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J. Biol. Chem., Vol. 277, Issue 47, 44593-44596, November 22, 2002

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ACCELERATED PUBLICATION
Regulation of TSC2 by 14-3-3 Binding^*

Yong Li, Ken Inoki, Raymond Yeung^§, and Kun-Liang Guan^¶

From the  Department of Biological Chemistry and ^¶ The Institute of Gerontology, University of Michigan Medical School, Ann Arbor, Michigan 48109 and the ^§ Department of Surgery, University of Washington, Seattle, Washington 98195

Received for publication, September 5, 2002, and in revised form, September 25, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for the inherited genetic disease of tuberous sclerosis complex. TSC1 and TSC2 form a physical and functional complex to regulate cell growth. Recently, it has been demonstrated that TSC1·TSC2 functions to inhibit ribosomal S6 kinase and negatively regulate cell size. TSC2 is negatively regulated by Akt phosphorylation. Here, we report that TSC2, but not TSC1, associates with 14-3-3 in vivo. Phosphorylation of Ser¹²¹⁰ in TSC2 is required for its association with 14-3-3. Our data indicate that 14-3-3 association may inhibit the function of TSC2 and represents a possible mechanism of TSC2 regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Tuberous sclerosis complex (TSC)¹ is a relatively common genetic disorder. TSC is caused by mutation in either the TSC1 (hamartin) or TSC2 (tuberin) gene, of which each contributes to ~50% of the genetic defects (1, 2). Studies of TSC patients and animal models support the hypothesis that TSC1 and TSC2 are tumor suppressor genes. Homozygous deletion of either TSC1 or TSC2 in mice produces an embryonic lethal phenotype, suggesting an essential function in development. As predicted, heterozygous deletion of either TSC1 or TSC2 in mice results in a significant increase of carcinomas in many tissues with a 100% incidence of renal carcinomas (3, 4).

Mutation of TSC1 or TSC2 results in similar phenotypes, suggesting that the two proteins function in the same pathway. Biochemical studies have shown that TSC1 and TSC2 form a stable complex (5). Genetic studies in Drosophila have demonstrated that TSC1·TSC2 plays a major negative role in the regulation of cell growth. Mutation of either TSC1 or TSC2 results in a significant increase of cell mass in Drosophila (6-8). Overexpression of either TSC1 or TSC2 in Drosophila produces little phenotype, while co-expression of both TSC1 and TSC2 causes a significant reduction of cell size. Furthermore, genetic epistatic studies indicate that TSC1·TSC2 acts downstream of the insulin receptor (6-8).

Recently, we and other groups have demonstrated that TSC1·TSC2 functions to inhibit S6K activation (9-14). In TSC1/ or TSC2/ cells, S6K is highly activated. S6K activation requires phosphorylation of multiple sites. Interestingly, TSC1·TSC2 specifically inhibits the phosphorylation of Thr³⁸⁹, but not phosphorylation of Thr⁴²¹ and Ser⁴²⁴ in S6K. Thr³⁸⁹ is the primary site phosphorylated by mTOR (mammalian target of rapamycin) (15). Furthermore, TSC1·TSC2 also inhibits phosphorylation of 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1), which is also an mTOR target. Both genetic data and biochemical data indicate that TSC1·TSC2 inhibits the function of mTOR (9, 11). Several groups, including ours, have shown that TSC2 is directly phosphorylated and inhibited by Akt (11-13). These studies provide an important link between TSC2 and growth factor signaling.

In this report, we show that TSC2 interacts with 14-3-3, but not the binding-defective 14-3-3 mutant. This interaction is dependent on the phosphorylation of TSC2. We have identified that Ser¹²¹⁰ of TSC2 is phosphorylated in vivo and is the primary binding site of 14-3-3. In contrast, mutation of all AKT phosphorylation sites in TSC2 had no effect on its interaction with 14-3-3. Overexpression of 14-3-3 enhanced phosphorylation of both S6K and 4E-BP1. Furthermore we demonstrated that interaction between TSC2 and 14-3-3 is also modulated by serum starvation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Antibodies and Plasmids-- Anti-phospho-S6K and anti-phospho-4E-BP1 were from Cell Signaling Inc. and anti-TSC2, anti-TSC2 blocking peptide, anti-14-3-3 (K-19), anti-14-3-3 (C-20), anti-14-3-3, anti-14-3-3, anti-14-3-3 were from Santa Cruz Biotechnology. Anti-HA and anti-Myc were from Covance; anti-FLAG and mouse IgG were purchased from Sigma. Rat TSC1 and TSC2 constructs were generously provided by Dr. Y. Xiong. HA-tagged S6K1 and all other DNA constructs including Myc-1433, Myc-1433-DN (dominant negative), and FLAG-4E-BP1 were laboratory stocks. Expressions of those plasmids are controlled by the pCMV promoter. Mutant constructs of TSC2 were created by PCR mutagenesis and verified by DNA sequencing.

