Tumor suppressor p53 protein is the transcription factor responsible for various genes including DNA repair, growth arrest, apoptosis and antiangiogenesis. Recently, we showed that clathrin heavy chain (CHC), which was originally identified as a cytosolic protein regulating endocytosis, is present in nuclei and functions as a coactivator for p53. Here, we determined the detailed p53-binding site of CHC and a CHC deletion mutant containing this region (CHC833-1406) behaved as a monomer in cells. Monomeric CHC833-1406 still had a higher ability to transactivate p53 than wild-type CHC although this CHC mutant no longer had endocytic function. Moreover, similar to wild-type CHC, monomeric CHC enhances p53-mediated transcription through the recruitment of histone acetyltransferase p300. Immunofluorescent microscopic analysis exhibited that CHC833-1406 is predominantly localized in nuclei, suggesting that there may be a certain regulatory domain for nuclear export in the C-terminus of CHC. Thus, the trimerization domain of CHC is not necessary for the transactivation of p53 target genes and these data provide further evidence that nuclear CHC plays a role distinct from clathrin-mediated endocytosis.
The p53 gene, in which most frequent mutations have been found in human cancers, encodes a protein that plays an important role in preventing tumorigenesis (Levine, 1997; Prives and Hall, 1999; Vogelstein et al., 2000; Vousden and Lu, 2002; Bourdon et al., 2003; Oren, 2003). Although it has been proposed that p53 has various functions such as transcriptional activation for cell cycle arrest and apoptosis (Bourdon et al., 2003; Oren, 2003), centrosome duplication (Tarapore and Fukasawa, 2002), homologous recombination (Linke et al., 2003), nucleotide excision repair (Seo et al., 2002) and transcription-independent mitochondria-mediated apoptosis (Mihara et al., 2003), transcriptional regulation by p53 is thought to be most important for the prevention of tumorigenesis because most p53 mutations in tumor cells are located in the central DNA-binding domain (Hollstein et al., 1991).
The expression level of p53 protein is tightly regulated by various E3 ubiquitin ligases including Mdm2 oncoprotein (Haupt et al., 1997; Honda et al., 1997; Kubbutat et al., 1997), Pirh2 (Leng et al., 2003), COP1 (Dornan et al., 2004) and ARF-BP1/Mule (Chen et al., 2005; Zhong et al., 2005), which bind p53 to degrade it (Brooks and Gu, 2006). In response to various genotoxic stresses such as γ-irradiation, UV and antitumor drugs, p53 is stabilized and activated through post-translational modification including phosphorylation, acetylation and methylation to promote the transcription of various genes responsible for DNA repair (p53R2 and GADD45), growth arrest (p21waf1) and apoptosis (Bax, Noxa, Puma and p53AIP1) (Levine, 1997; Prives and Hall, 1999; Vousden and Lu, 2002; Bourdon et al., 2003). Analysis of p53-mediated transcriptional mechanisms is important for the elucidation of tumorigenesis and the development of new antitumor drugs. Although many factors that contribute to the regulation of p53 activity have been reported (Prives and Hall, 1999; Ljungman, 2000; Samuels-Lev et al., 2001), the detailed mechanisms remain to be elucidated. We have recently reported that a serine-to-phenylalanine substitution of p53 at codon 46 (p53S46F) strongly increased the function of p53, leading to the stabilization of the interaction with clathrin heavy chain (CHC) to enhance p53-mediated transcription (Enari et al., 2006). In addition, it has recently been reported that adenovirus-mediated gene transfer of p53S46F suppresses the tumor growth of cancer cells more effectively than that of wild-type p53 in vivo (Nakamura et al., 2006).
In the clathrin-mediated endocytic pathway, clathrin is composed of a trimer of CHC and is associated with each light chain, called triskelion, and they further assemble to form a polyhedral cage-like structure (Kirchhausen, 2000). Polyhedral clathrin is recruited to the plasma membrane and promotes the internalization of certain receptors to take nutrients or to transduce correct intracellular signaling from receptors (McPherson et al., 2001). In addition, more recently, it has been reported that CHC plays a role in mitosis (Royle et al., 2005). CHC consists of an N-terminal β-propeller domain that is necessary for binding to adapter proteins for the internalization of various molecules, followed by seven α-helical repeat structures named ‘clathrin repeats’ (Kirchhausen, 2000). In the C-terminus, CHC possesses a trimerization domain essential for stable formation of the polyhedral clathrin structure (Wakeham et al., 2003). We recently showed that CHC was present in nuclei and that nuclear CHC formed a complex with both p53 and histone acetyltransferase p300 in response to DNA damage. Thus, we demonstrated that CHC has a novel function as a regulator of p53-mediated transcription in addition to the functions for endocytosis and mitosis (Enari et al., 2006). However, it remains elusive whether the trimerized formation of CHC, which is necessary for lattice formation to promote clathrin-dependent endocytosis, is required for p53 transactivation.
