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Human Molecular Genetics Advance Access originally published online on December 8, 2005
Human Molecular Genetics 2006 15(2):287-297; doi:10.1093/hmg/ddi444
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Hamartin, the tuberous sclerosis complex 1 gene product, interacts with polo-like kinase 1 in a phosphorylation-dependent manner

Aristotelis Astrinidis{dagger}, William Senapedis{dagger} and Elizabeth P. Henske*

Fox Chase Cancer Center, Philadelphia, PA 19111, USA

* To whom correspondence should be addressed at: Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111, USA. Tel: +1 2157282428; Fax: +1 2152141623; Email: elizabeth.henske{at}fccc.edu

Received September 28, 2005; Accepted November 30, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome caused by mutations in TSC1 and TSC2. Hamartin and tuberin, the products of TSC1 and TSC2, respectively, form heterodimers and inhibit the mammalian target of rapamycin. Previously, we have shown that hamartin is phosphorylated by CDC2/cyclin B1 during the G2/M phase of the cell cycle. Here, we report that hamartin is localized to the centrosome and that phosphorylated hamartin and phosphorylated tuberin co-immunoprecipitate with the mitotic kinase Plk1. Plk1 interacts with the N-terminus of hamartin (amino acids 1–880), which contains two potential Plk1-binding sites (T310 and S332). Phosphorylated hamartin interacts with Plk1 independent of tuberin with all three proteins present in a complex. A non-phosphorylatable hamartin mutant with an alanine substitution at residue T310 does not interact with Plk1, whereas a non-phosphorylatable hamartin mutant at residue S332 in conjunction with alanine mutations at the other CDC2/cyclin B1 sites (T417, S584 and T1047) does not impact hamartin binding to Plk1. Hamartin negatively regulates the protein levels of Plk1. Finally, Tsc1–/– mouse embryonic fibroblasts (MEFs) have increased number of centrosomes and increased DNA content, compared to Tsc1+/+ cells. Both phenotypes are rescued after pre-treatment with the mTOR inhibitor rapamycin. RNAi inhibition of Plk1 in Tsc1–/– MEFs failed to rescue the increased centrosome number phenotype. These data reveal a novel subcellular localization for hamartin and a novel interaction partner for the hamartin/tuberin complex and implicate hamartin and mTOR in the regulation of centrosome duplication.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tuberous sclerosis complex (TSC) is a tumor suppressor gene syndrome characterized by benign tumors in the brain, kidney, heart and skin, and neurological features including seizures, mental retardation and autism (1Go). TSC is caused by inactivating mutations in the TSC1 and TSC2 tumor suppressor genes (2Go,3Go). The protein products of TSC1 and TSC2, hamartin and tuberin, respectively, form heterodimers (4Go–6Go) that inhibit the mammalian target of rapamycin (mTOR). This is achieved via tuberin, which acts as a GTPase-activating protein toward the small GTPase Rheb (7Go,8Go). Both hamartin and tuberin have also been implicated in regulation of the cell cycle (9Go–11Go), probably mediated in part by the interaction of tuberin with the cyclin-dependent kinase inhibitor p27 (12Go,13Go). Mutations in either TSC1 or TSC2 lead to the same human disease, indicating that hamartin is essential for tuberin function; however, hamartin's precise role in the hamartin/tuberin complex is not completely understood.

We recently found that hamartin is phosphorylated by CDC2/cyclin B1 during the G2/M phase of the cell cycle (14Go). Mitotic kinases like CDC2/cyclin B1 often prime substrates for interaction with the mitotically active polo-like kinase 1 (Plk1) (15Go). Plk1 localizes to the centrosome during prophase and metaphase, moves to the mitotic spindle during anaphase and, finally, to the equatorial plate in cytokinesis (16Go,17Go). Increases in the level and activity of Plk1 coincide with the onset of mitosis (16Go). Polo-like kinases are key regulators of cell cycle progression, with roles in the G2 to M transition, centrosome maturation, spindle assembly and cytokinesis (reviewed in 18Go). A hallmark feature of Plks is the highly conserved C-terminal duplicated polo-box domain (PBD). The PBD mediates binding of Plk1 to phosphorylated substrates during mitosis and is required for centrosomal localization of Plk1 yet the protein(s) that target Plk1 to the centrosome are not known (15Go,19Go).

The core consensus motif recognized by the Plk1 PBD is S-pT/pS-P-X (15Go). Hamartin contains three potential Plk1-binding motifs that involve phosphorylation at residues T310, S332 and T1047. Of these, residue T310 is within the most stringent motif for PBD binding (20Go).

Hamartin's mitotic phosphorylation by CDC2/cyclin B1 and the presence of three potential Plk1-binding motifs led us to ask whether hamartin interacts with Plk1. Here, we report that hamartin localizes to the centrosome, which is the first specific subcellular localization reported for hamartin. A phosphorylated form of hamartin interacts with Plk1 and is dependent on hamartin residue T310, which lies within a Plk1 PBD-binding motif. The interaction of the hamartin/tuberin complex with Plk1 is hamartin-dependent, with all three proteins present in the complex. Hamartin depletion, either by RNA interference (RNAi) or in Tsc1–/– mouse embryonic fibroblasts (MEFs), increases Plk1 protein levels. Tsc1–/– MEFs have increased number of centrosomes, compared to their hamartin-proficient counterparts, suggesting that hamartin regulates centrosome duplication. Downregulation of Plk1 in Tsc1–/– MEFs by siRNA did not rescue the increased centrosome number phenotype. Finally, nocodazole-arrested Tsc1–/– MEFs, but not Tsc2–/– MEFs, have increased DNA content (>4 N). Both phenotypes are rescued after pre-treatment with rapamycin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hamartin localizes to the centrosome
To determine hamartin's localization during mitosis, Cos7 cells were treated with nocodazole and fixed in paraformaldehyde. Immunofluorescence confocal microscopy showed that hamartin and the centrosomal marker {gamma}-tubulin co-localize on the same plane of nocodazole-arrested cells (Fig. 1A). The centrosomal localization of hamartin was verified in HeLa cells (Fig. 1B). Pre-incubation of the anti-hamartin antibody (4Go) with the immunizing peptide completely removed the staining from the centrosomes (Fig. 1B). The centrosome staining was also observed with a commercially available hamartin antibody from Invitrogen (Carlsbad, CA, USA) (data not shown).



