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Human Molecular Genetics Advance Access originally published online on January 18, 2007
Human Molecular Genetics 2007 16(5):529-536; doi:10.1093/hmg/ddl485
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Evidence for a molecular link between the tuberous sclerosis complex and the Crumbs complex

Dominique Massey-Harroche1, Marie-Hélène Delgrossi1, Lydie Lane-Guermonprez1, Jean-Pierre Arsanto1, Jean-Paul Borg2,3,4, Marc Billaud5 and André Le Bivic1,*

1 IDBML, CNRS UMR6216, Case 907, Faculté des Sciences de Luminy, 13288 Marseille cedex 09, France, 2 Inserm, U599, Centre de Recherche en Cancérologie de Marseille and 3 Institut Paoli-Calmettes, Marseille F-13009, France, 4 Univ Méditerranée, Marseille F-13007, France and 5 LGMSC, CNRS UMR 5201, Domaine Rockefeller, 8 Av Rockefeller, 68373 Lyon cedex 08, France

* To whom correspondence should be addressed. Tel: +33 491269741; Fax: +33 491269748; Email: lebivic{at}ibdm.univ-mrs.fr

Received October 18, 2006; Revised December 8, 2006; Accepted December 30, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In human, mutations in tuberous sclerosis complex protein 1 or 2 (TSC1/2 or hamartin/tuberin) cause tuberous sclerosis characterized by the occurrence of multiple hamartomas. On the other hand, mutations in the Crumbs homolog-1 (CRB1) gene cause retinal degeneration diseases including Leber congenital amaurosis and retinitis pigmentosa type 12. Here we report, using a two-hybrid assay, a direct molecular interaction between TSC2 C-terminal part and PDZ 2 and 3 of PATJ, a scaffold member of the Crumbs 3 (CRB 3) complex in human intestinal epithelial cells, Caco2. TSC2 interacts not only with PATJ, but also with the whole CRB 3 complex by GST-pull down assays. In addition, TSC2 co-immunoprecipitates and co-localizes partially with PATJ at the level of the tight junctions. Furthermore, depletion of PATJ from Caco2 cells induces an increase in mammalian Target Of Rapamycin Complex 1 (mTORC1) activity, which is totally inhibited by rapamycin. In contrast, in the same cells, inhibition of phosphoinositol-3 kinase (PI-3K) by wortmannin does not abolish rpS6 phosphorylation. These functional data indicate that the Crumbs complex is a potential regulator of the mTORC1 pathway, cell metabolism and survival through a direct interaction with TSC1/2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Epithelial tissues must be able to integrate signals controlling junction remodeling, cell division and tissue overall architecture in order to avoid uncontrolled proliferation and tumor formation. Therefore, control of epithelial cell polarity and tissue homeostasis is a fundamental step in the development and maintenance of multicellular organisms. Works from the last decade have shown that several protein complexes that interact to establish boundaries between apical and lateral domains control epithelial cell polarity (1,2). Among these complexes, the Crumbs (CRB) complex is the only one to contain a transmembrane apical protein essential for junction biogenesis and stability in both Drosophila and mammals (3–6).

In Drosophila, there is only one CRB gene, and CRB mutants undergo massive ectodermal cell apoptosis after epithelial disorganization (7). In mammals, there are three CRB homologs. CRB1 is expressed in the brain and in the eye and is required for normal retinal function in both human and mouse (8–10). Mutations in CRB1 but not in CRB2 (also expressed in the retina) are responsible for severe retina degeneration diseases such as Leber congenital amaurosis and retinitis pigmentosa type 12 (8,11). In contrast, CRB3 is expressed in all epithelial tissues but it lacks the laminin and EGF-repeats found in other CRB family members (12). Recent work has shown that overexpression of CRB3 in MDCK, a renal epithelial cell line, induces polarity defects, delay in tight junction (TJ) formation and apical morphogenesis by mechanisms that are poorly understood (13,14). Nevertheless, it is known that CRB1 and 3 bind to the PDZ domain of Pals1 by its last four amino acids, ERLI. Pals1 is a MAGUK protein that is also necessary for TJ formation and epithelial polarity (15,16). Pals1 in turn recruits PATJ, a scaffolding protein, to the CRB complex through the interaction between the L27 N domain of Pals1 and the MRE domain of PATJ (16).