In this study, we determined the detailed p53-binding domain of CHC using various deletion mutants of CHC and revealed that CHC bearing residues from 833 to 1406 (CHC833-1406) lacking trimerization and clathrin light chain (CLC)-binding domains interacts with p53. Although CHC833-1406 does not oligomerize, it still has the ability to transactivate p53, indicating that the oligomerization of CHC, which is critical for endocytosis and mitosis, is not necessary for p53-mediated transcription. In addition to our previous observation (Enari et al., 2006), these results further support that CHC has an alternative function as a coactivator for p53 and a different role from the regulation of endocytosis and mitosis.
Trimerization domain of CHC is not required for interaction with p53 and the CLC-binding domain of CHC inhibits interaction with p53
CHC comprises the N-terminal globular domain, which is required for binding to various endocytic adapter proteins to regulate endocytic actions, seven repeat structures designated as clathrin repeats in the middle and C-terminal parts, followed by CLC-binding and trimerization domains in the C-terminus. We have previously determined the interaction domain between CHC and p53 and shown that p53 binds to CHC through the C-terminus of CHC (Enari et al., 2006); however, although the C-terminus of CHC is a major site for p53 binding, the other region of CHC is also required for full binding activity to p53 (data not shown). In order to determine the detailed region that binds to p53, PCR was used to engineer various C-terminal deletion mutants of CHC and they were expressed as 35S-labeled proteins by an in vitro transcription/translation system using rabbit reticulocyte lysates (Figure 1a). 35S-labeled CHC proteins were detected as expected bands by autoradiography (Figure 1b) and tested for binding to wild-type p53 fused to glutathione-S-transferase (GST-p53wt) in pull-down assays as described previously (Enari et al., 2006). Deletion of residues from 1675 to 1615 (CHC1-1634 and CHC1-1615) does not affect the binding of CHC to p53 (Figure 1b). In contrast, deletion to residues 1522 (CHC1-1522) reduced interaction with p53 (Figure 1b), whereas further deletion of CHC to residues 1406 (CHC1-1406) recovered p53 interaction, suggesting that the C-terminal region containing the trimerization domain on CHC is not necessary for binding to p53 and presumably competes for binding to CLC (Figure 1b).
To assess whether the CLC-binding ability of CHC lacking a trimerization domain increases, two isoforms of CLC proteins were prepared as GST-fusion proteins. The binding affinity of CHC deletion mutants lacking a trimerization domain to both isoforms of GST-CLC proteins is three to fourfold higher than that of wild-type CHC (Figure 1c), consistent with the fact that CHC lacking a trimerization domain has stronger binding activity to CLC than wild-type CHC as assayed using the yeast two-hybrid system (Chen et al., 2002). Thus, these results suggest that the increase of the binding affinity of CHC to CLC leads to the exclusion of p53 from the CHC complex. Mutants with further deletion to residues 1313, 1213 or 1073 had little binding ability to p53, indicating that a major p53-binding site of CHC is located between residues 1313 and 1406 although the N-terminal part of CHC is also required for full binding affinity to p53 (see below).
We next examined the effect of N-terminal deletion of CHC on binding to p53 using GST pull-down assays. As CHC has seven repeat structures (called clathrin repeats) in the middle and C-terminal parts and the predicted tertiary structure of these regions has been reported (Ybe et al., 1999), we constructed various CHC mutants bearing the N-terminal deletion to repeat 1 (aa 686–1675), repeat 2 (aa 833–1675) and repeat 3 (aa 979–1675) to assess which repeat structures are required for binding to p53 (Figure 1d). Analysis of GST pull-down assays revealed that CHC833-1675 had similar p53-binding affinity to wild-type CHC and that further deletion to repeat 3 greatly reduced interaction with p53 (Figure 1e). Furthermore, we generated N- and C-terminal deletions of CHC bearing the region from residues 833 to 1406 (CHC833-1406) and carried out a GST pull-down assay for this deletion mutant (Figure 1f). As expected, CHC833-1406 had full binding affinity to p53 compared to wild-type CHC (Figure 1g). Furthermore, by using the anti-FLAG antibody, we examined the interaction between FLAG-tagged p53 and hemagglutinin(HA)-tagged CHC833-1406 in cells. Immunoblot analysis showed that HA-tagged CHC833-1406 as well as HA-tagged full-length CHC was present in immunoprecipitates containing FLAG-tagged p53 from cell extracts of H1299 cells (Figure 1h), indicating that the region from residues 833 to 1406 on CHC, which corresponds to repeat 3–6, is a minimal p53-binding site for full binding affinity.
