2 Research Institute of Molecular Pathology (IMP), 1030 Vienna, Austria
Correspondence to Eugene M. Johnson Jr.: eugene.johnson{at}wustl.edu
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Introduction |
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c-Jun transcriptional activity is regulated by JNK-mediated phosphorylation of the NH2-terminal transactivation domain on Ser63 and Ser73 (Kallunki et al., 1996). JNK is part of a sequential kinase-signaling cascade involving three kinases. MAPK kinase activates JNK by dual Tyr/Thr phosphorylation. MAPK kinase activation is mediated by MAPK kinase kinases, including the mixed lineage kinase (MLK) family in neurons. Selective inhibition of the MLK family in sympathetic neurons by K252a analogue, CEP-1347, or dominant-negative MLKs demonstrates that MLKs mediate JNK activation after trophic factor deprivation, UV irradiation, and oxidative stress (Maroney et al., 1999, 2001; Harris et al., 2002).
Although activation of the JNK pathway is critical for regulating neuronal apoptosis, high levels of activated JNKs occur in neurons under normal conditions, indicating that JNK signaling may also be important for other metabolic processes in neurons (Harris et al., 2002; Besirli and Johnson, 2003). JNK isoforms (JNK1-3) are activated differentially in neurons under normal conditions and after a stress stimulus (Coffey et al., 2002). This isoform-specific activation presumably provides signaling specificity through phosphorylation of distinct substrates. Therefore, identification of proteins regulated by JNK-mediated phosphorylation is critical for understanding the consequences of JNK pathway signaling in neurons.
Despite high levels of activated JNKs, neurons contain low levels of c-Jun under normal conditions. During apoptosis, c-Jun is activated by increased transcription as well as JNK-mediated phosphorylation. Although often assumed to be the basis for the requirement for the JNK activity on neuronal death, the importance of c-Jun phosphorylation alone for neuronal apoptosis is not currently known because preventing JNK signaling by small molecule inhibitors CEP-1347 and SP600125 completely blocks both c-Jun transcription and phosphorylation (Besirli and Johnson, 2003). Similarly, dominant-negative overexpression or targeted deletion of c-Jun cannot separate c-Jun phosphorylation from expression. To determine whether c-Jun phosphorylation is a necessary event for neuronal apoptosis to proceed, we analyzed sympathetic neurons isolated from mice that carry a mutant c-jun allele (jun aa). This c-Jun mutant, JunAA, has alanines in the place of serines 63 and 73 and cannot be phosphorylated at these sites in its NH2-terminal transactivation domain. Neurons from JunAA mutant mice indeed showed resistance to both trophic factor deprivation and DNA damage. This resistance correlated with delayed expression of proapoptotic genes after trophic factor deprivation. The resistance of neurons from the JunAA mice, however, was surprisingly modest. In light of the complete protection provided by the JNK pathway inhibition, these data demonstrate that other critical JNK substrates must exist. We identify at least one component of the nuclear pore complex (NPC) as substrate of the JNK pathway during trophic factor deprivation. Moreover, trophic factor deprivation leads to JNK-mediated phosphorylation of additional nuclear proteins. These nuclear JNK pathway targets, including the Nup214 subunit of the NPC, may play important roles in neuronal death.
