From the Department of Biological Chemistry,
University of Michigan Medical School, and the ¶ Institute of
Gerontology, University of Michigan, Ann Arbor, Michigan
48109
Received for publication, January 27, 2003, and in revised form, February 5, 2003
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ABSTRACT |
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Tuberous sclerosis complex (TSC) is a genetic
disease caused by mutations in either TSC1 or TSC2 tumor suppressor
genes. TSC1 and TSC2 (also known as hamartin and tuberin, respectively)
form a functional complex and negatively regulate cell growth by
inhibiting protein synthesis. 14-3-3 binds to TSC2 and may inhibit TSC2
function. We have reported previously that phosphorylation of serine
1210 (Ser1210) in TSC2 is essential for 14-3-3 binding. Here we show that serum and anisomycin enhance the interaction
between TSC2 and 14-3-3 by stimulating phosphorylation of
Ser1210. Activation of p38 MAP kinase (p38) is essential
for the stimulating effect of serum and anisomycin although p38 is not
directly responsible for the phosphorylation of Ser1210 in
TSC2. Both in vitro and in vivo experiments
demonstrate that the p38-activated kinase MK2 (also known as MAPKAPK2)
is directly responsible for the phosphorylation of Ser1210.
Our data show that anisomycin stimulates phosphorylation of Ser1210 of TSC2 via the p38-MK2 kinase cascade.
Phosphorylation of TSC2 by MK2 creates a 14-3-3 binding site and thus
regulates the cellular function of the TSC2 tumor suppressor protein.
Tuberous sclerosis complex
(TSC)1 is an autosomal
dominant genetic disorder occurring in 1/6,000 individuals. Studies of
TSC patients demonstrate that mutation of either TSC1 or TSC2 is
responsible for TSC. Mutation of TSC1 and TSC2 each accounts for
~50% of TSC cases (1). TSC is characterized by the development of
benign hamartomas in many organs, including brain, kidney, heart, skin, and eyes. Although malignancy rarely develops in TSC hamartomas, brain
hamartomas produce the most serious clinical complication and often
result in mental retardation, seizures, and autism. Other symptoms
include renal dysfunction, dermatological abnormalities, and heart
problems (2).
Studies of TSC patients and animal models support the hypothesis that
TSC1 and TSC2 are tumor suppressor genes. Loss of function of either
the TSC1 or TSC2 gene product is the underlying molecular basis for the
pathogenesis of TSC (3, 4). Eker rats contain a heterozygous mutation
in TSC2 and have very high incidence of tumors, especially renal
carcinomas (5). Tumors in Eker rats are generated by mutation of the
wild type allele of the TSC2 gene. Homozygous deletion of either TSC1
or TSC2 in mice produces an embryonic lethal phenotype, demonstrating
an essential function in development (6, 7). Heterozygous deletion of
either TSC1 or TSC2 shows 100% incidence of renal carcinomas and a
significant increase of carcinomas in other tissues.
TSC1 and TSC2 gene products are also known as hamartin and tuberin,
respectively. Mutation in TSC1 or TSC2 results in similar phenotypes,
suggesting that the two proteins function in the same pathway (8). In
fact, TSC1 and TSC2 form a physical complex, and the TSC1·TSC2
complex is functionally important in vivo. Many disease-derived mutations in TSC2 weaken the complex formation with
TSC1 (9-12). However, the cellular functions of TSC1 and TSC2 were not
known until recent genetic studies in Drosophila demonstrated that TSC1 and TSC2 play a major negative role in the
regulation of cell growth (13-15). Mutations of either TSC1 or TSC2
significantly increase cell size in Drosophila. Genetic epistatic studies indicate that TSC1·TSC2 acts downstream from the
insulin receptor, which plays a major role in cell growth control and
cell size regulation. Phosphatidylinositol 3-kinase (PI 3-kinase) is a
major downstream effector of the insulin receptor. Activation of the PI
3-kinase-Akt pathway plays an important role in cell proliferation,
oncogenic transformation, cell survival, and cell size control
(16).
We have reported recently that TSC2 is a direct physiological target of
Akt. Akt phosphorylates TSC2 on multiple sites and inactivates TSC2
function (12). We identified that serine 939, serine 1086, serine 1088, and threonine 1422 of TSC2 are Akt-dependent phosphorylation sites (12). Similar observations were also made by
several other laboratories (17-20). TSC1·TSC2 functions to inhibit S6K, a positive regulator of translation, by decreasing the
phosphorylation state and activation of S6K. Similarly, TSC1·TSC2
activates eukaryotic initiation factor 4E-binding protein 1, a
negative regulator of translation, by inhibiting the phosphorylation of
this protein. Therefore, TSC1·TSC2 negatively regulates protein
synthesis. These observations provide an important molecular basis for
TSC1·TSC2 acting downstream from growth factor receptors to modulate
cell growth negatively. We and other groups have provided evidence that
TSC1·TSC2 inhibits the function of the mTOR (mammalian target of
rapamycin), which is the kinase directly upstream from S6K and
eukaryotic initiation factor 4E-binding protein 1 (12, 21, 22).
However, the mechanism of how TSC1·TSC2 inhibits mTOR is unknown, and
it is possible that TSC1·TSC2 may inhibit S6K independently of mTOR
(23).