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  		Figure 1. Hamartin localizes to the centrosome. (A) Cos7 cells were arrested in G2/M with 70 ng/ml nocodazole for 18.5 h. Cells were fixed in paraformaldehyde, permeabilized with 0.5% Tween-20, stained with hamartin antibodies (a and b) and co-stained with {gamma}-tubulin antibodies (a' and b'). The merged confocal images (a'' and b'') show that hamartin is co-localized with {gamma}-tubulin at the centrosomes (arrowheads) during metaphase (a'') and telophase (b''). (B) HeLa cells were treated with nocodazole, fixed in ice-cold methanol and permeabilized with 0.2% Triton X-100 in 1xPBS, then incubated with anti-hamartin (a and b) and anti-{gamma}-tubulin antibodies (a', b', c' and d'). These antibodies stained the centrosome of metaphase and telophase cells (a and b, respectively). The immunostaining was abolished when the anti-hamartin antibody was pre-incubated with the immunizing peptide (c and d). (C) NIH3T3 cells were treated with nocodazole for 18.5 h, then with cytochalasin B for 90 min. Centrosome fractions were isolated by a discontinuous sucrose gradient, the proteins resolved in a 7.5% Tris–HCl SDS/PAGE and immunoblotted. Hamartin and tuberin co-fractionated with {gamma}-tubulin (fractions 12–14).

 
Centrosomal fractionation was used to confirm the immunofluorescence results. Centrosomes from nocodazole-arrested NIH3T3 cells were initially purified over a sucrose cushion and further fractionated by ultracentrifugation in a discontinuous sucrose gradient. Hamartin was present primarily in fractions 12–14 (Fig. 1C), which are enriched in centrosomes as indicated by the increase in {gamma}-tubulin. Tuberin was also present in fractions 12–14, although the enrichment of tuberin in these fractions was less pronounced than hamartin or {gamma}-tubulin.

Phospho-hamartin co-immunoprecipitates with endogenous Plk1
Previously we reported that hamartin is threonine–proline phosphorylated by CDC2/cyclin B1 during G2/M arrest and during normal G2/M progression (14Go). Three of the five potential CDC2/cyclin B1 phosphorylation sites on hamartin (S-T310-P, S-S332-P and S-T1047-P) match the binding motif for the PBD of the mitotic kinase Plk1 (21Go). Therefore, we hypothesized that phosphorylated hamartin interacts with Plk1 during the G2/M phase of the cell cycle. We found that endogenous Plk1 co-immunoprecipitates with endogenous hamartin and tuberin from nocodazole arrested HEK293 cells (Fig. 2A). To confirm the specificity of the interaction, we performed Plk1 immunoprecipitations from vehicle or nocodazole-treated HEK293 cells. Hamartin was present only in the Plk1 immunoprecipitates from nocodazole-treated cells (Fig. 2B). More importantly, the hamartin that co-immunoprecipitated with Plk1 had slower electrophoretic mobility, suggesting that Plk1 interacts exclusively with phosphorylated hamartin.



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  		Figure 2. Plk1 interacts with phosphorylated hamartin. (A) HEK293 cells were treated with nocodazole (or vehicle control) for 18.5 h, lysed in PTY buffer, endogenous hamartin was immunoprecipitated and resolved in 3–8% Tris–acetate PAGE. The amount of Plk1 present in the hamartin immune complexes was increased in nocodazole-arrested cells. These data are representative of at least three independent experiments. (B) HEK293 cells were treated as in (A), endogenous Plk1 was immunoprecipitated and resolved in 3–8% Tris–acetate PAGE. Phosphorylated hamartin (arrowhead) co-immunoprecipitated with Plk1 only in the presence of nocodazole. These data are representative of three independent experiments. (C) HEK293 cells were transfected with myc-Plk1 and treated with nocodazole. Myc-immunoprecipitates were treated with CIAP and resolved in 3–8% Tris–acetate PAGE. Hamartin in untreated myc-Plk1 immunoprecipitates migrated more slowly (arrowhead). Treatment of the myc immunoprecipitates with CIAP restored the normal mobility of hamartin, indicating that the upshifted form of hamartin is phosphorylated. CIAP treatment increased the mobility of tuberin, indicating that the tuberin complexed with hamartin and Plk1 is also phosphorylated.

 
To confirm that the mobility shift of hamartin present in Plk1 immunoprecipitates is due to phosphorylation, myc-Plk1 was expressed in HEK293 cells, and the resulting myc immunoprecipitates were treated with calf intestinal alkaline phosphatase (CIAP). Treatment of the myc-Plk1 immunoprecipitates restored the normal electrophoretic mobility of hamartin (Fig. 2C), indicating that Plk1 interacts with a phosphorylated form of hamartin. Interestingly, CIAP treatment of the myc-Plk1 immunoprecipitates also increased the mobility of tuberin (Fig. 2C), demonstrating that the tuberin within the hamartin/Plk1 complex is also phosphorylated.

