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Human Molecular Genetics Advance Access originally published online on April 30, 2007
Human Molecular Genetics 2007 16(13):1541-1556; doi:10.1093/hmg/ddm103
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

p27Kip1 localization depends on the tumor suppressor protein tuberin

Margit Rosner1, Angelika Freilinger1, Michaela Hanneder1, Naoya Fujita3, Gert Lubec2, Takashi Tsuruo3 and Markus Hengstschläger1,*

1 Medical Genetics, Obstetrics and Gynecology and 2 Department of Pediatrics, Medical University of Vienna, Währinger Gürtel 18–20, 1090 Vienna, Austria and 3 Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan

* To whom correspondence should be addressed. Tel: +43 1404007847; Fax: +43 1404007848; Email: markus.hengstschlaeger{at}meduniwien.ac.at

Received February 2, 2007; Accepted April 12, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
p27Kip1 plays an important role in cell cycle regulation by inhibiting cyclin–CDK complex activity in the nucleus. p27Kip1 is regulated by its concentration as well as by its subcellular localization. Tuberin, encoded by the tuberous sclerosis tumor suppressor gene TSC2, is a potent negative cell cycle regulator. We show herein, that tuberin induces nuclear p27 localization by inhibiting its 14-3-3-mediated cytoplasmic retention. Tuberin interferes with 14-3-3's counteracting effects on p27-mediated cell cycle arrest. Akt-mediated phosphorylation of p27, but not of tuberin, negatively regulates tuberin's potential to trigger p27 nuclear localization. In G0 cells, tuberin binds p27 triggering downregulation of p27's binding to 14-3-3 and of its cytoplasmic retention. At transition to S phase p27 is phosphorylated by Akt, tuberin/p27 complex levels are downregulated and binding of p27 to 14-3-3 increases triggering cytoplasmic retention of p27. These findings demonstrate p27 localization during the mammalian cell cycle to be under the control of the tumor suppressor tuberin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The cyclin-dependent kinase (CDK) inhibitor, p27Kip1 (p27), accumulates in G0/G1 cells and is localized in the nucleus where it regulates CDKs. During the transition to S phase p27 is translocated to the cytoplasm and degraded by the ubiquitin–proteasome pathway. At G1 phase p27 is ubiquitylated by the cytoplasmic ubiquitin ligase KPC and degraded by the proteasome (1). At the G1 to S phase transition Skp2-containing E3 ubiquitin ligase recognizes p27 phosphorylated at T187 via CDK2 and promotes its degradation (2–6). In addition, Jab1 promotes CRM1-mediated nuclear export of p27 (7,8). p27 is also phosphorylated at S10, causing cytoplasmic localization and triggering progression to S phase (9–11). Recently, it was shown that Ras-dependent lung tumorigenesis is associated with cytoplasmic accumulation of p27 and S10 phosphorylation (12). 14-3-3 proteins bind to p27 through T198, when it is phosphorylated by Akt or p90 ribosomal protein S6 kinase (RSK), and trigger cytoplasmic p27 localization (13,14). In addition, 14-3-3 proteins sequester p27, phosphorylated within its nuclear localization signal (NLS) at T157 by Akt, from importin resulting in cytoplasmic retention of p27 due to downregulation of NLS/importin-dependent nuclear localization (15). Taken together, these data demonstrated that upon phosphorylation of p27 at T157 or T198, 14-3-3 sequesters p27 from importin resulting in the inhibition of nuclear import of p27. Phosphorylation of T157 or T198 of p27 by Akt was found to induce cytoplasmic localization of p27 in breast cancer (16–19).

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor syndrome that affects approximately 1 in 6000 individuals. It is characterized by the development of hamartomas in the kidneys, heart, skin and brain. The latter often cause seizures, mental retardation and developmental disorders, including autism (20). The tumor suppressor gene TSC1 encodes hamartin (21) and TSC2 encodes tuberin (22). TSC patients carry a mutant TSC1 or TSC2 gene in each of their somatic cells and loss of heterozygosity has been documented in a wide variety of TSC tumors. Inactivation of hamartin and tuberin causes a similar phenotype and tuberin and hamartin form a heterodimer, of which tuberin is assumed to be the functional component. A major function of the hamartin/tuberin complex is its role as a GTPase activating protein against Rheb (Ras homolog enriched in brain), which in turn regulates mTOR (mammalian target of Rapamycin) signaling. Tuberin is phosphorylated by several kinases, including the AMP-activated protein kinase (AMPK), Akt, extracellular signal-regulated kinase (ERK) and RSK, which regulate its activity (23–25). Overexpression of tuberin suppresses activation of p70S6K (p70 ribosomal protein S6 kinase) via mTOR (26–31).

Tuberin has also been implicated in cell cycle regulation. Downregulation of tuberin expression induces quiescent fibroblasts to enter the cell cycle and TSC2 –/– fibroblasts exhibit a shortened G1 phase. Overexpression of hamartin or tuberin triggers an increase in G1 cells and p27 protein levels. Tuberin negatively regulates the activity of CDK2 (32–34). Recently, tuberin was found to bind p27 and to inhibit its degradation via sequestering p27 from Skp2. Tuberin also triggers an upregulation of the amount of p27 bound to CDK2 (35). To mediate its cell cycle effects, p27 must be translocated into the nucleus where it inhibits CDK activity. In this study, we demonstrate that tuberin induces nuclear p27 localization via inhibiting its 14-3-3-mediated cytoplasmic retention, and we provide evidence for a model of how this activity of tuberin is regulated during the normal cell cycle.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tuberin regulates p27 nuclear/cytoplasmic localization independently of hamartin and of its potential to control mTOR activity
In embryonic fibroblasts, loss of tuberin causes downregulation of total p27 protein levels accompanied by a decrease of nuclear p27 (Fig. 1A). This finding prompted us to further investigate the effects of tuberin on p27 nuclear/cytoplasmic localization. However, since these TSC2 –/– cells exhibit a shortened G1 phase (33), they do not allow for a molecular analysis of this p27 deregulation separately from tuberin's effects on cell cycle. Therefore, in this study we used the HEK293 cell line, in which we found transient modulation of tuberin expression to regulate endogenous p27 protein levels before affecting cell cycle regulation. The transfection efficiency of HEK293 cells in this study was always around 80%, and GFP-cotransfection allowed to exclusively analyze the DNA distributions of transfected cells (Fig. 1B, C, F). Ectopic tuberin triggered not only upregulation but also nuclear localization of p27 in HEK293 cells (Fig. 1B).