The biochemical activity of TSC1 and TSC2 is largely unknown, although
GTPase-activating protein (GAP) activity has been implicated for
TSC2 (24-26). Phosphorylation of TSC1 and TSC2 is likely a major
mechanism involved in the regulation of TSC1 and TSC2. We have
demonstrated that TSC2 binds to 14-3-3 in a
phosphorylation-dependent manner (27). Furthermore, the
interaction between TSC2 and 14-3-3 is detected under physiological
conditions. Similar results have been reported by other research groups
(28-30). It has also been shown that 14-3-3 binding inhibits the
function of TSC2. Nellist et al. (29) reported that the
interaction between TSC2 and 14-3-3 is enhanced by
Akt-dependent phosphorylation of TSC2. Liu et
al. (30) reported that an Akt-dependent
phosphorylation site, Ser939, is required for 14-3-3 binding. However, our studies have shown that serum stimulates the
interaction between TSC2 and 14-3-3, and phosphorylation of
Ser1210 is responsible for 14-3-3 binding. Our data
indicate that Akt is not directly involved in the regulation of the
interaction between TSC2 and 14-3-3 (27). Studies reported by Shumway
et al. (28) are consistent with our observation. The
molecular mechanism of Ser1210 phosphorylation induced by
serum and anisomycin stimulation and the kinase responsible for
Ser1210 phosphorylation are the subjects of this study.
In this report, we show that MK2, also known as MAPKAPK2 (31), is
responsible for the phosphorylation of Ser1210 in TSC2, and
this phosphorylation increases 14-3-3 binding. We have demonstrated
that both serum and anisomysin, which activate p38 and S6K, stimulate
the phosphorylation of Ser1210 and enhance the interaction
between TSC2 and 14-3-3 via the p38 MAP kinase pathway. The S6K
activation by anisomycin is compromised in the TSC2 Antibodies and Plasmids--
Anti-TSC2 and anti-14-3-3 were from
Santa Cruz Biotechnology. Anti-HA and anti-Myc were from Covance.
Anti-p38 MAP kinase and
anti-phospho(Thr180/Tyr182)-p38 MAP kinase were
from Cell Signaling. Human TSC1 and rat TSC2 constructs were described
previously (12). HA-PRAK and Myc-MK2 constructs were generously
provided by Dr. J. Han (Department of Immunology, The Scripps Research
Institute, La Jolla, CA). GST-MNK1 constructs were generously provided
by Dr. J. A. Cooper (Fred Hutchison Cancer Center, Seattle, WA).
All other DNA constructs including pcDNA3- Myc-14-3-3 Cell Culture, Transfection, and Immunoprecipitation--
HEK293
cells were seeded and maintained in Dulbecco's modified Eagle's
medium containing 10% fetal bovine serum (FBS). EEF4 (TSC2+/+ cell line) (5) and EEF8 (TSC2 Metabolic Labeling and Two-dimensional Phosphopeptide
Mapping--
HEK293 cells were cotransfected with the various plasmids
using LipofectAMINE. The serum-starved cells were washed twice with phosphate-free Dulbecco's modified Eagle's medium and incubated with
0.25 mCi/ml 32Pi (ICN) for 4 h. HA-tagged
TSC2 was immunoprecipitated, resolved by SDS-PAGE, and transferred to a
polyvinylidene difluoride membrane. Phosphorylated TSC2 was visualized
by autoradiography. Phosphopeptide mapping was performed as described
previously (32).
Phosphorylation Site Mapping by Mass Spectrometry--
In
vivo labeled TSC2 was immunoprecipitated from HEK293 cells as
described above and resolved by SDS-PAGE and Coomassie Blue staining.
The TSC2 band was excised and in-gel digested with trypsin. The
resulting pool of tryptic peptides was fractionated using reverse phase
HPLC, and selected fractions were analyzed by matrix-assisted laser
desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry using a Voyager-DE Pro instrument (Applied Biosystems) in positive ion
mode. MALDI-postsource decay (PSD) was performed on selected parent
ions to observe neutral loss of the phosphate moiety and obtain partial
sequence information.
In Vitro Kinase Assay--
GST fusion protein containing an
11-amino acid fragment of TSC2 (TSC2F),
T1203ALYKSLSVPA1213, and the same
fragment with an Ala1210 mutation (TSC2F-M),
T1203ALYKSL-AVPA1213, were used
as substrates for the MK2 in vitro kinase assay. The oligonucleotides encoding the above peptides were synthesized. The
complimentary oligonucleotides were annealed and ligated into the
EcoRI- and XhoI-digested pGEX-KG vector. Each
clone was confirmed by DNA sequencing. GST and GST-TSC2F,
GST-TSC2F-M(S1210A) were expressed in the bacterial strain BL21 and
purified using glutathione-Sepharose 4B beads (Sigma) as described
(33).