However, the exact role of these two adaptor proteins in the building of TJs and epithelial homeostasis is still largely unknown. The function of PATJ is particularly intriguing because, although it consists of up to 10 PDZ domains that are protein–protein interaction modules, only a few proteins that are localized to TJs (Pals1, Claudin 1 and ZO-3) have been shown to bind directly to PATJ (5). In this study, we have identified tuberous sclerosis complex 2 (TSC2) as a new binding partner of PATJ. TSC2 and TSC1 are tumor-suppressor genes, and mutation in either gene causes TSC, a syndrome defined by the formation of multiple hamartomas in patients (17). TSC2 exhibits a GAP activity for the small G protein Rheb (18–20), and the TSC1/TSC2 complex has been shown to be a negative regulator of mammalian Target Of Rapamycin Complex 1 (mTORC1) and its targets and to control many cellular functions such as cell size, cell survival, apoptosis and cell cycle progression (21–24). We have also established that depletion of PATJ stimulates the mTORC1 pathway, thereby demonstrating for the first time that there is a molecular and functional link between a member of the TSC and the polarity-determining CRB complex.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PATJ interacts directly with TSC2
To uncover new cellular pathways that might be regulated by the CRB3 complex, we have chosen to investigate the role of PATJ because it is a scaffolding protein that could recruit many cellular partners through its PDZ domains. Interestingly, the first three PDZ domains of PATJ have no known interactors and are highly conserved in Drosophila melanogaster PATJ (25). To find new interactors, we have performed a two-hybrid screen of a human breast library, using the first three PDZ domains of PATJ as bait. Clone 43 (Cl43) was selected as positive and did not show any interaction with human p53 or lamin (not shown). Sequencing demonstrated that Cl43 encoded the C-terminal domain of TSC2 (Fig. 1A). TSC2 (tuberin) is a large cytosolic protein that interacts with TSC1 (hamartin) and consists of several domains including a GTPase-activating protein domain that stimulates the GTPase activity of a small G protein, Rheb (18–20). In mammals, mutations in either TSC1 or TSC2 genes induce tuberous sclerosis, a syndrome characterized by multiple hamartomas, and both TSC1 and TSC2 are considered tumor-suppressor genes (17). Since TSC2 interacts with a part of PATJ that contains three PDZ domains, we used the two-hybrid technique to test the interaction between Cl43 and PDZ domain 1, 2 or 3 (Fig. 1B). PDZ 2 and 3 bound to Cl43, and this interaction was suppressed when we deleted the last amino acids FTEFV of Cl43, suggesting that TSC2 binds to PATJ through a classical PDZ-binding motif. This binding however also needed the presence of the region of TSC2 downstream of amino acid 1538 probably for conformational reasons. This region, encompassing amino acids 1538–1758, showed, in fact, a weak binding to the first three PDZ domains of PATJ but none to PDZ2 or PDZ3 alone in contrast with the same construct containing the FTEFV sequence (see the last two rows in Fig. 1B). Thus we concluded that this upstream region acts in cooperation with the FTEFV motif to provide a strong binding to PDZ domains 2 and 3. We then confirmed that both PDZ domains 2 and 3 but not PDZ domain 1 were able to bind to the C-terminal part of TSC2 by expressing an HA-tagged Cl43 in Cos cells and incubating cell extracts on glutathione–sepharose beads coupled to fusion proteins made of GST and either PDZ domain of PATJ (Fig. 2A). Next, we asked whether endogenous TSC2 could bind to PDZ domains 2 and 3 of PATJ by incubating Caco2 cell extracts with glutathione–sepharose beads coupled to GST-PDZ 1, 2 or 3 fusion proteins (Fig. 2B). There was a specific binding of TSC2 to GST-PDZ domains 2 and 3 confirming the two-hybrid and GST-pull down data obtained with Cl43. Finally, we used a GST fused to Cl43 to pull down endogenous PATJ from Caco2 cells (Fig. 2C). Using affinity-purified polyclonal antibodies against PATJ, TSC2 was co-immunoprecipitated with PATJ (Fig. 3), indicating that the interaction between the endogenous proteins also occurs in Caco2 cells. In addition, TSC1 was also co-precipitated with PATJ, demonstrating that not only TSC2 itself but also the TSC complex was associated to PATJ (Fig. 3). One important question was whether PATJ bound alone to TSC, or the CRB3 complex was also engaged in the interaction. To test this hypothesis, we incubated Caco2 cell extracts with glutathione–sepharose beads coupled to GST-CRB3 cytoplasmic domain (GST-CRB3cyt). As shown in Figure 3B, TSC2 was precipitated specifically together with PATJ by GST-CRB3cyt but not by GST-CRB3cyt lacking the last amino acids ERLI of CRB3 that are required for the interaction with Pals1/PATJ (26). Pals1 and TSC1 also co-precipitated under these conditions (data not shown). This data demonstrated that PATJ and TSC2 interact while integrated in their own complexes.


