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• W2043456888 abstract "Article31 August 2006free access The tumor suppressor protein p53 is required for neurite outgrowth and axon regeneration Simone Di Giovanni Corresponding Author Simone Di Giovanni Laboratory for NeuroRegeneration and Repair, Hertie Institute for Clinical Brain Research, University of Tuebingen, Germany Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Chad D Knights Chad D Knights Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Mahadev Rao Mahadev Rao Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Alexander Yakovlev Alexander Yakovlev Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Jeannette Beers Jeannette Beers Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Jason Catania Jason Catania Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Maria Laura Avantaggiati Corresponding Author Maria Laura Avantaggiati Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Alan I Faden Alan I Faden Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Simone Di Giovanni Corresponding Author Simone Di Giovanni Laboratory for NeuroRegeneration and Repair, Hertie Institute for Clinical Brain Research, University of Tuebingen, Germany Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Chad D Knights Chad D Knights Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Mahadev Rao Mahadev Rao Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Alexander Yakovlev Alexander Yakovlev Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Jeannette Beers Jeannette Beers Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Jason Catania Jason Catania Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Maria Laura Avantaggiati Corresponding Author Maria Laura Avantaggiati Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Alan I Faden Alan I Faden Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA Search for more papers by this author Author Information Simone Di Giovanni 1,2, Chad D Knights3, Mahadev Rao3, Alexander Yakovlev2, Jeannette Beers2, Jason Catania3, Maria Laura Avantaggiati 3 and Alan I Faden2 1Laboratory for NeuroRegeneration and Repair, Hertie Institute for Clinical Brain Research, University of Tuebingen, Germany 2Department of Neuroscience, Georgetown University Medical Center, Washington DC, USA 3Lombardi Cancer Center, Georgetown University Medical Center, Washington DC, USA ‡These authors contributed equally to this work *Corresponding authors: Laboratory for NeuroRegeneration and Repair, Hertie Institute for Clinical Brain Research, University of Tuebingen, Otfried-Mueller Strasse 27, D-72076 Tuebingen, Germany. Tel.: +49 0 7071 29 80449; Fax: +49 0 7071 29 4521; E-mail: [email protected] Cancer Center, Georgetown University, 3970 Reservoir Road, Washington DC, 20057, USA. Tel.: +1 202 687 9199; Fax: +1 202 687 6402; E-mail: [email protected] The EMBO Journal (2006)25:4084-4096https://doi.org/10.1038/sj.emboj.7601292 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Axon regeneration is substantially regulated by gene expression and cytoskeleton remodeling. Here we show that the tumor suppressor protein p53 is required for neurite outgrowth in cultured cells including primary neurons as well as for axonal regeneration in mice. These effects are mediated by two newly identified p53 transcriptional targets, the actin-binding protein Coronin 1b and the GTPase Rab13, both of which associate with the cytoskeleton and regulate neurite outgrowth. We also demonstrate that acetylation of lysine 320 (K320) of p53 is specifically involved in the promotion of neurite outgrowth and in the regulation of the expression of Coronin 1b and Rab13. Thus, in addition to its recognized role in neuronal apoptosis, surprisingly, p53 is required for neurite outgrowth and axonal regeneration, likely through a different post-translational pathway. These observations may suggest a novel therapeutic target for promoting regenerative responses following peripheral or central nervous system injuries. Introduction Axonal injuries induce delayed biochemical alterations that may result in either cell death or survival/restoration, including successful target re-innervation (Yakovlev and Faden, 1995; Kalb and Strittmatter, 2000; Dumont et al, 2001; Makwana and Raivich, 2005). Functional recovery reflects, in part, the number of surviving cells and fiber tracts, the extent of neural plasticity, and the presence of a permissive environment for regeneration. Such processes are orchestrated by time-regulated gene expression changes, with an earlier phase associated with inflammation, extension of axonal damage and cell death, and a later one linked to axon outgrowth and regeneration (Kubo et al, 2002; Bareyre and Schwab, 2003; Di Giovanni et al, 2003, 2005a; Glanzer et al, 2004). Some of the genes