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Results |
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DNA damageinduced neuronal apoptosis is inhibited by c-Jun mutation
To determine whether c-Jun phosphorylation is also important for sympathetic neuronal apoptosis after DNA damage, we exposed jun aa/aasympathetic neurons to the DNA-damaging agent Ara-C (Besirli et al., 2003). Wild-type neurons were modestly more susceptible to Ara-C exposure compared with jun +/aa and jun aa/aa neurons (Fig. 3, P < 0.05 for 48 h). Similar results were obtained with another DNA-damaging drug, i.e., topoisomerase II inhibitor etoposide (unpublished data). Similar to sympathetic neurons, DNA damage caused a significant increase in both c-Jun levels and NH2-terminal c-Jun phosphorylation in cerebellar granule neurons (Fig. S1 A available at http://www.jcb.org/cgi/content/full/jcb.200501138/DC1). This induction was comparable to c-Jun activation seen after the removal of trophic factors K+ and serum from cerebellar granule neurons. c-Jun activation during DNA damageinduced sympathetic neuronal death is largely independent of the JNK pathway (Besirli and Johnson, 2003). In contrast, MLK-inhibitor CEP-11004 or JNK-inhibitor SP600125 completely prevented DNA damageinduced c-Jun transcription and phosphorylation in cerebellar granule neurons (Fig. S1 A). In addition, MLK inhibition by CEP-11004 inhibited DNA damageinduced death in cerebellar granule neurons (Fig. S1 B; P < 0.001 at 44 h), in contrast to lack of any prosurvival effect of MLK or JNK inhibitors in DNA-damaged sympathetic neurons. This block was transient, similar to the partial saving effect of MLK inhibition after K+ deprivation of cerebellar granule neurons (Harris and Johnson, 2001). Cerebellar granule neurons isolated from jun aa/aa mice were also more resistant to DNA damage. Treatment with 40 µM etoposide killed 50% of jun +/aa neurons after 44 h, whereas only 35% of jun aa/aa cerebellar granule neurons died at the same time point (Fig. S2; P < 0.01; available at http://www.jcb.org/cgi/content/full/jcb.200501138/DC1). This resistance to DNA damage in mutant neurons was similar to the effect of JNK pathway inhibition by CEP-11004. These results demonstrate that NH2-terminal c-Jun phosphorylation was important, but not absolutely required, for neuronal apoptosis induced by DNA damage.
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The BH3-only Bcl-2 family member PUMA is induced in neurons after trophic factor withdrawal and DNA damage
PUMA, first identified as a BH3-only proapoptotic protein from the Bcl-2 family, is activated after cellular stress including DNA damage and serum deprivation (Han et al., 2001; Nakano and Vousden, 2001; Yu et al., 2001). Because two other BH3-only Bcl-2 family members, Bim and DP5, are activated during sympathetic neuronal death, we analyzed protein extracts from NGF-deprived neurons for a potential induction of PUMA. Sympathetic neurons showed a rapid and substantial increase in PUMA expression after NGF deprivation (Fig. 5 A). To determine whether PUMA induction is a generalized neuronal response to trophic factor deprivation, we examined cerebellar granule neurons, which depend on both depolarizing concentrations of potassium and presence of serum for in vitro survival. When potassium was removed from the culture medium of 7-DIV cerebellar granule neurons, PUMA expression was increased by 6 h and remained elevated for 24 h (Fig. 5 B). PUMA is also activated in cells undergoing DNA damageinduced death (Han et al., 2001; Nakano and Vousden, 2001). Exposure to the topoisomerase-II poison etoposide causes c-Jun activation and apoptosis in sympathetic neurons, presumably by creating extensive amounts of double-stranded DNA breaks (Besirli and Johnson, 2003). Similar to trophic factor deprivation, etoposide-induced DNA damage led to rapid PUMA induction in sympathetic neurons (Fig. 5 C). Because the expression of other BH3-only proteins are regulated by c-Jun during neuronal death, we compared PUMA induction in jun +/+ and jun aa/aasympathetic neurons. Similar to Bim (Fig. 4 B), PUMA expression was regulated by c-Jun, as lack of c-Jun NH2-terminal phosphorylation delayed PUMA induction after trophic factor deprivation (Fig. 5 D). The delay in PUMA expression was not as significant as that observed for Bim expression, suggesting that other transcription factors also regulate PUMA gene transcription during neuronal apoptosis.
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Discussion |
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c-Jun phosphorylation on Ser63 and 73 is important, but not necessary, for neuronal apoptosis
The importance of c-Jun activity for neuronal death has been implicated in several in vitro systems (Estus et al., 1994; Ham et al., 1995; Behrens et al., 1999; Palmada et al., 2002). However, other studies in nonneuronal systems suggest that c-Jun expression can be a protective measure against cellular injury and may actually prevent death (Potapova et al., 2001). Data presented in this paper further demonstrate that c-Jun is a proapoptotic factor in sympathetic and cerebellar granule neurons. Importantly, our results also show that phosphorylation of c-Jun promoted neuronal apoptosis, but it was not absolutely necessary for death. Because the JunAA protein is still functional, whether in vivo deletion of c-Jun from sympathetic and cerebellar granule neurons would completely prevent apoptosis after trophic factor deprivation or DNA damage remains to be determined.