In vitro kinase assays were performed using standard
experimental conditions as described previously (34). Kinase assays were carried out at 37 °C for 30 min in a reaction volume of 30 µl, containing immunoprecipitated MK2, purified GST-TSC2F,
GST-TSC2F-M(S1210A), 250 µM ATP, and 20 µCi of
[ p38 MAP Kinase Is Required for the Interaction between TSC2 and
14-3-3--
Our previous study has demonstrated that phosphorylation
of Ser1210 of TSC2 is necessary and sufficient for its
interaction with 14-3-3 (27). This observation is consistent with the
report by Shumway et al. (28). However, Nellist et
al. (20) and Liu et al. (30) reported that 14-3-3 binds
to different sites, and the interaction is stimulated by Akt
phosphorylation. To understand the molecular mechanism of TSC2
regulation, we tested the effect of several pharmacological kinase
inhibitors. TSC2 was cotransfected with 14-3-3 into HEK293 cells.
Interaction between TSC2 and 14-3-3 was determined by
immunoprecipitation of TSC2 and subjected to immunoblot with
anti-Myc-14-3-3. We have observed that serum stimulates the interaction
between TSC2 and 14-3-3. Serum is known to activate numerous kinases
including the ERK, PI 3-kinase, and p38 MAP kinase, therefore
inhibitors of these kinase pathways were examined. We found that
SB203580, which is a p38-specific inhibitor (35), significantly
inhibited the interaction between TSC2 and 14-3-3 (Fig.
1A). SB203580 inhibits the
interaction in a dose-dependent manner, consistent with a
role for p38 MAP kinase. In contrast, inhibition of the MEK-ERK pathway
by PD98059, which is a MEK inhibitor, and the inhibition of PI 3-kinase
by wortmannin had no effect on the interaction between TSC2 and 14-3-3 (Fig. 1A). The effectiveness of PD98059 and wortmannin was
confirmed by the inhibition of ERK and Akt, respectively (data not
shown). These data demonstrate that Akt does not regulate the
interaction between TSC2 and 14-3-3. The effect of SB203580 was
confirmed by the blocking the interaction of endogenous TSC2 and 14-3-3 (Fig. 1B). Inclusion of SB203580 significantly decreased the
amount of TSC2 in the 14-3-3 immunoprecipitated complex. These data
demonstrate that p38 MAP kinase is likely to play a role in the
phosphorylation of TSC2 and stimulate the interaction between TSC2 and
14-3-3.
Anisomycin Stimulates the Interaction between TSC2 and
14-3-3--
Anisomycin has been well characterized as a potent
activator of p38 MAP kinase (36). We determined whether anisomycin may modulate the phosphorylation of TSC2 and stimulate the interaction between TSC2 and 14-3-3. As expected, anisomycin treatment caused activation of p38 MAP kinase as determined by immunoblotting with and
anti-phospho-p38 antibody, which recognizes the phosphorylated active
form of p38 (Fig. 1, C and D).
Coimmunoprecipitation experiments demonstrated that anisomycin
treatment caused a significant increase of the interaction between TSC2
and 14-3-3 (Fig. 1C). Furthermore, anisomycin treatment also
induced a mobility shift of TSC2, indicating that anisomycin may induce
phosphorylation of TSC2. Interestingly, the time course of interaction
between TSC2 and 14-3-3 closely correlates with the activation of p38
and the mobility shift of TSC2 (Fig. 1C). A
dose-dependent response of anisomycin was also examined
(Fig. 1D). Again, the dose-dependent effect of
anisomycin on the interaction between TSC2 and 14-3-3 closely
correlates with the effect of anisomycin on the p38 activation and the
mobility shift of TSC2. The above observations strongly indicate that
anisomycin activates p38, induces phosphorylation of TSC2, and enhances
the interaction between TSC2 and 14-3-3.
p38 MAP Kinase Plays an Important Role in TSC2 Phosphorylation in
Response to Anisomycin Treatment--
To test further the role of p38
in the regulation of the interaction between TSC2 and 14-3-3, p38 was
activated by coexpression with an active MKK3 mutant (MKK3-DE), which
directly phosphorylates and activates p38 (37). The interaction between
TSC2 and 14-3-3 was examined in HEK293 cells cotransfected with MKK3-DE
and p38. We observed that activation of p38 by MKK3-DE increased the
interaction between TSC2 and 14-3-3 (Fig.
2A). Coexpression of MKK3-DE
and p38 also induced a significant mobility shift of TSC2 similar to
those caused by anisomycin (Fig. 2A, compare lanes
1, 3, and 4). To test further the role of
p38 in FBS- and anisomycin-stimulated interaction between TSC2 and
14-3-3, we tested the effect of dominant negative p38-KM, which is a
kinase-inactive mutant and functions as a dominant negative by blocking
endogenous p38 activation. Coexpression of p38-KM blocked the effect of
both FBS and anisomycin on the interaction between TSC2 and 14-3-3 (Fig. 2A). Furthermore, the dominant negative p38-KM blocked
the mobility shift of TSC2 induced by anisomycin. These data indicate
that endogenous p38 is likely to have an important role in the
phosphorylation of TSC2 in response to FBS and anisomycin
stimulation.
The effect of anisomycin on the mobility shift of TSC2 and the
interaction between TSC2 and 14-3-3 indicate that anisomycin stimulates
TSC2 phosphorylation, including phosphorylation of Ser1210.
Therefore, in vivo 32P labeling and
two-dimensional phosphopeptide mapping were performed. Consistent with
our previous studies, TSC2 is phosphorylated on multiple tryptic
peptides (Fig. 2B). We observed that anisomycin treatment
induced phosphorylation of four tryptic peptides, peptides 1-4 (only
the relevant section of the two-dimensional phosphopeptide map is
shown) (Fig. 2B, compare a and b).