Figure 1
Figure 1
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  		Figure 1. Tuberin affects nuclear p27 levels in an NLS-dependent manner. (A) Total lysates and cytoplasmic and nuclear protein fractions of logarithmically growing TSC2 +/+ and TSC2 –/– embryonic fibroblasts were analyzed for p27 protein levels. Purity of fractions was proven by co-analyzing {alpha}-tubulin and topoisomerase IIß. In addition, immunocytochemical p27 analyses were performed in these cells. (B) Logarithmically growing HEK293 cells were transiently transfected with empty pcDNA3 vector or pcDNA3-wild-type human TSC2 (FLAG-tagged). Total lysates and cytoplasmic and nuclear protein fractions were analyzed for p27 protein levels. After 48 h, DNA distributions (means±SD) of GFP-cotransfected cells were cytofluorometrically studied. (C) Cells transfected with TSC2 alone or with TSC2 together with TSC1 were analyzed for p27 protein amounts and DNA distributions (upon GFP-cotransfection). (D) HEK293 cells were transiently transfected with empty pcDNA3 vector, or pcDNA3 wild-type human TSC2, or pcDNA3 harboring the TSC2 mutant R611Q, which is known not to bind to hamartin. After 48 h, total lysates and cytoplasmic and nuclear protein fractions were analyzed for p27 protein levels. (E) TSC1 –/– cells were transfected with FLAG-tagged TSC2 as described above and p27 protein levels were analyzed by western blot of total lysates. (F) p27 protein amounts and DNA distributions (of GFP co-transfected cells) were analyzed in HEK293 cells upon 48 h TSC2 siRNA treatment. (G) HEK293 cells were treated with TSC2 siRNA, with and without blocking mTOR activity via rapamycin treatment, and p27 protein amounts were analyzed in total lysates, cytoplasmic and nuclear fractions. (H) Logarithmically growing Rat1 fibroblasts were transiently transfected with EGFP, wild-type EGFP-p27, the NLS-mutant EGFP-p27R166A and wild-type TSC2. After 48 h ectopic expressions were confirmed by western blotting. Transfected Rat1 cells with only nuclear expression of EGFP-tagged p27 were identified and counted under the microscope. The amount of cells with only nuclear p27 is given as percentage of EGFP-positive cells (300 EGFP-positive cells were analyzed per experiment). Means±SD of two independent experiments are presented. In case of the NLS-mutant EGFP-p27R166A almost no cells with only nuclear p27 were detected (0–1.5%). In addition, DNA distributions of the so-transfected cells were cytofluorometrically analyzed.

 
Since tuberin and hamartin are interacting partners (see Introduction), we next wanted to investigate the role of hamartin for this regulation. We found co-overexpression of hamartin to mediate additive effects on this tuberin-dependent regulation (Fig. 1C). Still, the question whether binding to hamartin is essential for tuberin's effects on p27 remained elusive. Accordingly, we next ectopically expressed the tuberin R611Q mutant, known not to bind to hamartin (29). Our finding that this mutant triggered the same effects on p27 compared with wild-type tuberin, demonstrated that tuberin's effects on p27 are independent of its potential to bind hamartin (Fig. 1D). Furthermore, we found that tuberin can regulate p27 expression even in TSC1 –/– cells (Fig. 1E), once again proving tuberin-mediated p27 regulation to be independent of hamartin.

To analyze more physiological conditions we studied the effects of downregulation of endogenous intracellular tuberin (mimicking the situation of hamartomas of TSC patients harboring loss of heterozygosity of TSC2). Downregulation of p27 observed in total lysates upon TSC2 siRNA treatment was accompanied by a decrease of nuclear p27 (Fig. 1F).

Since tuberin is a potent negative regulator of mTOR activity (see Introduction), we next tested whether this potential plays a role for tuberin's effects on p27. We found downregulation of tuberin via siRNA to still affect p27 expression and localization even under conditions of rapamycin-induced block of mTOR activity (Fig. 1G). These data demonstrate that the tuberin-mediated effects on p27 are independent of its potential to regulate mTOR.

p27 nuclear localization is known to be regulated in an NLS-dependent manner (see Introduction). To investigate whether tuberin affects the NLS-mediated p27 transport, we investigated the NLS p27R166A mutant, which was shown to diminish NLS-dependent p27 nuclear import (36). For these experiments we used Rat1 cells, since we were not able to express these porcine p27 proteins at adequate levels in other cells. Transfection efficiency of Rat1 cells was always approximately 25%, and the GFP-tag on the used p27 constructs allowed us to exclusively analyze transfected cells. Upon transfection 78.5% of the EGFP-positive cells expressed EGFP-p27wt only in the nucleus. Due to the mutation in the NLS, 0–1.5% (almost undetectable levels) of the EGFP-positive cells expressed EGFP-p27R166A only in the nucleus (and in contrast to wt p27 this construct does not arrest Rat1 cells in G0/G1). Co-overexpression of tuberin increased the amount of cells with nuclear localization of EGFP-p27wt from 78.5 to 88.5% and had no effects on EGFP-p27R166A (P < 0.05; Student's t-test). Under these experimental conditions ectopic tuberin again did not mediate negative cell cycle effects (Fig. 1H). These data demonstrate that tuberin regulates p27 nuclear amounts in an NLS-dependent manner.

Tuberin counteracts 14-3-3's potential to mediate cytoplasmic p27 localization
Nuclear/cytoplasmic p27 localization is known to be regulated by 14-3-3 (see Introduction). From the data described above, it was now of interest to investigate a putative role of tuberin for the 14-3-3-mediated p27 regulation. In agreement with earlier observations (see Introduction), we found Akt-mediated 14-3-3 binding to downregulate the nuclear levels and upregulate cytoplasmic levels of ectopic FLAG-tagged p27 (Fig. 2A, lanes 3, 4 and 8, 9). Ectopic tuberin alone is also sufficient to induce nuclear localization of FLAG-tagged p27 (Fig. 2B, lanes 2, 4 and 8, 10).


Figure 2
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  		Figure 2. Tuberin abolishes 14-3-3-mediated cytoplasmic p27 localization. (A) In HEK293 cells cytoplasmic and nuclear localization of ectopically transfected FLAG-tagged p27 was analyzed upon transfections as indicated. Ectopic protein expressions were confirmed by western blot analyses. DNA distributions of so-transfected cells were cytofluorometrically analyzed. (B) Cytoplasmic and nuclear localization of ectopically transfected FLAG-tagged p27 was analyzed upon transfection with plasmids as indicated. (C) Ecoptic FLAG-p27 was detected immunocytochemically in cells transfected as described in (A). Cells with only nuclear expression of FLAG-p27 were identified and counted under the microscope. The amount of cells with only nuclear p27 is given as percentage of FLAG-p27-positive cells (100 FLAG-p27-positive cells were analyzed per experiment). Means±SD of five independent experiments are presented (P < 0.05; Student's t-test).