CHC binding to p53 correlates with p53 transactivation
When the wild-type CHC construct was cotransfected with p53 expression vector in p53-null H1299 cells, the transactivation of various p53-responsive promoters including p21waf1, Noxa, p53R2 and p53AIP1 promoters, was markedly enhanced compared with those transfected with p53 alone (Enari et al., 2006). Under these conditions, we tested the effect of CHC833-1406 on p53 transactivation of various p53-target promoters (p53AIP1, Noxa and p21waf1 promoters). Reporter assays using various p53-target promoters showed that the p53-dependent transactivation of all promoters used was enhanced by the expression of CHC833-1406 in both p53-null H1299 and Saos-2 cells (Figures 2a and b), indicating that CHC833-1406 lacking the C-terminal region containing a trimerization domain has similar or rather higher activity to transactivate p53 compared with wild-type CHC. To confirm the effect of CHC833-1406 on its transcriptional activity, the induction of endogenous p53 target genes was monitored by reverse transcriptase-coupled semiquantitative PCR (RT–PCR). RT–PCR experiments showing that the induction of p53-responsive genes was enhanced by the coexpression of CHC833-1406 with p53 up to similar or slightly higher values compared with that by full-length CHC (Figure 2c), although p53 protein level was nearly the same, as judged by immunoblotting (Figure 2d).
The region of CHC from residues 833 to 1406 functions as a monomer
CHC forms a homo-trimer through the trimerization domain located in the C-terminus of CHC and the homo-trimer further assembled to form a cage-like structure responsible for endocytic function; therefore, we assessed whether CHC833-1406 lacking a trimerization domain was indeed present as a monomer under our conditions. To ascertain this, we first carried out an immunoprecipitation assay with various FLAG- and HA-tagged CHC constructs. FLAG- or HA-tagged wild-type CHC or both constructs were transfected into cells and cell lysates were immnoprecipitated with anti-HA antibody. The immune complex was eluted and coprecipitated FLAG-tagged CHC in the eluates was detected by immunoblotting with anti-FLAG antibody. As shown in Figure 3a, FLAG-tagged wild-type CHC was able to associate with HA-tagged wild-type CHC. On the other hand, CHC833-1406 lacking a trimerization domain had no or little ability to associate with any CHC proteins and did not form a homo-oligomer (Figures 3a and b). To further characterize the polymeric status of CHC833-1406, we performed chemical cross-linking experiments, using bis[sulfosuccinimidyl]suberate (BS3), a bi-functional chemical cross-linker with 11.4 Å arm length, which was chosen because for cross-linking of CHC, dimethyl suberimidate (DMS) with a similar arm length (11.0 Å) has previously been used (Kirchhausen and Harrison, 1981). When recombinant CHC1073-1675 protein purified from Escherichia coli was reacted with BS3 at a concentration of 2 mM, it was cross-linked and the trimerized formation of CHC1073-1675 was detected (Figure 3c). In contrast, CHC833-1406 devoid of a trimerization domain was not oligomerized even when treated with BS3 and was detected as a monomer, as determined by silver staining (Figure 3c). These data indicate that CHC833-1406, with the ability to transactivate p53, is present as a monomer.
Effect of CHC833-1406 on clathrin-mediated endocytosis
Given that CHC833-1406 did not form an oligomer, we next asked if this CHC mutant could affect clathrin-mediated endocytosis to exclude the possibility that p53 transactivation by CHC is due to the dysfunction of clathrin-dependent endocytosis. Ligand-induced internalization of transferrin receptor occurs via clathrin-mediated endocytosis and the system of transferrin-bound transferrin receptor internalization has been used to analyze clathrin-mediated endocytosis. Using this system, we examined the effect of a monomeric CHC mutant on clathrin-mediated endocytosis. For this experimental procedure, each construct, wild-type CHC or CHC833-1406, was transfected into cells in the absence or presence of the expression vector of short-hairpin RNA (shRNA) against the 3′-untranslated region of CHC mRNA (shRNA-CHCunt) to down-regulate endogenous CHC expression and to see the effect of ectopically expressed CHC on endocytosis. When cells were transfected with wild-type CHC construct without shRNA-CHCunt, the uptake of fluorescent-labeled transferrin slightly increased compared with the control (Figure 4a, Column 1 vs 2), although the amount of cell surface-bound transferrin was almost the same as the control (Figure 4b, Column 1 vs 2). In cells transfected with control vector plus shRNA-CHCunt, the endocytosis of transferrin was severely impaired and decreased by up to 50% (Figure 4a, Column 4), whereas when cells were transfected with wild-type CHC construct plus shRNA-CHCunt, the severe defect of endocytosis by shRNA-CHCunt was recovered up to 90% (Figure 4a, Column 5). Endocytic activity on transferrin uptake well correlates with the expression level of CHC (Figure 4c). Under these conditions, the effect of CHC833-1406 on transferrin endocytosis was similar to that of an empty vector (Figures 4a and b) although the expression of CHC833-1406 and transferrin receptor is similar to that of full-length CHC (Figure 4c), indicating that CHC833-1406 has no or little ability to affect endocytic action.