Peptide 1 contains the Ser1210 phosphorylation (27);
peptide 5 contains the Ser939 phosphorylation (12). Neither
activation of p38 (by anisomycin or MKK3-DE+p38 transfection) nor
inhibition by SB203580 caused a significant effect on peptide 5, which
is the Akt-dependent Ser939-phosphorylated
peptide (Fig. 2B). Inclusion of SB203580 efficiently blocked
the effect of anisomycin (Fig. 2B, compare b and
c). Coexpression of MKK3-DE or MKK3-DE+p38 also increased
TSC2 phosphorylation on the same four phosphopeptides that are enhanced
by anisomycin, indicating that p38 plays a major role in TSC2
phosphorylation in response to anisomycin (Fig. 2B, compare
a, b, d, and e).
Furthermore, coexpression of the dominant negative p38-KM blocked the
effect of MKK3-DE (Fig. 2B, compare d and
f). Our phosphopeptide mapping experiment clearly implicates
p38 in TSC2 phosphorylation. Taken together, our data demonstrate that
the p38 MAP kinase plays a major role in the phosphorylation of
Ser1210 in response to anisomycin stimulation.
Our results are inconsistent with the report by Liu et al.
(30) that Ser939 is the 14-3-3 binding site. We examined
the interaction of TSC2-S939A mutation. Our data clearly demonstrate
that phosphorylation of Ser939 is not responsible for
14-3-3 binding (Fig. 2C). Mutation of Ser939 to
alanine (S939A) had no effect on the interaction with 14-3-3. Further,
the interaction between the TSC2-S939A mutant and 14-3-3 is stimulated
by anisomycin (Fig. 2C). Phosphorylation of
Ser939 is not affected by anisomycin, SB203580, MKK3, and
p38-KM, whereas the interaction between TSC2 and 14-3-3 is altered by
these treatments (Figs. 1A and 2, A and
B).
To examine the role of TSC2 in S6K activation by anisomycin, we
examined the TSC2 MK2 Stimulates the Interaction between TSC2 and 14-3-3--
p38
MAP kinase is a proline-directed kinase that phosphorylates Ser/Thr
adjacent to a proline residue (38). However, Ser1210 in
TSC2 is followed by a valine residue, therefore Ser1210 is
unlikely to be a direct phosphorylation target of p38. These observations suggest that Ser1210 in TSC2 is likely to be
phosphorylated by a kinase downstream from p38. p38 has been shown to
activate several protein kinases including MK2, PRAK, and MNK1 (31, 34,
39). We tested the effect of these three kinases individually on the
interaction between TSC2 and 14-3-3. Coexpression of the wild type MK2
significantly increased the coimmunoprecipitation between TSC2 and
14-3-3 (Fig. 3A). The effect
of MK2 requires its kinase activity because KM, which has the catalytic
essential lysine replaced by a methionine, failed to stimulate the
interaction between TSC2 and 14-3-3. Furthermore, the constitutively
active MK2-EE mutant, which has the activation phosphorylation sites
replaced by acidic glutamic residues and possesses high kinase
activity, increased the interaction between TSC2 and 14-3-3 compared
with wild type MK2 (Fig. 3A). It is worth noting that MK2,
especially the constitutively active MK2-EE mutant, induced a mobility
shift of TSC2 (Fig. 3A, compare lanes
5/6 and 7/8). The above results
support a role of MK2 in TSC2 phosphorylation.
We also examined the effect of another kinase activated by p38, PRAK
(34). Cotransfection of PRAK wild type, KM, and constitutively active
mutant (182D), which has the activating phosphorylation site replaced
by an acidic aspartic residue, had no effect on the interaction between
TSC2 and 14-3-3 (Fig. 4B).
Consistently, PRAK coexpression did not induce a mobility shift of
TSC2. MNK1 is particularly interesting because it has been implicated
in regulation of translation and is also activated by p38 (39). We
performed similar experiments with MNK1 and found that coexpression of
MNK1 affected neither the interaction between TSC2 and 14-3-3 nor the
mobility shift of TSC2 (Fig. 4C). Therefore, we conclude that MK2 plays an important role in TSC2 phosphorylation and the interaction with 14-3-3, whereas neither PRAK nor MNK1 is involved.
MK2 Functions Downstream from p38 to Regulate the Interaction
between TSC2 and 14-3-3--
The importance of MK2 in the regulation
of the interaction between TSC2 and 14-3-3 was examined using the
dominant negative MK2-KM. We observed that MK2-KM effectively blocked
the interaction between TSC2 and 14-3-3 in response to stimulation by
either FBS or anisomycin (Fig. 4A). MK2-KM decreased the
interaction even below the basal level (Figs. 3A and
4A, compare lanes 1, 4, and 5). These results suggest that the endogenous MK2 plays a
role in the interaction between TSC2 and 14-3-3 and that MK2 mediates the effect of FBS and anisomycin.
The fact that MK2 is activated by p38 indicates that MK2 acts
downstream from p38 to regulate TSC2 and SB203580 should not block the
effect of MK2. We performed experiments to test this hypothesis.