related to neurite and axon outgrowth and regeneration play a dual role in both cell death and in cell survival following central nervous system (CNS) or peripheral nerve injury (Benowitz and Routtenberg, 1997; Hughes et al, 1999; Emery et al, 2003). One example of this duality of effects is provided by the transcription factor c-jun, which has been implicated in cell death and nerve degeneration in various models of CNS and peripheral nerve injury, but more recently has also been shown to be required for axonal regeneration following nerve transection (Raivich et al, 2004). Another transcription factor with pleiotropic functions is the tumor suppressor protein p53, which serves as a key determinant of cell fate following exposure to a variety of insults (Schuler and Green, 2005; Vousden and Prives, 2005). p53 functions as a DNA-binding, sequence-specific transcription factor that activates the expression of multiple genes, causing either reversible cell-cycle arrest or apoptosis. The proapoptotic activity of p53 often occurs when cells are exposed to severe genotoxic stress, whereas its effects on cell-cycle arrest may occur after moderate DNA damage, or after nutrient depletion (Bates et al, 1999; Jones et al, 2005). The transcriptional activity of p53 is increased in neuronal precursors of the developing mouse brain, whereas it is reduced in cells undergoing terminal differentiation (Rogel et al, 1985; Schmid et al, 1991). However, high levels of p53 mRNA have also been detected in the developing brain in areas showing little or no apoptosis (Gottlieb et al, 1997; Komarova et al, 1997), and a percentage of p53-deficient mice exhibit neuronal abnormalities, particularly defects in neural tube closure (Armstrong et al, 1995). Studies in neuronal-like cells have also suggested that p53 plays a role in cell survival following NGF administration in PC-12 cells, and that the interaction of p53 with neuronal-specific transcription factors, such as Brn-3a, may determine a shift from cell death to neuronal survival (Hudson et al, 2004, 2005). Thus, because of its dual actions on cell cycle and cell death, p53 plays a versatile role in the regulation of cellular growth and differentiation. We have recently employed temporal gene expression profiling after experimental spinal cord injury (SCI) in rats (Di Giovanni et al, 2005b), through which we identified a cluster of temporally correlated genes, including factors known to promote neurite outgrowth and axonal regeneration. This cluster includes the actin-binding protein Coronin 1b, and the GTPase Rab13, which we showed to be required for physiological neurite outgrowth in PC-12 cells and dorsal root ganglion neurons (Di Giovanni et al, 2005b). The temporally coordinated activation of these genes suggested that their expression might be regulated by a common transcription factor. In this study, we have explored this possibility, and we demonstrate that p53 regulates the expression of both Coronin 1b and Rab13. Moreover, p53 is required for neurite outgrowth and maturation in cultured neurons, as well as for axonal regeneration following facial nerve transection. These activities are mediated by acetylation of p53 at position K320, which we have recently shown to be involved in p53-mediated resumption of proliferation and cell survival. Results In silico promoter analysis and experimental evidence identify p53 as a transcription factor for Coronin 1b and Rab13 We recently demonstrated that the actin-binding Coronin 1b and the small GTPase Rab13 are temporally coregulated following experimental SCI during the phase of nerve regeneration and recovery (Di Giovanni et al, 2005b). Importantly, these genes are also coexpressed with the proneurite outgrowth and proaxonal regeneration protein Gap-43 (growth-associated protein-43) in axons after injury, and are required for the promotion of neurite outgrowth in PC-12 cells as well as in primary neurons (Di Giovanni et al, 2005b). These findings led us to hypothesize that their expression may be regulated by common transcription factors. In order to verify this hypothesis, we performed an ‘in silico’ computer-based analysis of the predicted promoters of Rab13 and Coronin 1b genes. We found that rat Coronin 1b and Rab13 have multiple common p53 transcription binding sites (TBS). Significantly, such p53-binding motifs were conserved in both the mouse and human genes, thus implying that they are physiologically relevant (Figure 1A). Figure 1.p53 binds to the TBS of Coronin 1b and Rab13, and regulates their expression. (A) Shown is a schematic illustrating the TBS for p53 on Coronin 1b and Rab13. DNA sequences and location from the transcription starting site are also reported. MS stands for matrix similarity, and a score between 0.7 and 1 is considered highly