| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Radiobiology Division, National Cancer Center Research Institute, Tokyo, Japan
2 SORST, Japan Science and Technology Agency (JST), 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan
3 Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan
4 Department of Neuroscience, Okayama University, Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
One of the most characterized receptors to study the mechanisms of clathrin-mediated endocytosis of receptors is a system of epidermal growth factor (EGF)-induced internalization of EGF receptor (EGFR). EGF triggers the recruitment of various adaptor proteins to EGFR through post-translational modifications such as phosphorylation and ubiquitination (Levkowitz et al. 1998; Citri & Yarden 2006). Activated EGFR is internalized and degraded by endocytic regulators including AP2, CHC, Grb2, dynamin-2, Cbl, Eps15, Epsins, STAM, Hrs, TSG101 and ESCRT to terminate signals from EGFR (Sebastian et al. 2006). It has been proposed that the route of internalized receptors is individually distinguished even though their internalization is mediated by clathrin. Ligand-induced internalized receptors including EGFR and LDLR travel to early endosome along with microtubules, whereas internalized transferrin receptor (TfnR) migrates slower in a microtubule-independent manner (Lakadamyali et al. 2006). Although many proteins including clathrin-coated vesicles have so far been identified by proteomic analyses (Blondeau et al. 2004; Borner et al. 2006), studies have been unsuccessful in identifying regulators contributing to the early events of EGFR internilization because of the difficulty of isolating clathrin-coated pits.
The tumor-suppressor protein p53, which is encoded by the most frequently mutated gene in human cancers, mainly functions in the nucleus as a transcription factor that activates the transcription of various target genes including DNA repair, cell cycle arrest, apoptosis and anti-angiogenesis (Levine 1997; Prives & Hall 1999; Vousden & Lu 2002; Bourdon et al. 2003; Oren 2003). However, several reports from other groups have demonstrated that p53 is present not only in the nucleus to promote the transcription of anti-tumor-responsive genes but also in cytosol, mitochondria and centrosomes to prevent tumorigenesis through other mechanisms (Moll & Zaika 2001; Tarapore & Fukasawa 2002). It has been shown that p53 directly binds to the mitochondrial outer membrane and induces mitochondria-mediated apoptosis (Dumont et al. 2003; Mihara et al. 2003; Chipuk et al. 2004). On the other hand, p53 is also targeted to centrosomes for the prevention of aberrant centrosome duplication during cell cycle progression (Tarapore & Fukasawa 2002; Shinmura et al. 2006). While there have been huge numbers of reports regarding p53 functions in the nucleus, the functions of cytosolic p53, in particular at the plasma membrane, remain to be analyzed.
In recent studies, we have shown that p53 interacts with clathrin heavy chain (CHC) in the nucleus, and nuclear CHC is required for p53-mediated transcription (Enari et al. 2006). As CHC was originally identified as a cytosolic protein that regulates clathrin-mediated endocytosis, it is assumed that p53 could bind to CHC in cytosol and might regulate clathrin-mediated endocytosis through the association with CHC.
In this paper, we show that p53 associates with CHC not only in nuclei but also in cytosol, and co-localizes with CHC at the plasma membrane in normal human fibroblast and human cancer cells. Surprisingly, p53 also co-localizes with EGF and CHC at the plasma membrane in response to EGF treatment. Moreover, co-immunopurification assay revealed that p53 is contained in the EGFR complex including endocytic regulators. In addition, the ablation of p53 expression by RNA interference (RNAi) causes the delayed uptake of EGF and transduces the aberrant signaling from EGFR, suggesting that p53 regulates the endocytosis of EGFR to transduce accurate signaling from its receptor.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
We have recently shown that CHC is also present in nuclei and interacts with p53 to promote p53-mediated transcription (Enari et al. 2006). Based on our knowledge that nuclear CHC binds to p53, we tested whether CHC could also interact with p53 in cytosol. For co-immunoprecipitation experiments, either wild-type p53 construct or empty vector was transfected into p53-null H1299 cells, and cytosolic and nuclear fractions from transfected cells were prepared by homogenization with a Dounce homogenizer. Both fractions were immunoprecipitated with control IgG or anti-CHC antibody, followed by immunoblotting with anti-p53 antibody. As shown in Fig. 1A, p53 was present in cytosolic fractions and co-immunoprecipitated with cytosolic CHC as well as nuclear CHC. We also examined whether a physical interaction could be detected using normal human fibroblast TIG-7 cells expressing endogenous wild-type p53 (Fig. 1B). Immunoprecipitation analysis showed that p53 was present in immunoprecipitates containing CHC. In addition, when cytosolic and nuclear extracts from p53-positive A549 and MCF-7 cells were immunoprecipitated with anti-CHC antibody, p53-CHC interaction was also detected as efficiently as seen in TIG-7 cells (data not shown), indicating that p53 physically associates with CHC not only in nuclei but also in cytoplasm.