Plk1 interacts with phospho-hamartin independent of tuberin
To determine whether the Plk1/tuberin interaction is dependent on the hamartin/tuberin interaction, HisXpress-tagged hamartin was expressed in HEK293 cells either with wild-type tuberin or with mutant R611Q tuberin. R611Q is one of the most frequent mutations in TSC patients and does not interact with hamartin (22Go). Co-expression of TSC1 with TSC2–R611Q increased the interaction between hamartin and Plk1 when compared with co-expression of TSC1 with wild-type TSC2 (Fig. 3A, compare third and first lanes, respectively). These results suggest that the hamartin/Plk1 interaction is not dependent on the interaction of hamartin with tuberin. Plk1 co-immunoprecipitated with His-tagged R611Q tuberin to a lesser extent (Fig. 3A, fourth lane) compared with His-tagged wild-type tuberin (second lane). These results suggest that hamartin mediates the interaction between Plk1 and the hamartin/tuberin complex. Although Nellist et al. (22Go) previously found that tuberin-R611Q did not interact with hamartin, in our experiments a significant amount of hamartin was present in the His-tuberin-R611Q immunoprecipitates (Fig. 3A). This difference between our results and Nellist et al. likely reflect the much lower expression in the Nellist et al. study, compared to our expression levels in Figure 3A.



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  		Figure 3. Plk1 interacts with phospho-hamartin independent of tuberin. (A) HEK293 cells were transfected with HisXp–TSC1/TSC2, TSC1/HisXp–TSC2, HisXp–TSC1/TSC2–R611Q or TSC1/HisXp–TSC2–R611Q, then treated with nocodazole for 18.5 h. His immunoprecipitates were resolved in 4–12% Bis–Tris PAGE and immunoblotted with anti-Xpress, anti-Plk1 and anti-hamartin antibodies. Plk1 co-immunoprecipitated with HisXp-hamartin (first and third lanes). The amount of Plk1 in the immunoprecipitates was increased in cells expressing TSC2–R611Q. Immunoprecipitates of His-Xp–tuberin or His-Xp–tuberin-R611Q contained comparatively less Plk1 (second and fourth lanes). (B) Nocodazole-treated whole cell lysates from Tsc1–/– MEFs with stable re-expression of hamartin (clones T3 and T9) and vector-control clone (P2) were immunoblotted for tuberin, hamartin, Plk1 and ß-actin as a loading control. (C) Endogenous Plk1 was immunoprecipitated from the MEF clones shown in (B). Hamartin co-immunoprecipitated with Plk1 from clones T3 and T9. Tuberin was absent from the Plk1 immune complexes in the MEFs lacking hamartin (P2). Mouse IgG was used as a control.

 
To confirm that Plk1 interacts with the hamartin/tuberin complex in a hamartin-dependent manner, we re-introduced hamartin in Tsc1–/– MEFs and created stable cell lines. Expression levels of hamartin and tuberin in a vector-control Tsc1–/– cell line (P2) and in two hamartin re-expressing cell lines (T3 and T9) are shown in Figure 3B. As reported previously (23Go), re-expression of hamartin increased the stability of tuberin. Tuberin was present in the Plk1 immunoprecipitates from hamartin-expressing clones T3 and T9 but not the vector-control clone P2 (Fig. 3C), indicating that the Plk1/tuberin interaction is not direct and is mediated by hamartin.

The N-terminus of hamartin binds Plk1
To identify the domains through which hamartin interacts with Plk1, HEK293 cells were transfected with His-tagged full-length TSC1 (amino acids 1–1164), N-terminal TSC1 (N-TSC1, amino acids 1–880) or C-terminal TSC1 (C-TSC1, amino acids 511–1164) (Fig. 4A and B). Plk1 co-immunoprecipitated with the full-length and N-terminal hamartin constructs (Fig. 4B), but not with the C-terminal hamartin construct. Tuberin co-immunoprecipitated with all three hamartin constructs (Fig. 4B). These results suggest that the hamartin/Plk1 interaction is mediated by hamartin amino acids 1–510.



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  		Figure 4. Hamartin residue T310 is required for Plk1 interaction. (A) Diagram of hamartin deletion constructs (N-TSC1, amino acids 1–880 and C-TSC1, amino acids 511–1164). Hamartin contains five potential CDC2/cyclin B1 phosphorylation sites (T310, S332, T417, S584 and T1047). Three of these sites (T310, S332 and T1047) are within potential Plk1 PBD-binding motifs. (B) HEK293 cells were transfected with HisXp, HisXp–TSC1, HisXp–N-TSC1 (amino acids 1–880) and HisXp–C-TSC1 (amino acids 511–1164), then treated with nocodazole for 18.5 h. His immunoprecipitates were resolved in 3–8% Tris–acetate PAGE and immunoblotted with antibodies to Plk1, Xpress and tuberin. Endogenous Plk1 was present in the immunoprecipitates of full-length and N-terminal hamartin (second and third lanes), but not in the immunoprecipitates of C-terminal hamartin (fourth lane) or the vector control. All hamartin constructs appeared to be phosphorylated, resulting in upshifted forms (arrowheads). (C) HEK293 cells were transfected with HisXp–TSC1, HisXp–TSC1-T310A, His–TSC1-4A (S332A/T417A/S584A/T1047A quadruple mutant) or HisXp–TSC1-5A (T310A/S332A/T417A/S584A/T1047A quintuple mutant), then treated with nocodazole for 18.5 h. His immunoprecipitates were resolved in 4–12% Bis–Tris PAGE and immunobloted for Xpress, tuberin and Plk1. Endogenous Plk1 co-immunoprecipitated with wild-type TSC1 and TSC1-4A (second and fourth lanes), but not with TSC1-T310A (third lane), TSC1-5A (fifth lane) or vector control.