Monomeric CHC enhances p53-mediated apoptosis
Wild-type CHC enhances p53-mediated apoptosis when cells are cotransfected with CHC and p53 expression vectors or when the CHC construct is transfected into p53-positive cells (Enari et al., 2006); therefore, we next addressed whether the monomeric form of CHC has the ability to enhance p53-mediated apoptosis. As caspase activation is a well-characterized marker for apoptosis, caspase-3/7 activation was measured using a fluorogenic substrate. When monomeric CHC833-1406 construct was cotransfected with p53 expression vector, increased caspase-3/7 activity was detected at a higher level than those cotransfected with wild-type CHC and p53 constructs (Figure 5a), although the expression level of p53 between these samples was similar (Figure 5b). Furthermore, we also confirmed this result by monitoring the cleavage of poly-ADP ribose polymerase-1 (PARP-1), which is known as a death substrate (Figure 5c), indicating that monomeric CHC still has the ability to promote p53-mediated apoptosis.
p53 transactivation by monomeric CHC mediates p300
We have previously shown that CHC transactivates p53 through the recruitment of histone acetyltransferase p300 (Enari et al., 2006). To investigate whether monomeric CHC833-1406 interacts with p300, an immunoprecipitation assay was performed. HA-tagged CHC833-1406 was present in the immunoprecipitates including FLAG-tagged p300 as well as HA-tagged full-length CHC (Figure 6a). To examine whether CHC833-1406 as well as wild-type CHC cooperates with p300 to stimulate p53-mediated transcriptional activity, a reporter assay using p53AIP1 promoter was used as described above. The ectopic expression of wild-type p300 and p53 with wild-type CHC in p53-null cells greatly enhanced p53 transactivation compared to p53 alone (Figure 6b). Similarly with wild-type CHC, CHC833-1406 enhanced p300-mediated p53 transactivation (Figure 6b). Furthermore, p300DN (residues 1514 to 1922), which works as a dominant-negative p300 for p53-mediated transcription, inhibited CHC833-1406-mediated p53 transactivation (Figure 6c), indicating that the enhancement of p300-mediated p53 transactivation by the monomeric form of CHC is almost equivalent to that by wild-type CHC.
Increased nuclear localization of monomeric CHC
We previously showed that approximately 5% of endogenous CHC is present in nuclei and is important for the induction of p53-target genes. To examine the effect of the deletion of the trimerization domain in CHC protein on cellular localization, we performed confocal immunofluorescent microscopic analysis. HA-tagged wild-type CHC and CHC833-1675 bearing a trimerization domain were mainly localized in cytoplasm and exhibited a similar localization pattern to endogenous CHC (Figures 7a and b and data not shown). However, HA-tagged CHC833-1406 lacking a trimerization domain predominantly accumulated in nuclei and exhibited increased nuclear localization (Figures 7a and b), suggesting that the C-terminal region containing the trimerization domain of CHC is crucial to keep CHC in the cytoplasm. CHC833-1406 may induce p53-mediated apoptosis more efficiently than full-length CHC (Figure 5) due to elevated nuclear accumulation caused by C-terminal deletion of CHC.
Leptomycin B is an inhibitor of nuclear export mediated by CRM1, a nuclear export factor, and blocks the nuclear export of various nucleocytoplasmic shuttle proteins bearing leucine-rich nuclear export signal (NES) sequences (Kumar et al., 2006). To address whether the nuclear accumulation of CHC is dependent on the classical nuclear export system, we tested the effect of leptomycin B on the nuclear export of CHC. Treatment of cells with leptomycin B induced the nuclear accumulation of p53 fused to NES sequences derived from an inhibitor of cAMP-dependent protein kinase (PKI), which is well known to depend on CRM1 (Figure 7c). In contrast, leptomycin B had no or little effect on the nuclear accumulation of CHC (Figures 7c and d), indicating that the nuclear localization of CHC is mediated by an unidentified nuclear export system and is likely to utilize a distinct nuclear export system from the conventional method.