|
We next investigated the subcellular localization of p53 in cells to address whether p53 co-localizes with CHC. For localization of p53, we used normal human fibroblast TIG-7 cells because TIG-7 cells had large and flat cytoplasm, and it is easy to distinguish p53 localization in cytoplasm. Confocal microscopic analysis revealed that a small amount of p53 localizes in cytosol under normal culture conditions though most p53 localizes in nuclei (Fig. 2). Interestingly, some p53 (in 10%–20% of total cells) co-localized with CHC and was present at the plasma membrane, as judged by overlays with a staining pattern of CHC (Fig. 2).
|
Clathrin-mediated endocytosis of EGFR is a well-established system to study endocytic mechanisms; therefore, we next examined whether the localization of p53 is changed by treatment with EGF. Cells were incubated in serum-free medium for 3 h, treated without or with EGF on ice, and indirect immunofluorescent analysis was performed using anti-p53 (FL393) and anti-CHC (TD.1) antibodies. In cells without EGF treatment, most p53 did not exhibit co-localization with CHC at the plasma membrane in serum-starved cells (unlike cells incubated under normal culture conditions), suggesting that the removal of serum containing various growth factors leads to the decreased localization of p53 with CHC at the plasma membrane (Fig. 3A). Surprisingly, when cells were stimulated with EGF on ice, p53 as well as CHC was recruited to the plasma membrane, suggesting that the recruitment of p53 to the plasma membrane occurs in an energy-independent manner (Fig. 3A). A temperature shift to 37 °C induced the internalization of EGFR, in which p53 and CHC were recruited and the rapid dissociation of p53 and CHC from coated vesicles was observed (Fig. 3A). The fluorescent signal from p53 in immunofluorescence was confirmed to be p53 itself by RNAi-directed ablation of p53 expression (Fig. 3B). Similar results were obtained by using different anti-p53 antibodies (DO-1 and pAB421) (Supplementary Fig. S1).
|
To assess the effect of down-regulation of CHC gene expression by RNAi on EGF-induced translocation of p53 to plasmalemmal sites, two small-interfering RNAs (siRNAs) for different sequences in chc mRNA were synthesized and used. Because complete depletion of CHC expression caused cell death and cell growth arrest, CHC expression was partially reduced by restricted amounts of siRNA. CHC down-regulation by RNAi caused the decreased EGF-induced p53 translocation (Supplementary Fig. S3), suggesting that CHC is required for the EGF-induced recruitment of p53 to plasma membrane.
Depletion of p53 by RNAi delays EGFR internalization
The co-localization of p53 with CHC at the site, where endocytosis occurs promoted us to investigate the role of p53 in clathrin-mediated endocytosis. To test the effect of p53 on clathrin-mediated endocytosis, we knocked down p53 expression using RNAi. For endocytic activity, the uptake of 125I-labeled EGF into TIG-7 cells was quantified. When cells were infected with lentiviruses encoding short-hairpin RNA (shRNA) against p53, the expression level of p53 was reduced more than 90% compared with control cells (Fig. 4A). These cells were starved in serum-free medium for 3 h, treated with 125I-labeled EGF on ice to saturate 125I-labeled EGF binding to EGFR on the cell surface, and uptake of 125I-labeled EGF was measured by a temperature shift to 37 °C at various time points. In p53-depleted cells, the internalization of 125I-labeled EGF was delayed compared with that of mock-infected control cells (Fig. 4B, upper panel). We also tested internalization assay using additional two different siRNAs for p53. The rate of EGF uptake was dramatically reduced by down-regulation of p53 expression using other siRNAs, as similar as that in stable p53-depleted cells (Supplementary Fig. S4). To further confirm this, we performed an internalization assay for EGFR using human lung carcinoma A549 cells expressing wild-type p53 under the same conditions as described above. The p53 expression was suppressed using a synthetic siRNA (Fig. 4C). In p53-depleted cells, uptake of 125I-labeled EGF was obviously decreased by half (Fig. 4D, upper panel).