 
In co-overexpression experiments we found that tuberin triggers upregulation of nuclear p27 and downregulation of cytoplasmic p27 even in cells with high ectopic Akt and 14-3-3 protein expression (Fig. 2A, lanes 4, 5 and 9, 10). In the experiment presented in Figure 2A ectopic p27 is expressed to such high levels that tuberin, in contrast to its effects on endogenous p27 levels (Figs 1A–G, 3I), cannot further upregulate them anymore (proven also in Figs 3A–E, 4A, 5E). Still, tuberin affects nuclear/cytoplasmic p27 localization under these experimental conditions (Fig. 2A).


Figure 3
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  		Figure 3. Tuberin downregulates 14-3-3/p27 complex levels. (A) HEK293 cells were transfected as indicated. After 48 h immunoprecipitations of ectopic p27 or ectopic 14-3-3 were performed and analyzed by immunodetection of 14-3-3, tuberin and p27. Short exposures not presenting endogenous tuberin are shown to allow comparison of ectopic tuberin expression. Ectopic Akt expression and Akt-mediated p27 phosphorylation was proven by western blot analysis of lysates. The asterisk indicates a background band. (B) p27 immunoprecipitates were analyzed by immunodetection of 14-3-3 protein in cells transfected as indicated. (C) The effects of ectopic expression of TSC2 alone and of TSC2 together with TSC1 on the interaction of p27 and 14-3-3 were compared as described in (A). (D) HEK293 cells were treated with siRNA specific for TSC2. After 48 h, 14-3-3/p27 complex levels were analyzed via immunoprecipitation as described in (A). (E) The effects of TSC2 siRNA treatment on complex formation between ectopic p27 and endogenous 14-3-3 were analyzed in cells with insulin-mediated activation of endogenous Akt. (F) HEK293 cells were transfected with FLAG-tagged wild-type p27, myc-tagged activated Akt, with increasing amounts of HA-14-3-3 expression plasmid (lane 2: 0.5 µg; lane 3: 0.5 µg; lane 4: 1 µg; lane 5: 3 µg; lane 6: 3 µg) and with different amounts of TSC2 expression plasmid (lanes 3–5: 0.5 µg; lane 6: 3 µg). After 48 h, ectopic p27 was immunoprecipitated and 14-3-3 and tuberin was immunodetected. Ectopic protein expressions were confirmed by western blot analysis of lysates. (G) HEK293 cells were transfected as indicated and 14-3-3/p27 complex levels were analyzed on p27 immunoprecipitates. Ectopic protein expressions were confirmed by western blot analysis of lysates. (H) HEK293 cells were transfected as indicated. Ectopic HDAC4 was immunoprecipitated and immunodetection was performed for ectopic HA-14-3-3 and FLAG-HDAC4. Ectopic protein expressions were confirmed by western blot analysis of lysates. (I) HEK293 cells were transfected as indicated and the amounts of phospho-Akt S473, total Akt protein, phospho-mTOR S2448, total mTOR protein and p27 protein were analyzed after 48 h.

 


Figure 4
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  		Figure 4. Functional interactions of tuberin, 14-3-3 and p27 in cell cycle regulation. Logarithmically growing HeLa cells were transfected as indicated. (A) After 48 h ectopic protein expression was confirmed by western blot analysis. (B) Cells were co-transfected with a GFP expression vector. After 48 h DNA distribution of GFP-positive cells was cytofluorometrically analyzed upon propidium iodide staining. Means±SD of five independent experiments are presented. The observed differences were determined to be statistically relevant (P < 0.05; Student's t-test). (C) DNA replication of HeLa cells transfected as indicated was analyzed by immunocytochemical BrdU incorporation assays. The presented results are the average of three independent experiments±SD (at least 300 cells were counted for each experiment).

 


Figure 5
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  		Figure 5. The role of p27 phosphorylation by Akt. (A) HEK293 cells were transfected as indicated (Akt K179M is a kinase-dead mutant used as a control) and endogenous p27 protein expression and DNA distributions were analyzed after 48 h. (B) In serum-starved cells transfected with and without TSC2, Akt activity was induced by insulin and p27 protein levels were analyzed in cytoplasmic and nuclear fractions. (C) HEK293 cells were transfected as indicated. Phosphorylation of FLAG-p27 by endogenous Akt and FLAG-p27 protein levels were analyzed by western blotting. The asterisk indicates a background band. In addition, phosphorylation of FLAG-p27 by endogenous Akt and FLAG-p27 protein levels were analyzed by immunoblotting after immunoprecipitation of FLAG-p27. As a control endogenous Akt activity in the cells was downregulated by 24 h treatment with 50 µM PI3K inhibitor LY294002. (D) HEK293 cells were transfected as indicated. After 48 h 14-3-3/p27 complex levels were analyzed via immunoprecipitation. Phosphorylation of FLAG-p27 by Akt, FLAG-p27 protein levels and ectopic Akt protein levels were also analyzed by western blotting of lysates. (E) Tuberin/p27 complex levels were analyzed by immunoprecipitations in HEK293 cells transfected as indicated. A short exposure not presenting endogenous tuberin is shown to allow comparison of ectopic tuberin expression. (F) HEK293 cells were transfected as indicated. Phosphorylation of FLAG-p27 by Akt, FLAG-p27 protein levels and ectopic Akt protein levels were analyzed by western blotting of lysates. The asterisk indicates a background band. (G) The levels of tuberin/p27 complexes and of 14-3-3/p27 complexes were analyzed by immunoprecipitations in HEK293 cells transfected as indicated. A total lysate was co-loaded on the gel. A short exposure not presenting endogenous tuberin after immunoprecipitation is shown to allow comparison of ectopic tuberin expression. The asterisk indicates a background band. (H) HEK293 cells were transfected as indicated. 14-3-3/p27 complex levels were analyzed by immunoprecipitations.

 
Taken together these findings demonstrate that (i) tuberin's effects on p27 protein levels and on p27 localization are separable; (ii) tuberin and Akt +14-3-3 mediate opposing effects on p27 localization and (iii) ectopic tuberin overcomes the potential of Akt +14-3-3 to mediate cytoplasmic p27 localization. The latter has been confirmed by immunocytochemical analyses showing that upon Akt +14-3-3 overexpression less cells harbor FLAG-p27 in the nucleus and that ectopic tuberin negatively regulates these effects of 14-3-3 on the amount of cells with only nuclear p27 (Fig. 2C; P < 0.05; Student's t-test).

Tuberin negatively regulates the interaction of 14-3-3 and p27
Ectopic Akt activity triggers phosphorylation of p27 and upregulation of the interaction between p27 and 14-3-3 proteins (Fig. 3A, lanes 3 and 5; and see introduction). As the HA-14-3-3 R56, 60A mutant, which loses its ligand binding ability (13), failed to bind to p27, the binding to 14-3-3 is proven to be specific. This mutant was also found to harbor almost no affinity to tuberin (Fig. 3A, lanes 7, 8).