Cell Culture, Transfection, and Immunoprecipitation-- HEK293 cells and Phoenix (retrovirus packaging cells) were seeded and maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS). LExF2 (TSC2/ cell line) were cultured in DMEM/F-12 containing 10% FBS (16). Transfections were performed using LipofectAMINE^TM 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 indicated antibodies. Immunocomplexes were subjected to SDS-PAGE.

Metabolic Labeling and Two-dimensional Phosphopeptide Mapping-- HEK293 cells were co-transfected with the indicated plasmids. The serum-starved cells were washed twice with phosphate-free DMEM and incubated with 0.25 mCi/ml [³²P]orthophosphate (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 (11).

Stable Expression of TSC2 in LExF2 Cells by Retrovirus Infection-- The TSC2 cDNA was subcloned to the retrovirial vector pPGS-CMV-CITE-Neo. The vectors containing TSC2 were transfected into the Phoenix retrovirus packaging cell line using the calcium phosphate method (Profection Kit, Promega). 48 h post-transfection, retrovirus produced by the Phoenix cells was used for infection of LExF2 cells. LExF2 cells stably expressing TSC2 were selected for and maintained by G418 (300 µg/ml).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Interaction between TSC1·TSC2 and 14-3-3-- We have previously found that TSC2 is negatively regulated by AKT phosphorylation (11). Akt recognition sequences often overlap with putative 14-3-3 binding sites (17, 18). 14-3-3 has been shown to regulate the function of many cellular proteins via a direct association (19). We tested whether TSC2 interacts with 14-3-3 in HEK293 cells. Our data showed that 14-3-3 co-precipitated with HA-TSC2 (Fig. 1a). In contrast, a dominant negative 14-3-3, which is unable to bind target proteins (20), did not associate with TSC1·TSC2 (Fig. 1a). Reciprocal immunoprecipitation confirmed that TSC2 could be immunoprecipitated with 14-3-3 (Fig. 2a). 14-3-3 is a family of highly related proteins with numerous isoforms. We found that HEK293 cells express 14-3-3 , , , and . Our results showed that TSC2 interacts with all endogenous 14-3-3 isoforms tested present in HEK293 cells (Fig. 1b). These results suggest that TSC2 interacts with different 14-3-3 isoforms.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Interaction between TSC2 and 14-3-3. a, interaction between transfected TSC1·TSC2 and 14-3-3. HEK293 cells were transfected with 100 ng of HA-TSC2, Myc-TSC1, FLAG-14-3-3, FLAG-14-3-3--DN as indicated. Cell lysates were immunoprecipitated with anti-HA or mock IgG as indicated. The immunoprecipitates were blotted with anti-HA for HA-TSC2 and anti-FLAG for FLAG-14-3-3. b, TSC2 interacts with different endogenous 14-3-3 isoforms. HA-TSC2 and Myc-TSC1 were transfected in HEK293 cells. HA-TSC2 was immunoprecipitated with HA antibody or mock-IgG as indicated. The presence of HA-TSC2 and 14-3-3 in the immunoprecipitates were detected by specific antibodies. Western blots (WB) with isoform-specific 14-3-3 antibodies are indicated. c, interaction of endogenous TSC2 and 14-3-3 is shown. HEK293 cell lysates were immunoprecipitated with anti-TSC2 antibody. For competition, the anti-TSC2 antibody was preincubated with competing peptide prior to immunoprecipitation. The presence of 14-3-3 was detected by Western blot with anti-14-3-3 (K-19) antibody. IP, immunoprecipitation.

View larger version (38K): [in this window] [in a new window]   Fig. 2.   TSC2, but not TSC1, interacts with 14-3-3. a, binding of TSC2 with co-transfected 14-3-3. HEK293 cells were transfected with 100 ng of Myc-TSC1, HA-TSC2, and FLAG-14-3-3 as indicated. The immunoprecipitates were blotted with anti-HA for HA-TSC2, anti- Myc for Myc-TSC1, and anti-FLAG for FLAG-14-3-3. b, binding of TSC2 with endogenous 14-3-3. HEK293 cells were transfected with Myc-TSC1, HA-TSC2, or both plasmids. Co-immunoprecipitation of 14-3-3 was detected by the anti-14-3-3 (K-19) antibody. IP, immunoprecipitation.