Figure 1
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  		Figure 1. PATJ interacts with TSC2. (A) Schematic representation of the molecular organization of human PATJ and TSC2. The gray boxes represent the PDZ domains in PATJ. In TSC2, the conserved domains are LZ, leucine zipper domain; CC, coiled-coil domain; TAD, transcription activator domain; GAP, GTPase-activating domain. The constructs that were derived from PATJ and used as baits in the two-hybrid system are indicated. Cl43, TSC2 clone found by a two-hybrid screen with PDZ 123. Numbers stand for the amino acids starting from the ATG. (B) Results from the two-hybrid assays between PATJ and TSC2 constructs. Row: PATJ constructs used as baits as shown in (A). Column: TSC2 constructs used as preys. ND, not determined; ++, strong growth and coloration in a selective medium; +, weaker growth or coloration in the same medium. +/–, very weak growth or coloration in the same medium; –, no growth and coloration in the same medium.

 


Figure 2
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  		Figure 2. TSC2 interacts with PATJ in vivo. (A) GST-pull down assays with fusion proteins made of GST and the PDZ domains of PATJ. Cos7 cells were transfected with Cl43 tagged with the hemagglutinin (HA) epitope at the N-terminus (+) or with a non-relevant cDNA (–), and after 48 h, cells were lyzed and cell extracts were incubated with the beads coupled to GST-fusion proteins. Bound HA-Cl43 was detected using anti-HA antibodies by western blotting after SDS–PAGE and electro-transfer. The respective amounts of GST-fusion protein used in the binding assay are shown by Ponceau Red staining. Input: 1/60th of the total cell extract. (B) Endogenous TSC2 binding to GST-fusion proteins made of GST and PDZ domains of PATJ. (C) Endogenous PATJ binding to GST-fusion proteins made of GST and Cl43 of TSC2. In (B) and (C), Caco2 (TC7) cells were lyzed and cell extracts were incubated as in (A). TSC2 and PATJ were revealed using specific antibodies. Caco2: 1/60th of the total cell extract. *, GST or GST-fusion protein.

 


Figure 3
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  		Figure 3. TSC interacts with PATJ and CRB3 complex. (A) Caco2 (TC7) cells were lyzed and cell extracts were incubated with affinity-purified antibodies against PATJ (PATJ IP) or with rabbit anti-mouse antibodies (CT) coupled to G-protein sepharose beads. PATJ or TSC2 or TSC1 were then revealed with specific antibodies and anti-rabbit antibodies coupled to peroxidase. Caco2: 1/60th of the total cell extract. Blot: antibodies used to detect either PATJ or TSC2 or TSC1 (or the two last: TSC) on cell extracts or immunoprecipitates. (B) Caco2 cell extracts were incubated with GST-fusion proteins made of GST and cytoplasmic domain of CRB3. ERLI, last four amino acids of CRB3 deleted in CRB3 {Delta}ERLI. The respective amounts of GST-fusion protein used in the binding assay are shown by Ponceau Red staining. Caco2: 1/60th of the total cell extract. TSC2 and PATJ were revealed using specific antibodies. *, GST-fusion protein.

