The p38 and MK2 Kinase Cascade Phosphorylates Tuberin, the Tuberous Sclerosis 2 Gene Product, and Enhances Its Interaction with 14-3-3*

Yong LiDagger §, Ken InokiDagger §, Panayiotis VacratsisDagger , and Kun-Liang GuanDagger ||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-/- 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.

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EXPERIMENTAL PROCEDURES
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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-14-3-3beta , pcDNA3-HA-p38, pcDNA3-HA-MKK3-DE were laboratory stocks. Expressions of those plasmids are controlled by the pCMV promoter.

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-/- 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.

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 [gamma -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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. 


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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.

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.


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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.

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-/- 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.

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.


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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.

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.


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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.

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 -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 [gamma -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.


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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 [gamma -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.

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 (-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

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.

    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

    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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