|
Ablation of p53 alters EGFR signaling
It has recently been suggested that EGF-induced endocytosis of EGFR regulates the spatial and temporal coordination of accurate signaling hierarchy (Shilo 2005; Kholodenko 2006); therefore, we next asked if the downstream signaling pathways of EGFR are altered by the ablation of p53 expression. In response to EGF stimulation, EGFR is dimerized and autophosphorylated, and activated EGFR transduces signals to multiple pathways including the Ras–MAPK pathway, the PI3K–Akt pathway and the JNK–SAPK pathway (Logan et al. 1997; Weston et al. 2004). Therefore, we examined the effect of p53 on the signaling pathways for EGFR using phosphorylation-specific antibodies to determine the kinetics of the activation of signaling pathways. Immunoblot analysis revealed that the expression levels of EGFR and CHC proteins were not changed and EGFR was tyrosine-phosphorylated in response to EGF treatment irrespective of p53 expression (Fig. 5A); however, EGF-dependent activation of Akt and JNK was greatly up-regulated in p53-depleted cells (Fig. 5B). In contrast, no significant differences were observed in MAPK activity (Fig. 5B).
|
p53 interacts with EGFR complex in response to EGF treatment
To understand the mechanism by which p53 regulates EGFR internalization, we next analyzed the physical association of p53 with EGFR complex upon EGF treatment. Biotin-labeled EGF was used to stimulate EGFR on cell surfaces and to purify a complex containing biotin-labeled EGF/EGFR from cell lysates by streptavidin-conjugated magnetic beads. Cells were starved in serum-free medium for 3 h, treated without or with biotin-labeled EGF on ice for 1 h, and then incubated for indicated times. These cells treated with EGF were immediately placed on ice and homogenized in hypotonic buffer to prepare a fraction containing cytosol and plasma membrane for immunopurification of an EGF-signaling complex. Interestingly, when eluates were immunoblotted with the various antibodies indicated, not only endocytic regulators but also p53 were co-purified with EGFR complex in response to EGF stimulation (Fig. 6A), and p53 transiently associated with EGFR complex unlike endocytic regulators tested (Fig. 6A), suggesting that p53 may act as a catalyst regulating clathrin-mediated endocytosis. In parallel experiments, we checked if p53 still co-localized with CHC in immunofluorescence under same conditions. Even though cells were incubated for various time periods, p53 still co-localized with CHC at plasma membrane (Fig. 6B), being consistent with the data that p53 is included in EGFR complex in response to EGF stimulation. We next examine the effect of partial ablation of CHC gene expression by RNAi on EGF-induced p53–EGFR complex formation. CHC down-regulation by RNAi caused the reduced p53–EGFR complex formation (Fig. 6C), supporting our findings that CHC contributes to EGF-induced recruitment of p53 at plasma membrane.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Confocal immunofluorescent microscopic analysis shows that co-localization of p53 with EGF–EGFR is observed in cells treated with fluorescent-labeled EGF on ice; however, p53 is rarely detected in the EGF–EGFR complex under the same conditions (Fig. 6). This discrepancy may be caused because the interaction of p53 with EGFR complex is easily dissociated by biochemical fractionation. On the other hand, the association of p53 with EGFR complex was transiently detected at very early time points (Fig. 6A), at which p53 still co-localized with EGF–EGFR–CHC complex (Fig. 6B and data not shown), suggesting that the binding affinity between p53 and EGFR complex increases in an energy-dependent and unknown mechanisms. Presumably, incubation of EGF-treated cells at 18 °C leads to the stabilization of p53–EGFR complex association through energy-dependent modifications or conformational changes of EGFR, p53 or its associated proteins at the plasma membrane. Thus, our findings suggest that p53 regulates the internalization of EGFR by binding to it or associated proteins.