Although CHC was originally identified as a regulator of clathrin-mediated endocytosis, vesicle transport and protein sorting, we have recently demonstrated that a small amount of CHC is present in nuclei and is required for p53-mediated transcription (Enari et al., 2006). Cytosolic CHC is known to be important to trimerize and form a cage-like structure during endocytic action. Moreover, it has recently been reported that the trimerization of CHC is required for mitosis (Royle and Lagnado, 2006); however, it remains elusive whether the trimerization of nuclear CHC is necessary for p53-mediated transcription.
In this study, we generated various CHC deletion mutants and determined the detailed region responsible for interaction with p53. In vitro binding analysis using various CHC mutants lacking N- or/and C-terminal regions revealed that the region between residues 833 and 1406 is required for interaction with p53 and that the trimerization domain of CHC is not required for p53 binding (Figure 1). This region corresponds to clathrin repeat 3–6, for which there has been no report regarding binding protein, although part of this region overlapped with CLC binding (Enari et al., 2006). These data are consistent with our previous observation (Enari et al., 2006) that both CLC and p53 bind mutually exclusively to clathrin repeat 6 in this region. Furthermore, the analysis of p53 transactivation using reporter assay indicates that CHC interactions with p53 are required for and correlate with enhanced p53 transactivation (Figure 2).
We recently noted a considerable similarity of the N-terminal transactivation domain of p53 around Ser46 to the CHC-binding region of CLC, and a critical Trp108 residue (bovine CLCa) for CHC binding conserved in p53 (Chen et al., 2002; Enari et al., 2006). Presumably, CLC competes with p53 through this homologous region, in particular through clathrin repeat 6. The tertiary structure of p53-CHC complex remains to be determined and it will give a clue to resolve the mechanisms by which CHC transactivates p53.
It is known that the trimerization domain of CHC is required for endocytic function, and the dysregulation of endocytosis may cause various global alterations including cell survival, cell growth and cell death signalings. To exclude the possibility that p53 transactivation by CHC833-1406 is caused by affecting clathrin-mediated endocytosis, we first performed a coimmunoprecipitation assay. As expected, this assay indicates that neither endogenous CHC nor CHC containing a C-terminal trimerization domain was coimmunoprecipitated with FLAG-tagged CHC833-1406 (Figure 3). Furthermore, an assay for the cellular uptake of transferrin supports that CHC833-1406 actually does not affect clathrin-mediated endocytosis (Figure 4). The results demonstrate that CHC833-1406 is present in cells as a monomeric form and has the ability to transactivate p53 in the absence of affecting clathrin-mediated endocytosis (Figure 4). Thus, these findings provide further evidence that CHC plays a distinct role from known CHC functions such as endocytosis and mitosis; however, is monomeric CHC present in cells? Gel filtration and glycerol gradient experiments from various reports suggest that some population of both endogenous and ectopically expressed CHC appears to exist as a monomer in cells, although the authors have not mentioned the polymeric status of CHC (Kim and Kim, 2000; Barth and Holstein, 2004; Royle and Lagnado, 2006). The importance of endogenous monomeric CHC exhibited by hydrodynamic methods remains unclear and further investigation will be required to make sure that monomeric CHC in cells plays an important role in regulating p53-mediated transcription.
Although it has so far been reported that numerous proteins bind to the N-terminal domain of CHC (Kirchhausen, 2000; Dell'Angelica, 2001; Lafer, 2002), there is no report regarding the association with the region identified here as a p53-binding domain corresponding to repeat 3–6. Some endocytic proteins, including β-arrestins, epsin1 and eps15, translocate from cytoplasms to nuclei in response to leptomycin B or physiological cognate ligands for G protein-coupled receptors and have been implicated to function as transcriptional modulators for certain target genes (Vecchi et al., 2001; Kang et al., 2005). As most endocytic regulators bind to CHC at the N-terminal domain that is not required for p53 transactivation, they may not regulate p53-mediated transcription through CHC binding; however, we cannot role out the possibility that certain endocytic regulators may modulate other transcriptional factors in collaboration with nuclear CHC.