To demonstrate that TSC2 interacts with 14-3-3 under physiological conditions, we immunoprecipitated endogenous TSC2 from 293 cells. Western blot with the anti-14-3-3 (K-19) antibody, which recognizes all 14-3-3 isoforms, indicated that TSC2 associates with 14-3-3 under physiological conditions (Fig. 1c). Preincubation of the TSC2 antibody with a competing peptide completely eliminated TSC2 and the co-precipitated 14-3-3 (Fig. 1c). These results demonstrated that TSC2 is associated with 14-3-3 under physiological conditions and suggest that 14-3-3 may play a role in the regulation of TSC2.

TSC2, but Not TSC1, Interacts with 14-3-3-- TSC1 and TSC2 form a physical and functional complex in vivo. To determine whether the TSC1·TSC2 complex, TSC1 or TSC2 alone interacts with 14-3-3, HEK293 cells were transfected with these constructs, and co-immunoprecipitation studies were performed. Immunoprecipitation of 14-3-3  did not bring down TSC1 alone. Co-precipitation of 14-3-3 and TSC1 was observed only when TSC2 was present in the transfection (Fig. 2a), indicating that TSC1 alone cannot interact with 14-3-3. In contrast, 14-3-3 co-immunoprecipitated with TSC2 regardless of the presence of TSC1. These results demonstrate that 14-3-3 can interact with TSC2 alone or the TSC1·TSC2 complex. The interaction between transfected TSC2, but not TSC1, and endogenous 14-3-3 further confirmed that 14-3-3 interacts with TSC2 or with the TSC1·TSC2 complex, but not with TSC1 (Fig. 2b). Interestingly, 14-3-3 interacts with TSC2 stronger than with the TSC1·TSC2 complex (Fig. 2). We tested the effect of 14-3-3 on the TSC1·TSC2 complex and found that 14-3-3 had no significant effect on the complex formation between TSC1 and TSC2 (data not shown).

Mapping of the 14-3-3 Interaction Domain in TSC2-- Serial deletions of TSC2 were constructed to locate the domain responsible for 14-3-3 interaction. Our results showed that fragments 1-608, 1-1080, 1-1200, and 1321-1765 did not bind 14-3-3, while fragments 1-1320, 1101-1320, 1080-1765 interacted with 14-3-3 at a level similar to the wild type TSC2 (Fig. 3a). These data demonstrate that the 14-3-3 binding site in TSC2 is localized between residues 1101 and 1320. It has been well established that 14-3-3 binds to phosphorylated residues with a consensus recognition sequence (18). We first tested whether the Akt phosphorylation sites are required for 14-3-3 binding. The TSC2-6A mutant, which has all six predicted Akt phosphorylation sites (residues 939, 1086, 1088, 1378, 1422, 1756) substituted by alanine (11), still binds to 14-3-3 at a level no different from wild type TSC2 (Fig. 3b). This observation shows that the 14-3-3 binding site in TSC2 is different from the Akt phosphorylation sites.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Mapping the 14-3-3 binding domain in TSC2. a, deletion mapping of the 14-3-3 binding domain in TSC2. Various HA-tagged TSC2 deletion constructs were transfected into HEK293 cells and immunoprecipitated with anti-HA antibody. The 14-3-3 present in the immunoprecipitates was detected with anti-14-3-3 (K-19) antibody. TSC2 deletions in the immunoprecipitates were also detected with anti-HA Western blot in which the horseradish peroxidase-conjugated protein A was used as a secondary antibody to avoid the detection of IgG. b, Ser1210 in TSC2 is required for interaction with 14-3-3. The four predicted 14-3-3 binding sites were mutated individually or in combination as indicated. TSC2-6A contains mutations of all putative AKT sites. These mutants were transfected in HEK293 cells and immunoprecipitated. Co-precipitation of endogenous 14-3-3 was detected by anti-14-3-3 (K-19) Western blot. IP, immunoprecipitation.

Sequence analysis by scansite (www.scansite.mit.edu) (21) predicts that TSC2 contains several putative 14-3-3 binding sites. We created single and double mutations by substituting the top four predicted 14-3-3 binding sites (Fig. 3b). Our data demonstrated that Ser¹²¹⁰ is essential for 14-3-3 binding, while mutations of the other putative sites had no effect on 14-3-3 binding (Fig. 3b). These data are completely consistent with the deletion data that fragment 1101-1320 contains the 14-3-3 binding site. We further mutated Ser¹²¹⁰ in the fragment 1101-1320 and confirmed that Ser¹²¹⁰ is essential for 14-3-3 binding (Fig. 3b). Therefore, TSC2 utilizes Ser¹²¹⁰ as the primary 14-3-3 binding site.