As described in the Introduction, 14-3-3 binding to Akt-phosphorylated p27 triggers cytoplasmic retention of p27. We here found that ectopic tuberin increased the interaction between tuberin and p27 and between tuberin and 14-3-3 (Fig. 3A), and downregulated 14-3-3/p27 complex levels in cells with high ectopic Akt activity (Fig. 3A) and under endogenous Akt activity conditions (Fig. 3B). Under the here used experimental conditions ectopic expression of tuberin's binding partner hamartin had only weak additive effects (Fig. 3C).

To analyze more physiological conditions (mimicking the situation of TSC2-associated hamartomas) we showed that downregulation of endogenous tuberin via TSC2 siRNA treatment triggered an increase of 14-3-3/p27 complex levels in cells with high ectopic Akt activity (Fig. 3D).

To choose an even more physiological setting we next wanted to investigate the effects of downregulated endogenous tuberin on p27 complex formation with endogenous 14-3-3 under conditions of modulated endogenous Akt activity. We induced the interaction between ectopic p27 and endogenous 14-3-3 by upregulation of endogenous Akt activity via insulin treatment of serum-starved cells. Under these conditions we also found that TSC2 siRNA upregulates the interaction between 14-3-3 and p27. Ectopic expression of p27 in these experiments allowed us to separate tuberin's effects on this complex formation from its well-known effects on endogenous p27 protein levels (Fig. 3E).

As a logical consequence of the experiments described above it was then of interest to prove that 14-3-3 and tuberin indeed compete for binding to p27. This assumption was proven by our finding that 14-3-3/p27 complex levels or tuberin/p27 complex levels could be upregulated by titrating increasing amounts of 14-3-3 or tuberin, respectively (Fig. 3F). Tuberin was found to bind to all 14-3-3 isoforms (37–40). To investigate whether tuberin's potential to bind to 14-3-3 could play a role for its effects on p27, we examined the tuberin S1210A mutant, which was shown to be unable to bind 14-3-3 proteins (38). This mutant downregulated 14-3-3/p27 complex levels with lower efficiency (Fig. 3G).

Taken together, these findings demonstrate that tuberin and 14-3-3 compete for interaction with p27. In addition, our data suggest that tuberin's potentials to bind 14-3-3 and to downregulate 14-3-3/p27 complex formation are associated.

Binding to 14-3-3 was shown to mediate cytoplasmic retention not only of p27, but also of the forkhead transcription factor FKHRL1, of the Cdc25C protein phosphatase and of the histone deacetylases 4 and 5 (HDAC4 and 5) (15,41 and references therein). We found that tuberin does not influence the complex levels of 14-3-3/HDAC4 (Fig. 3H) showing that tuberin does not ubiquitously affect 14-3-3-mediated regulations.

Akt phosphorylates and negatively regulates tuberin (see Introduction). However, feedback regulation of Akt by tuberin has also been demonstrated. Tuberin negatively regulates p70S6K. Phosphorylation by p70S6K downregulates insulin receptor substrate proteins to activate Akt. Accordingly, long-term effects of tuberin could lead to activation of Akt (42,43). Furthermore, mTOR is a part of two distinct multiprotein complexes, mTORC1 and mTORC2. Tuberin blocks Rheb to activate mTORC1 (44). But recently, mTORC2 was shown to be blocked by Rheb (45). mTORC2 was earlier demonstrated to phosphorylate and activate Akt (46). Accordingly, tuberin can also induce Akt activity via activation of mTORC2 (45). Since Akt phosphorylation promotes 14-3-3/p27 binding, a tuberin-mediated upregulation of Akt via these two pathways could not provide a causative molecular mechanism for tuberin's negative effects on this complex. Still, it was of interest to investigate whether tuberin affects Akt activity under the here chosen experimental conditions. We found that tuberin downregulates 14-3-3/p27 complex levels (Fig. 3A) without affecting Akt activity (Fig. 3I). Under these experimental conditions 48 h ectopic tuberin expression in HEK293 cells does not deregulate the cell cycle distribution (compare Fig. 1B and C).

Taken together all data presented so far demonstrate that tuberin negatively regulates the potential of 14-3-3 to bind to Akt-phosphorylated p27 and to induce its cytoplasmic localization by competing with 14-3-3 for p27 binding and without influencing Akt activity.

14-3-3's counteracting effects on p27's cell cycle arrest are regulated by tuberin
HEK293 cells were used for the experiments described above, since 48 h transient modulation of tuberin has no effects on the cell cycle distribution in these cells (compare Fig. 1B, C, F) allowing to separately analyze tuberin's effects on p27 regulation. We next wanted to study the cell cycle effects of these molecules. For these experiments we chose the HeLa cell line, since it is also an immortalized cell line, in which we found the transfection efficiency (always approximately 75%) and the transient overexpression levels of tuberin, 14-3-3 and p27 to be very similar to those obtained in HEK293. In addition, we already knew from former studies that 48 h transient expression of these molecules affects HeLa cell cycle regulation (47). Transiently expressed p27 caused an increase of G0/G1 cells from 47.2 to 57.6% in GFP-positive transfected cells (Fig. 4B) and a downregulation of BrdU incorporation (Fig. 4C). Our finding that 14-3-3 downregulates the amount of cells with nuclear p27 suggested that 14-3-3 could mediate positive effects on proliferation of so-transfected cell pools. Transfection experiments demonstrated that 14-3-3 counteracted the p27-induced cell cycle arrest. In these experiments tuberin diminished these effects of 14-3-3 on p27-modulated cell proliferation (Fig. 4; P < 0.05; Student's t-test). In addition, BrdU incorporation assays revealed that ectopic Akt negatively affects p27 cell cycle arrest and that tuberin counteracts this potential of Akt (Fig. 4C). Here, it might be noteworthy that ectopic tuberin cannot further arrest cells, which are already arrested by high ectopic p27 (Fig. 4C).

In summary, the data presented so far provide evidence that 14-3-3 binding to Akt-phosphorylated p27 triggers its cytoplasmic retention and prevents a p27-mediated cell cycle arrest. Tuberin harbors the potential to interfere with these effects of 14-3-3 on p27 localization and on p27's anti-proliferative activity.

Tuberin's effects on p27 are regulated by Akt-mediated phosphorylation of p27
The here found potential of tuberin to trigger nuclear localization of p27 is suggested to be part of the underlying mechanism of how this tumor suppressor mediates cell cycle arrest. This mechanism very likely contributes to the proliferative activity of cells without functional tuberin, such as the cells that develop to hamartomas in TSC patients. Still, p27 localization is strictly cell cycle regulated (see Introduction), whereas tuberin protein levels are constant throughout the mammalian cell cycle (32,34). Accordingly, it remained unclear how tuberin's effects on p27 localization are regulated throughout the normal cell cycle. Akt phosphorylation of p27 is a prerequisite for its binding to 14-3-3 and its cytoplasmic retention. Furthermore, Akt phosphorylation of tuberin mediates negative effects on specific tuberin-mediated functions (see Introduction). Accordingly, it was of interest to test the role of modulated Akt activity in cells with constant tuberin protein levels. Indeed, under such experimental conditions we found tuberin's potential to trigger increased nuclear p27 to be negatively affected by high Akt activity. This observation was made upon overexpression of activated Akt (Fig. 5A) and upon insulin-mediated induction of endogenous Akt activity in serum-starved cells (Fig. 5B).