significant. p53 matching P-value for the described TBS is highly significant. For details about interpretation of these data see: http://www.genomatix.de. (B) ChIP assays demonstrate in vivo interaction of WTp53 with DNA-binding elements derived from the human Coronin 1b and Rab13 promoters. H1299-WTp53 cells were left untreated (−) or treated for 48 h (48 h) with tetracycline to induce the expression of native p53. Following induction of p53, cells were crosslinked with formaldehyde and immunoprecipitated with a polyclonal antibody recognizing p53. The ‘input’ samples represent equal fractions of extract collected prior to precipitation from which DNA was extracted and used as a control. WAF1 (p21) promoter region was used as a positive control for p53 DNA binding. (C) Data from real-time RT–PCR experiments show increased mRNA expression levels for Coronin 1b and Rab13 in H1299 cells with tetracycline p53-inducible system 8 h after p53 activation. Similar results were obtained in PC-12 cells (data not shown). (*t-test P-values <0.01; **P-values <0.001). (D) Protein expression data from immunoblotting experiments at the 24 h time point in H1299 cells that have a p53 tetracycline-inducible expression system (p53 tet-ind) and in PC-12 cells (p53 WT, siRNA, DN) (similar data were obtained in 293 cells, not shown). Induction (p53 tet-ind, and p53 WT) or repression (p53DN and p53 siRNA) of p53 activity, respectively, triggers or suppresses expression of Coronin 1b and Rab13. Download figure Download PowerPoint To assess whether p53 can bind to these promoter regions in vivo and within a chromatin environment, chromatin immunoprecipitation (ChIP) assays were performed. For these experiments, we took advantage of a p53 null cell line (H1299), where the expression of p53 is controlled by a tetracycline-inducible system. As shown (Figure 1B), p53 is bound to the ‘in silico’ predicted TBS of endogenous Coronin 1b and Rab13 in vivo. Moreover, in human embryonic kidney cells (293), in rat pheocromocytoma PC-12 cells, and in the p53-inducible H1299 cells, both messenger RNAs and protein levels of Coronin 1b and Rab13 were coordinately upregulated following p53 expression (Figures 1C and D). Indeed, neither Coronin 1b nor Rab13 were expressed in H1299 cells in the absence of tetracycline, and overexpression of p53 in 293 or PC-12 cell lines resulted in a marked induction of their levels (Figure 1D). Conversely, either a p53-specific RNAi or a p53 dominant-negative construct (p53DN), previously shown to inhibit p53-dependent transcription (Yakovlev et al, 2004), nearly completely abrogated the expression of these two proteins in response to NGF treatment. Thus, the expression levels of Coronin 1b and Rab13 are regulated by p53. p53 and its target genes Coronin 1b and Rab13 are expressed sequentially during neurite outgrowth The above results lead to the prediction that a correlation should exist between p53 expression and the levels of Coronin 1b or Rab13 during neurite outgrowth and neuronal maturation. To test this possibility, we used PC-12 cells treated with NGF or rat embryonic primary cortical neurons (RCN) exposed to neuronal medium (B27). NGF mediates neurite outgrowth and cytoskeleton remodeling in PC-12 cells, whereas cultured RCN undergo maturation, neurite elongation, sprouting and expression of postmitotic neuronal-specific proteins in the presence of B27. In PC-12 cells exposed to NGF, we observed an early induction of nuclear p53, which was paralleled by elevations in the expression levels of Coronin 1b and Rab13, as well as of Gap-43 (Figure 2A and B). The latter is a cytoskeleton-bound protein found in growth cones and commonly used as a marker of neurite outgrowth, maturation and regeneration (Figure 2A). Figure 2.p53, Coronin 1b and Rab13 are coregulated during neurite outgrowth. (A) Protein expression temporal profiling in PC-12 cells after NGF. Western blot shows early induction (4–24 h) of the expression of p53, Rab13, Coronin 1b and Gap-43 after NGF. Bar graphs show densitometry data (the expression level for each protein and time point represents the intensity of each band of the immunoblot as a ratio value to β-actin levels). (B) Immunocytochemistry shows increased nuclear expression of p53 in PC-12 cells 24 h after NGF. (C) Protein temporal profiling for MDM2 by immunoblotting shows marked suppression by 4 h following NGF in PC-12 cells. (D) Immunoblotting shows protein expression temporal profiling in E16 RCN during maturation in Neurobasal medium. p53 expression is highest at day 2 corresponding to the highest increase in expression for Coronin 1b, Rab13, and Gap-43. Bar graphs show densitometry data (the expression level for each protein and time point represents the intensity of each band of the immunoblot as a ratio value to β-actin