CHC is a very abundant protein in cells and most CHC is present in cytoplasm. However, as we have previously reported in recent paper (Enari et al., 2006), approximately 5% of CHC is present in nuclei and required for p53-mediated transcription. Furthermore, immunoelectorn microscopic analysis revealed that CHC is actually present in intact cells, as described previously (Enari et al., 2006). To further show this physiological relevance, we quantified how many CHC and p53 molecules are present in nuclei. We purified recombinant CHC and p53 from cells ectopically tansfected with FLAG-tagged CHC or p53 and the concentrations of purified proteins were determined by CBB staining by comparing with known concentrations of bovine serum albumin (BSA). The amounts of CHC and p53 in the cytosolic and nuclear fractions were calculated using the purified CHC and p53. These experiments revealed that 4.74 × 105 molecules of CHC per nucleus is roughly present, on the other hand, 0.34 × 105 molecules of p53 per nucleus is present in DNA-damaged cells, as determined by NIH image Version 1.6.1 densitometry (unpublished data). Thus, nuclear CHC is more abundant than DNA damage-accumulated nuclear p53 even though the amounts of CHC existing in nuclei is only 5% of total CHC protein, supporting that CHC functions as a coactivator for p53.
Our previous observation that a small fraction of CHC is present in nuclei suggests that a certain part of CHC is required for retention to cytoplasm or the prevention of nuclear import. To explore the mechanism by which the nuclear transport of CHC is regulated, leptomycin B, a specific inhibitor of CRM1/Exportin1-dependent nuclear export, was used in this study. Unfortunately, leptomycin B did not have any effect on the localization of CHC, suggesting that the nuclear transport of CHC is mediated in a CRM1/Exportin1-independent manner (Figure 7). Surprisingly, however, the deletion of the C-terminal region (residues 1523–1675) on CHC leads to elevated nuclear accumulation, suggesting that CHC has the potential to enter the nucleus and that there might be strong nuclear export or cytoplasmic retention sequences in the C-terminal region containing the trimerization domain of CHC. So far, seven nuclear export factors including CRM1, CAS, Exp-t, Exp-4, -5, -6 and -7 have been identified (Kutay and Guttinger, 2005). Also, some transcription factors are retained in the cytoplasm by certain proteins associated with actin and microtubule cytoskeletons (Ziegler and Ghosh, 2005). In future, it will be necessary to elucidate the mechanism for the nuclear transport of CHC to identify which factor is responsible for the cytoplasmic retention of CHC.
In summary, we have defined the minimal p53-binding region of CHC and demonstrated that monomeric CHC still has the ability to transactivate p53. As monomeric CHC has little effect on clathrin-mediated endocytosis and predominantly accumulates in the nucleus to enhance p53-mediated transcription, it can possibly be utilized as an antitumor drug without any side effects.
Materials and methods
Cell culture and transfection
Human lung carcinoma H1299 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. Human cervical carcinoma HeLa cells and human osteosarcoma Saos-2 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37°C in a 5% CO2 atmosphere.
For transfection, cells were plated at 80–90% confluency the day before transfection and transfected with LipofectAmine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.
Plasmids and antibodies
FLAG-tagged p53, HA-tagged and untagged full-length CHC expression plasmids were described previously (Enari et al., 2006). cDNAs encoding each CHC fragment were amplified by PCR and cloned into pcDNA3.1 (Invitrogen) or pCAGGS (Niwa et al., 1991) with and without an HA epitope and confirmed by DNA sequencing. For the expression of shRNA against the 3′-untranslated region of CHC mRNA, synthetic oligoDNAs, 5′-IndexTermGATCCCCAGAGCACCATGATTCCAATTTCAAGAGATTGGAATCATGGTGCTCTTTTTTGGAAA-3′ and 5′-IndexTermAGCTTTTCCAAAAAGAGCACCATGATTCCAATTCTCTTGAAATTGGAATCATGGTGCTCTGGG-3′, were annealed and cloned into pSUPER vector (Brummelkamp et al., 2002). Anti-Mdm2 antibody (Ab1) was purchased from Calbiochem. Anti-p21 antibody (SX.118) was purchased from BD Pharmingen (Tokyo, Japan). Horseradish peroxidase-conjugated anti-p53 antibody (DO-1) was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-PARP and anti-HA (6E2) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-FLAG antibody (M2) was purchased from Sigma (St Louis, MO, USA). Antitransferrin receptor antibody was purchased from Zymed (San Francisco, CA, USA).