Next, we wanted to investigate whether Akt phosphorylation of p27 affects its ability to bind to tuberin. We found that a human p27 T198A mutation, which diminishes phosphorylation of p27 by Akt (Fig. 5C) and binding of p27 to 14-3-3 (Fig. 5D), increases p27's binding to tuberin (Fig. 5E). The p27 S10A mutation, which has low effects on phosphorylation of p27 by Akt (Fig. 5C), still binds 14-3-3 (Fig. 5D) and does not influence p27's binding to tuberin (Fig. 5E). These data indicate that Akt phosphorylation of p27 negatively regulates p27's binding to tuberin. Further support for this notion comes from our finding that the human p27 1–188 mutant, lacking T198, harbors properties very comparable to p27 T198A (Fig. 5F, G). We have already reported that the p27 T187A mutation, which has only very weak effects on Akt-mediated phosphorylation of p27 (Fig. 5F), does not influence p27's binding to tuberin (35).

To investigate the role of Akt phosphorylation of tuberin we made use of the tuberin SATA mutant harboring mutations of the Akt phosphorylation sites S939 and T1462 (30). Mutation of the Akt phosphorylation sites does not affect tuberin's potential to regulate 14-3-3/p27 complex levels (Fig. 5H).

Taken together, these data suggest that phosphorylation of p27, but not phosphorylation of tuberin, by Akt interferes with tuberin's potential to bind to p27.

Tuberin's effects on p27 localization are conserved in human and mouse
Phosphorylation of human p27 at T157 or T198 is required for Akt-induced binding of p27 to 14-3-3 and p27 cytoplasmic retention. T157 is not conserved in mouse p27. T198 in human p27 (193-LRRRQT-198) has homology to the residue T197 in mouse and rat p27 (193-LRRQT-197) (14,15). Accordingly, it was of interest to prove that the here reported mechanism is conserved between human and mouse p27. We found that mouse p27 also interacts with 14-3-3 and that Akt phosphorylation of mouse p27 positively affects this binding (Fig. 6A). In the NIH3T3 cell line (transfection efficiency was always approximately 55%) tuberin triggered nuclear localization of endogenous mouse p27 and an increase of G0/G1 cells. This potential of tuberin was again negatively regulated by high ectopic Akt activity (Fig. 6B). Interestingly, the relative amount of nuclear anti-proliferative p27 compared with cytoplasmic p27 appeared to be lower in NIH3T3 cells than in HEK293 cells. This could be correlated with a shorter G1 phase of the faster proliferating rodent cells compared with the human cell line (Fig. 6C, and data not shown). Most importantly, in this study we found that Akt phosphorylates both, human and mouse p27, and positively affects the interaction of human and mouse p27 with 14-3-3. In addition, Akt opposes tuberin's potential to trigger nuclear localization of both, human and mouse p27.


Figure 6
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  		Figure 6. Tuberin also regulates rodent p27. (A) HEK293 cells were transfected with HA-tagged wild-type 14-3-3 {theta}, myc-tagged activated Akt and murine wild-type p27. Murine p27 (human p27 is of larger size) and HA-14-3-3 were analyzed by immunoblotting after immunoprecipitation of HA-14-3-3. HA-14-3-3, myc-tagged Akt, murine p27 and Akt-mediated phosphorylation of murine p27 were also analyzed by western blotting of lysates. (B) NIH3T3 mouse fibroblasts were transiently transfected with FLAG-tagged wild-type TSC2 with or without myc-tagged activated Akt. Overexpression of tuberin and Akt was confirmed by western blot analyses of lysates and DNA distributions were analyzed after 48 h. Cytoplasmic and nuclear protein fractions were analyzed for p27 expression. (C) Cytoplasmic (C) and nuclear protein fractions (N) of logarithmically growing HEK293 cells and NIH3T3 cells were prepared. Equal protein amounts of these fractions and of total lysates (TL) were analyzed for expression of p27 via western blotting. (D) In HEK293 cells endogenous p27/tuberin complex formation was analyzed by detection of tuberin on p27 immunoprecipitates using total lysates (TL), cytoplasmic (C) and nuclear (N) fractions.

 
In this report we provide evidence that tuberin negatively regulates 14-3-3 binding to p27 and 14-3-3-triggered cytoplasmic p27 retention. 14-3-3 is a cytoplasmic protein and tuberin can be found in both, cytoplasm and nucleus (see Introduction and Figs 2A, 6D). Accordingly, it was interesting to learn that the complex of endogenous tuberin and endogenous p27 exists in both, the cytoplasm and the nucleus (Fig. 6D).

The cell cycle regulation of p27, tuberin and 14-3-3
An important proof for our model was to demonstrate the endogenous 14-3-3/p27 complex formation and the endogenous tuberin/p27 complex formation (the latter together with endogenous nuclear p27 localization) to be conversely regulated throughout the normal cell cycle. NIH3T3 cells were analyzed to investigate whether this molecular mechanism of p27 regulation is conserved in human and mouse. In addition, since NIH3T3 cells, in contrast to HEK293 or HeLa cells, can be serum arrested in G0 and restimulated, they represent an optimal model to investigate cell cycle regulations of endogenous proteins. Logarithmically growing NIH3T3 cells (Fig. 7, log) were serum-starved for 48 h in 0.5% FCS (ss) and restimulated with 10% serum. This approach once again proved that cyclin D1 and cyclin A protein levels are induced during the transition to S phase, p27 is downregulated upon entry into the S phase, and tuberin and 14-3-3 protein levels are constant throughout the cell cycle. Akt activity (detected by phospho-Akt S473) is low in G0 and immediately upregulated upon serum-induced re-entry into the cell cycle. Accordingly, this experiment allowed us to investigate Akt's effects on endogenous tuberin and p27 regulations, by modulating endogenous Akt activity (Fig. 7).


Figure 7
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  		Figure 7. Cell cycle regulation of endogenous p27, tuberin and 14-3-3. Logarithmically growing NIH3T3 fibroblasts (log) were arrested by serum-starvation for 48 h (ss). At the indicated time points of serum re-stimulation cells were harvested and cytofluorometrically analyzed for DNA distribution. Cyclin D1, cyclin A, p27, tuberin, 14-3-3, phospho-Akt S473 and Akt protein levels were analyzed by western blotting. Akt-mediated phosphorylation of p27 (p-p27), tuberin/p27 complex levels and 14-3-3/p27 complex levels were analyzed by immunoblotting after immunoprecipitation of p27. In addition, p27 expression was analyzed in cytoplasmic and nuclear fractions. The fractionated protein extracts have been loaded and immunoblotted twice. Different exposures of p27 immunodetections are presented (A and B).