levels). (E, F) Immunocytochemistry shows nuclear expression of p53 (E) and at the cortical cytoskeleton (colocalize with tubulin) for Coronin 1b and Rab13 (F) in RCN. Download figure Download PowerPoint The levels of p53 are regulated predominantly at a post-translational level, via a regulatory feedback with the ubiquitin ligase MDM2. Thus, we examined whether the expression of MDM2 changes during neurite outgrowth. We found that a marked reduction of MDM2 protein levels preceded p53 activation (Figure 2C). Such downregulation might explain, at least in part, the observed p53 induction in response to growth factors. In RCN, the expression of p53 peaked in the nuclei 2 days after the addition of growth factors, and this coincided with the time of greatest increase of Coronin 1b, Rab13, and Gap-43 protein levels (Figure 2D–F) at the cortical cytoskeleton. Therefore, p53 seems to act as a switch to turn on Coronin 1b and Rab13 expression levels in RCN, and its baseline expression levels appear to be sufficient to maintain Coronin 1b and Rab13 protein levels over longer periods of time. Alternatively, p53 post-translational modifications such as acetylation, which might increase the transcriptional activity of p53, as well as other transcription factors, might be involved in the sustained, long-term expression of Coronin 1b and Rab13. Thus, in different experimental systems there is a tight temporal correlation between the pattern of expression of p53 and that of Coronin 1b and Rab13. p53 is required for neurite outgrowth To further evaluate the potential role of p53 for neurite outgrowth, we first studied the effects of inhibiting p53 activity with the p53DN construct. The read-outs for these experiments were the NGF-mediated induction of neurite outgrowth in PC12 cells, and neurite outgrowth and maturation in cultured embryonic RCN. Stably transfected p53DN PC12 cells or RCN infected with a p53DN-lentiviral vector were treated with growth factors, and their phenotypes were analyzed. As it can be seen, inhibition of p53 signaling blocked the NGF-mediated increase of Coronin 1b and Rab13 expression (Figure 3A). Strikingly, in contrast to naive PC-12 cells, the p53DN-transfected cells did not produce neurites following NGF treatment but, rather, demonstrated neurite length and number similar to that of untreated cells (Figure 4B and C). Similar results were also obtained by inhibiting p53 expression with a specific siRNA (data not shown). Likewise, expression of the p53DN in RCN markedly reduced the expression of both Coronin 1b and Rab13 (Figure 4A, Supplementary Figure 1), and significantly inhibited neurite outgrowth and branching (Figure 4B and C). Gap-43 levels were also decreased in cells expressing the p53DN construct. Thus, in the absence of p53, RCN cells fail to complete the maturation program in response to growth factors. Figure 3.p53 is required for neurite outgrowth in PC-12 cells and modulates the expression of Coronin 1b and Rab13. (A) Immunoblotting shows protein temporal expression profiling in WT PC-12 cells versus p53DN stably transfected cells after NGF. Increase in protein expression for Coronin 1b and Rab13 in WT is repressed in p53DN cells. (B) Immunofluorescence documents that p53DN stable-transfected PC-12 do not develop neurites following NGF (72 h), whereas WT PC-12 develop long neurites. (C) Bar graphs show that p53DN cells exposed to NGF have neurite length comparable to untreated PC-12 (number of experiments: 3, total number of cells: about 300, average values: *t-test P-values <0.001) Download figure Download PowerPoint Figure 4.p53 is required for neurite outgrowth and maturation in embryonic rat cortical neurons and influences the expression of Coronin 1b and Rab13. (A) Immunoblotting shows inhibition of protein expression for Coronin 1b and Rab13, following infection with p53DN lentivirus at day 1 in RCN. Reduction in protein levels for Gap-43 suggests impaired maturation. (B) Immunofluorescence at day 5 in RCN after infection with p53DN or eGFP lentivirus. Shown is the inhibition of neurite outgrowth and branching when p53DN (V5+β-3-tubulin) is expressed in the nucleus versus eGFP-infected cell (eGFP+β-3-tubulin). (C) Bar graphs show the inhibitory effects of p53DN infection (days 1–5) on RCN neuritic network (number of experiments: 3, total number of cells: about 240, average values: *t-test P-values <0.01; **P-values <0.001). Download figure Download PowerPoint To more definitely assess the impact of p53-mediated control of Coronin 1b and Rab13 on neurite outgrowth, a rescue experiment was subsequently performed. In PC-12 cells stably transfected with p53DN, transfection of Coronin 1b and Rab13, which localized at high levels at the cortical cytoskeleton and at the growth cone (Figure 5A, Supplementary Figure 2), significantly