 
Hamartin residue T310 is responsible for the hamartin/Plk1 interaction
CDC2/cyclin B1 phosphorylates serine/threonine residues within both consensus (K/R-S/T-P-X-K/R) (24Go) and non-consensus motifs (S/T-P) (25Go). Hamartin contains a total of five potential CDC2/cyclin B1 sites: three consensus sites (predicted phosphorylation at residues T417, S584 and T1047) and two non-consensus sites (T310 and S332) (Fig. 4A). Of these sites, three (T310, S332 and T1047) are part of potential Plk1-binding motifs (15Go,20Go). The N-terminus of hamartin, which interacts with Plk1, contains two potential CDC2/cyclin B1 phosphorylation and Plk1 PBD-binding sites at residues T310 and S332. Amino acid T310 is within the most stringent consensus PBD-binding motif as defined by Elia et al. (20Go).

To identify the hamartin residues mediating binding to Plk1, three His-tagged non-phosphorylatable hamartin alanine mutants were created: a TSC1-T310A single mutant, TSC1-4A (S332A/T417A/S584A/T1047A) and TSC1-5A (T310A/S332A/T417A/S584A/T1047A). The hamartin alanine mutants were expressed in HEK293 cells and the cells arrested with nocodazole. We found that endogenous Plk1 co-immunoprecipitated with wild-type hamartin and TSC1-4A (Fig. 4C). In contrast, Plk1 did not co-immunoprecipitate with the hamartin mutants TSC1-5A or TSC1-T310A. These data suggest that hamartin T310 is a major determinant for Plk1 binding.

Hamartin expression negatively regulates Plk1 protein levels
Using western immunoblotting, we observed that Plk1 protein levels were decreased in Tsc1–/– MEFs with stable hamartin re-introduction (clones T3 and T9), compared with the vector-control stable clone P2 (Fig. 3B). Similar results were seen in Tsc1–/– MEFs which have 2.2-fold higher Plk1 expression than Tsc1+/+ cells (Fig. 5A). To verify that hamartin expression affects Plk1 protein levels, we used an RNAi approach. U2OS cells were transfected with siRNA for TSC1, TSC2 and PLK1, and the protein levels were assayed by western immunoblotting. Silencing of TSC1 or TSC2 increased Plk1 protein levels in G2/M-arrested cells by 2.4- and 1.9-fold, respectively (Fig. 5B). Silencing of PLK1 slightly decreased the hamartin and tuberin protein levels.



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  		Figure 5. Plk1 expression is regulated by hamartin. (A) Tsc1+/+ and Tsc1–/– MEFs were treated with nocodazole (or vehicle control) for 16 h, then lysed in PTY buffer. Lysates were resolved in 4–12% Bis–Tris PAGE and immunoblotted for hamartin, tuberin, Plk1 and ß-actin (loading control). Densitometry values (arbitrary units) are indicated under the Plk1 immunoblot. (B) U2OS cells were transfected with control siRNA, TSC1, TSC2 or PLK1 siRNA. After 48 h, the cells were treated with nocodazole for 16 h, lysed in PTY buffer, proteins resolved in 4–12% Bis–Tris PAGE and immunoblotted for tuberin, hamartin, Plk1, pT389-p70S6K, pS235/236-S6, pT70-4E-BP1 and ß-actin (loading control). Densitometry values (arbitrary units) are indicated under the Plk1 immunoblot. Silencing of Plk1 decreased the protein levels of hamartin and tuberin. PLK1 siRNA decreased the phosphorylation of p70S6K, S6 and 4E-BP1. The additional bands above pT389-p70S6K represent cross-reactivity of the antibody with p85S6K.

 
RNAi-mediated Plk1 in U2OS cells decreased the phosphorylation of p70S6K at residue T389, ribosomal protein S6 at residues S235/S236 and 4E-BP1 at residue T70, both in nocodazole-arrested U2OS cells (Fig. 5B) and in asynchronous cells (data not shown).

Hamartin regulates centrosome number in an mTOR-dependent manner
Because Plk1 is implicated in centrosome maturation (26Go), we tested whether hamartin is required for maintenance of proper centrosome number. We found that 59.5% of Tsc1–/– MEFs have multiple (more than two) centrosomes, compared to 34.4% of Tsc1+/+ MEFs (P<0.05, Fig. 6A and 6B). We hypothesized that the increased centrosome number was caused by the aberrant mTOR activation observed in Tsc1–/– MEFs. Pre-treatment of Tsc1–/– MEFs with 2 nM of the mTOR inhibitor rapamycin reduced the percentage of cells with multiple centrosomes from 49.6 to 21.8% (Fig. 6C, P<0.05). In Tsc1+/+ MEFs, rapamycin pre-treatment had only a minimal effect, reducing the percentage of cells with multiple centrosomes from 23.7 to 17.4% (P>0.05).



