the mean and S.D. from three independent assays performed in
duplicate. UVA-induced p70S6K activity was significantly
inhibited (*, p < 0.01) by rapamycin (100 mM) compared with corresponding positive controls.
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Fig. 5.
Inhibition of UVA-induced activation and
phosphorylation of p70S6K at all four sites by PD98059 or
DNM-ERK2. JB6 Cl 41 or DNM-ERK2 cells were cultured for 24 h
in each well of a six-well plate until 90% confluence was reached. The
cells were starved for 48 h in 0.1% FBS MEM and then harvested 15 or 30 min after UVA irradiation or a combination of UVA and
pretreatment with PD98059 at the doses indicated. Total and
phosphorylated p70S6K, ERKs, and p38 kinase as well as 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 similar independent
experiments. A shows that PD98059 blocks UVA-induced
phosphorylation of ERKs and p70S6K at Thr389,
Ser411, and Thr421/Ser424 (30 min).
B shows that DNM-ERK2 suppresses phosphorylation of ERKs and
p70S6K at Thr389/Ser411 and
Thr421/Ser424 (harvested at 30 min).
D, each bar represents the mean and S.D. from
three independent assays performed in duplicate. UVA (160 kJ/m2)-induced p70S6K activity was
significantly inhibited (**, p < 0.001) by PD98059 (25 µM) (C) or DNM-ERK2 (D) compared
with corresponding positive controls.
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Fig. 6.
Inhibition of UVA-induced activation and
phosphorylation of p70S6K at Thr389 but not
Ser411 or Thr421/Ser424 by DNM-p38
and SB202190. JB6 Cl 41 or DNM-p38 cells were treated as described
in Fig. 5. The cells were irradiated with UVA either following SB202190
pretreatment or with no pretreatment. Total and phosphorylated
p70S6K, p38 kinase, and ERKs as well as p70S6K
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 similar independent
experiments. A shows that SB202190 inhibits UVA-induced
phosphorylation of p38 kinase and p70S6K at
Thr389 but not Ser411 or
Thr421/Ser424 (harvested at 30 min).
B, DNM-p38 also blocks phosphorylation of p38 kinase and
p70S6K at Thr389 but not at Ser411
or Thr421/Ser424 (30 min). Each bar
indicates the mean and S.D. from three independent assays performed in
duplicate. UVA (160 kJ/m2)-induced p70S6K
activity was significantly inhibited by SB202190 (1.0 µM), PD169316 (0.5 µM) (C), or
DNM-p38 (D) compared with corresponding positive controls
(*, p < 0.05; **, p < 0.01).
/
and
Jnk2
/
knockout cells to assess whether JNKs play a role
in activation and phosphorylation of p70S6K induced by UVA.
Our data showed that UVA-induced phosphorylation of p70S6K
at Ser411 and Thr389, but not at
Thr421/Ser424, was inhibited by DNM-JNK1
compared with control Cl 41 cells (Fig.
7A), and the phosphorylation
at the same two sites was attenuated in Jnk1
/
and
Jnk2
/
knockout cells compared with wild-type Jnk+/+ cells (Fig. 7B). Also, p70S6K
activation by UVA was partially inhibited (p < 0.05)
in DNM-JNK1 cells (Fig. 7C) and almost totally blocked
(p < 0.001) in Jnk1
/
and
Jnk2
/
cells (Fig. 7D), as compared with those
in corresponding control cells. In addition, UVA-stimulated
phosphorylation and activation of JNKs was blocked in DNM-JNK1 cells
(Fig. 7A and data not shown), and corresponding
phosphorylation and activation of JNKs was attenuated in
Jnk1
/
or Jnk2
/
cells (Fig. 7B
and data not shown). These results suggest that p70S6K
activation and phosphorylation at Ser411 and
Thr389, but not at Thr421/Ser424,
may be required for JNK pathway activation when cells are exposed to
UVA.
View larger version (63K):
[in a new window]
Fig. 7.
Inhibition of UVA-induced activation and
phosphorylation of p70S6K at Thr389 and
Ser411 but not at Thr421/Ser424 in
DNM-JNK1 and Jnk1 /
and Jnk2
/
cells. JB6 Cl 41 or DNM-JNK1 cells were treated as described
in the legend to Fig. 5. Preparation and treatment of primary embryo
fibroblasts of wild-type Jnk+/+ and Jnk1
/
or
Jnk2
/
knockout mice and analysis of p70S6K
and JNKs and their phosphorylated proteins as well as of
p70S6K kinase activity were performed as described under
"Experimental Procedures." The sample membrane was stripped and
reprobed with different antibodies. This is one of three similar
independent experiments. The images show that DNM-JNK1
inhibits UVA-induced phosphorylation of JNKs and p70S6K at
Thr389 and Ser411 but not
Thr421/Ser424 (A) and also partially
suppresses p70S6K kinase activity following exposure to UVA
(160 kJ/m2) (C). Moreover, phosphorylation of
JNKs and p70S6K at Thr389 and
Ser411, but not Thr421/Ser424
(B), as well as p70S6K kinase activity induced
by UVA (80 kJ/m2) (D) was also almost completely
attenuated in Jnk1
/
and Jnk2
/
cells. Each
bar represents the mean and S.D. from three independent
experiments performed in duplicate. UVA-induced p70S6K
activity in DNM-JNK1, Jnk1
/
, or Jnk2
/
cells was significantly inhibited compared with corresponding positive
controls (*, p < 0.05; **, p < 0.001).