Ser63/73 phosphorylation is not required for neuronal c-Jun induction during apoptosis
Mutation of c-Jun NH2-terminal phosphorylation sites significantly inhibited neuronal apoptosis. The underlying cause for this is most likely due to the impaired induction of proapoptotic c-Jun transcriptional targets. One of these targets is c-jun, the expression of which is autoregulated (Angel et al., 1988). Under normal conditions, c-Jun exists in neurons at low levels with no detectable NH2-terminal phosphorylation despite high levels of constitutive JNK activity. One suggested model is that during stress, isoform-specific JNK activation or altered JNK localization to specific cell compartments causes NH2-terminal phosphorylation of c-Jun on Ser63 and Ser73, increasing transcriptional activity (Coffey et al., 2002). As a result, NH2-terminal phosphorylation activates the c-Jun autoregulatory cycle, leading to more c-jun transcription and NH2-terminal phosphorylation. Our results show that this is not the sole mechanism of c-Jun activation during neuronal apoptosis, because c-Jun expression was impaired, but not completely prevented in JunAA neurons. Several explanations may exist for this induction in the absence of NH2-terminal phosphorylation at Ser 63 and 73. First, JunAA protein is still functional at some low level and can induce c-Jun transcription, though inefficiently. Second, two other known phosphorylation sites in the NH2-terminal region, Thr91 and Thr93, exist. Phosphorylation of these residues by JNKs or other kinases may be sufficient to activate c-Jun, and this may explain the residual activity of the JunAA mutant. Finally, besides c-Jun, other JNK substrates may regulate c-Jun expression, such as other Jun, Fos, or ATF family members. Trophic factor deprivation induces the expression of other AP-1 transcription factors, including JunB, c-Fos, and ATF-2 (Estus et al., 1994; Eilers et al., 2001). ATF-2 is phosphorylated by JNKs, leading to its transcriptional activation. ATF-2 regulates c-jun transcription in sympathetic neurons after trophic factor deprivation (Eilers et al., 2001). The ability of the JNK pathway to activate c-Jun in jun aa/aa neurons strongly suggests that JNKs may be modulating the activity of several transcription factors that concomitantly regulate the c-jun promoter.
Expressions of BH3-only Bcl-2 family members Bim and PUMA are regulated by c-Jun during neuronal death
Bcl-2 family members are critical for making life-or-death decisions in neurons. The proapoptotic Bcl-2 family protein Bax is necessary for neuronal death in both sympathetic and cerebellar granule neurons (Deckwerth et al., 1996; Miller et al., 1997). Bax activity is regulated by the BH3-only members of the Bcl-2 family. Two BH3-only proteins, Bim and DP5, are increased in sympathetic and cerebellar granule neurons during cell death (Harris and Johnson, 2001; Putcha et al., 2001). Expression of both Bim and DP5 is regulated by MLKJNK signaling and blocked by pharmacological inhibitors of this pathway (Harris and Johnson, 2001). In addition, c-Jun is important for Bim induction in vitro because dominant-negative c-Jun inhibits Bim expression (Whitfield et al., 2001). Our results confirm these findings, showing that Bim expression is attenuated in neurons carrying a hypomorphic c-Jun protein. We also identified another BH3-only protein that is up-regulated during neuronal death. PUMA/Bbc3, first identified as a proapoptotic protein in thymocytes and tumor cells, is induced by DNA damage, dexamethasone treatment, or serum deprivation (Han et al., 2001; Nakano and Vousden, 2001). In primary murine thymocytes, PUMA expression is dependent on p53 activity after DNA damage, but not after dexamethasone treatment or serum deprivation (Han et al., 2001). In the work reported here, both trophic factor deprivation and DNA damage induced PUMA expression in sympathetic and cerebellar granule neurons. PUMA expression occurred well before the terminal stages of death, similar to Bim and DP5. The delay of PUMA expression in JunAA neurons suggests that c-Jun activity contributes to PUMA induction during neuronal death. Identification of yet another BH3-only proapoptotic protein (in addition to Bim and DP5) induced during neuronal death demonstrates the redundancy built into the apoptotic machinery in neurons and probably accounts for the modest phenotype associated with the deletion of Bim or DP5 (Putcha et al., 2001; Imaizumi et al., 2004).