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  		Figure 6. Hamartin-deficient MEFs have increased centrosome number which is rescued by rapamycin. (A) Tsc1+/+ and Tsc1–/– MEFs were arrested in G1/S with 500 µM hydroxyurea for 40 h, fixed in methanol, stained for {gamma}-tubulin and the nuclei counterstained with DAPI. Indirect immunofluorescence was used to observe the centrosomes (arrowheads). Multiple centrosomes are observed in the Tsc1–/– MEFs. (B) Quantitation of the results from (A). Centrosomes were counted from more than 100 cells by two independent investigators blinded to the experimental conditions. Results are representative of three independent experiments. About 59.5% of Tsc1–/– MEFs have multiple (more than two) centrosomes, compared with 34.4% of Tsc1+/+ MEFs (asterisk indicates statistical significance at P<0.05). (C) Tsc1+/+ or Tsc1–/– MEFs were pre-treated for 24 h with 2 nM rapamycin (closed boxes) or vehicle control, then arrested in G1/S with 500 µM hydroxyurea in the presence of rapamycin and centrosome number counted as in (B). Results are representative of three independent experiments. Multiple (more than two) centrosomes were observed in 21.8% of rapamycin-pre-treated Tsc1–/– MEFs, compared with 49.6% of untreated cells (asterisk indicates statistical significance at P<0.05). (D) Tsc1–/– MEFs were transfected with 100 nM control or Plk1 siRNA for 72 h. Densitometry values (arbitrary units) are indicated under the Plk1 immunoblot. Plk1 protein levels from Plk1 siRNA-transfected Tsc1–/– MEFs were 20% of the control siRNA-transfected cells. (E) Tsc1–/– MEFs were transfected with 100 nM control or Plk1 siRNA for 24 h, then arrested in G1/S with 500 µM hydroxyurea in the presence of siRNAs, fixed and stained as in (A) and the centrosome number counted from 300 cells. Results are averages of two independent experiments. About 73.7% of Plk1 siRNA-transfected cells had multiple (more than two) centrosomes, compared with 50.1% of control siRNA-transfected cells (asterisk indicates statistical significance at P<0.05). In all graphs, error bars represent standard deviation.

 
We then tested the hypothesis that the increase in the centrosome number observed in Tsc1–/– MEFs is Plk1-dependent. Plk1 siRNA reduced the Plk1 protein levels at 20% of control siRNA-transfected cells (Fig. 6D). Unexpectedly, 73.7% of Tsc1–/– MEFs transfected with Plk1 siRNA showed increased centrosome number, compared with 50.1% of control siRNA-transfected Tsc1–/– MEFs (Fig. 6E, P<0.05). These results suggest that the increase in centrosome number observed in hamartin-deficient cells is mTOR-dependent and Plk1-independent.

Hamartin-deficient cells have increased DNA content which is rescued by rapamycin
Centrosome multiplicity is associated with genomic instability and abnormal mitosis. As we observed that the Tsc1–/– MEFs have increased number of centrosomes, we asked whether they might also have mitotic defects. We found that 36.8% of nocodazole-arrested Tsc1–/– MEFs have increased DNA content (>4 N), compared with 24.7% of Tsc1+/+ MEFs (P<0.01, Fig. 7A and B). Tsc1–/– MEFs released from the nocodazole block failed to progress to a normal cell cycle even 24 h after release (data not shown). Pre-treatment of the Tsc1–/– MEFs with 2 nM rapamycin for 24 h prior to the nocodazole block was sufficient to rescue the increase in DNA content from 66.7 to 17.3% (P<0.05, Fig. 7C and D). Treatment of the Tsc1–/– MEFs with 2 nM rapamycin did not cause a G1 arrest (G1 fraction 50.9% in untreated cells versus 49.4% in treated cells, Fig. 7C).



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  		Figure 7. Hamartin-deficient MEFs have increased DNA content which is rescued by rapamycin. (A) Exponentially growing (asynchronous) Tsc1+/+ or Tsc1–/–MEFs were treated with 70 ng/ml nocodazole for 16 h and the DNA content (FL2A) was measured by fluorescence-activated cell sorting (FACS). Arrowheads indicate the G1 and G2/M fractions of the cell population. The region of increased DNA content (>4 N) was used for quantitation of the aneuploid fraction of cells. (B) Quantitation of the fraction of cells with increased DNA content. About 36.8% of Tsc1–/– MEFs have >4 N DNA content, compared with 24.7% of Tsc1+/+ MEFs (asterisk indicates statistical significance at P<0.01). Averages of four independent experiments. (C) Exponentially growing (asynchronous) Tsc1–/– MEFs were pre-treated with 2 nM rapamycin for 24 h, then treated with 70 ng/ml nocodazole for 16 h in the presence of rapamycin and subjected to FACS analysis. (D) About 66.7% of untreated Tsc1–/– MEFs have >4 N DNA content, compared with 17.3% of rapamycin-treated Tsc1–/– MEFs (asterisk indicates statistical significance at P<0.05). Averages of three independent experiments. (E) Exponentially growing (asynchronous) Tsc2+/+ p53–/– or Tsc2–/– p53–/– MEFs were treated with 70 ng/ml nocodazole for 16 h, then subjected to FACS analysis. (F) About 46.6% of nocodazole-treated Tsc2–/– p53–/– MEFs had increased (>4 N) DNA content, compared with 37.1% of Tsc2+/+ p53–/– MEFs (P>0.05). Results are averages of two independent experiments. In all graphs, error bars represent standard deviations.