 
In G0 phase (Fig. 7, ss) p27 is not phosphorylated by Akt (p-p27) and has high affinity to tuberin (IP p27/tuberin detection), which inhibits p27's binding to 14-3-3 (IP p27/14-3-3 detection) and p27 cytoplasmic retention (p27/nucleus). At cell cycle re-entry p27 is phosphorylated by Akt, which is followed by downregulation of tuberin/p27 complex formation and by upregulation of binding of p27 to 14-3-3 and p27 nuclear import is downregulated. These data provide additional evidence for our here proposed model: In G0 tuberin interacts with p27 downregulating cytoplasmic retention of p27 via 14-3-3. p27 can localize to the nucleus and inhibit CDK activity. At cell cycle re-entry p27 is phosphorylated via Akt. This phosphorylation downregulates the binding of p27 to tuberin and leads to upregulation of 14-3-3/p27 complex levels and cytoplasmic retention of p27 via 14-3-3. Nuclear p27 decreases and CDK activity drives cells into S phase (Fig. 8).


Figure 8
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  		Figure 8. The role of tuberin for p27 localization. In G0 cells tuberin binds p27 triggering downregulation of p27's binding to 14-3-3 and of its cytoplasmic retention. p27 is localized in the nucleus. At the transition from G0 phase to S phase p27 is phosphorylated by Akt, tuberin/p27 complex levels are downregulated, and binding of phosphorylated p27 to 14-3-3 increases triggering cytoplasmic retention of p27.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The earlier observation that high ectopic levels of p27 cannot affect proliferation of TSC2 –/– cells (33) suggested that, besides regulation of p27 protein levels, tuberin positively regulates another mechanism, which is essential for p27 activity. Here we demonstrate that tuberin affects p27 nuclear localization.

Considering the wide spectrum of 14-3-3 proteins (with all their interacting proteins) it was striking to see, that one protein, tuberin, harbors the potential to revert 14-3-3-mediated cytoplasmic p27 localization and cell cycle stimulation. Tuberin negatively regulates the potential of 14-3-3 to bind to Akt-phosphorylated p27 and to induce its cytoplasmic localization by competing with 14-3-3 for p27 binding without influencing Akt activity. In this study, these conclusions were drawn from tuberin overexpression experiments as well as upon siRNA downregulation of endogenous tuberin levels. In addition, that endogenous p27 localization depends on endogenous tuberin levels has also been confirmed in TSC2 –/– and TSC2 +/+ fibroblasts. Finally, the entire model has been confirmed by studying cell cycle regulation of endogenous proteins.

Tuberin was found to bind to all 14-3-3 isoforms (37–40). A tuberin mutant, which was shown to be unable to bind 14-3-3 proteins, still harbored the potential to downregulate 14-3-3/p27 complex levels but did so with lower efficiency compared with wild-type tuberin. These data demonstrate that binding of tuberin to 14-3-3 plays a role for tuberin's effects on the 14-3-3/p27 complex formation, but is not essential.

Earlier, our group has shown that tuberin negatively affects p27 degradation (35). We now demonstrate that tuberin still delocalizes p27 even when p27 is ectopically overexpressed to such high levels that its protein levels cannot be upregulated by tuberin anymore. These findings prove that these two functions of tuberin can be separated.

We earlier reported that tuberin, but not hamartin, binds to p27 and that hamartin cannot regulate p27 levels in the absence of tuberin (35). Now we found downregulation of tuberin expression (in TSC2 –/– cells or upon siRNA treatment) to be sufficient to deregulate p27 localization. These experiments mimic the situation of hamartomas of TSC patients harboring loss of heterozygosity of TSC2. Such cells may fail to arrest because the CDK inhibitor p27 is inactivated. We have already reported that loss of endogenous tuberin is sufficient to drive a G0 arrested cell into the cell cycle (32). In this study we have also investigated the role of hamartin for tuberin's effects on p27 regulation. We found that a tuberin mutant R611Q, which is known not to bind to hamartin, still affects p27 with the same efficiency compared with wild-type tuberin. In addition, we demonstrated that tuberin can regulate p27 expression even in TSC1 –/– cells. These data show that tuberin's effects on p27 are independent of its potential to bind hamartin.

Astrocytes and fibroblasts of TSC2 +/– mice exhibit decreased p27 expression (48,49). The finding that the TSC genes are involved in regulation of the mTOR signaling network already initiated clinical trials for the treatment by rapamycin, a negative regulator of mTOR, of renal tumors and lung cysts found in TSC. The here reported results strengthen the argument that p27 or CDKs could also be considered targets for hamartoma therapeutics in TSC (50). Further understanding of the role of the TSC genes has also implications for sufferers from other cancers that may involve the TSC proteins. TSC gene mutations occur in sporadic bladder cancer (51,52). Most interestingly, in bladder tumors TSC gene mutations are correlated with reduced p27 expression (53). Cytoplasmic localization of p27 due to phosphorylation of T157 or T198 and inactivation of p27 was found in breast cancer (16–19). Recently, it was demonstrated that tuberin and hamartin expression is downregulated in breast cancer (54). Deletions in the TSC gene regions are also found at significant frequency in ovarian and gall bladder carcinoma and non-small-cell carcinoma of the lung (55).

We previously reported that three different pathogenic tuberin mutants still affected p27 protein levels and cell proliferation (56). Another pathogenic tuberin mutation was found to not affect its potential to upregulate p27 protein levels and to downregulate proliferation. A fifth pathogenic mutant lost the potential to upregulate p27, but still affected proliferation (57). The here reported data allow new interpretation of these earlier findings. In addition to cell cycle control, p27 has been implicated in the regulation of a wide variety of different cellular processes, such as apoptosis, cell growth, or tumorigenesis (50,58). It is possible that specific mutations in the TSC2 gene affect p27 localization and cell proliferation, whereas other mutations could affect p27 stability, which could be involved in the regulation of, e.g. apoptosis (or vice versa). Although a major function of the hamartin/tuberin complex is assumed to be its role in regulating p70S6K, 5 of 15 analyzed pathogenic tuberin mutants were reported to still downregulate p70S6K activity (59). It could be assumed that one pathogenic mutation causes a loss of p27 control and does not affect tuberin's potential to regulate p70S6K, whereas other mutations could affect p70S6K but not p27. The fact that the same mutants can behave differently with respect to different cellular processes has been demonstrated by our earlier finding that pathogenic tuberin mutants, which can still regulate p27 stability and proliferation, lost the capacity to regulate cell size (47).