Activated RTKs including EGFR, insulin receptor and TrkA are rapidly internalized and eventually delivered to the lysosome to down-regulate signaling from cell-surface receptors (Di Fiore & Gill 1999; Wiley & Burke 2001). It has been shown that p53 associates with TrkA (high-affinity receptor for nerve growth factor) in vitro and in vivo (Montano 1997), implying that p53 is involved in its signal transduction pathways, possibly through association with TrkA. Our findings, that signal transduction from EGFR was aberrant when p53 expression was suppressed (Fig. 5B) and that p53 was co-purified with EGF–EGFR complex (Fig. 6), suggest that p53 functions as a spatial and temporal coordinator of correct signaling from EGFR at the plasma membrane. The magnitude and kinetics of signal activation in RTK pathways are important for homeostasis. Once this system is dysregulated, it presumably causes various diseases including cancer (Le Roy & Wrana 2005). In the case of EGFR, prevention of EGFR internalization by the suppression of factors responsible for EGFR internalization facilitates cell transformation (Levkowitz et al. 1998). Our observations provide the possibility that p53 also coordinates signals from growth factor receptor through the regulation of clathrin-mediated endocytosis to prevent cell transformation. Thus, our findings provide a new insight on p53 as a tumor suppression mechanism and for the possibility that cytosolic p53 participates in the regulation of clathrin-mediated endocytosis in a transcription-independent manner although we cannot exclude the possibility that p53 indirectly regulates these events via the induction of p53-target genes. To address the effect of p53 on the recruitment of endocytic proteins to EGFR complex, we examined immunopurification experiments using p53-depleted cells; however, as far as we assayed whether various endocytic proteins including CHC, AP-2 and Grb2 are contained in the EGFR complex in p53-depleted cells, no alteration of complex formation of these proteins was observed (data not shown). Presumably, p53 depletion by RNAi causes the altered association or expression of other endocytic proteins. Further analysis will be required to understand the direct contribution of cytosolic p53 to clathrin-mediated endocytosis.
Why are p53-deficient mice viable without any developmental defect (Donehower et al. 1992) although p53 regulates clathrin-mediated EGFR internalization? One possible explanation is that the regulation of EGFR internalization by p53 may be restricted to specific areas in specific tissues. Even though hierarchical signaling from EGFR is aberrant in restricted cells, p53-deficient mice would not exhibit lethality. Indeed, various mice deficient in endocytic regulators such as Hip1, c-Cbl and Parkin responsible for EGFR internalization are viable without obvious abnormalities (Murphy et al. 1998; Naramura et al. 1998; Rao et al. 2001; Goldberg et al. 2003). Thus, the dysregulation of EGFR internalization may not cause developmental lethality although the function is required for homeostasis in adults to prevent diseases. Alternatively, we also cannot rule out the possibility that the mechanism by which p53 regulates clathrin-mediated EGFR internalization is specific to the human system. Further investigation will be necessary to elucidate the physiological roles of cytosolic p53 in clathrin-mediated endocytosis.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Human lung carcinoma H1299 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). H1299 cells transfected with FLAG-tagged p53 construct were prepared as described previously (Enari et al. 2006). Normal human fibroblast TIG-7 cells and human lung carcinoma A549 cells were grown in DMEM containing10% FBS. Anti-p53 (DO-1) and anti-CHC (X.22) antibodies were purchased from Oncogene Science, Cambridge, MA. Anti-CHC (clone 23), anti-EGFR (clone 13) and anti-AP-2 (clone 8) antibodies were purchased from BD Biosciences, Rockville, MD. Anti-Lamin A/C (N-18), anti-β-Tubulin (H-235), anti-Grb2 (C-23), anti-JNK (FL), anti-p53 (FL-393) and horseradish peroxidase (HRP)-conjugated anti-p53 (DO-1) antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-phospho-MAPK (Thr202/Tyr204), anti-MAPK, anti-phospho-Akt (Thr308), anti-Akt and anti-phospho-JNK (Thr183/Tyr185) antibodies were purchased from Cell Signaling Technology, Danvers, MA. Anti-PTEN (6H2.1) antibody was from Cascade BioScience, Winchester, MA. Anti-phospho-tyrosine (4G10) antibody was purchased from Upstate Biotechnology, Lake Placid, NY. Anti-FLAG (M2) antibody was purchased from Sigma, St Louis, MO. HRP-conjugated NeutrAvidin was obtained from Pierce, Rockford, IL and HRP-conjugated secondary antibodies were from Amersham Biosciences, Uppsala, Sweden.