View larger version (34K):
[in a new window]
Fig. 8.
Co-immunoprecipitates of p70S6K
and PI 3-kinase or possibly PDK1 are phosphorylated and activated by
active MAPKs in vitro. After starvation of JB6
cells for 48 h, the cell lysates were subjected to
immunoprecipitation (IP) with rabbit anti-
p70S6K polyclonal antibody. A, the immune
complexes were incubated with or without MAPKs including ERK1, ERK2,
JNK1, JNK2, or p38 kinase (Upstate Biotechnology) in the amounts
indicated. Then the reactions were analyzed by Western immunoblotting
with mouse anti-phosphospecific p70S6K monoclonal antibody
(Ser411) or rabbit anti-phosphospecific p70S6K
polyclonal antibodies (Thr421/Ser424). Shown is
one of three similar independent experiments. B, the
IP-p70S6K proteins were incubated in vitro in
kinase buffer with MAPKs and the S6 peptide as a substrate of
p70S6K. The addition of bovine serum albumin instead of
MAPKs was used as an internal control. C, the S6 peptide was
incubated with or without MAPKs in kinase buffer containing protein A/G
plus Sepharose. B, each bar represents the mean
and S.D. from three independent assays performed in duplicate.
Immunoprecipitates of p70S6K were significantly activated
(*, p < 0.05; **, p < 0.01) in the
presence of the different MAPKs versus the control
containing no MAPK or the control containing no immunoprecipitated
p70S6K (p < 0.0001). D,
IP-p70S6K and IP-PI 3-kinase were precipitated from
UVA-irradiated or nonirradiated cell lysates and then subjected to the
PI 3-kinase assay as described under "Experimental Procedures."
This is one of three similar independent experiments. E and
F, p70S6K, IP-PDK1, and IP-Akt from
UVA-irradiated or nonirradiated cell lysates were precipitated and then
subjected to Western immunoblotting analysis with anti-PDK1, anti-Akt,
or phospho-Akt (Ser473) antibodies, respectively. One of
three similar independent experiments is shown.
View larger version (22K):
[in a new window]
Fig. 9.
Partial reactivation of PP1-dephosphorylated
IP-p70S6K preparations by ERKs. A, the
p70S6K preparations from serum-starved JB6 Cl 41 cells were
immunoprecipitated with p70S6K antibody and then
dephosphorylated by PP1 (0.5 unit) treatment (72, 73). Then the PP1
activity was inhibited by the addition of NaF (10 mM).
Subsequently, PP1-deactivated p70S6K preparations were
incubated with ERK1 (10 ng/µl), ERK2 (10 ng/µl), p38 kinase (10 ng/µl), JNK1 (50 milliunits/µl), or JNK2 (50 milliunits/µl) or
without MAPKs (as a control), and an S6 kinase activity assay was
performed as described under "Experimental Procedures." Each
bar indicates the mean and S.D. from two independent
experiments performed in duplicate. Partial reactivation of PP1-treated
p70S6K by ERKs is different (*, p < 0.05)
from that of corresponding control by no MAPKs. B shows
concise procedures of the above mentioned experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
p85), further suggesting that UVA-stimulated activation and
phosphorylation at Thr389 may be regulated by the PI
3-kinase pathway but that the phosphorylation at the autoinhibitory
domain S/T-P sites (Ser411 and
Thr421/Ser424) occurs through PI
3-kinase-independent pathways.
/
and Jnk2
/
cells compared with their
corresponding control cells, suggesting that activation of the JNK
pathway may be involved in UVA-stimulated p70S6K
activation. Moreover, UVA-stimulated phosphorylation of
p70S6K at Ser411 but not
Thr421/Ser424 was inhibited in DNM-JNK1 cells
and also attenuated in Jnk1
/
and Jnk2
/
cells. These results indicated that prior phosphorylation at
Ser411 but not Thr421/Ser424 may
occur through activation of JNKs and facilitate a second phosphorylation at Thr389 and subsequent activation of
p70S6K.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Wei-Ya Ma for help with some details of the experimental methods 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. Hormel Institute, University of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.: 507-437-9640; Fax: 507-437-9606; E-mail: zgdong@smig.net.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M009047200
2 Nomura, M., and Dong, Z., manuscript in preparation.
3 Liu, G., Zhang, Y., Bode, A. M., Ma, W. Y., and Dong, Z., manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: UVA, UVB, and UVC, ultraviolet light A, B, and C, respectively; p70S6K, p70/p85 ribosomal S6 kinases; S/T-P, proline-directed Ser/Thr; TPA, 12-O-tetradecanoylphorbol-13-acetate; EGF, epidermal growth factor; PI, phosphatidylinositol; PDK1, 3-phosphoinositide-dependent protein kinase 1; mTOR, mammalian target of rapamycin; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p38, p38 MAPK or p38 kinase; PP1, protein phosphatase 1; RSK, p90 ribosomal S6 kinase(s); MEM, Eagle's minimum essential medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; DNM, dominant negative mutant; MOPS, 4-morpholinepropanesulfonic acid; p-, phospho-; np-, nonphospho-.
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