In both sympathetic and cerebellar granule neurons, the lack of NH2-terminal c-Jun phosphorylation on Ser63 and 73 retarded, but did not prevent, cell death. Therefore, NH2-terminal c-Jun phosphorylation by JNKs is important, but not absolutely necessary, for neuronal apoptosis. Because JunAA protein is a c-Jun hypomorph, but not a complete functional null, c-Jun activity may still be critical for promoting neuronal death in jun aa/aa mice. This is supported by the delayed expression of proapoptotic c-Jun transcription targets. In the absence of any c-Jun activity, these genes may never get transcribed and apoptosis may be completely suppressed without these important cell death mediators. If c-Jun is necessary for neuronal apoptosis, the phenotype of c-Junnull neurons should mirror that of Bax-deficient neurons. Bax expression is absolutely required for trophic factor deprivation-induced death and sympathetic ganglia of bax-null mice contain twice as many neurons (Deckwerth et al., 1996). Although not as impressive as the bax-null mice, at birth JunAA mice also appear to have more neurons in their sympathetic ganglia (Fig. 1 B). The difference between bax deletion and c-jun mutation on the number of neurons in a SCG is not surprising because JunAA neurons eventually die after NGF deprivation. The role of c-Jun during naturally occurring or stress-induced cell death may also be different in distinct neuronal populations. In adult mice, conditional deletion of the neuronal c-jun gene causes a significant decrease in axotomy-induced death of facial nucleus motor neurons, but the total number of motor neurons is increased by only 20% (Raivich et al., 2004), which is significantly less than the 51% increase seen in Bax-null mice (Deckwerth et al., 1996). In addition, conditional c-jun knockout mice had no significant increase in the number of dorsal root ganglion sensory neurons. Analysis of SCGs isolated from conditional c-Jun knockout mice may allow a determination of whether c-Jun is necessary for the developmental programmed cell death of sympathetic neurons, resembling the cell death phenotype of Bax-null SCG.
The NPC subunit Nup214 is recognized by the phospho-c-Jun Ser73 antibody in a JNK-dependent manner specifically after NGF deprivation of sympathetic neurons
Inhibition of the MLKJNK signaling completely prevents NGF deprivationinduced death of sympathetic neurons. In contrast, lack of NH2-terminal c-Jun phosphorylation delays death only transiently. These findings indicate that MLKJNK signaling regulates other proteins critical for neuronal programmed cell death after trophic factor deprivation. One of these proteins is Bim. Ser65 phosphorylation of Bim potentiates its proapoptotic function during NGF deprivationinduced death of sympathetic neurons (Putcha et al., 2003). To identify additional JNK pathway targets, we used the rabbit polyclonal antibody against Ser73-phosphorylated c-Jun. This antibody proved to be a useful tool in identifying downstream targets of the JNK pathway. The loss of detectable bands on Westerns upon treatment with CEP-1347 indicates that this antibody acted as a general JNK substrate antibody, showing immunoreactivity against other neuronal proteins that were either constitutively phosphorylated in sympathetic neurons or were specifically phosphorylated after NGF deprivation. Increased phosphorylation or expression of these proteins after NGF deprivation was dependent on the JNK signaling and failed to occur in the presence of selective MLK-inhibitor CEP-1347. Database searches using the Ser73 motif identified Nup358, a 270-kD protein on SDS-PAGE, as a potential JNK substrate. Although Nup358 comigrated with the >250-kD phospho-c-Jun Ser73 immunoreactive band in whole cell lysates (resolution of the gel is poor above the 250-kD marker), immunoprecipitated Nup358 was not detectable with phospho-c-Jun Ser73 antibody. This may indicate that Nup358 is not phosphorylated after NGF deprivation. Alternatively, Mab414 antibody could have immunoprecipitated only the nonphosphorylated form of Nup358 if the phosphorylation changes the conformation of Nup358 or otherwise inhibits Mab414 recognition (e.g., by facilitating the association of Nup358 with another protein that blocks antibody binding). Unlike Nup358, the same immunoprecipitation experiments definitively showed that the related Nup214 became immunoreactive for the phospho-c-Jun antibody only after NGF deprivation. This result indicates that NPC was specifically phosphorylated by JNK during neuronal apoptosis.