Immunoprecipitation and immunoblotting
H1299 cells (1.8 × 106) were transfected with FLAG-tagged and/or HA-tagged CHC plasmids and harvested at 21 h after transfection. Cells were lysed with lysis buffer (50 mM Tris–HCl at pH 7.2, 250 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM MgCl2, 0.1% Nonidet P-40, 0.1 mM DTT, 10 μg ml−1 antipain, 10 μg ml−1 pepstatin, 10 μg ml−1 chymostatin, 10 μg ml−1 leupeptin, 10 μg ml−1 E-64, 10 μg ml−1 aPMSF, 1 mM Na3VO4 and 10 mM NaF) for 20 min on ice and the lysate was clarified by centrifugation at 20 000 g for 20 min. For immunoprecipitation of Flag-tagged protein, the lysate was immunoprecipitated with 10 μl of M2-agarose beads (Sigma) for 2 h and washed three times with lysis buffer. Immunoprecipitates were eluted with 3 × Flag peptide (Sigma) at 4 °C for 30 min, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotted with the indicated antibodies. For immunoprecipitation of HA-tagged proteins, anti-HA antibody (12CA5) and protein G Sepharose (GE Healthcare, Tokyo, Japan) were used. Bound proteins were eluted by boiling at 100 °C for 5 min and subjected to SDS–PAGE, followed by immunoblotting. For immunoblotting, proteins in SDS polyacrylamide gels were transferred to a polyvinylidene fluoride (PDVF) membrane (Millipore, Bedford, MA, USA), the membranes were blocked with 5% nonfat skim milk in tris buffered saline with Tween (TBS-T) buffer (20 mM Tris–HCl at pH 7.6 and 137 mM NaCl and 0.1% Tween 20) for 1 h, sequentially probed with primary antibodies overnight at 4 °C, and a secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The antigens were visualized by ECL chemiluminescence (GE Healthcare).
Recombinant proteins and chemical cross-linking
For the expression of various recombinant CHC proteins, cDNAs encoding CHC fragments 1073–1675, 1073–1406 and 833–1406 were cloned into pGEX-6P vector and transformed into E. coli DH10B cells. These bacteria harboring each pGEX-CHC plasmid were grown in Luria Broth medium until OD600 reached 0.6–0.8. The expression of GST-CHC was induced by the addition of isopropyl-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1 mM and continuous culture at 30 °C for 3 h. Cells were spun and disrupted by sonication at 4 °C and the lysates were centrifuged at 20 000 g at 4 °C for 10 min. The resultant supernatants were mixed with glutathione-Sepharose 4B (GE Healthcare) for 1 h at 4 °C. The beads were washed four times with 25 mM Hepes-NaOH at pH 7.5, 150 mM NaCl, 0.1 mM DTT and recombinant CHC proteins were eluted with eight units of PreScission protease (GE Healthcare) at 4 °C overnight.
For chemical cross-linking, each purified CHC fragment was diluted in 25 mM Hepes-NaOH (pH 7.5) 150 mM NaCl to a concentration of 20 μg ml−1. To provide adequate buffering capacity, 1/5 volume of 1 M triethanolamine at pH 8.5 was added. A chemical cross-linker BS3 (Pierce Chemical Co., Rockford, IL, USA) was dissolved in 5 mM sodium citrate (pH 5.0) and used for this assay within 2 min. One volume of 10 mM BS3 was mixed with four volumes of protein samples and incubated at 30 °C for 10 min. The cross-linking reaction (10 μl) was terminated by the addition of 1/5 volume (2 μl) of 1 M Tris–HCl at pH 6.8. Samples were boiled for 2 min, separated by SDS–PAGE and visualized by silver staining.
To analyze the effect of CHC fragments on endocytic activity, HeLa cells (3 × 105) were transfected with 1 μg of pSUPER-CHC plus 2 μg of pCAGGS-CHC vectors in combination with 50 ng of pmaxGFP vector (Amaxa), a transfection marker. Three days after transfection, cells were incubated in DMEM containing 0.1% BSA for 3 h, followed by treatment with 20 μg ml−1 Alexafluor-594-conjugated transferrin (AF594-transferrin, Molecular Probes) for 8 min at 37 °C. Cells were rapidly chilled by extensive washing with ice-cold phosphate-buffered saline (PBS) and then AF594-transferrin bound on the cell surface was removed by washing with ice-cold acid-washing buffer containing 0.2 M acetic acid (pH 4.5) and 0.5 M NaCl. These cells were trypsinized, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and resuspended in 0.1% BSA in PBS. Relative fluorescence was quantified by flow cytometric analysis (Beckton Dickinson, Franklin, NJ, USA) for internalized AF594-transferrin.
To measure transferrin receptors present on the cell surface, cells were first detached using PBS containing 1 mM EDTA and incubated on ice with 20 μg ml−1 AF594-transferrin for 20 min. Cells were then washed once with ice-cold PBS, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and then resuspended in 0.1% BSA in PBS. Relative fluorescence was quantified by flow cytometric analysis for surface-bound AF594-transferrin.