Fractionation
Cytosolic and nuclear fractions were extracted, as described previously, with some modifications (Dignam et al. 1983). Briefly, cells were collected by centrifugation at 600 g for 5 min, washed with ice-cold PBS and cell pellets were suspended in hypotonic buffer (20 mM HEPES–KOH, pH 7.9, 10 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM Na3VO4, 10 mM NaF, 10 µg/mL antipain, 10 µg/mL pepstatin, 10 µg/mL chymostatin, 10 µg/mL leupeptin, 10 µg/mL E-64, 10 µg/mL aPMSF) and incubated for 10 min on ice. The cells were homogenized with 15 strokes of a Dounce homogenizer. The supernatants were collected by centrifugation at 600 g for 5 min and used as cytosolic fractions. To prepare nuclear extracts, the pellets were washed twice with hypotonic buffer and resuspended in hypertonic buffer (20 mM HEPES–KOH, pH 7.9, 420 mM NaCl, 0.2 mM EDTA, 10% glycerol, 0.5 mM DTT, 1 mM Na3VO4, 10 mM NaF, 10 µg/mL antipain, 10 µg/mL pepstatin, 10 µg/mL chymostatin, 10 µg/mL leupeptin, 10 µg/mL E-64, 10 µg/mL aPMSF) and incubated for 30 min on ice. After centrifugation at 20 000 g for 20 min, the supernatants were collected as nuclear fractions.
Immunoprecipitation and immunoblotting
Cytosolic and nuclear extracts were diluted with lysis buffer (50 mM Tris–HCl, pH 7.2, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM DTT, 1 mM Na3VO4, 10 mM NaF, 10 µg/mL antipain, 10 µg/mL pepstatin, 10 µg/mL chymostatin, 10 µg/mL leupeptin, 10 µg/mL E-64, 10 µg/mL aPMSF) and supplemented to final concentrations of 100 mM NaCl and 0.1% Tween-20, respectively. Both fractions were immunoprecipitated with anti-CHC (X.22) or anti-FLAG (M2) antibody-coupled protein A-Sepharose beads (Amersham Biosciences). Immunoprecipitates were then washed 3 times with lysis buffer containing 50 mM NaCl and 0.1% Tween-20, and eluted with SDS sample buffer. Immunoprecipitates were resolved by 5%–20% gradient SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-craft, North Wales, PA) and transferred to PVDF membranes (Millipore, Bedford, MA), which were sequentially incubated with primary and secondary antibodies, and the immune-complex was detected using ECL Western blotting detection reagents (Amersham Biosciences).
EGF or transferrin treatment
Cells were starved in serum-free medium (DMEM containing 0.1% bovine serum albumin (BSA)) for 3 h at 37 °C in a CO2 incubator, washed extensively with pre-chilled serum-free medium, placed on ice for 10 min and then changed to pre-chilled serum-free medium containing EGF or transferrin (various concentrations according to the purpose described below), followed by further incubation on ice for 1 h. For immunofluorescence and immuno-electron microscopy, 2 µg/mL AlexaFluor488-labeled EGF (AF488-EGF, Molecular Probes, Eugene, OR) and 100 ng/mL human EGF (Roche Applied Science, Basel, Switzerland) were used, respectively. For internalization assay, 1 ng/mL 125I-labeled human EGF (Amersham Biosciences) or 500 ng/mL 125I-labeled transferrin (Perkin Elmer, Boston, MA) were used. For immunopurification of EGFR complex, 100 ng/mL biotin-labeled EGF (Molecular Probes) was used. To examine EGFR downstream signaling pathways, cells were starved in serum-free medium overnight, treated with 100 ng/mL human EGF, washed twice with pre-chilled serum-free medium to remove unbound EGF and then kept on ice or warmed to 18 °C or 37 °C for various time periods.
