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First published online 13 December 2005
doi: 10.1242/jcs.02699
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Research Article |
1 National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China
2 Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA
Author for correspondence (e-mail: gj{at}pku.edu.cn)
Accepted 19 September 2005
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Summary |
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is mediated by its upstream kinase and associated proteins. Here we identify a new nuclear protein, NP60, which regulates the activation of p38 in response to sorbitol treatment. NP60 specifically binds to p38, but not to JNK and ERK, in vitro and in vivo. Co-transfection of NP60 leads to the phosphorylation and activation of p38, and subsequently results in the phosphorylation and activation of activating transcription factor 2. The phosphorylation of p38 induced by NP60 requires upstream activity of p38 MAP kinase, MAP kinase kinase 6 (MKK6) or MKK4. Our results indicate that NP60 mediates stress activation of p38 and regulates p38 signaling in a specific way.
Key words: NP60, p38 phosphorylation, Signaling, Sorbitol
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Introduction |
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; Fanger et al., 1997; Su and Karin, 1996). Several independent MAP kinase pathways have been identified in mammals including the extracellular regulatory protein kinase (ERK) (Robinson and Cobb, 1997), Jun N-terminal kinase (JNK) (Derijard et al., 1994; Kyriakis et al., 1994) and p38 MAP kinase pathway (Han et al., 1994; Han et al., 1993). The activation of p38 requires upstream kinase that phosphorylates p38 by dual phosphorylation within a Thr-X-Tyr site. MAP kinase kinase 3 (MKK3) and MKK6 are the upstream kinases of p38 (Cuenda et al., 1996; Derijard et al., 1995; Han et al., 1996; Moriguchi et al., 1996; Raingeaud et al., 1996). In addition, JNK upstream kinase MKK4 can also phosphorylate p38 in vivo and in vitro (Derijard et al., 1995; Kayali et al., 2000). MAP kinase kinases are located in the cytoplasm and the phosphorylation of p38 in the cytoplasm by upstream kinases could result in the translocation of p38 into the nucleus (New et al., 2003; Seternes et al., 2002). The molecular mechanism for the translocation of p38 is not fully understood. Some evidence suggests that the activation of p38 also occurs in nuclei (New et al., 2003; Seternes et al., 2002). However, this hypothesis has not well been addressed.
The prototypical model of MAP kinase activation is a cascade of three layers of kinase reaction: the MAPK is activated by upstream MAP kinase kinase (MAPKK), which is activated by further upstream MAP kinase kinase kinase (MAPKKK) (Pearson et al., 2001). The ERK family of MAPKs is activated by the MEK1 and MEK2 MAPKK (Pearson et al., 2001), JNK by MKK4 and MKK7, and p38 by MKK3, MKK4 and MKK6 (Brancho et al., 2003; Derijard et al., 1995; Han et al., 1996; Raingeaud et al., 1996). The MAPKKKs in the ERK pathway include Raf-1, A-Raf, B-Raf and Mos (Davis, 2000; Fanger et al., 1997). MEKKs, MLKs, TAKs and ASK1 function as MAPKKKs in the JNK pathway. Many of the MAPKKKs that activate the JNK pathway also regulate the p38 pathway (Ono and Han, 2000). Recently, an alternative MAPK activation model has been proposed. It demonstrates that the scaffold protein TAB1 can induce p38 autophosphorylation and leads to its activation. This process is independent of MAPKK (Ge et al., 2002; Ge et al., 2003).