 
TSC2 is a cytosolic protein while PATJ is strongly associated with TJs and occasionally with the apical membrane. To uncover a possible co-localization between the two proteins, we performed double immunofluorescence experiments in which Caco2 cells were first incubated alive with a detergent-containing buffer to remove free TSC2 from the cytosol and then subsequently fixed and treated for standard double immunofluorescence with antibodies directed against a TJ marker, occludin (27) or either TSC2 or PATJ. A direct double staining for TSC2 and PATJ could not be performed since the two antibodies are from the same species. TSC2 was found at the level of TJs co-localizing with occludin similar to PATJ (Fig. 4A). As this TSC2 association with TJs has never been observed before, we further confirmed this subcellular localization by performing immunogold labeling of TSC2 on ultrathin sections of human colon or Caco2 cells. Gold particles were associated with the region of TJs (Fig. 4B), a region where we have previously shown that PATJ also accumulates (26). Thus, combined, these data show that the CRB3 complex and TSC can interact in Caco2 cells at the level of TJs.


Figure 4
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  		Figure 4. TSC2 is found at the level of TJs. (A) Caco2 cells were permeabilized with SDS and then fixed and treated for double indirect immunofluorescence either with rabbit antibodies against TSC2 (in green) or with PATJ (in blue) and mouse antibodies against occludin (Occ) (in red). Bar: 10 µm. Arrowheads point at TJs. (B) Human colon and Caco2 ultrathin sections were labeled with rabbit anti-TSC2 antibodies, followed by anti-rabbit antibodies coupled to gold particles (6 nM). The arrow points at TSC2 labeling. Bracket, TJ area; mv, microvilli; Ap, apical membrane. Bar: 100 nM.

 
PATJ regulates the mTORC1 cascade
The direct interaction observed between PATJ and TSC2 prompted us to investigate its cellular consequences. Active TSC2, in complex with TSC1, inhibits the activity of Rheb that activates mTORC1 (23). The signaling pathways acting upstream of TSC/mTORC1 are the PI-3K/Akt cascade, the LKB1/AMPK cascade and the MEK/ERK1/2 cascade (Fig. 5). Although Akt and ERK1/2 phosphorylate TSC2 on different residues (for example, S540 and S666 for ERK1/2, and S939 and T1462 for Akt), the consequence in both cases is that TSC is inactivated. This inactivation in turn stimulates the mTORC1 activity (28–30). Conversely, LKB1 activates AMPK, which phosphorylates TSC2 on T1227 and S1345 and activates it, thus counteracting the Akt and ERK1/2 inhibition (31). This activity can be assayed by analyzing ribosomal protein S6 (rpS6) phosphorylation: activated TSC inhibits rpS6 phosphorylation through the downregulation of the S6-Kinase (S6-K) (32).


Figure 5
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  		Figure 5. Scheme of the TSC/mTORC1 cascade with inhibitors (in italics) and kinases controlling TSC2 phosphorylations (P) and its relationships with the CRB3 complex. Simple lines are for molecular interactions.

 
To determine whether PATJ could modify the activity of TSC2 in Caco2 cells, we used several clones of Caco2 cells that were downregulated for PATJ (PATJ KD) by stable expression of an shRNA (33). These PATJ KD cells showed an abnormal intracellular redistribution of CRB3 and Pals1, two other members of the CRB3 complex, and diffusion on the lateral membrane of ZO-3 and occludin, the two members of TJs (33). Specific antibodies directed against a phosphorylated form of rpS6 were used to probe cell lysates from control (CT, Cl8) and PATJ KD (Cl 4 and 12) cells (Fig. 6A and B). In PATJ KD clones, there was a 2–3-fold increase of rpS6 phosphorylation, indicating that the mTORC1 pathway was upregulated when PATJ was depleted, whereas the total amount of TSC2 (as estimated by western blotting) was the same in CT (cl8) and PATJ KD (cl4) cells (not shown). This finding was confirmed in six independent clones of PATJ KD Caco2 cells out of eight in total (data not shown). To ensure that this increase was the consequence of mTORC1 activation, we used a rapamycin treatment to block mTORC1 activity (34). Indeed, treatment of both CT and PATJ KD cells with rapamycin suppressed phosphorylation of rpS6 (Fig. 7A), indicating that the increase of rpS6 phosphorylation in PATJ KD cells was mTORC1 dependent. Thus we concluded that loss of PATJ expression downregulated TSC function, leading to the activation of mTORC1 either directly or indirectly. To test a possible indirect effect of loss of PATJ on TSC function, we measured AMPK phosphorylation levels in control and PATJ KD cells. Indeed, there was a decrease in AMPK phosphorylation in PATJ KD cells (cl 4 and 12), indicating that changes in AMPK activity could play a role in the elevation of rpS6 phosphorylation (Fig. 7B).