restored the differentiated phenotype to a similar extent with and without previous administration of NGF, suggesting that NGF signaling is completely blocked in p53DN stably expressing cells and that Coronin 1b and Rab13 act downstream of p53 in such pathway (Figure 5A and B). These results support the conclusion that p53 is required for neurite outgrowth, and that such effects largely reflect p53-mediated modulation of Coronin 1b and Rab13 expression. Figure 5.Coronin 1b and Rab13 rescue neurite outgrowth in p53DN cells. (A) Overexpression of Coronin 1b-GFP or Rab13-GFP in p53DN PC-12 cells exposed or not to NGF compared to control GFP-transfected cells. Both proteins are localized at the cortical cytoskeleton and at the growth cone and partially rescue neurite outgrowth. p53DN is localized in the nucleus (V5), as shown by colocalization with the nuclear marker Hoechst. (B) Bar graphs show neurite measurements, which demonstrate the effects of Coronin 1b and Rab13 on neurite outgrowth in p53DN cells compared to control (number of experiments: 3, total number of cells: about 240, average values: *t-test P-values <0.01; **P-values <0.001). Download figure Download PowerPoint Specific acetylation of p53 at lysine 320 preferentially induces Coronin 1b and Rab13 expression, binds to their promoter elements and promotes neurite outgrowth The highly undifferentiated human (NT2/D1) teratocarcinoma cells differentiate into several cell types, including neurons, following retinoic acid (RA) administration, and develop neuritic and synaptic networks. Thus, they are a very good model to study the molecular mechanisms of neurite outgrowth (Pleasure et al, 1992). Importantly, it was shown previously that although p53 transcriptional activity is required for NT2/D1 cellular differentiation, RA treatment induces a paradoxical reduction of p53 total protein levels (Curtin et al, 2001; Curtin and Spinella, 2005). This observation suggests that specific post-translational modifications may be required to regulate p53 activity, especially in conditions where the total levels of the protein are diminished. We have recently shown that in epithelial cells, specific acetylation of p53 at lysine 320 (K320) favors cell survival, unlike acetylation of lysine 372 (K372), which promotes cell death (Knights et al, 2006). Therefore, we examined whether specific acetylation events of p53 occur during neuronal maturation and neurite outgrowth. To this end, we first measured total p53 protein levels following RA treatment in NT2-D1 cells. Consistent with previous studies, we found that RA induced indeed a reduction of total p53 levels, which corresponded with the time of expression of the neuronal and neurite outgrowth markers β-3 tubulin and Gap-43. This also coincided with a net increase in the levels of expression of the p53 targets Coronin 1b and Rab13 (Figure 6A). Despite the lower p53 levels induced by RA, acetylation of K320 but not of K372 of p53 was markedly increased, and RA treatment also clearly induced the expression levels of the acetyltransferase PCAF, which is known to acetylate p53 at K320 (Knights et al, 2006). Figure 6.Acetylated p53 at lysine 320 is induced following RA during neurite outgrowth, binds to promoter regions of Coronin 1b and Rab13, and preferentially triggers their expression levels. (A) Immunoblotting shows induction of p53K320 and its acetyltransferase PCAF, but not of p53K372, following RA administration (5 days) in NT2-D1 cells (left). Coronin 1b and Rab13 are also induced by RA along with the neuronal and neurite outgrowth markers β-3-tubulin and Gap-43. (B) Protein expression data from immunoblotting experiments at the 24 and 48 h time point in p53 null cell line (H1299) where the expression of native p53 or of p53 acetylation mutants at position 320 (p53K320Q) and 372 (p53K372Q) was reconstituted with a tetracycline-inducible system. Lysine (K) in place of glutamine (Q) mimics the effects of a constitutive acetylation. Expression of the p53K320Q mutant versus K372Q preferentially triggers the expression of Coronin 1b and Rab13. (C) ChIP assays demonstrate in vivo interaction of WTp53 and of lys320-p53 with DNA-binding elements derived from the human Coronin 1b and Rab13 promoters. H1299-WTp53 cells were left untreated (−) or treated for 48 h (48 h) with tetracycline to induce the expression of native p53 with or without overexpression of full-length PCAF. Following induction of p53, cells were crosslinked with formaldehyde and immunoprecipitated with a polyclonal antibody recognizing p53 or lys320-p53. The ‘input’ samples represent equal fractions of extract collected prior to precipitation from which DNA was extracted and used as a control. WAF1 (p21) promoter region was used as a positive control for p53 DNA binding. Preferential