Immunofluorescence
Cells plated on a 4-well chamber slide (NalgeNunc, Rochester, NY) were starved in serum-free medium and treated with EGF or AF488-EGF as described above. EGF-treated cells were placed on ice to stop the reaction of EGF-induced EGFR-mediated endocytosis. The cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 for 5 min on ice, washed with PBS and blocked with 3% BSA for 30 min, followed by sequential incubation with an antibody against p53 (FL393), CHC (X.22) or Grb2 (C-23) for 1 h at room temperature and with AF488- or AF594-conjugated secondary antibody (Molecular Probes). Confocal imaging was performed using an ECLIPSE E600 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a Radiance 2000 imaging system (Bio-Rad, Hercules, CA).
Internalization assays
Ligand uptake assays were performed as described previously, with several modifications (Hinrichsen et al. 2003; Motley et al. 2003). In brief, cells grown on 12-well plates were starved in serum-free medium and treated with 125I-labeled human EGF or 125I-labeled transferrin on ice for 1 h, washed twice with pre-chilled serum-free medium and then further incubated with pre-warmed serum-free medium at 37 °C for various periods. The cell surface-bound 125I-labeled EGF and transferrin were collected by incubation twice with pre-chilled acid buffer (0.2 M acetic acid at pH 4.5, 0.5 M NaCl) on ice for 2–5 min. On the other hand, internalized 125I-labeled EGF and transferrin were collected by extraction twice with extraction buffer (0.1 M Tris–HCl at pH 8.0, 1% SDS). The radioactivity in each sample was quantified using a gamma counter (Cobra, Packard, Meriden, CT).
Immunopurification of EGFR complex
Cells were starved in serum-free medium and treated with biotin-labeled EGF as described above. Biotin-labeled EGF-treated cells were lysed with hypotonic buffer containing 0.1% Triton X-100 on ice for 10 min to fractionate cytosolic and nuclear extracts. Biotin-labeled EGF–EGFR complex was immunopurified using Dynabeads M-280 Streptavidin (Dynal, Oslo, Norway) without a centrifugation step (to exclude the loss of fractions containing heavy membrane by centrifugation even at a low speed). Immunopurified complex was washed 3 times with hypotonic buffer and eluted with SDS sample buffer at room temperature.
RNAi knockdown
Small-interfering RNAs (siRNAs) directed to human p53 (For siRNA sequences, see Supplementary information) and its negative control were purchased from Invitrogen, Carlsbad, CA and transfected in A549 cells with Lipofectamine 2000 according to the manufacturer's protocol. The siRNAs directed to human CHC were purchased from Qiagen, Venlo, the Netherlands for sequences #1 (5'-GUAAUCCAAUUCGAAGACCTT-3') and Sigma for sequences #2 (5'-AGAGCACCAUGAUUC CAAUTT-3') and transfected in TIG-7 cells as described above. For the generation of p53-knockdown TIG-7 cells, lentiviruses derived from pLenti6/V5-DEST lentiviral expression vector (Invitrogen), engineered so that shRNA targeted to p53 is expressed in cells, were infected in TIG-7 cells, blasticidinS-resistant cells were selected and used for assays.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
* Correspondence: Emails: ytaya{at}gan2.res.ncc.go.jp; menari{at}gan2.res.ncc.go.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Blondeau, F., Ritter, B., Allaire, P.D., Wasiak, S., Girard, M., Hussain, N.K., Angers, A., Legendre-Guillemin, V., Roy, L., Boismenu, D., Kearney, R.E., Bell, A.W., Bergeron, J.J. & McPherson, P.S. (2004) Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling. Proc. Natl. Acad. Sci. USA 101, 3833–3838.