Coexpression of MKK3-DE and p38 stimulated the interaction between TSC2
and 14-3-3. Treatment with SB203580 inhibited the effect of MKK3-DE+p38
(Fig. 4B, compare lanes 1-3 and
4-6). In contrast, SB203580 did not significantly inhibit
the enhancement of the interaction between TSC2 and 14-3-3 by MK2-EE
expression (Fig. 4B, compare lanes 7/8
and 9/10). In addition, SB203580 blocked the
mobility shift of TSC2 induced by MKK3-DE+p38 but not that induced by
MK2-EE (Fig. 4B). These results demonstrate that MK2 acts
downstream from p38 to regulate the phosphorylation of TSC2.
MK2 Phosphorylates Ser1210 of TSC2--
The above data
strongly indicate that MK2 plays a positive role in the phosphorylation
of Ser1210 of TSC2 and stimulates the interaction with
14-3-3. However, our results do not distinguish whether MK2 directly
phosphorylates TSC2, or indirectly activates another downstream kinase
and/or inhibits a downstream phosphatase. We analyzed the sequence
surrounding Ser1210 of TSC2 and found that it resembles the
consensus phosphorylation sites of several known MK2 substrates (Fig.
5A) (40-44). The
conserved hydrophobic residue at position
We have shown that activation of p38 and MK2 increases the interaction
between TSC2 and 14-3-3. To test whether phosphorylation of
Ser1210 is essential for the stimulation by MK2, we
examined the TSC2-S1210A mutant. The TSC2-S1210A mutant cannot interact
with 14-3-3, and coexpression of MKK3-DE+p38 or MK2-EE did not
stimulate the interaction between TSC2 and 14-3-3 (Fig. 5C).
These results demonstrate that phosphorylation of Ser1210
in TSC2 is required for the p38- and MK2-dependent
stimulation of the interaction between TSC2 and 14-3-3. Interestingly,
FBS, anisomycin, MKK3-DE+p38, and MK2-EE can still induced a mobility shift of the TSC2-S1210A mutant (Fig. 5C), suggesting that
these signals stimulate phosphorylation of additional sites in TSC2 as
shown in the two-dimensional phosphopeptide mapping (Figs. 2B and 5D).
To determine whether MK2 indeed stimulates phosphorylation of
Ser1210 of TSC2 in vivo, we performed
two-dimensional phosphopeptide mapping. Cotransfection of MK2-EE
increased phosphorylation of peptide 1 (Fig. 5D,
a and b). Anisomycin treatment stimulated phosphorylation of four peptides (c). Coexpression of the
MK2-KM dominant negative mutant significantly blocked the
phosphorylation of peptides 1, 2, and 4 (d). The above data
demonstrate that MK2 mediates the effect of anisomycin to phosphorylate
Ser1210 of TSC2 in vivo. It is worth nothing
that peptide 5 (containing Ser939) is not affected by
anisomycin, MK2-EE, or MK2-KM, further supporting that phosphorylation
of Ser939 in TSC2 is not involved in 14-3-3 binding.
Mutation of Ser1210 in TSC2 results in a specific loss of
phosphopeptide 1 (Fig. 5D, e and f),
suggesting that phosphorylation of Ser1210 is responsible
for peptide 1. However, it is possible that peptide 1 may not contain
Ser1210 phosphorylation. Mutation of Ser1210
may alter phosphorylation of another residue that is responsible for
peptide 1, or this mutation may alter the migration of the peptide 1. To demonstrate further that TSC2 is phosphorylated at
Ser1210 in vivo, a mass spectrometry approach
was utilized. TSC2 was immunoprecipitated from HEK293 cells and
subjected to SDS-PAGE. After in-gel trypsin digestion, the TSC2 tryptic
peptides were fractionated by HPLC, and fractions were analyzed by
MALDI-TOF. A TSC2 tryptic peptide mass value of 1,742 corresponding to
amino acids 1208-1224 containing a single phosphorylation moiety was observed in linear mode and processed further by MALDI-PSD to obtain
partial sequence information (Fig. 5E). The most striking feature of the fragmentation pattern was the neutral loss of the phosphate group ( We observed that anisomycin significantly increases the
phosphorylation of Ser1210 and enhances the interaction
between TSC2 and 14-3-3. Similar results were obtained in cells treated
with serum. These results demonstrate that the interaction between TSC2
and 14-3-3 is regulated and likely to have an important physiological
function. The phosphorylation of Ser1210 in TSC2 is
supported by mutagenesis/phosphopepetide mapping experiments and is
confirmed by mass spectrometry data. Shumway et al. (28) reported that 14-3-3 binding inhibits TSC2 function. Consistently, we
observed that expression of 14-3-3 increases S6K phosphorylation. These
results are consistent with the fact that both serum and anisomycin
activate S6K. We propose a model in which MK2 is directly responsible
for the phosphorylation of Ser1210 in TSC2 in response to
extracellular stimuli such as serum and anisomycin (Fig.
5F). Our study provides a biochemical mechanism for how TSC2
is regulated by cellular signaling pathways.