In Saccharomyces cerevisiae, the STE11 MAPKKK is involved in both the pheromone-response pathway and the osmoregulatory pathway (Marcus et al., 1994; Posas and Saito, 1997). However, there is little or no crosstalk between these two pathways. The formation of a signaling complex is one of the mechanisms that determine the signaling specificity. As a scaffold protein, STE5 in the mating pathway and Pbs2 in the osmoregulatory pathway assist signaling components to form a multi-protein complex, which prevents inappropriate crosstalk (Marcus et al., 1994; Posas and Saito, 1997). Mammals use a similar mechanism to specify the signaling. It has been demonstrated that JIP-1 functions as a scaffold protein for the JNK signaling pathway; JIP-1 tethers MLK, MKK7 and JNK into a complex (Whitmarsh et al., 1998); MP1 functions as a scaffold protein for the ERK pathway and facilitates MEK1 to ERK1 signaling (Schaeffer et al., 1998). Osm was identified as a p38 scaffold protein that enables Rac, MEKK3, MKK3 and p38 to form a signaling complex (Uhlik et al., 2003).
In the present study, we describe a new role for NP60 in the regulation of p38 activity. NP60 is a nuclear protein with a molecular mass of 60 kDa that specifically interacts with p38. Transfection of NP60 leads to the phosphorylation of endogenous and exogenous p38, and subsequently results in the activation of activating transcription factor 2 (ATF2). It mediates the stress activation of p38 in response to sorbitol treatment. The induction of p38 phosphorylation by NP60 requires p38 upstream kinase. NP60 does not have kinase activity. Our findings suggest that NP60 functions as a regulator in stress activation of p38 and the p38 signaling pathway.
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Results |
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, we screened a library constructed from human gastrointestinal tract tissue using the yeast two-hybrid system (Ge et al., 2002). We identified four clones encoding NP60 from 1.5x107 transformants. NP60 is composed of 15 exons and located on human chromosome 16p13.3. The ORF of NP60 was 1659 bp coding for 553 amino acids with a predicted molecular mass of 60.5 kDa (Fig. 1A,B). The protein sequence analysis revealed that NP60 was not homologous with any known protein in GenBank (original name for this clone was n-PAC, gene ID: AY352585 and protein ID: AAQ57265). It contained several motifs, such as two nuclear localization sequences (NLS), a PWWP (Pro-Trp-Trp-Pro) domain and an AT-hook at the N-terminus, and an NAD-binding sequence spanning more than 200 amino acids at the C-terminus (Fig. 1C). The sequence and motif analysis indicated that NP60 was a newly identified protein.
NP60 localizes only in nuclei
To examine the expression of NP60, total protein and RNA from 293T, RAW264.7, HUVEC, HL-60 and Jurkat cells were prepared. The expression of NP60 was analyzed by western blot and RT-PCR. NP60 protein in RAW264.7 cells was easily detected with a molecular mass of 60 kDa as predicated from its amino acid sequence (Fig. 2A). It was hard to detect NP60 protein in other cell lines although its transcript was detected by RT-PCR (Fig. 2B). The presence of an NLS in NP60 prompted us to check its localization in the cell. Cytoplasmic protein and nuclear protein from RAW264.7 cells were carefully prepared. Western blotting was performed using NP60 antibody. As shown in Fig. 2C, endogenous NP60 was seen only in nuclei. We then transfected 293T cells with NP60. Cytoplasmic and nuclear proteins from transfected cells were carefully prepared and used for western blot assay. This showed that ectopic NP60 was only observed in nuclei (Fig. 2D). Histoimmunological assay was also performed after transfection to view the subcellular localization of NP60. It clearly showed that NP60 located in the nuclei; no signal was detected in cytoplasm (Fig. 2E).