NPC is a large protein assembly that penetrates the nuclear membrane and is the only known channel between the cytoplasm and the nucleus (Suntharalingam and Wente, 2003). NPC mediates the bidirectional permeability of the nucleus and facilitates nucleocytoplasmic exchange. Small molecules can diffuse through NPC in and out of the nucleus, but proteins that are larger than 40 kD require active transport. Although the primary function of NPC is to regulate the nucleocytoplasmic exchange, recent studies demonstrate that NPC function is also important for other physiological process such as mitosis, gene expression, and cell death (Talcott and Moore, 1999). Deletion of Nup358 leads to defects in chromosome segregation and kinetochore structure, indicating that some components of NPC are involved in mitosis (Salina et al., 2003). During apoptosis, Nup153, Nup358, and Nup214 are cleaved by caspases. Other non-Nup components of NPC, such as RanGTP and karyopherins, redistribute across the nuclear envelope during cell death before caspase activation and limit the permeability of NPC (Ferrando-May et al., 2001).
Both Nup358 and Nup214 are located in the cytoplasmic fibril compartment of NPC, which also contains two other domains, i.e., central core and the nuclear basket. Karyopherins recognize nuclear import and export signals in their specific protein cargo (Weis, 2003). During nuclear import karyopherincargo complex interacts with the FG repeat domains of the cytoplasmic fibril Nups such as Nup358 (Weis, 2003). JNK phosphorylation of Ser or Thr residues in this region may alter the kinetics of karyopherincargo binding to cytoplasmic Nups. Alternatively, JNK-dependent phosphorylation may change the tertiary structure of the Nups and modify the nucleocytoplasmic permeability.
One of the immediate effects of the reduced nucleocytoplasmic transport is the accumulation of mRNA in the nucleus. A similar process may be occurring in neuronal cells undergoing trophic factor deprivation-induced apoptosis. Sympathetic neurons show reduced metabolic activity after trophic factor deprivation (Martin et al., 1988). This loss of metabolism is largely prevented by the inhibition of the JNK pathway by CEP-1347 (Harris et al., 2002), which maintains protein and mRNA synthesis rates in NGF-deprived sympathetic neurons. The loss of nuclear permeability due to JNK-mediated NPC phosphorylation may be one of the underlying causes of the reduced metabolic activity and trophic status of sympathetic neurons after trophic factor deprivation. Phosphorylation of NPC may be causing the buildup of the neuronal mRNA in the nucleus. This hypothesis is consistent with our inability to demonstrate detectable changes in the activity of protein translation machinery in sympathetic neurons after NGF deprivation (unpublished data), suggesting that at least part of the underlying defect may not be inherent to the protein translation machinery but may be a direct result of impaired nuclear mRNA export. Therefore, inhibition of the JNK pathway may be beneficial for sympathetic neurons by inhibiting the induction of proapoptotic genes such as c-jun and bim and protecting the integrity of NPC and nucleocytoplasmic exchange to maintain neuronal metabolism.