Nellist et al. (29) have also reported that TSC2 interacts
with 14-3-3 in a phosphorylation-dependent manner. However,
these authors conclude that 14-3-3 binds to several regions of TSC2, and Akt phosphorylation is responsible for the interaction between TSC2
and 14-3-3. Recently, Liu et al. (30) showed that the Akt phosphorylation site Ser939 is responsible for 14-3-3 binding. However, our results demonstrate that 14-3-3 binds to a single
phosphorylation site (Ser1210) in TSC2, and Akt is not
involved in the phosphorylation of this site. This conclusion is
supported by a report by Shumway et al. (28). We
demonstrated that the p38-MK2 kinase cascade is responsible for the
phosphorylation of Ser1210 in TSC2 and the increase of
interaction between TSC2 and 14-3-3 in response to serum or anisomycin treatment.
The obvious discrepancy between our data and those published by Nellist
et al. and Liu et al. could be the result of
different methods utilized in the protein-protein interaction studies
by different groups (27-30). Nellist et al. analyzed the
interaction between TSC2 and 14-3-3 based on in vitro
binding of purified GST-14-3-3, whereas we determined the binding by
examining the interaction of transfected TSC2 with endogenous 14-3-3. The in vitro experiments by Nellist et al. (29)
and Liu et al. (30) may artificially drive the interaction
between TSC2 and GST-14-3-3. For example, TSC2 may have several
nonspecific weak interaction sites for 14-3-3. The high concentration
of purified GST-14-3-3 used in the in vitro binding
experiments may detect these nonspecific interactions. Liu et
al. determined the 14-3-3 binding sites in TSC2 by using
competition of TSC2 phosphopeptides in vitro. The Ser939 phosphopeptide may display a nonspecific weak
interaction with 14-3-3. However, the present of high concentration of
Ser939 phosphopeptide may nonspecifically occupy all
available GST-14-3-3 in the in vitro binding reaction,
therefore competing for TSC2 binding. Our data demonstrate
unequivocally that neither Akt kinase nor Ser939
phosphorylation of TSC2 is responsible for the interaction with 14-3-3. The evidence that phosphorylation of Ser1210 in TSC2 is
responsible for the interaction with 14-3-3 (from this study and our
previous report) is summarized below (27). First, deletion mutations
show that TSC2 fragments containing the Ser1210 can bind
14-3-3, whereas fragments without Ser1210 cannot. Second,
mutation of the single Ser1210 to alanine completely
eliminates the 14-3-3 binding. Third, inhibition of the PI 3-kinase-Akt
pathway has no significant effect on the interaction between TSC2 and
14-3-3. Fourth, inhibition of p38 by SB203580 inhibits the interaction.
Coexpression of p38-KM decreases the interaction between TSC2 and
14-3-3. Fifth, serum and anisomycin stimulate the phosphorylation of
Ser1210 and increase the binding of 14-3-3. Anisomycin does
not stimulate Akt activation (data not shown). Sixth, phosphorylation
of Ser939 is not affected by anisomycin, MKK3-DE/p38, MK2,
and SB203580, although these treatments significantly increase or
decrease the interaction between TSC2 and 14-3-3. Finally, MK2 directly
phosphorylates Ser1210 in TSC2, which is important for the
interaction with 14-3-3. Expression of the dominant negative MK2-KM
blocks the interaction between TSC2 and 14-3-3. In addition, data by
Shumway et al. (28) also support that phosphorylation of
Ser1210 is essential for the interaction between TSC2 and
14-3-3. Taken together, we conclude that phosphorylation of
Ser1210 in TSC2 by the p38-MK2 kinase cascade modulates the
interaction between TSC2 and 14-3-3. Akt-dependent
phosphorylation of TSC2 does not directly regulate this interaction.
Activation of p38 by anisomycin causes TSC2 phosphorylation on multiple
sites. Our data indicate that MK2 affects mainly the phosphorylation of
the peptide containing Ser1210 but has limited effect on
the phosphorylation of other anisomycin-inducible phosphorylation
sites. These results further indicate that regulation of TSC2 by
phosphorylation is a rather complex event. Further studies to map all
of the p38-dependent phosphorylation sites in TSC2 are
required to understand the significance of the other phosphorylation
sites induced by anisomycin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
EEF8 cells, suggesting an important role of TSC2 in anisomycin-induced S6K activation. The p38 MAP kinase stimulates phosphorylation of
Ser1210 through the activation of MK2, which is directly
phosphorylated and activated by p38. Our study provides a molecular
mechanism of Ser1210 phosphorylation of TSC2 in response to
serum and anisomycin stimulation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
pcDNA3-HA-p38, pcDNA3-HA-MKK3-DE were laboratory stocks.
Expressions of those plasmids are controlled by the pCMV promoter.
/
cell line) were cultured in Dulbecco's modified Eagle's medium and
F-12 medium containing 10% FBS. Transfections were performed using
LipofectAMINE reagent (Invitrogen) following the manufacturer's instructions. Transiently transfected cells were lysed in lysis buffer
(10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1%
Nonidet P-40, 1% Triton X-100, 50 mM NaF, 2 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml
leupeptin, 10 µg/ml aprotinin) and immunoprecipitated with the
specific antibodies. Protein A- or protein G-agarose was used to
precipitate the immunocomplexes. The immunoprecipitates were subjected
to SDS-PAGE.