 
We next asked whether the mitotic defect observed in the hamartin-deficient MEFs is also present in tuberin-deficient MEFs. About 46.6% of nocodazole-arrested Tsc2–/– p53–/– MEFs had increased (>4 N) DNA content, compared to 37.1% of Tsc2+/+ p53–/– MEFs (Fig. 7E and F). This difference was not statistically significant (P>0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Relatively little is known about the function and regulation of hamartin, especially when compared with the rapidly expanding knowledge of tuberin's function and regulation. We report here that hamartin is centrosomally localized and that Plk1 is a novel interacting partner for the hamartin/tuberin complex. Interaction of the hamartin/tuberin complex with Plk1 appears to be mediated by phosphorylated hamartin, as tuberin is not present in Plk1 immunoprecipitates from Tsc1–/– MEFs. We found that the N-terminus of hamartin (amino acids 1–880) interacts strongly with Plk1, whereas the C-terminus (amino acids 511–1164) does not, indicating that the hamartin/Plk1 interaction domain resides within the first 510 amino acids of hamartin. Both constructs interact with tuberin, suggesting either that hamartin has two distinct tuberin-interaction domains (amino acids 1-510 and 881-1164), or that the tuberin-interaction domain lies within hamartin amino acids 511–880. Our findings complement the yeast two-hybrid work of Hodges et al. (6Go), which suggested that hamartin contains multiple tuberin-interaction domains at the N-terminus (residues 1–430). Hamartin's C-terminus interacted weakly with tuberin in the yeast system, but strongly in the mammalian cells. To our knowledge, these yeast two-hybrid results have never been validated in mammalian cells. Additional work is needed to define the domains through which hamartin interacts with tuberin in mammalian cells.

To identify the residues responsible for the hamartin/Plk1 interaction, we created non-phosphorylatable alanine mutants of hamartin at the potential CDC2/cyclin B1 phosphorylation sites T310, S332, T417, S584 and T1047. We found that a single alanine mutation at hamartin residue T310 was sufficient to disrupt the hamartin/Plk1 interaction, suggesting that Plk1 binds hamartin when it is phosphorylated at threonine 310. A quadruple non-phosphorylatable hamartin alanine mutant (S332A/T417A/S584A/T1047A) did not affect the interaction with Plk1. This indicates that phosphorylation at these four sites is not required for Plk1 interaction nor are they required as priming phosphorylation events for T310 phosphorylation. Tuberin is present in the immune complexes of the wild-type and all hamartin mutants suggesting that overall folding and stability of the protein was maintained and the hamartin/tuberin complex is not influenced by the hamartin/Plk1 interaction.

We found that Plk1 protein levels increased in Tsc1–/– MEFs, and after siRNA silencing of either TSC1 or TSC2, indicating that loss of hamartin or tuberin leads to increased expression or decreased degradation of Plk1. Plk1 expression is often increased in cancer (27Go–29Go), and a specific Plk1 inhibitor was recently reported (30Go). The ubiquitin ligase CHFR (checkpoint with forkhead and ring finger domains) is believed to regulate the proteasome-dependent degradation of Plk1 (31Go), although the exact molecular mechanisms for the regulation of Plk1 levels during the cell cycle are not completely understood. Additional studies are needed to identify the mechanism of the increased Plk1 protein in hamartin-null cells and to determine whether Plk1 inhibition has potential benefits in the treatment of TSC.

Plk1 is pivotal for centrosome maturation (32Go), and depletion of Plk1 by siRNA has been reported to decrease the number of centrosomes in G1/S phase arrested cells (26Go). Our finding that hamartin localizes to the centrosome and that Plk1 interacts with the hamartin/tuberin complex suggests that hamartin may play a functional role in the regulation of centrosome maturation and/or mitotic progression. In agreement with this hypothesis, we found that the Tsc1–/– MEFs have increased percentage of cells with multiple centrosomes, compared with Tsc1+/+ MEFs. Pre-treatment of the Tsc1–/– MEFs with low doses of rapamycin rescued this phenotype, suggesting that centrosome duplication is regulated by the hamartin–tuberin–mTOR pathway. To our surprise, siRNA-mediated silencing of Plk1 in the hamartin-deficient MEFs not only failed to decrease, but also increased the percentage of cells with multiple centrosomes. These results indicate that the increased centrosome number observed in the Tsc1–/– MEFs is Plk1-independent. The increase in centrosome number after Plk1 siRNA was unexpected because of the work of Liu and Erikson (26Go) who demonstrated that RNAi-mediated inhibition of PLK1 in U2OS cells decreases the multiplicity of centrosomes. This could mean that centrosome amplification is controlled through different mechanisms in different cell types.

We also found that nocodazole-arrested Tsc1–/– MEFs have increased fraction of cells with >4 N DNA content, a phenotype that is rescued after pre-treatment with rapamycin. In Tsc2–/– p53–/– MEFs, we observed an increase in the >4 N DNA content fraction of cells compared with Tsc2+/+ p53–/– MEFs; however this increase was not statistically significant. At this point, it is unclear whether the differences in mitotic defects between the Tsc1–/– and the Tsc2–/– p53–/– MEFs represent separable functions of hamartin and tuberin or p53 dependence. Expression of a Plk1 kinase dead mutant (K82M) or a deletion mutant that does not contain the kinase domain (Plk1{Delta}N) causes mitotic arrest with increase in the >4 N DNA content and failure of cytokinesis (19Go), apparently caused by a spindle checkpoint defect. It is possible that the >4 N DNA content phenotype we observed in the hamartin-null MEFs involves similar pathways to bypass a spindle checkpoint.