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NP60 interacts with p38
Since NP60 was selected from a yeast two-hybrid library using p38 as bait, it indicated that NP60 could interact with p38. To further verify this interaction, we first used an ELISA method. Bacterially expressed NP60 and its different mutants, and ATF2 were coated on the plate. They were then incubated with bacterially expressed p38. The interaction was assayed by p38 antibody. As shown in Fig. 3A, NP60 interacted with p38. The binding activity disappeared when the AT hook sequence was removed. The binding activity of NP60 to p38 was stronger than that of ATF2 to p38. We then used pull-down assay to further test the interaction in vitro. NP60 was incubated with p38 in buffer. The reaction complex was then pulled down using either glutathione Sepharose 4B or Ni-NTA agarose. The precipitates were resolved on SDS-PAGE and probed with p38 antibody or NP60 antibody by western blot assay after transferring to the membrane. It showed that p38 could pull down NP60 and NP60 pulls down p38 (Fig. 3B,C). We also tested the interaction of NP60 mutants with p38. Full-length NP60, and PWWP and NAD binding motif deletion mutants of NP60 maintained the binding activity to p38 whereas the AT hook deletion mutant lost this binding activity (Fig. 3D). The in vitro binding assay strongly demonstrated the interaction between NP60 and p38. Next, we performed co-immunoprecipitation assays to test the interaction in vivo. Vectors that express myc-NP60 and Flag-p38 were co-transfected into 293T cells. Total protein was extracted after transfection and precipitated with either myc antibody or Flag antibody. The precipitation was examined by immunoblotting with either p38 antibody or myc antibody. It showed that NP60 existed in the complex precipitated by Flag antibody (data not shown) whereas p38 existed in the complex precipitated by myc antibody (Fig. 3E). We also examined if NP60 could interact with another two MAPKs, JNK1 and ERK2 in co-immunoprecipitation experiments. These results showed that NP60 did not interact with either JNK or ERK (Fig. 3F), indicating that NP60 interaction is specific to p38.
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activation, we co-transfected myc-NP60 with p38 into 293T cells and examined first the phosphorylation of p38 by immunoblotting using anti-phospho-p38 antibody. Co-expression of NP60 and p38 in cells led to the phosphorylation of p38 compared with the control, although the phosphorylation level of p38 was lower than that induced by MKK6b (Fig. 4A). Expression of transfected NP60 also resulted in the phosphorylation of endogenous p38 at a level comparable with that of MKK6b (Fig. 4B). However, the effect of NP60 on phosphorylation of endogenous JNK1 or ERK2 could not be detected (data not shown).
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PWWP lacking the PWWP sequence, to test the effect on the phosphorylation of p38. Expression of PWWP with p38 in 293T cells was also able to induce the phosphorylation of p38 (Fig. 4C). We then tested another two NP60 mutants with the deletion of AT-hook domain and NAD-binding domain. These two mutants were co-transfected with p38 into 293T cells, respectively. Neither of the mutants could induce the phosphorylation of p38 (data not shown). It indicated that the middle AT-hook domain and the C-terminal NAD-binding domain were required for NP60 to induce the phosphorylation of p38.
To determine the specificity of p38 phosphorylation induced by NP60, we co-transfected myc-NP60 with Flag-JNK1 and Flag-ERK2 into 293T cells, respectively. The phosphorylation of JNK1 or ERK2 was examined by immunoblotting using anti-phospho-JNK antibody or anti-phospho-ERK antibody. It showed that NP60 had no effect on phosphorylation of either JNK1 or ERK2 (Fig. 4D,E).
MAPKs can be activated by different stress stimuli. We then addressed which stress could trigger the activation of p38 through NP60. Sorbitol is a known stress factor that activates p38 and other MAPKs (Ge et al., 2002). To look the effect of NP60 on the activation of p38 in response to sorbitol stress, we silenced the endogenous NP60 by siRNA. Endogenous NP60 was successfully interrupted by siRNA in 293T cells (Fig. 4F). These cells were then treated with sorbitol. Total proteins were extracted after treatment and subjected to western blot assay using anti-phospho-p38 antibody. It showed that silencing of NP60 blocks the activation of p38 by sorbitol treatment (Fig. 4G), and the activation of JNK and ERK was not impaired. It indicated that NP60 mediates sorbitol stress activation specifically for p38.