Phosphorylated Ser¹²¹⁰ of TSC2 Is the 14-3-3 Binding Site-- We wanted to test whether the interaction between 14-3-3 and TSC2 requires the phosphorylation of TSC2. GST-TSC2 was expressed and purified from transfected HEK293 cells. Purified GST-TSC2 was treated with -phosphatase. Dephosphorylation of GST-TSC2 is evident by an increased electrophoretic mobility of the protein (Fig. 4a). The purified GST-TSC2 was incubated with immunoprecipitated Myc-14-3-3 and the co-precipitation of GST-TSC2 by Myc-14-3-3 was determined. Treatment with phosphatase completely eliminated the interaction between GST-TSC2 and Myc-14-3-3 (Fig. 4a).

View larger version (69K): [in this window] [in a new window]   Fig. 4.   Phosphorylated Ser1210 of TSC2 is the primary 14-3-3 binding site. a, phosphorylation of TSC2 is required for interaction with 14-3-3. GST-TSC2 was purified from transfected HEK293 cells and dephosphorylated with -phosphatase as indicated. The immunoprecipitated Myc-14-3-3 (from 300 µg of transfected cell lysates) was incubated with the purified GST-TSC2 (~10 ng). GST-TSC2 present in the anti-Myc-14-3-3 immunoprecipitates was detected by Western blot. b, Ser1210 of TSC2 is phosphorylated. TSC2 and the TSC2/S1210A mutant was transfected into HEK293 cells and labeled with [32P]phosphate. Two-dimensional phosphopeptide mapping was performed. The circle indicates the phosphopeptide absent in the mutant, but present in wild type TSC2. c, inhibition of S6K in the LExF2 cells by TSC2. The LExF2 TSC2/ cells were infected with wild type TSC2 or TSC2 (S1210A) mutant, and stably infected cells were selected. The phosphorylation status of endogenous S6K was determined by phospho-specific antibodies (pS6K(T389)). d, serum starvation decreased interaction between TSC2 and 14-3-3. HA-TSC2-transfected HEK293 cells were treated with PD98059 (50 µM, 90 min), wortmannin (100 nM, 30 min), rottlerin (5 µM, 60 min), or serum starvation (16 h). Co-immunoprecipitation of 14-3-3 was determined. e, expression of 14-3-3 enhanced phosphorylation of S6K and 4E-BP1. Increasing amounts of Myc-14-3-3 were transfected into HEK293 cells with HA-S6K or FLAG-4E-BP1. Basal phosphorylation of transfected S6K and 4E-BP1 was determined by anti-phospho-S6K and anti-phospho-4E-BP1. The expression of Myc-14-3-3 was also determined. IP, immunoprecipitation.

To directly demonstrate the phosphorylation status of Ser¹²¹⁰ in TSC2, we performed in vivo ³²P labeling and two-dimensional phosphopeptide mapping of TSC2. The TSC2/S1210A mutant eliminated a single phosphopeptide spot depicted by the arrow in Fig. 4b, while the rest of phosphopeptides were unchanged (Fig. 4b). These results strongly indicate that Ser¹²¹⁰ is an in vivo phosphorylation site in TSC2.

Binding of 14-3-3 may modulate the cellular function of TSC2. We have shown that one of the physiological functions of TSC1·TSC2 is to inhibit S6K activation. In TSC2/ LExF2 cells, S6K is highly activated. The abilities of wild type and 14-3-3 binding-defective mutant TSC2 to inhibit S6K were tested in the TSC2/ cells. We observed that both wild type and the 14-3-3 binding-defective TSC2 could inhibit S6K (Fig. 4c). These data indicate that 14-3-3 binding may not modulate the ability of TSC2 to inhibit S6K. However, the lack of a difference between the wild type and the mutant TSC2 could be due to the fact that the majority of the expressed TSC2 is not phosphorylated on Ser¹²¹⁰, therefore, free of 14-3-3 binding. Our two-dimensional phosphopeptide mapping data also indicates that the majority of TSC2 is not phosphorylated on Ser¹²¹⁰, because the intensity of this phosphopeptide is significantly weaker compared with the Akt phosphorylation site Ser⁹³⁹ (Fig. 4b) (11).