Figure 6
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  		Figure 6. The mTOR pathway is upregulated in PATJ-KD Caco2 cells. (A) PATJ KD (Cl4 and Cl12) and CT Caco2 cells (Cl8) were grown for 4 days after confluence and lyzed and probed with antibodies against PATJ, phosphorylated rpS6 (P-rpS6) or total rpS6 (tot-rpS6). (B) Relative levels of phosphorylated rpS6 in PATJ KD (Cl4 and Cl12) and CT Caco2 cells (Cl8) as shown in (A). n = 3.

 


Figure 7
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  		Figure 7. Contribution of signaling pathways in PATJ KD cells. (A) PATJ KD (Cl4) and CT Caco2 cells (Cl8) were grown for 4 days after confluence and treated with rapamycin (+) or not (–) to block mTORC1. Signal from cells without treatment is overexposed to show the absence of rpS6-P in rapamycin-treated cells. Cell extracts were then probed with antibodies against phosphorylated rpS6 (P-rpS6) or total rpS6 (tot-rpS6). (B) PATJ KD (Cl4 and Cl12) and CT Caco2 cells (Cl8) were grown for 4 days after confluence and lyzed and probed with antibodies against total AMPK (tot-AMPK) and phosphorylated AMPK (P-AMPK). (C) PATJ KD (Cl4, Cl12) and CT Caco2 cells (Cl8) were grown for 4 days after confluence and treated with either wortmannin (Wort) (an inhibitor of PI3K) or U0126 (an inhibitor of MEK 1/2), or with both wortmannin and U0126. Cell extracts were then probed with antibodies against phosphorylated rpS6 (P-rpS6) or total rpS6 (tot-rpS6). The exposure shown is overexposed to better detect the extent of rpS6-P decrease in drug-treated cells. n = 3.

 
Given that MEK1/2- and Akt- signaling pathways negatively regulate TSC activity, we wanted to know whether these pathways were modified in PATJ KD cells. CT and PATJ KD cells were treated either with wortmannin, an inhibitor of PI3K, or with U0126, an inhibitor of MEK1/2 (Fig. 7C). U0126 treatment strongly diminished rpS6 phosphorylation in both CT and PATJ KD cells, indicating that the MEK1/2 activity was involved in the control of mTORC1 activity in these cells. Wortmannin treatment almost abolished rpS6 phosphorylation in CT cells, but not in PATJ KD cells, suggesting that an alternative to PI3K-Akt pathway has been activated in Caco2 cells. When the cells were treated with wortmannin and U0126 in combination, rpS6 phosphorylation was decreased in both PATJ KD and CT cells as it was in U0126-treated cells. There was however a residual activity in PATJ KD cells, which was not observed in CT cells that might contribute to the increase in mTORC1 activity observed in PATJ KD cells. This residual activity on rpS6 phosphorylation resistant to wortmannin treatment was observed in six independent PATJ KD clones out of eight, suggesting that it was correlated to the depletion of PATJ in these cells (not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In Drosophila, the CRB complex, comprising CRB, Stardust (Sdt) and DPATJ, is essential for the stability of the newly formed adherens junctions (AJs) and for the ectodermal cell survival (5). Several studies have shown that there is a homologous complex made of CRB3, Pals1 and PATJ in mammalian epithelial cells (35). This CRB3 complex in vertebrates is necessary for the proper assembly of TJs since overexpression of CRB3 or PATJ delayed the establishment of functional TJs in MDCK cells (13,14), and knockdown of Pals1 or PATJ induced TJ and polarity defects in MDCK (13,36) or Caco2 cells (33). CRB3 is a short transmembrane protein that binds to the PDZ domain of Pals1 by its cytoplasmic last amino acids ERLI (16). Pals1 recruits PATJ by its L27 N domain that interacts with the N-terminal MRE domain of PATJ (35). Both CRB3 and Pals1 are able to interact with Par-6 to connect two protein complexes essential for the establishment of the apical membrane and junctions (14,37). On the other hand, PATJ binds to ZO-3 and Claudin-1 by its sixth and eighth PDZ domains, respectively (38), to anchor the CRB3 complex to TJs (33). Despite the existing functional analyzes and the currently known network of interactions, a complete identification of all the roles of the CRB3 complex in epithelial morphogenesis has yet to be unraveled.