Upstream MKK is required for NP60 to induce the phosphorylation of p38
The induction of p38 phosphorylation by NP60 in vivo prompts us to address if NP60 phosphorylates p38 directly. NP60 expressed by 293T cells was isolated by immunoprecipitation. It was then incubated with bacterially expressed p38 in an appropriate buffer containing [
-32P]ATP with or without SB203580. NP60 did not have kinase activity for p38, because the phosphorylation did not increase in the presence of NP60 (Fig. 5A). The phosphorylation signal should be from the autoactivation of p38, because the autoactivation of p38 was completely blocked by SB203580, even in the presence of NP60. We further tested the effect of phosphorylation of ATF2 by NP60 with p38. Ectopic NP60 expressed by 293T cells was isolated by immunoprecipitation and incubated with p38 and ATF2 in an appropriate buffer containing [-32P]ATP. The phosphorylation of ATF2 was visualized by autoradiography. The result revealed that NP60 did not affect the phosphorylation of ATF2 by p38 (Fig. 5B). It indicated that NP60 was not an upstream kinase for p38.
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autoactivation in a MAPKK-independent manner (Ge et al., 2002). Therefore, we tried to determine the mechanism by which NP60 induced the phosphorylation of p38. Since the intrinsic activity of p38 is required for TAB1-mediated p38 activation, we first tested whether NP60 could induce p38 phosphorylation when the intrinsic activity of p38 was inhibited. A p38 mutant, p38(M) that has no enzyme activity due to the substitution of an amino acid in the ATP-binding site (Ge et al., 2003), was used for the test. The intrinsic activity of p38 could also be impaired by its specific inhibitor SB203580 (Ge et al., 2003). The inhibitor was also used in the experiments. Induction of p38 phosphorylation by NP60 was not affected by the presence of SB203580 (Fig. 6A). Co-expression of NP60 with p38(M) still induced the phosphorylation of p38(M), although this mutant had no enzymatic activity. These results indicated that the phosphorylation of p38 induced by NP60 was not through its autoactivation mechanism.
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The upstream kinases of p38 include MKK3, MKK6 and MKK4. Mutation forms of these MAPKKs such as MKK3(A), MKK6(A) and MKK4(A) can function as dominant-negative mutants. We used these mutants to examine whether they have an effect on the phosphorylation of p38 induced by NP60. NP60 and p38 were co-transfected with either MKK6b(A) or MKK4(A) into 293T cells. The phosphorylation of p38 was analyzed by immunoblotting assay. It showed that the phosphorylation of p38 was inhibited in the presence of either MKK6b(A) or MKK4(A) (Fig. 6B). We then examined whether the phosphorylation of p38 by MAPKKs could be enhanced in the presence of NP60. MKK4 or MKK6b were co-transfected with NP60 and p38 into 293T cells. The phosphorylation level of p38 was analyzed by immunoblotting. It was found that NP60 enhanced MKK4 activity for the phosphorylation of p38 in the cells co-transfected with NP60 and MKK4 compared with the control that was transfected with MKK4 alone. It did not have an effect on MKK6b activity in the cells co-transfected with NP60 and MKK6b (Fig. 6C).
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signaling pathway could lead to the phosphorylation of downstream target proteins such as ATF2, MEF2C and many others. To examine whether the phosphorylation of p38 induced by NP60 results in its activation and subsequently mediates the signaling, we performed an ATF2 report assay. This report system is composed of the chimeric transcription factor GAL-ATF2 and the GAL4-driven luciferase reporter gene. The transcriptional activity of GAL-ATF2 could be increased if activation of p38 induced by NP60 could transduce the signaling downstream. The GAL4-driven luciferase reporter construct, the GAL-ATF2 fusion protein and p38 and NP60 were transfected into 293T cells in different combinations. Luciferase activity was examined 24 hours after transfection. Luciferase activity was increased in the cells transfected with the combination of p38 and NP60, although the activity level was lower than in the cells transfected with p38 and MKK6b together (Fig. 7A). To confirm this activity, p38 and NP60 were co-transfected into 293T cells. Ectopic p38 was isolated by immunoprecipitation after transfection. It was then used for in vitro kinase assay using ATF2 as a substrate. The increase of p38 phosphorylation induced by NP60 was correlated with the increase of ATF2 phosphorylation (Fig. 7B). It indicated that NP60 regulated the p38 signaling pathway. |
Discussion |
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that directly lead to the phosphorylation of p38 include enzymes such as MKK6, MKK3 and MKK4 (Cuenda et al., 1996; Derijard et al., 1995; Han et al., 1996; Kayali et al., 2000; Moriguchi et al., 1996; Raingeaud et al., 1996), and the non-enzymatic adapter, TAB1 (Ge et al., 2002). Although activation and regulation of the p38 MAPK pathway has been well established, some processes are still not fully understood. To highlight the p38 signaling pathway, emphasis should be placed on the identification of p38-interacting proteins. In this report, we characterized a p38-interacting protein, NP60.