Sequences surrounding Ser¹²¹⁰ have limited resemblance to PKA and PKC- phosphorylation sites. Inhibition of PKA (data not shown) or PKC- by rottlerin had no effect on the interaction between TSC2 and 14-3-3 (Fig. 4d). We also observed that inhibition of the phosphatidylinositol 3-kinase-Akt pathway by wortmannin and the ERK pathway by PD90589 had no effect on the interaction (Fig. 4d), which suggests that the Akt phosphorylation sites are not involved. Interestingly, serum starvation resulted in a visible reduction of association between TSC2 and 14-3-3 (Fig. 4d). The above data indicate that the complex formation between TSC2 and 14-3-3 may be modulated by cell growth status.

Increased Phosphorylation of S6K and 4E-BP1 by 14-3-3-- To test the effect of 14-3-3 on downstream effectors of TSC2, we examined the phosphorylation of S6K and 4E-BP1. Phosphorylation of these two proteins was inhibited by TSC1·TSC2. We discovered that co-expression of 14-3-3 elevated the Thr³⁸⁹ phosphorylation of S6K (Fig. 4e). Similarly, expression of 14-3-3 also enhanced the basal phosphorylation of 4E-BP1 (Fig. 4e). These observations indicate that 14-3-3 may negatively regulate the functions of TSC2.

The cellular functions of the TSC1·TSC2 tumor suppressor gene products have just begun to be elucidated. TSC1·TSC2 plays an important role in cell growth regulation and cell size control. Recent studies have demonstrated the TSC2 protein was phosphorylated and inhibited by Akt-dependent phosphorylation (11-13). In this report, we showed that TSC2 binds to 14-3-3 under physiological conditions. We have mapped a single site, Ser¹²¹⁰, in TSC2 responsible for binding with 14-3-3. The binding of 14-3-3 requires the phosphorylation of Ser¹²¹⁰. During the preparation of this manuscript, Nellist et al. (22) also reported that TSC2 interacts with 14-3-3. However, they showed that 14-3-3 binds to multiple sites in TSC2 and concluded that the Akt phosphorylation sites in TSC2 are responsible for 14-3-3 binding (22). We have no obvious explanation why the data by Nellist et al. (22) are dramatically different from ours. Nevertheless, our data clearly indicate that one major 14-3-3 binding site exists in TSC2, and Akt phosphorylation sites are not responsible for 14-3-3 binding.

Overexpression of 14-3-3 results in elevated phosphorylation of S6K and 4E-BP1. In contrast, overexpression of TSC1·TSC2 suppresses the phosphorylation of these two proteins, indicating 14-3-3 and TSC1·TSC2 have opposite effects on S6K activation. Our results indicate that 14-3-3 binds to phosphorylated TSC2 and may suppress its activity. This interpretation is consistent with the fact that the interaction between TSC2 and 14-3-3 is decreased under serum-starved conditions. Serum starvation is predicted to activate TSC2 and suppress cell growth. The dissociation of 14-3-3 may partly contribute to TSC2 activation and S6K inhibition under conditions of serum starvation. However, we cannot exclude the possibility that the effect of 14-3-3 on S6K and 4E-BP1 may not be mediated by TSC2. It has been reported that 14-3-3 may positively modulate the function of TOR in yeast (23). 14-3-3 has also been shown to interact with mTOR (24). Therefore, 14-3-3 may regulate S6K and 4E-BP1 through multiple targets. Future studies to identify the kinase responsible for phosphorylation of Ser¹²¹⁰ will provide new insights into the mechanism of TSC2 regulation.

	ACKNOWLEDGEMENTS

We thank Tianqing Zhu for technical assistance, Haris Vikis and Jen Aurandt for critical reading of the manuscript, and Yue Xiong for communication of unpublished information.

	FOOTNOTES

^* This work was supported by grants from the National Institutes of Health and the Walther Cancer Institute and by a McArthur 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.

To whom correspondence should be addressed: Dept. of Biological Chemistry, University of Michigan, 1301 E. Catherine St., Ann Arbor, MI 48109. Tel.: 734-763-3030; Fax: 734-763-4581; E-mail: kunliang@umich.edu.

Published, JBC Papers in Press, October 2, 2002, DOI 10.1074/jbc.C200510200

	ABBREVIATIONS

The abbreviations used are: TSC, tuberous sclerosis complex; S6K, ribosomal S6 kinase; mTOR, mammalian target of rapamycin; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; GST, glutathione S-transferase; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PKA, protein kinase A; PKC, protein kinase C.

	REFERENCES

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