In summary, we demonstrated that NH2-terminal c-Jun phosphorylation by the JNK pathway was important, but not necessary, for neuronal death after trophic factor deprivation or DNA damage. The resistance of mutant sympathetic neurons to NGF deprivation in the absence of c-Jun phosphorylation was consistent with delayed expression of proapoptotic genes c-Jun, Bim, and PUMA. The complete suppression of sympathetic neuronal death by JNK pathway inhibition, but not lack of NH2-terminal c-Jun phosphorylation, indicated that other JNK substrates were required for neuronal apoptosis. It was found that phospho-c-Jun Ser73 antibody was an effective experimental tool in identifying downstream substrates of the JNK pathway. Using this antibody, we identified Nup214 subunit of the NPC as a target of the JNK pathway during trophic factor deprivation. In addition to Nup214, the phospho-c-Jun Ser73 antibody detected other nuclear JNK pathway targets that remain to be identified. Similar to c-Jun and Bim, JNK-mediated regulation of NPC and possibly other nuclear proteins may be important for neuronal programmed cell death.
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Materials and methods |
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Neuronal cultures
Primary sympathetic neuronal cultures were prepared from postnatal day (P) 0P1 rat or mouse SCG by using previously described methods (Johnson and Argiro, 1983; Deckwerth and Johnson, 1993). Cultures were maintained in AM50 medium (90% minimum essential medium [Invitrogen], 2 mM glutamine, 10% FBS [Hyclone], 50 ng/ml 2.5S NGF, 20 µM fluorodeoxyuridine, 20 µM uridine, 100 U/ml penicillin, and 100 U/ml streptomycin), supplemented with 3.3 µg/ml aphidicolin (A.G. Scientific) to reduce the number of nonneuronal cells. Total number of neurons from each animal was determined by counting all viable neurons in three to four wells containing 1/51/7 of a SCG at 612 DIV. Because SCGs of JunAA mice had significantly more sympathetic neurons in initial experiments, higher dilution of SCG per well was used in later experiments. To deprive neurons of NGF, 5-DIV neurons were washed three times with AM0 (AM50 medium lacking NGF) and fed with fresh AM0 containing 0.01% anti-NGF antiserum. Anti-NGF treatment was stopped by rinsing the cultures three times with AM0 and adding back fresh AM50 for 57 d. To induce neuronal DNA damage, the culture medium was replaced with fresh AM50 containing 10 µM of topoisomerase-IIinhibitor etoposide. In neuronal cultures used to harvest protein or measure somal diameter, 50 µM of broad-spectrum caspase inhibitor, BAF (Enzyme Systems), was included in all treatment conditions to inhibit neuronal death.
Cerebellar granule cell culture protocol was described in detail previously (Miller et al., 1997). In brief, P6P8 mouse or rat cerebellum was dissected, dissociated by 1 mg/ml trypsin for 15 min, followed by mechanical trituration. Cells were plated onto poly-L-lysinecoated dishes (Nunc) and were maintained in K25+S medium (Eagle's basal medium, 25 mM potassium chloride, 10% dialyzed FBS, 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin). 3.3 µg/ml aphidicolin was added 1 or 2 d later to reduce the number of nonneuronal cells. At 7 DIV, the cells were washed twice with K5S medium (K25+S medium without the serum and with only 5 mM potassium) and treated with K5S or fresh K25+S.
Neuronal survival
The number of viable sympathetic neurons was assessed after fixing the cultures with 4% PFA (Fischer Scientific) in PBS and staining with crystal violet. Neurons were scored as viable by a naive observer if the crystal violet-positive cells had large, well-defined cellular outlines. Dead neurons and debris stain faintly or show no staining with crystal violet. The percentage of viable neurons was calculated by dividing the number of crystal violet-positive neurons at each time point by the total number of neurons in NGF-maintained, untreated sister cultures.
Immunocytochemistry
Cultures were fixed with fresh 4% PFA in PBS, washed with Tris-buffered saline (TBS: 0.1 M Tris-HCl, pH 7.6, 0.9% NaCl), and incubated in blocking solution (5% normal goat serum in TBS, containing 0.3% Triton X-100) for 1 h at RT. The cultures were then incubated with phospho-c-Jun (Ser73; 1:1000; Cell Signaling) antibody in antibody solution (1% normal goat serum in TBS, containing 0.3% Triton X-100) overnight at 4°C. The cultures were next washed three times with TBS and incubated in antibody solution containing Cy-3labeled secondary antibody (1:400; Jackson ImmunoResearch Laboratories) for 4 h at 4°C and counterstained with 1 µg/ml bisbenzimide (Hoechst 33258; Molecular Probes). After four washes with TBS, the cultures were mounted for fluorescence microscopy. Sympathetic neurons were visualized with a Zeiss Axiophot microscope.