NP60 is a newly identified protein with several well-characterized motifs, such as an NLS, PWWP, AT hook and an NAD-binding region (Fig. 1). It localizes only in the nuclei (Fig. 2) and interacts specifically with p38 (Fig. 3). PWWP has been considered a domain-mediating protein-protein interaction (Stec et al., 2000). It has also been identified to mediate DNA-protein interaction (Qiu et al., 2002). The data from in vitro binding assays demonstrates that this domain is not required for the interaction of NP60 and p38 as removal of this domain has no effect (Fig. 3A), nor does it affect the induction of p38 phosphorylation (Fig. 4C). It seems that the AT hook motif is required for the interaction, because truncated NP60 lacking an AT hook region lost its binding activity to p38, resulting in the failure of induction of p38 phosphorylation (data not shown). The AT hook was originally identified to be a motif involved in DNA-protein interaction (Aravind and Landsman, 1998). We show here that it may also mediate protein-protein interaction. However, its action in the interaction of NP60 with p38 needs further investigation.
Co-expression of NP60 with p38 leads to the phosphorylation of p38 (Fig. 4). It prompted us to address whether NP60 has kinase activity on the phosphorylation of p38. It turns out that NP60 cannot phosphorylate p38 directly (Fig. 5A). Induction of p38 phosphorylation by NP60 is not mediated by the p38 autoactivation mechanism because SB203580 does not inhibit the reaction (Fig. 6A), and the mutant of p38, p38(M) can also be phosphorylated by NP60 in vivo (Fig. 6A); it requires upstream kinase (Fig. 6B). Although it was shown that TAB1 induces p38 autoactivation in a MAPKK-independent manner (Ge et al., 2002), our results show that NP60 functions differently, because dominant-negative MKK6 and MKK4 inhibited p38 phosphorylation induced by NP60. Moreover, SB203580 had no effect on p38 phosphorylation by NP60. It is interesting to find that NP60 can enhance the activity of MKK4 on p38 (Fig. 6C). These data together suggest that NP60 may induce the phosphorylation of p38 through MKK4. It could be an alternative p38 signaling pathway mediated by MKK4 activation. Extensive study on the mechanism of this unique pathway are required if this is the case.
The phosphorylation of p38 can be induced in response to different extracellular stresses. It is interesting to find that NP60 can mediate sorbitol-stressed activation of p38 (Fig. 4G). We successfully disrupted the NP60 expression in 293T cells by siRNA (Fig. 4F), which allowed us to use this cell model for physiological tests. Disruption of NP60 in 293T cells leads to a blockage of the induction of p38 phosphorylation by sorbitol stress. It suggests that NP60 plays a role in the regulation of p38 signaling during some stress responses.
The prototypical model of MAPK activation has three layers of phosphoryl transfer reaction. A series of reports demonstrate that scaffold proteins mediate MAPK activation by tethering the components of one specific pathway into a complex (Marcus et al., 1994; Posas and Saito, 1997; Schaeffer et al., 1998; Uhlik et al., 2003; Whitmarsh et al., 1998). For example, MP1 has been identified as a scaffold protein in the ERK pathway (Schaeffer et al., 1998), as has JIP in the JNK pathway (Whitmarsh et al., 1998). We have tested whether NP60 functions as a scaffold protein in the MKK4-mediated p38 pathway. However, we fail to find the interaction of NP60 with MKK4 or MKK6. It is more likely that NP60 regulates p38 activation in a specific way that needs further investigation. Nevertheless, we demonstrate here that NP60 is a novel nuclear protein that regulates the activity of p38. We identify a new p38-interacting protein in nuclei, which will help us to further understand the p38 pathway.