RT–PCR assay was performed essentially as described in Enari et al. (2006). Briefly, total RNA was isolated using an RNeasy mini kit (QIAGEN, Valencia, CA, USA) and five micrograms of total RNA were reverse-transcribed with Superscript II first-strand synthesis kit (Invitrogen) using an oligo-dT primer. Reverse-transcribed products were used for semiquantitative PCR. Primer sequences and PCR programs were described in Enari et al. (2006). PCR products were resolved by 2% agarose gel electrophoresis and visualized by ethidium bromide.
Reporter assay was performed essentially as described previously (Enari et al., 2006). In brief, H1299 cells (4 × 104) or Saos-2 cells (3 × 104) plated on 24-well plates were transfected with 1–5 ng of pC53-SN3, 200 ng of pcDNA3.1-CHC, 200 ng of pCMVβ-p300 and 150 ng of the indicated reporter vectors in combination with 10 ng of phRG-TK encoding Renilla luciferase as an internal control. At 24 h after transfection, cells were harvested and luciferase activity was quantified by a dual luciferase assay system (Promega, Madison, WI, USA) according to the manufacture's instructions.
For caspase-3/7 assay, H1299 cells (2 × 105) were transfected with 50 ng of pC53-SN3 with or without 2 μg of pcDNA3.1-CHC. After 17 h incubation, cells were harvested and caspase-3/7 activity in the cell lysates was measured by CaspACE assay kit using fluorogenic substrates (Promega) according to the manufacture's instructions.
In vitro binding assay
To analyze the interaction between CHC and p53, GST-pull-down assay was carried out as described previously (Enari et al., 2006). In brief, bacterial lysates containing p53 fused to GST (GST-p53) were incubated with glutathione-Sepharose beads (GE Healthcare) and washed extensively with binding buffer (50 mM Tris–HCl at pH7.2, 250 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2 mM MgCl2, 0.1% Tween 20, 0.1 mM DTT, 10 μg ml−1 antipain, 10 μg ml−1 pepstatin, 10 μg ml−1 chymostatin, 10 μg ml−1 leupeptin, 10 μg ml−1 E-64, 10 μg ml−1 aPMSF, 1 mM Na3VO4, 10 mM NaF). 35S-labeled full-length CHC and its deletion derivatives were synthesized using an in vitro transcription/translation-coupled reticulocyte lysate system (Promega). Lysates containing 35S-labeled CHC proteins were diluted in 12 volumes of binding buffer and incubated with the above beads immobilized with GST-p53 at 4 °C for 2 h. After washing with 1 ml of binding buffer, bound proteins were eluted by boiling in SDS sample-loading buffer for 5 min, subjected to 10% SDS–PAGE and analysed by autoradiography.
H1299 cells (2 × 104) were plated in an 8-well Labtek II chamber (NalgeNunc, Tokyo, Japan). The next day, cells were transfected with 400 ng of pcDNA3.1-HA-CHC and incubated for 24 h. Cells were fixed with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 and blocked with 3% BSA in PBS. The chamber was sequentially incubated with anti-HA antibody for 1 h at room temperature and AlexaFluor488- or AlexaFluor594-labeled secondary antibody (Molecular Probes, Eugene, OR, USA), and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA). Confocal immunofluorescence was performed using an ECLIPSE E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a Radiance 2000 imaging system (Bio-Rad, Hercules, CA, USA). To quantify the subcellular localization of CHC, more than 100 cells per sample were counted.
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We thank Drs T Nagase and M Ohishi (Kazusa DNA Research Institute) for the full-length CHC clone (KIAA0034), Dr I Kitabayashi (National Cancer Center Research Institute) for FLAG-tagged and HA-tagged p300 constructs, Dr B Vogelstein (Johns Hopkins University) for the WWP-Luc reporter plasmid, Drs H Arakawa and Y Nakamura (The University of Tokyo) for the reporter vector containing p53AIP1 promoter, and Drs T Shibue and T Taniguchi for the reporter vector of Noxa promoter (The University of Tokyo), respectively. This work was supported by MEXT, KAKENHI (17013088) and a Grant-in-Aid from the Ministry of Health, Labor and Welfare for the 3rd Term Comprehensive 10-year Strategy for Cancer Control (to YT).
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Ohmori, K., Endo, Y., Yoshida, Y. et al. Monomeric but not trimeric clathrin heavy chain regulates p53-mediated transcription. Oncogene 27, 2215–2227 (2008) doi:10.1038/sj.onc.1210854
- clathrin heavy chain
- gene transcription
- tumor suppressor