Loss of functional tuberin via mutation, as it occurs in TSC hamartomas, can lead to inactivation of p27 activity. In addition, tuberin is inactivated and activated via phosphorylation by different enzymes, including AMPK, Akt, ERK and RSK. Akt phosphorylation of S939 and T1462 negatively affects tuberin's potential to regulate p70S6K (23–25). We demonstrated here that mutations of these Akt phosphorylation sites do not affect tuberin's potential to regulate 14-3-3/p27 complex levels. In addition, we found that downregulation of tuberin via siRNA still affects p27 expression and localization even under conditions of rapamycin-induced block of mTOR activity. Furthermore, we observed that a tuberin mutant R611Q, which is unable to affect mTOR (29), still regulates p27. These data now clearly demonstrate that tuberin-mediated effects on p27 are independent of tuberin's potential to regulate mTOR. These findings are in agreement with our recently published result that mutations of the Akt-phosphorylation sites within tuberin, which increase tuberin's potential to affect mTOR, do not affect tuberin-mediated regulation of p27 expression (60). It will be of great interest for the future to investigate, whether phosphorylation by AMPK, ERK or RSK affects tuberin's potential to regulate p27 stabilization and/or p27 localization.

During transition into S phase p27 nuclear localization is downregulated, whereas tuberin protein levels remain constant. Accordingly, tuberin's potential to trigger nuclear localization of p27 must be inactivated. We found increased Akt activity to negatively affect tuberin's potential to trigger p27 nuclear localization. Whereas Akt phosphorylation of tuberin appears not to be involved in this regulation, we provide evidence that phosphorylation of p27 by Akt is of relevance. We found that a p27 T198A mutation diminishes phosphorylation by Akt and binding of p27 to 14-3-3, but increases p27's binding to tuberin. Cell cycle analyses indicated that Akt phosphorylation of p27 during the G0/G1-S phase transition downregulates tuberin/p27 binding and upregulates 14-3-3/p27 binding. Binding to 14-3-3 causes cytoplasmic retention of p27 allowing CDK-mediated S phase entry (Fig. 8).

TSC2 –/– embryonic fibroblasts exhibit a shorter G1 phase and harbor less p27 in the nucleus (33, this report). Furthermore, as already mentioned, antisense inhibition of tuberin expression induces quiescent fibroblasts to enter the cell cycle (32). Our here presented findings suggest that tuberin's potential to trigger p27 nuclear localization is an important gatekeeper of G0/G1. Inactivation of this function is involved in the regulation of the transition into S phase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells, cell culture, flow cytometry
HEK293 cells (human embryonic kidney), HeLa cells (human cervical carcinoma), NIH3T3 cells (mouse fibroblasts), Rat1 cells (rat fibroblasts), TSC1 +/+ and TSC1 –/– mouse embryonic fibroblasts (obtained from D. Kwiatkowski), and TSC2 +/+ and TSC2 –/– embryonic fibroblasts, derived from the Eker rat model (33), were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. For cytofluorometric DNA analyses cells were fixed by rapid submersion in ice-cold 85% ethanol. After overnight fixation at – 20°C, DNA was stained with 0.25 mg/ml propidium iodide, 0.05 mg/ml RNase, 0.1% Triton X-100 in citrate buffer, pH 7.8. For transient transfection experiments using co-transfected GFP-spectrin as a reporter, a total of 10.000–40.000 GFP-positive single cells were selectively gated. DNA distribution was analyzed on a Beckton Dickinson FACScan. In HEK293 cells starved in 0.1% serum for 24 h endogenous Akt activity was induced by adding 2µg/ml insulin (Sigma) for 20 min. mTOR activity was blocked by treatment with 50 nM rapamycin (Calbiochem) for 36 h.

Transfections
All transfection experiments were performed transiently and cells were analyzed after 48 h. The following plasmids were used: pcDNA3-TSC2wt (human), pcDNA3-TSC2R611Q (human), pcDNA3-FLAG-TSC2wt (human), the FLAG-tagged TSC2 SATA mutant (S939A/T1462A) (human), pcDNA3-HA-TSC2wt (rat), pcDNA3-HA-TSC2 S1210A (rat), pcDNA3-TSC1wt (human), pEGFP, pEGFP-p27wt (porcine), pEGFP-p27 R166A (porcine), pHM6-HA-14-3-3 {theta} wt (human), pHM6-HA-14-3-3 {theta} R56,60A (human), pHM6-HA-14-3-3 {varepsilon} wt (human), pUSE-myc-Akt (activated mouse Akt), pUSE-myc-Akt K179M (kinase dead mouse Akt), pFLAG-CMV-2-p27wt (human), pFLAG-CMV-2-p27 S10A (human), pFLAG-CMV-2-p27 T187A (human), pFLAG-CMV-2-p27 T198A (human), pcDNA3-FLAG-p27 1–188 (human), pcDNA3-p27wt (mouse), pcDNA3-FLAG-HDAC4wt (human), GFP-spectrin expression vector or empty vector controls. HEK293 cells were transfected with Lipofectamine 2000, NIH3T3 and HeLa cells using Lipofectamine and Lipofectamine Plus reagents (all Invitrogen). FuGene 6 (Roche) was applied for the transient transfection of Rat1 fibroblasts.

For western blotting experiments of total lysates and fractionated proteins, transfections were performed in 6-well plates using 1–1.3 mg of total cDNA (depending on the transfection reagent used). For western blotting experiments of proteins after immunoprecipitation, transfections were performed in dishes with a diameter of 60 mM using 2 mg of total cDNA. Whenever multiple plasmids were co-transfected the ratio was 1:1.

siRNA treatment specific for TSC2
RNA silencing was achieved using human TSC2 siRNA (Cell Signaling or Dharmacon) at a final concentration of 25–200 nM. siRNA was delivered to the cells using Lipofectamine 2000 reagent (Invitrogen) following the transfection protocol provided by the manufacturers. Analyses were performed after 48 h.

Cytoplasmic and nuclear fractionation
Cell pellets were lysed in five packed cell volume buffer F1 containing 20 mM Tris, pH 7.6, 50 mM 2-mercaptoethanol, 0.1 mM EDTA, 2 mM MgCl2, 1 mM PMSF supplemented with protease inhibitors (2 mg/ml aprotinin, 2 mg/ml leupeptin, 0.3 mg/ml benzamidin chloride, 10 mg/ml trypsin inhibitor) for 2 min at room temperature and subsequent incubation on ice for 10 min. Thereafter, NP-40 was added at a final concentration of 1% (v/v) and lysates were homogenized by passing through a 20-gauge needle for three times. Nuclei were pelleted by centrifugation at 600 g for 5 min at 4°C and supernatant containing cytoplasmic proteins was collected and stored at – 80°C. Remaining nuclei were washed three times in buffer F1 containing 1% NP-40. During the last wash nuclei were stained with trypan blue and microscopically examined for number, purity and integrity. The nucleic pellets were lysed in buffer containing 20 mM Hepes, pH 7.9, 0.4 M NaCl, 2.5% glycerol, 1 mM EDTA, 1 mM PMSF, 0.5 mM NaF, 0.5 mM Na3VO4, 0.5 mM DTT, supplemented with protease inhibitors by repeated freezing and thawing. Supernatants containing soluble nucleic proteins were collected by centrifugation at 25 000 g for 20 min and stored at – 80°C.