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Materials and Methods |
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, -p38(M), -JNK1, -ERK2, MKK3b, MKK6b, MKK4, MKK3b(A), MKK6b(A) and MKK4(A) were all gifts (Ge et al., 2002; Ge et al., 2003). Plasmid used for the generation of siRNA of NP60 was mU6pro vector (Yu et al., 2002). The target sequence was 5'-GCTGTGGATGCTGTTGAAG-3' (19 bp) with TTCG as the loop structure.
RT-PCR analysis
Total RNA was isolated from 293T, HUVEC, HL60 and Jurkat cells using the Total RNA Isolation Kit (Promega, Madison, WI). Reverse transcription was performed with SuperScript II RT (Invitrogen, Rockville, MA). Primers for amplification of NP60 by PCR were, 5'-GAGTCTAGTACCGTGAAGGG-3' (forward) and 5'-GATCCCTTGCAGCACACCAC-3' (reverse). PCR for the amplification of human ß-actin transcript was performed using the primers: 5'-CACCAACTGGGACGACAT-3' (forward) and 5'-CATACTCCTGCTTGCTGATC-3' (reverse).
Construct of NP60 expression vectors
The full-length coding region of NP60 was amplified by PCR and subcloned into PET30a (Novagen), pGEX4T-1 (Promega, Madison, WI), pcDNA6A/myc-His (Invitrogen, Rockville, MA) and pCMV-Myc (Clontech, Palo Alto, CA). The mutants of NP60 were amplified by PCR using full-length NP60 as a template and subcloned into pGEX4T-1 (Promega, Madison, WI) and pcDNA3.1(-) vector (Invitrogen, Rockville, MA).
Antibody preparation
Full-length NP60 cDNA was subcloned into PET30a (Novagen). The plasmid was transformed into E. coli BL21(DE3). Recombinant protein with a His tag was induced in the presence of 1 mM IPTG and purified by Ni2+-NTA agarose resin (Qiagen) under denaturing conditions according to the manufacturer's protocol. Polyclonal anti-NP60 serum was generated by the immunization of rabbits with purified His-NP60 recombinant protein.
Transfection and cell treatment
293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 0.1% gentamycin. Fresh medium was added 4 hours before transfection, which was performed using the standard calcium phosphate precipitation method (Sambrook, 1989). In some experiments, cells were treated with 2 µM SB203580 4 hours after transfection, or with 500 mM sorbitol for 30 minutes.
Preparation of cellular proteins
Cells were lysed in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 2 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). The lysate was centrifuged at 15,000 g for 10 minutes at 4°C. Total cellular proteins were collected in the supernatant. For the preparation of cytoplasmic and nuclear protein, cells were resuspended in Buffer A (10 mM HEPES-KOH, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF, 0.5 mM Leupeptin, pH 7.9) and incubated on ice for 10 minutes. Lysates were centrifuged at 7000 g for 10 minutes at 4°C and soluble cytoplasmic proteins collected in the supernatant. The pellet was resuspended in Buffer B (20 mM HEPES-KOH, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM PMSF, 0.5 mM leupeptin, pH 7.9) and incubated on ice for 30 minutes. The supernatant nuclear proteins were collected by centrifugation at 15,000 g for 10 minutes at 4°C.
Immunoprecipitation analysis
Total cell extracts were incubated with antibodies at 4°C overnight. Protein G Sepharose beads were added and incubated at 4°C for another 2 hours. Beads were washed four times with lysis buffer after incubation. Precipitates were resolved by 12% SDS-PAGE and transferred to PVDF membrane for western blot assay.