Borner, G.H., Harbour, M., Hester, S., Lilley, K.S. & Robinson, M.S. (2006) Comparative proteomics of clathrin-coated vesicles. J. Cell Biol. 175, 571–578.
Bourdon, J.C., Laurenzi, V.D., Melino, G. & Lane, D. (2003) p53: 25 years of research and more questions to answer. Cell Death Differ. 10, 397–399.[CrossRef][Medline]
Chipuk, J.E., Kuwana, T., Bouchier-Hayes, L., Droin, N.M., Newmeyer, D.D., Schuler, M. & Green, D.R. (2004) Direct activation of Bax by p53 mediates mitochondrial membrane permeabilization and apoptosis. Science 303, 1010–1014.
Citri, A. & Yarden, Y. (2006) EGF-ERBB signalling: towards the systems level. Nat. Rev. Mol. Cell Biol. 7, 505–516.[CrossRef][Medline]
Di Fiore, P.P. & Gill, G.N. (1999) Endocytosis and mitogenic signaling. Curr. Opin. Cell Biol. 11, 483–488.[CrossRef][Medline]
Dignam, J.D., Lebovitz, R.M. & Roeder, R.G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.
Donehower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J., Montgomery, C.A.J., Butel, J.S. & Bradley, A. (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356, 215–221.[CrossRef][Medline]
Dumont, P., Leu, J.I., Della Pietra, A.C. 3rd, George, D.L. & Murphy, M. (2003) The codon 72 polymorphic variants of p53 have markedly different apoptotic potential. Nat. Genet. 33, 357–365.[CrossRef][Medline]
Enari, M., Ohmori, K., Kitabayashi, I. & Taya, Y. (2006) Requirement of clathrin heavy chain for p53-mediated transcription. Genes Dev. 20, 1087–1099.
Floyd, S. & De Camilli, P. (1998) Endocytosis proteins and cancer: a potential link? Trends Cell Biol. 8, 299–301.[CrossRef][Medline]
Goldberg, M.S., Fleming, S.M., Palacino, J.J., et al. (2003) Parkin-deficient mice exhibit nigrostriatal deficits but not loss of dopaminergic neurons. J. Biol. Chem. 278, 43628–43635.
Hinrichsen, L., Harborth, J., Andrees, L., Weber, K. & Ungewickell, E.J. (2003) Effect of clathrin heavy chain-and 
-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells. J. Biol. Chem. 278, 45160–45170.
Jiang, X., Huang, F., Marusyk, A. & Sorkin, A. (2003) Grb2 regulates internalization of EGF receptors through clathrin-coated pits. Mol. Biol. Cell 14, 858–870.
Kholodenko, B.N. (2006) Cell-signalling dynamics in time and space. Nat. Rev. Mol. Cell Biol. 7, 165–176.[CrossRef][Medline]
Lakadamyali, M., Rust, M.J. & Zhuang, X. (2006) Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell 124, 997–1009.[CrossRef][Medline]
Le Roy, C. & Wrana, J.L. (2005) Clathrin- and non-clathrin-mediated endocytic regulation of cell signalling. Nat. Rev. Mol. Cell Biol. 6, 112–126.[CrossRef][Medline]
Levine, A.J. (1997) p53, the cellular gatekeeper for growth and division. Cell 88, 323–331.[CrossRef][Medline]
Levkowitz, G., Waterman, H., Zamir, E., Kam, Z., Oved, S., Langdon, W.Y., Beguinot, L., Geiger, B. & Yarden, Y. (1998) c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor. Genes Dev. 12, 3663–3674.
Logan, S.K., Falasca, M., Hu, P. & Schlessinger, J. (1997) Phosphatidylinositol 3-kinase mediates epidermal growth factor-induced activation of the c-Jun N-terminal kinase signaling pathway. Mol. Cell. Biol. 17, 5784–5790.