Our data show for the first time a direct link between TSC, a major inhibitor complex of mTORC1, and the CRB complex. It is worth to point out that these two complexes when mutated are responsible for human diseases such as retina degeneration and tumor formation. How can PATJ be involved in the regulation of the mTORC1 pathway? One possibility is that PATJ, through its multiple TJ partners, connects signaling pathways. In fact, PATJ binds to ZO-3 (38), whereas ZO-3 binds to occludin (39), which is delocalized from TJs to the lateral membrane in PATJ KD Caco2 cells (33). It has been shown that occludin interacts with the regulatory subunit p85 of PI3K (40) and thus a delocalization of occludin along the lateral membrane could modify the PI3K-Akt activity onto TSC2 in PATJ KD Caco2 cells. This hypothesis is a likely one since we observed that wortmannin treatment has a different outcome in PATJ KD versus control cells. This data indicate that there may be an uncoupling between PI3K/Akt and TSC2 in PATJ KD cells. This uncoupling between TJ and TSC components could also be responsible for the decreased levels of AMPK phosphorylation in PATJ KD cells. Alternatively, the loss of PATJ binding to TSC2 could facilitate ERK1/2 phosphorylation on TSC2, leading to the activation of mTORC1 since the phosphorylation sites are located in the C-terminal part of TSC2 where PATJ interacts with TSC2 (28), but there are so far no phospho-specific antibodies for these phosphorylation sites. Furthermore, the molecular mechanisms leading to functional activation of TSC are far from being elucidated. These two mechanisms could act in parallel and it will be interesting to investigate the expression of PATJ in tumors from TSC patients to evaluate whether PATJ could contribute to disease progression through mTORC1 activation.

There are now more and more examples of links between complexes involved in cell polarity and junction formation and complexes involved in cell survival and proliferation. TSC1 and TSC2 form a complex, and mutations in either lead to the human disease TSC that is similar to the Peutz-Jegher syndrome in that it is an autosomal-dominant condition resulting in hamartomas. Another disease associated with hamartomas is Cowden's disease originating from mutations in PTEN, a phosphatase regulating the PKB/Akt pathway upstream of TSC. It is thus obvious that LKB1, TSC and PTEN are regulating the mTOR cascade after growth factor stimulation (41). What is more exciting in the view of our data is that LKB1 (or Par-4) is also involved in the determination of cell polarity in human intestinal cells (42), whereas PTEN has been shown to interact with Par-3 (43,44), another essential protein for the establishment of cell polarity and epithelial junctions (45). Thus the interaction between PATJ and TSC2 belongs to a network of interconnection between cell polarity and junctional complexes and the TOR pathway, thereby potentially involving in tissue homeostasis and tumor growth.