Protein kinase assay
Flag-p38 and myc-NP60 were co-transfected into 293T cells. Total protein was precipitated using M2 FLAG antibody or myc antibody after transfection. The precipitate was washed four times with lysis buffer and twice with kinase reaction buffer (25 mM Tris-HCl, pH 7.5, 5 mM ß-glycerophosphate, 10 mM MgCl2, 2 mM DTT, 0.1 mM Na3VO4) followed by resuspension in 50 µl kinase reaction buffer. It was then used for the kinase assay. The reaction was initiated by either adding 4 µg ATF2 fusion protein or p38 as a substrate to the precipitate in the presence of 250 µM ATP with or without 10 µCi [-32P]ATP, and terminated after 30 minutes at 30°C by adding SDS sample buffer. The reaction complex was resolved by 12% SDS-PAGE. The phosphorylation of ATF2 fusion protein was analyzed by western blot assay using an antibody against phospho-ATF2 (Thr-71) or by autoradiography.
Reporter assay
Reporter gene assay was performed using the dual luciferase system (Promega). 293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 0.1% gentamycin. GAL4-responsive luciferase plasmid with NP60 and p38 or with MKK6b and p38 were co-transfected into 293T cells. The total amount of plasmid for each transfection was adjusted with empty vector. Luciferase activity was measured 24 hours later after transfection following the manufacturer's instructions.
Immunohistochemistry assay
293T cells transfected for 16 hours, were subjected to fluorescence analysis. Cells were grown on coverslips, washed with PBS and fixed in 4% paraformaldehyde for 15 minutes. Cells were permeabilized with 0.1% Triton for 5 minutes. Nonspecific binding was blocked with 3% BSA/PBST (0.1% Triton X-100 in PBS, pH 7.6) for 1 hour. His-tagged NP60 was detected using anti-His mouse monoclonal antibody (1:200 dilution) and FITC-conjugated goat anti-mouse IgG (1:100 dilution). Nuclei were stained with DAPI (1:10,000 dilution). Immunofluorescence was visualized at 100x magnification using an Olympus BX51 microscope.
Pull-down assay
Bacterially expressed GST, GST-NP60 protein and truncated NP60 mutant proteins were purified by affinity purification with glutathione Sepharose 4B agarose. Purified GST-tagged proteins (10 µg) were incubated with 10 µl Glutathione Sepharose 4B agarose for 2 hours at 4°C, followed by adding 1 µg purified His-p38 and 10 µg BSA, and incubated at 4°C overnight. The mixture was washed four times with 0.1 M Tris-HCl pH 8.0. The pellet was resuspended in Laemmli buffer after centrifugation and subjected to SDS-PAGE. Western blotting was performed with the anti-p38 mouse monoclonal antibody. Similarly, 10 µg purified His-p38 or BSA was incubated with Ni-NTA agarose for 2 hours at 4°C. Then, 1 µg GST-NP60 and 10 µg BSA were added and incubated over night at 4°C. Western blotting was then performed with the anti-NP60 rabbit polyclonal antibody.
ELISA assay for protein interaction
One µg purified, bacterially expressed GST, GST-ATF2, GST-NP60 and truncated NP60 mutant proteins was diluted in 100 µl 100 mM Tris-HCl pH 8.0, and coated on 96-well plates for 2 hours at 37°C. Non-specific binding was blocked by adding 200 µl of 2% BSA/PBST (0.1% Tween20 in PBS, pH 7.6) for 1 hour at 37°C followed by adding 0.1 µg purified His-p38 diluted in 100 µl of 2% BSA/PBST, and incubated at 4°C overnight. Anti-p38 mouse monoclonal antibody (1:1000 dilution) and HRP-conjugated goat anti-mouse IgG (1:1000 dilution) were then added after washing three times, and samples were incubated for 1 hour at 37°C. After incubation with 100 µl TMB substrate solution at room temperature for 15 minutes, the reaction was stopped with 50 µl of 2 M H2SO4. The optical density at 450 nm was measured immediately by spectrophotometer.
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Acknowledgments |
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Footnotes |
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References |
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