Mihara, M., Erster, S., Zaika, A., et al. (2003) p53 has a direct apoptogenic role at the mitochondria. Mol. Cell 11, 577–590.[CrossRef][Medline]
Moll, U.M. & Zaika, A. (2001) Nuclear and mitochondrial apoptotic pathways of p53. FEBS Lett. 493, 65–69.[CrossRef][Medline]
Montano, X. (1997) p53 associates with trk tyrosine kinase. Oncogene 15, 245–256.[CrossRef][Medline]
Motley, A., Bright, N.A., Seaman, M.N. & Robinson, M.S. (2003) Clathrin-mediated endocytosis in AP-2-depleted cells. J. Cell Biol. 162, 909–918.
Mukherjee, S., Ghosh, R.N. & Maxfield, F.R. (1997) Endocytosis. Physiol. Rev. 77, 759–803.
Murphy, M.A., Schnall, R.G., Venter, D.J., Barnett, L., Bertoncello, I., Thien, C.B., Langdon, W.Y. & Bowtell, D.D. (1998) Tissue hyperplasia and enhanced T-cell signalling via ZAP-70 in c-Cbl-deficient mice. Mol. Cell. Biol. 18, 4872–4882.
Naramura, M., Kole, H.K., Hu, R.J. & Gu, H. (1998) Altered thymic positive selection and intracellular signals in Cbl-deficient mice. Proc. Natl. Acad. Sci. USA 95, 15547–15552.
Oren, M. (2003) Decision making by p53: life, death and cancer. Cell Death Differ. 10, 431–442.[CrossRef][Medline]
Polo, S. & Di Fiore, P.P. (2006) Endocytosis conducts the cell signaling orchestra. Cell 124, 897–900.[CrossRef][Medline]
Prives, C. & Hall, P.A. (1999) The p53 pathway. J. Pathol. 187, 112–126.[CrossRef][Medline]
Rao, D.S., Chang, J.C., Kumar, P.D., Mizukami, I., Smithson, G.M., Bradley, S.V., Parlow, A.F. & Ross, T.S. (2001) Huntingtin interacting protein 1 is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors. Mol. Cell. Biol. 21, 7796–7806.
Sebastian, S., Settleman, J., Reshkin, S.J., Azzariti, A., Bellizzi, A. & Paradiso, A. (2006) The complexity of targeting EGFR signalling in cancer: from expression to turnover. Biochim. Biophys. Acta 1766, 120–139.[Medline]
Shilo, B.Z. (2005) Regulating the dynamics of EGF receptor signaling in space and time. Development 132, 4017–4027.
Shinmura, K., Bennett, R.A., Tarapore, P. & Fukasawa, K. (2006) Direct evidence for the role of centrosomally localized p53 in the regulation of centrosome duplication. Oncogene 26, 1–6.[CrossRef][Medline]
Sorkin, A. & Carpenter, G. (1993) Interaction of activated EGF receptors with coated pit adaptins. Science 261, 612–615.
Sorkin, A. & Von Zastrow, M. (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat. Rev. Mol. Cell Biol. 3, 600–614.[CrossRef][Medline]
Stahl, P. & Schwartz, A.L. (1986) Receptor-mediated endocytosis. J. Clin. Invest. 77, 657–662.[CrossRef][Medline]
Stambolic, V., MacPherson, D., Sas, D., et al. (2001) Regulation of PTEN transcription by p53. Mol. Cell 8, 317–325.[CrossRef][Medline]
Tarapore, P. & Fukasawa, K. (2002) Loss of p53 and centrosome hyperamplification. Oncogene 21, 6234–6240.[CrossRef][Medline]
Vieira, A.V., Lamaze, C. & Schmid, S.L. (1996) Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274, 2086–2089.
Vousden, K.H. & Lu, X. (2002) Live or let die: the cell's response to p53. Nat. Rev. Cancer 2, 594–604.[CrossRef][Medline]
Weston, C.R., Wong, A., Hall, J.P., Goad, M.E., Flavell, R.A. & Davis, R.J. (2004) The c-Jun NH2-terminal kinase is essential for epidermal growth factor expression during epidermal morphogenesis. Proc. Natl. Acad. Sci. USA 101, 14114–14119.
Wiley, H.S. & Burke, P.M. (2001) Regulation of receptor tyrosine kinase signaling by endocytic trafficking. Traffic 2, 12–18.[Medline]
Received: 11 October 2007
Accepted: 8 January 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