-32P]ATP. Reactions were terminated by the addition
of Laemmli sample buffer. Reaction products were resolved by SDS-PAGE,
and the extent of protein phosphorylation was visualized by autoradiograph.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 1.
The p38 MAP kinase inhibitor SB203580 blocks
the stimulating effect of serum and anisomycin on the interaction
between TSC2 and 14-3-3. A, inhibition of p38 but not
PI 3-kinase or MEK decreases the interaction between TSC2 and 14-3-3. HA-TSC2 was cotransfected with Myc-14-3-3 into HEK293 cells. Cell
lysates were immunoprecipitated (IP) with anti-HA for TSC2
and subjected to immunoblot with anti-Myc for the coimmunoprecipitated
14-3-3. The treatments of various inhibitors are indicated. Cells were
treated with 0.1, 0.5, 2, and 5 µM SB203580 (lanes
2-5, respectively). 20 µM PD98059 and 50 nM wortmannin treatments are indicated. TSC1 is coexpressed
with TSC2 in all experiments (Figs. 1-5), but the cotransfection of
TSC1 is not indicated in the figures. B, SB203580 inhibits
the interaction between endogenous TSC2 and 14-3-3. HEK293 cells were
treated with 1 and 5 µM SB203580 (lanes 2 and
3, respectively). Cell lysates were immunoprecipitated with
anti-14-3-3 and subjected to immunoblot with anti-TSC2 for the
coimmunoprecipitated TSC2. C, time-dependent
effect of anisomycin. HEK293 cells were transfected as indicated and
stimulated with 5 µg/ml anisomycin for the indicated time.
Coimmunoprecipitation of HA-TSC2 and Myc-14-3-3 was performed.
Phosphorylation of p38 was detected by
anti-phospho(Thr180/Tyr182)-p38 MAP kinase
antibody. D, dose-dependent effect of
anisomycin. Experiments are similar to those in C.
View larger version (66K):
[in a new window]
Fig. 2.
p38 plays an important role in
anisomycin-stimulated TSC2 phosphorylation. A, serum and
anisomycin-stimulated interaction between TSC2 and 14-3-3 is mediated
by p38. HA-TSC2 and Myc-14-3-3 were transfected with or without
HA-MKK3-DE+HA-p38 or p38-KM. Coimmunoprecipitation (IP) of
TSC2 and 14-3-3 was performed. Treatments with FBS and anisomycin are
indicated. The expression levels of Myc-14-3-3, p38-KM, p38, and
MKK3-DE were detected by immunoblot. B, p38 mediates TSC2
phosphorylation in response to anisomycin stimulation as determined by
two-dimensional phosphopeptide mapping. HEK293 cells were transfected
with TSC2 and labeled with 32P. Cotransfection with p38 or
treatment with anisomycin or SB203580 is indicated. In vivo
labeled HA-TSC2 was immunoprecipitated and subjected to digestion with
trypsin. The digested peptides were resolved by two-dimensional
phosphopeptide mapping. The four spots that are induced by
anisomycin treatment are circled in b. The
peptide containing Ser1210 is indicated by an
arrow. Spot 5 is the Akt-dependent
Ser939-containing peptide. B, TSC2-S939A mutant
has no effect on 14-3-3 binding. Myc-14-3-3 was cotransfected with
HA-TSC2-WT (wild type TSC2), HA-TSC2-S939A, and HA-TSC2-S1210A,
respectively. Coimmunoprecipitations of TSC2 and 14-3-3 were
performed.
/
EEF8 cells. In the control
TSC2+/+ EEF4 cells, anisomycin induced a robotic S6K
phosphorylation. In contrast, no significant S6K phosphorylation by
anisomycin was observed in the TSC2
/
EEF8 cells (data
not shown). These observations support a role of TSC2 in S6K activation
in response to anisomycin treatment.
View larger version (28K):
[in a new window]
Fig. 3.
MK2 but not PRAK or MNK1 stimulates the
interaction between TSC2 and 14-3-3. A, effect of MK2
on the interaction between TSC2 and 14-3-3. Cotransfections of MK2 wild
type (WT), constitutively active mutant (EE), and
dominant negative mutant (KM) are indicated. Cell lysates
were immunoprecipitated (IP) with anti-HA for HA-TSC2 and
blotted with anti-Myc for Myc-14-3-3. B, PRAK does not
affect the interaction between TSC2 and 14-3-3. Experiments are similar
to those in A. WT, 182D, and
KM denote the wild type, constitutively active mutant, and
the dominant negative mutant of PRAK, respectively. C, MNK1
does not affect the interaction between TSC2 and 14-3-3. Experiments
are similar to those in A. WT, T2D2,
and T2A2 denote the wild type, constitutively active mutant,
and dominant negative mutant of MNK1, respectively.
View larger version (39K):
[in a new window]
Fig. 4.
MK2 acts downstream from p38 to modulate the
interaction between TSC2 and 14-3-3. A, dominant negative
MK2 blocks the effect of serum and anisomycin. The interaction between
TSC2 and 14-3-3 was examined in the presence of dominant negative MK2
(Myc-MK2-KM) in response to FBS or anisomycin stimulation as indicated.