For co-immunoprecipitation experiments of cytoplasmic and nuclear proteins cells were fractionated as described above with the exception that 2-mercaptoethanol was omitted from buffer F1.

Immunoblotting and immunoprecipitation
For preparing total lysates cells were washed with PBS, collected by scraping and lysed in buffer containing 20 mM Hepes, pH 7.9, 0.4 MM NaCl, 2.5% glycerol, 1 mM EDTA, 1 mM PMSF, 0.5 mM NaF, 0.5 mM Na3VO4 supplemented with 2 µg/ml aprotinin, 2 µg/ml leupeptin, 0.3 µg/ml benzamidin chloride, 10 µg/ml trypsin inhibitor by repeated freezing and thawing. Supernatants were collected by centrifugation and stored at – 80°C. Protein concentrations were determined using the Bio-Rad protein assay with bovine serum albumin as the standard. Proteins were run on an SDS–polyacrylamide gel and transferred to nitrocellulose. Blots were stained with Ponceau-S to visualize the amount of loaded protein. For immunodetection, antibodies specific for the following proteins were used: 14-3-3 ß (K-19, Santa Cruz), 14-3-3 ß (H-8, Santa Cruz), Akt (Cell Signaling), phosphorylated Akt (S473) (Cell Signaling), phospho-(Ser/Thr) Akt substrate (Cell Signaling), c-jun (H-79, Santa Cruz), c-myc (9E10; Pharmingen), cyclin A (H-432, Santa Cruz), cyclin D1 (M-20, Santa Cruz), FLAG (M2, Sigma), HA (3F10, Roche), p27 (C-19, Santa Cruz), p27 (57, Transduction Laboratories), tuberin (C-20, Santa Cruz), hamartin (Cell Signaling), mTOR (Cell Signaling), phosphorylated mTOR (S2448) (Cell Signaling), ribosomal protein S6 (Cell Signaling), phosphorylated ribosomal protein S6 (S235/6) (Cell Signaling), topoisomerase IIß(Transduction Laboratories; human specific), topoisomerase IIß(H-286, Santa Cruz; human and rodent specific), {alpha}-tubulin (Ab-1, Calbiochem), ß-actin (AC-15, Sigma).

The following primary antibodies of three different species were used: (i) rabbit polyclonal and monoclonal antibodies were detected using anti-rabbit IgG, a HRP-linked whole antibody from donkey (NA934, GE Healthcare) or anti-rabbit IgG, a HRP-linked whole antibody from goat (L42007, Caltag Laboratories); (ii) mouse monoclonal antibodies were detected using anti-mouse IgG, a HRP-linked whole antibody from sheep (NA931, GE Healthcare); (iii) rat monoclonal antibodies were detected using anti-rat IgG, a HRP-linked whole antibody from goat (NA935, GE Healthcare). Signals were detected with the enhanced chemiluminescence method.

For immunoprecipitations proteins were prepared as described above. Briefly, crude cell extracts (300 µg to 3 mg) were precleared with 40 µl Protein G-Sepharose beads for 30–60 min at 4°C and incubated with the primary antibodies anti-FLAG (M2, Sigma), anti-HA (3F10, Roche), anti-p27 (C-19, Santa Cruz) or anti-p27 (57, Transduction Laboratories). After complex formation at 4°C (at least for 2 h) immunoprecipitates were washed twice with buffer containing 50 mM Tris–HCl; pH 8.0; 1% NP-40; 150 mM NaCl; 10 mM ß-glycerophosphate; 1 mM NaF; 0.1 mM Na3VO4; 0.2 mM PMSF supplemented with protease inhibitors.

Immunocytochemistry
HEK293 cells were seeded onto chamber slides at a density of 3 x 104/slide 24 h before transfection. Forty-eight hours after transient transfection cells on slides were washed with PBS, fixed in 4% paraformaldehyde for 10 min at room temperature, treated with 0.1% Triton X-100 for 15 min and blocked for non-specific binding in PBS containing 3% non-fat, dry milk for another 30 min. For imunocytochemical analyses logarithmically growing TSC2 +/+ and TSC2 –/– embryonic fibroblasts were fixed, permeabilized and blocked following the same protocol. After 1 h incubation with anti-FLAG antibody (M2, Sigma) or anti-p27 antibody (57, Transduction Laboratories), FLAG-p27- or p27-positive cells were visualized by incubation with the secondary antibody anti-mouse IgG-TRITC conjugated (T2402, Sigma) or with Alexa Fluor 594 anti-mouse IgG (A11032, Molecular Probes). After washing in PBS, slides were rinsed in DAPI-staining solution (1 µg/ml).

Twenty-four hours after seeding of Rat1 fibroblasts at a density of 6.6 x 103/slide, cells were transiently transfected with a total amount of 0.2 µg pEGFP, pEGFP-p27wt or pEGFP-p27 R166A with and without cotransfection of TSC2. After 48 h slides were washed with PBS, fixed in 2% paraformaldehyde for 5 min, washed again and nuclei were counterstained with DAPI.

BrdU incorporation assay
The DNA Replication Assay Kit (Upstate) was used according to the manufacturers instructions. Briefly, cells were grown on slides, transfected with the indicated plasmids, labeled for 30 min with 10 µM BrdU and fixed in 95% ethanol/5% glacial acetic acid. Cells were labeled with a monoclonal antibody against BrdU and a Cy2-conjugated secondary antibody. Cell nuclei were identified by DAPI staining. Cells were counted under the microscope, at least 300 for each experiment. BrdU-positive cells are given as a percentage of the total amount of cells counted in each experiment. Presented results are the average of three experiments±SD.

Microscopy
Fluorescence images were analyzed on a conventional microscope equipped with a video camera and a fluorescence attachment (model Axioplan 2 imaging with FluoArc, Zeiss) using a Plan-Neofluor 40 x oil immersion (NA 1.3) objective or a Plan-Apochromat 63 x oil immersion (NA 1.4) objective. Cytovision software CV 2.81 was used for image acquisition and Adobe Photoshop 7.0 was used for further processing.

Statistical analyses
The significance of the observed differences was determined by Student's t-test (paired, two-tailed) using Graph-Pad INSTAT software. P-values > 0.05 are defined as not significant.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Drs A.J. Beavis, J. Blenis, M. Eilers, D. Halley and M. Nellist, K. Hirano, D. Kwiatkowski, B. Manning, V. Ramesh, S. Shumway, X-J. Yang, R. Yeung and Y. Yoneda for reagents. This research has been supported by the Red Bull Company, Salzburg, Austria and by the FWF Austrian Science Fund (P18894 [GenBank] -B12).

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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