At least two other tumor suppressor genes (BRCA1 and p53), both of which function in part to maintain genome stability, have centrosomal localization (33Go). Moreover, Plk1 phosphorylates the DNA damage checkpoint proteins Chk2 (34Go) and BRCA1 (35Go,36Go), suggesting that the centrosomes play a significant role in maintenance of genomic stability through the cross-talk of spindle and DNA damage checkpoint pathways with those of mitotic progression (37Go). Whether hamartin regulates genomic stability is not known. dTsc1 and dTsc2 mutant Drosophila cells have multiple cell cycle defects, including elevated levels of mitotic cyclins, even in post-mitotic cells (38Go). The underlying mechanisms are not completely understood. TSC patient-derived cells exhibit prolonged S phase of the cell cycle (39Go). Although not extensively studied, there are numerous reports of chromosomal instability in TSC tumors, including multiple complex translocations (40Go–42Go), trisomies (43Go) and chromosome losses (44Go). Whether these defects are linked to the hamartin/Plk1 interaction is not known yet.

Mitotic progression is a complex biological process involving the co-ordinated activation, inactivation and changes in expression of key proteins. During late G2, Plk1 directly phosphorylates and activates the phosphatase CDC25C, which in turn dephosphorylates and activates CDC2. Cyclin B1 expression increases in late G2 and the formation of CDC2/cyclin B1 complexes allows mitotic entry. We propose a model in which the interaction between the hamartin/tuberin complex and Plk1 regulates mitotic protein synthesis locally (Fig. 8). Local protein translation is believed to be important in all organisms, with Drosophila and Xenopus the most studied and understood (reviewed in 45Go). For example, in Xenopus embryos, centrosomally localized protein translation of cyclin B1 seems to be regulated by cytoplasmic polyadenylation element-binding protein and maskin, impacting the mitotic apparatus and cell division (46Go). In our proposed model, the interaction between Plk1 and the hamartin/tuberin complex regulates local mTOR activity, resulting in increased mRNA translation of cyclin B1 and other key proteins in or around the centrosomes, thus promoting mitotic entry.



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  		Figure 8. Proposed model for the role of Plk1/hamartin/tuberin interaction in regulation of local protein synthesis. The Plk1 interaction with hamartin regulates mTOR-dependent local protein synthesis in or around the centrosomes, with consequent centrosome duplication and increase in cyclin B1 mRNA translation during late G2. Simultaneously, Plk1 activates CDC2 via the direct phosphorylation of CDC25C. The formation of CDC2/cyclin B1 complex allows mitotic entry.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cloning and site-directed mutagenesis
Wild-type human TSC1 coding region was subcloned in pcDNA3.1+/His (Invitrogen) and the pMSCVneo retroviral vector (BD Biosciences, Mountain View, CA, USA). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). PLK1 cDNA (IMAGE clone 2822226, ATCC, Manassas, VA, USA) was sub-cloned in pCMV/Tag3 vector (Stratagene) using high fidelity PCR. All cDNAs were fully sequence-confirmed.

Cell treatments and generation of stable cell lines
HEK293, Cos7, HeLa and U2OS cells (all from ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. NIH3T3 cells (ATCC) were cultured in DMEM with 10% calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine. Tsc1+/+, Tsc1–/–, Tsc2+/+ p53–/– and Tsc2–/– p53–/– MEFs (the gift of Dr David Kwiatkowski, Brigham and Women's Hospital, Boston, MA, USA) (47Go) were cultured in DMEM, 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine and 100 µM non-essential amino acids. Tsc1–/– MEFs were retrovirally transduced with human TSC1/pMSCVneo as previously described (48Go). Stable clones were selected over 2 weeks by addition of 500 µg/ml G418 in the growth medium. Expression of hamartin was assayed by western immunoblotting.

Cells were arrested in G2/M using 70 ng/ml nocodazole (Sigma, St Louis, MO, USA) for 18.5 h. For quantitation of centrosome number, cells were arrested in G1/S using 500 µM hydroxyurea (Sigma) for 40 h. Where indicated, cells were treated with 2 nM rapamycin (Biomol, Plymouth Meeting, PA, USA) for 24 h.

Transfections were performed using Lipofectamine 2000 (Invitrogen). RNA silencing was achieved using human TSC1, TSC2 or PLK1 or mouse Plk1 SMARTPool siRNAs (Dharmacon, Lafayette, CO, USA) at a final concentration of 100 nM. siRNAs were delivered to the cells using Transit-TKO transfection reagent (Mirus Bio Corporation, Madison, WI, USA).

Antibodies
Immunoprecipitations were performed with anti-hamartin, anti-Plk1 (Inivtrogen) or anti-TetraHis (Qiagen, Valencia, CA, USA) antibodies. Rabbit or mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as control. Western immunoblotting was performed with anti-hamartin, anti-Plk1, anti-Xpress (Invitrogen), anti-tuberin C20 (Santa Cruz Biotechnology), anti-ß-actin (Sigma) and anti-pS235/236-S6, anti-pT389-p70S6K and anti-pT70-4E-BP1 (Cell Signaling, Beverly, MA, USA). Immunofluorescence was performed with anti-hamartin (4Go) (Invitrogen) and anti-{gamma}-tubulin (Sigma).