IP, immunoprecipitation. B, SB203580 blocks the
effect of MKK3-DE+p38 but not that of MK2. The effect of p38 and MK2 on
the interaction between TSC2 and 14-3-3 and the mobility shift of TSC2
were examined in the presence of 5 µM SB203580.
Increasing amounts of MKK3-DE+p38 plasmids were transfected in
lanes 1-3 and 4-6.
5 and a positive charged
residue at
3 are present in TSC2 (45). These observations indicate that Ser1210 may be a direct phosphorylation site of MK2.
This possibility was tested directly by in vitro
phosphorylation experiments. The TSC2 fragment (residues 1203-1213)
was expressed as a GST fusion protein (GST-TSC2F) and purified. MK2 was
immunoprecipitated from transfected HEK293 cells and used to
phosphorylate GST-TSC2F in the presence of
[
-32P]ATP. We observed that MK2 phosphorylated
GST-TSC2F but not GST alone (Fig. 5B). As a negative
control, the kinase-inactive MK2-KM did not phosphorylate GST-TSC2F. To
confirm that Ser1210 is the in vitro
phosphorylation site, Ser1210 was mutated to alanine, and
phosphorylation of the mutant protein GST-TSC2F-M was determined. MK2
failed to phosphorylate GST-TSC2F-M (Fig. 5B), demonstrating
that Ser1210 is the target of direct in vitro
phosphorylation by MK2.
View larger version (48K):
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Fig. 5.
Phosphorylation of Ser1210 by
MK2. A, the sequences surrounding Ser1210
are similar to the MK2 consensus phosphorylation sites. The conserved
residues are shown in bold. The MK2 consensus sequences are
shown on top. SRF, serum-responsive factor;
eEF2, eukaryotic elongation factor 2; 5LO,
5-lipoxygenase; HSP27, heat shock protein 27;
CREB, cAMP-responsive element-binding protein;
LSP1, lymphocyte-specific protein 1. All sequences are known
human MK2 substrates. B, MK2 directly phosphorylates a
peptide containing Ser1210. Amino acid residues of 1203-1213 TSC2 were expressed as a GST fusion,
GST-TSC2F. GST-TSC2F-M denotes the mutation of Ser1210 by
alanine. Myc-MK2 was transfected into HEK293 cells and
immunoprecipitated. An in vitro kinase reaction was
performed in the presence of [ -32P]ATP. The
kinase-inactive MK2-KM and GST were included as negative controls. Note
that the GST is in fact larger than the GST-TSC2F because the original
pGEX-KG vector contains a longer linker in the multicloning site which
is replaced by the TSC2 fragment (residues 1203-1213). C,
MK2-stimulated interaction between TSC2 and 14-3-3 requires
Ser1210 in TSC2. TSC2 and 14-3-3 were cotransfected in
HEK293 cells. The treatment of FBS and anisomycin or cotransfection
with MKK3-DE+p38 and MK2-EE are indicated. D, the
constitutively active MK2-EE and the dominant negative MK2-KM cause an
increase and decrease of Ser1210 phosphorylation,
respectively. Two-dimensional phosphopeptide mapping was performed as
Fig. 2B. Serum starvation or anisomycin treatment and
cotransfection with MK2 mutants are indicated. The phosphopeptide spot
containing Ser1210 is indicated by arrows.
E, MALDI-PSD mass spectrum of a TSC2 phosphopeptide. The y
and b fragment ions and the neutral loss of the phosphate group are
labeled. The tryptic peptide corresponding to residues 1208-1224 of
TSC2 is indicated. F, model for TSC2 phosphorylation by the
p38-MK2 kinase cascade in response to FBS and anisomycin
stimulation.
H3PO4 =
98
m/z,
PO3 =
80
m/z), indicating that the parent ion was
phosphorylated. Furthermore, the mass values of the y and b fragment
ions allowed unambiguous assignment of the phosphate group to
Ser1210 of TSC2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Drs. J. Cooper for providing the
MNK1 plasmids and J. Han for providing the MK2 and PRAK plasmids; Huira
Chong for experiments with the TSC2/
EEF8 cells and
critical reading of the manuscript.
![]() |
FOOTNOTES |
---|
* This work is supported by grants from the National Institutes of Health and the Walther Cancer Institute and by a MacArthur fellowship (to K. L. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
To whom correspondence should be addressed: Dept. of
Biological Chemistry, University of Michigan Medical School, 1301 E. Catherine St., Ann Arbor, MI 48109. Tel.: 734-763-3030; Fax:
734-763-4581; kunliang{at}umich.edu.
Published, JBC Papers in Press, February 11, 2003, DOI 10.1074/jbc.M300862200
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ABBREVIATIONS |
---|
The abbreviations used are: TSC, tuberous sclerosis complex; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; HPLC, high performance liquid chromatography; KM, kinase mutant; MALDI, matrix-assisted laser desorption/ionization; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MK2, MAP kinase-activated protein kinase-2, also known as MAPKAPK2; MKK3, MAP kinase-activated protein kinase kinase-3; MNK1, MAP kinase-interacting kinase 1; p38, p38 mitogen-activated protein kinase; PI 3-kinase, phosphatidylinositol 3-kinase; PRAK, p38-regulated/activated protein kinase; PSD, postsource decay; S6K, ribosomal S6 kinase; TOF, time of flight.
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