Finally, an interesting point is that in human, mutations in CRB1 lead to severe retina degeneration by unknown mechanisms and it will be of interest to study the potential interaction between the CRB1 complex in the retina and TSC since TSC regulates cell survival and apoptosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell culture, antibodies and drug treatments
A clone of Caco2 cells (TC7) was grown as described (31) and used to produce clones containing either the plasmid peGFP-N2 (BD Bioscience Clontech, Palo Alto, CA, USA) with a U6 promoter (clone 8) or the same plasmid in which an shRNA against human PATJ mRNA was introduced (clones 4 and 12) as described recently (33). Stable clones were maintained in G418 (0.5 mg/ml) supplemented culture medium. Confluent cells were grown for 16 h without serum and then treated for 2 h with either rapamycin (100 nM), wortmannin (100 nM) or U0126 (25 µM).

Rabbit polyclonal antibodies against PATJ were used as before (25). Mouse monoclonal antibodies against tubulin and hemagglutinin were from Sigma (St Louis, MO, USA) and against occludin were from Zymed (South San Francisco, CA, USA). Rabbit polyclonal antibodies against TSC2 (SC-893 C-20) were from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA, USA). The following antibodies were purchased from Cell Signaling Technology: polyclonal rabbit antibodies against rpS6 (2212), phospho-S6 RP (S235/236) (2211) and AMPK-{alpha} (2532), and rabbit Mab 40H9 against phospho-AMPK-{alpha} (T172).

Two-hybrid screen
The first three PDZ domains of hPATJ (amino acids 2–455) were expressed in the pGKT7 vector and used to screen a human breast library in pACT2 in YAH109 yeast strain (BD Bioscience Biotech). Around 600 000 transformed clones (growing in the absence of tryptophane and leucine) were plated and 192 clones were harvested after selection in the absence of tryptophane, leucine and histidine and in the presence of 10 mM 3-aminotriazole. Sixty clones were sorted by their digestion pattern and two positive and specific clones were identified as hTSC2 after sequencing.

Immunoprecipitations, western blotting and GST-pull down assays
Caco2 cell extracts were prepared and analyzed by western blotting as described (26). Bands were revealed using the Lumilight kit (Roche Diagnostics, Meylan, France) and quantified using ImageQuant (Amersham Bioscience, Orsay, France) software. For immunoprecipitations, Caco2 cells were lyzed in buffer L (150 mM NaCl, 50 mM Tris HCL, pH 8.0, 50 mM NaF, 0.5% Nonidet P40, 2 mM EDTA, 1 mM Na3VO4) and processed as described before with proteases inhibitors (14). For GST pull-downs, 1% Triton X-100 extracts of COS-7 cells transfected with HA-tagged C-terminal part of TSC2 (amino acids 1329–1763) were incubated overnight with glutathione–sepharose beads (30 µl) coated with GST-hPATJ PDZ domain fusion proteins or GST alone (40 µg), and after washes, bound proteins were analyzed by western blotting after SDS–PAGE.

Immunofluorescence and immunoelectron microscopy
For immunofluorescence experiments, clones of Caco2 cells were seeded at near confluency on Transwell filters (24 mM in diameter; Corning, NY, USA), maintained for 8–10 days in culture and processed as described (13). Images were taken using a Zeiss 510 Meta confocal microscope (Zeiss, Le Pecq, France). For immunoelectron microscopy, Caco2 cells and human colon biopsies (Paoli Calmette Institute, Marseille, France) were processed by the cryosubstitution method, as previously described (33). After immunogold labeling, ultrathin sections were observed with Zeiss 912 electron microscope (Zeiss).


   ACKNOWLEDGEMENTS
 
We would like to thank J.P. Chauvin (IBDML, Marseilles) for his help with the immuno-electron microscopy and P. Rashbass (University of Sheffield, UK) for critical reading of the manuscript. We thank all the scientists who provided antibodies or plasmids to us. We also acknowledge the technical facilities of the IBDML. This work was supported by grants from Fondation de France (to A.L.B.), ACI BCMS, Cancéropôle PACA (to A.L.B. and J.-P.B.), Ligue Nationale contre le Cancer (to A.L.B., J.-P.B. and M.B.), ARC network (to A.L.B. and M.B.) and CNRS 6216. L.L.-G.'s fellowship was supported by Fondation de France.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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