Western blot analysis
Neuronal cultures were rinsed twice with cold PBS, lysed in reducing sample buffer (125 mM Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 4% SDS, 0.1% bromophenol blue, and 20% glycerol), boiled for 5 min, and stored at 20°C until use. Proteins were separated by SDS-PAGE on Tris-Glycine mini-gels (Invitrogen) and transferred to Immobilon-P PVDF membranes (Millipore). Blots were blocked for 1 h at RT with TBST (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween 20) containing 5% nonfat dry milk or BSA and incubated overnight at 4°C with primary antibody diluted in blocking solution recommended by manufacturer. The following primary antibodies were used: c-Jun (0.25 µg/ml; BD Transduction Labs), phospho-c-Jun (Ser73; 1:1,000; Cell Signaling), Bim (1:1,000; Stressgen), PUMA (1:250; Axxora), Mab414 (1 µg/ml; Covance), and tubulin (1:50,000; Sigma-Aldrich). After washing, blots were incubated for 1 h at RT with HRP-linked secondary antibodies (Cell Signaling) diluted 1:2,5001:10,000 in blocking solution. The blots were washed three times with TBST and developed with a chemiluminescent substrate (Supersignal; Pierce Chemical Co.). To strip and reprobe blots, the membranes were incubated in 100 mM glycine (pH 2.5) twice for 25 min and then washed with TBST; Western analysis was repeated. A Bio-Rad Laboratories ChemiDoc system with QuantiOne Software was used to quantify the immunoblots. All values were normalized against the values obtained for tubulin-loading controls.
Immunoprecipitation
Neuronal cultures were rinsed twice with cold PBS and lysed in NP-40 immunoprecipitation buffer (Tris-buffered saline, pH 7.4, 1% Nonidet P-40, 10% glycerol, protease inhibitors, and 1 mM sodium orthovanadate, 1 mM sodium fluoride, 50 mM ß-glycerophosphate, 1 mM DTT, and 50 mM NaF) with gentle rocking at 4°C. The detergent extracts were cleared of insoluble debris and nuclei by centrifugation at 13,000 g in a refrigerated microcentrifuge for 10 min. The cleared lysates were immunoprecipitated with 1 µg of Mab414 antibody or normal mouse IgG.
Subcellular fractionation
Sympathetic neurons were harvested into isotonic fractionation buffer (250 mM sucrose, 0.5 mM EDTA, 20 mM Hepes, 500 µM sodium orthovanadate, pH 7.2) supplemented with protease inhibitors (inhibitor cocktail complete; Roche Molecular Biochemicals) and centrifuged at 900 g for 5 min. The pellet was resuspended into 500 µl of fractionation buffer, homogenized with a ball-bearing homogenizer, and centrifuged at 900 g for 5 min to remove nuclei. The nuclear pellet and the postnuclear supernatant were resuspended to equivalent volumes with reducing sample buffer and evaluated by Western blotting.
Online supplemental material
DNA damageinduced c-Jun activation is MLK and JNK dependent in cerebellar granule neurons (Fig.S1). DNA damageinduced death of cerebellar granule neurons is delayed in the absence of NH2-terminal c-Jun phosphorylation (Fig. S2). Phosphorylation of c-Jun detected by antiphospho-c-Jun Ser63 antibody in wild-type and jun aa/aa neurons (Fig. S3). Online supplemental materials are available at http://www.jcb.org/cgi/content/full/jcb.200501138/DC1.
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Acknowledgments |
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This work was supported by National Institutes of Health grants R37AG-12947 and RO1NS38651.
Submitted: 10 May 2005
Accepted: 21 June 2005
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References |
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