Immunofluorescence
Cells were plated on cover slips, fixed with either 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in 1xphosphate-buffered saline (PBS) or ice-cold methanol (Sigma) for 10 min at room temperature, permeabilized with 0.2% Triton X-100 in 1xPBS and stained with the primary antibodies in blocking buffer (1xPBS, 3% BSA, 0.2% Triton X-100). Secondary antibodies were conjugated with either AlexaFluor488 or AlexaFluor594 (Invitrogen). The nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma). Images were obtained using either a DXM1000 digital camera (Nikon, Tokyo, Japan) or an MRC600 confocal microscope (Bio-Rad, Hercules, CA, USA).

To determine centrosome number, cells were arrested in G1/S with 500 µM hydroxyurea (Sigma) for 40 h, fixed in methanol and stained for {gamma}-tubulin (Sigma).

Sucrose gradient fractionation
NIH3T3 cells were used to isolate centrosomes as previously described (49Go). Cells were grown on five 150 mm plates, treated with 70 ng/ml nocodazole for 18.5 h, then with cytochalasin B (Sigma) for 90 min prior to lysis. Cells were washed consecutively in the following solutions: 1xPBS, 0.1xPBS/8% sucrose and 8% sucrose. Cells were lysed in 7 ml of sucrose fractionation buffer [1 mM Tris–HCl pH 8, 0.5% Triton X-100, 0.1% ß-mercaptoethanol, 50 µM PMSF, 1 mM Na3O4V, protease and phosphatase inhibitor cocktails (Sigma)]. After cell debris was removed by centrifugation, the supernatant was filtered through a 40 µm pore diameter nylon mesh, placed on top of a 1 ml 60% sucrose cushion and centrifuged at 12 000 r.p.m. for 40 min at 4°C in an HB-4 swing-bucket rotor (Sorvall, Asheville, NC, USA). All but 2 ml above the sucrose cushion were removed and discarded. The remaining lysate was mixed with the sucrose cushion to make a 20% sucrose solution with enriched centrosomes. The enriched centrosome lysate was placed on top of a discontinuous sucrose gradient range of 40, 50, and 70% sucrose in (10 mM PIPES pH 6.8, 1 mM EDTA, 0.1% Triton X-100, 0.1% ß-mercaptoethanol). The gradient was centrifuged at 25 000 r.p.m. for 80 min at 4°C in an SW50.1 swing-bucket ultracentrifuge rotor (Beckman-Coulter, Fullerton, CA, USA). Fractions (250 µl each) were collected from the bottom up and washed with 10 mM PIPES pH 7.2. The fractions were centrifuged at 14 000 r.p.m. at 4°C, the pellets resuspended in 40 µl of SDS loading buffer and analyzed by SDS/PAGE and western immunoblotting.

Immunoblotting and immunoprecipitation
Cells were lysed in PTY buffer (50 mM HEPES, 50 mM NaCl, 5 mM EDTA, 1% Triton X-100, 50 mM NaF, 10 mM Na4P2O7, 1 mM Na3O4V, 10 µg/ml phenylmethanesulfonyl fluoride) supplemented with protease and phosphatase inhibitor cocktails (Sigma). Protein concentration was determined by the Bradford method (Bio-Rad). Twenty-five micrograms of total lysate were resolved in 7.5 or 4–20% Tris–HCl (Bio-Rad), 3–8% Tris–Acetate or 4–12% Bis-Tris (Invitrogen) PAGE gels, then transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA).

One milligram of total protein cell lysate was used for each immunoprecipitation. Five micrograms of antibody or IgG control was added to each sample and rotated at 4°C for 1 h. Fifty microlitres of Protein-A agarose beads (Invitrogen) were added to each sample and rotated at 4°C overnight. For myc immunoprecipitation, anti-myc-conjugated agarose beads (BD Biosciences) were incubated with lysate overnight. The beads were washed three times with 500 µl PTY buffer. Forty microlitres of SDS loading buffer was added to each sample. Twenty microlitres of each sample was analyzed by PAGE and western immunoblotting.

For phosphatase treatment of the immunoprecipitates, the agarose beads were washed three times with PTY buffer, three times with dephosphorylation buffer (50 mM Tris–HCl pH 7.5, 1 mM MgCl2) then incubated in 100 µl dephosphorylation buffer for 10 min at 37°C. Thirty units of CIAP (Amersham Biosciences, Piscataway, NJ, USA) was added in each sample and incubated for an additional 30 min at 37°C. The beads were washed three times with dephosphorylation buffer, resuspended in 40 µl of loading buffer and analyzed by PAGE and western immunoblotting.

Fluorescence activated cell sorting
Exponentially growing cells were treated as indicated, trypsinized, washed once in ice-cold growth medium and once in 1xPBS and fixed overnight in 1 ml 70% ethanol at –20°C. Cells were washed once in 1xPBS and stained in (70 µM propidium iodide, 30 mM sodium citrate pH 7.0, 10 mg/ml RNAse A) at 37°C for 30 min. Flow cytometry analysis was performed using a Becton-Dickinson FACScan machine and CellQUEST DNA Acquisition software.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr David Kwiatkowski (Brigham and Women's Hospital, Boston, MA, USA) for providing the Tsc1+/+, Tsc1–/–, Tsc2+/+ p53–/– and Tsc2–/– p53–/– MEFs and to Drs Erica Golemis and Jonathan Chernoff for critical review of this manuscript. We thank the Fox Chase Cancer Center Cell Imaging Facility for their assistance with confocal microscopy. Funding was provided by The LAM Foundation (Cincinnati, OH, USA), the Tuberous Sclerosis Alliance (Silver Spring, MD, USA) and the NIH (DK 51052).

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger}The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back


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