binding to Coronin 1b and Rab13 promoter elements of lys320-p53 is visible following the overexpression of PCAF. Download figure Download PowerPoint To test the possibility that acetylation of K320 is specifically responsible for the regulation of the expression levels of Coronin 1b and Rab13, we used a p53 null cell line (H1299) where the expression of native p53 or of p53 acetylation mutants at positions 320 (p53K320Q) and 372 (p53K372Q) was reconstituted with a tetracycline-inducible system. Substitution of lysine (K) for glutamine (Q) mimics the effects of a constitutive acetylation (Knights et al, 2006), likely because acetylation neutralizes the positive charge of lysine, whereas glutamine recapitulates this effect. The results of these experiments were striking. Indeed, the p53K320Q acetylation mimic strongly induced the expression of Coronin 1b and Rab13, whereas p53K372Q had only a minimal effect (Figure 6B). To gain more confidence that truly acetylated p53 at lysine 320 (acetyl-K320 p53) is capable of a direct interaction with the p53-binding sequences contained in the promoter regions of Coronin 1b and Rab13, we performed a ChIP assay in cells expressing native p53. As it can been seen, the comparison of ChIP reactions performed with the antibody recognizing total p53 (FL-393), and the antibody recognizing exclusively p53 populations acetylated at position 320 (anti-acetyl-320), showed that acetylated p53 binds to these promoters with very high affinity. In addition, overexpression of the acetyltransferase PCAF resulted in a net increase in binding of acetylated p53 to these promoters. Thus, collectively, these results demonstrate that p53 populations acetylated at lysine 320 bind to the promoter regions of Coronin 1b and Rab13 (Figure 6C), and regulate the expression of these two genes. Then, to explore whether p53K320 is really important for neuronal maturation and neurite outgrowth, NT2-D1 cells were transfected with the plasmid expressing the p53 acetylation mimics p53K320Q or p53K372Q. The expression of Gap-43 and of the p53 target genes Coronin 1b and Rab13 was then examined. Strikingly, we found that only cells transfected with p53K320Q, but not with p53K372Q expressed Gap-43, and displayed increased expression of Coronin 1b and Rab13 (Figure 7A). In order to address the specificity of the effect of the p53K320Q mimic, a p53K320R mutant, where lysine was replaced with arginine, was employed in similar experiments and results were compared to cells treated with RA-GFP or transfected with GFP only. Arginine has the same charge as lysine, but it prevents acetylation, and therefore is a useful control for the glutamine substitution (see for example, Knights et al, 2006). In this case, the expression levels of Coronin 1b and Rab13, while induced by RA, were comparable to control cells transfected with GFP (Figure 7B). Thus, these results strongly support the notion that p53 molecules acetylated at position 320 are specifically responsible for the regulation of Coronin 1b and Rab13 during neurite outgrowth. Figure 7.Mimicking p53 acetylation mutant at lysine 320 promotes expression of Coronin 1b and Rab13 along with the proneurite outgrowth protein Gap-43 in NT2-D1 cells. (A) Overexpression of the p53K320Q mutant versus K372Q is performed by transfecting NT2-D1 cells. Immunoblotting shows induction of the proneurite outgrowth proteins Gap-43, Coronin 1b and Rab13 only in the K320Q after 5 days in culture. GFP-transfected cells are used as negative control. Bar graphs show densitometry data (the expression level for each protein and time point represents the intensity of each band of the immunoblot as a ratio value to β-actin levels). (B) Overexpression of the p53K320R mutant versus the effects of RA in NT2-D1 cells. Immunoblotting shows induction of the proneurite outgrowth proteins and p53 targets Coronin 1b and Rab13 only in the RA-treated cells after 5 days in culture. p53K320R does not induce protein expression changes compared to GFP-transfected cells, which are used as negative control. Bar graphs show densitometry data (the expression level for each protein and time point represents the intensity of each band of the immunoblot as a ratio value to β-actin levels). Download figure Download PowerPoint Finally, to validate the concept that p53 acetylation at lysine 320 plays a role" @default.
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• W2043456888 date "2006-08-31" @default.
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• W2043456888 title "The tumor suppressor protein p53 is required for neurite outgrowth and axon regeneration" @default.
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• W2043456888 doi "https://doi.org/10.1038/sj.emboj.7601292" @default.
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