Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA
Author for correspondence (e-mail: savraham{at}bidmc.harvard.edu)
Accepted 3 August 2005
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Summary |
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Key words: NRP/B, Nuclear matrix, BTB/POZ domain, Neuronal differentiation, Neurite outgrowth
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Introduction |
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We have previously discovered and characterized NRP/B (nuclear-matrix-restricted protein/brain) (Kim et al., 1998) that is expressed in the nucleus as a component of the nuclear matrix in neuronal cells. As reported during the sequence analysis of the human genome (McPherson et al., 2001
; Venter et al., 2001
), NRP/B is a member of a growing family of proteins that contains two major structural elements: a BTB/POZ domain in the N-terminus and a kelch motif in the C-terminus. The finding that the kelch motif and BTB/POZ-domain-containing proteins are highly abundant in the human genome, suggests the importance of these motifs as components of genes that are highly conserved during evolution. NRP/B mRNA (5.5 kb), also termed ENC-1 (Hernandez et al., 1997
), encodes an unusually evolutionarily conserved protein between human and mouse (Hernandez et al., 1997
; Kim et al., 1998
).
Interestingly, a gene termed PIG 10 (p53-induced protein 10) was identified by overexpression of wild-type p53 in DLD1 colon cancer cells (Polyak et al., 1997). PIG 10, which contains multiple mutations and deletions, is a variant form of NRP/B/ENC-1. Elevated expression of PIG 10 in colon cancers appears to occur in a p53-independent fashion (Polyak et al., 1997
). NRP/B is predominantly expressed in human fetal and adult brain. It is specifically expressed in neurons but not in primary astrocytes (Kim et al., 1998
). During mouse embryogenesis, NRP/B/ENC-1 mRNA expression is upregulated in the nervous system. NRP/B/ENC-1 is expressed early in gastrulation in the prospective neuroectodermal region of the epiblast, and later in development, throughout the nervous system. NRP/B expression is highly dynamic and after neurulation, preferentially defines the prospective cortical area (Hernandez et al., 1997
; Kim et al., 1998
).
Although the expression of NRP/B/ENC-1 is most abundant in fetal brain, NRP/B is also expressed in fetal kidney, lung, heart and liver (Kim et al., 2000) and this expression is diminished significantly in adult organs (Kim et al., 1998
). Thus, NRP/B may play a role during differentiation in other cell lineages. Indeed, the expression of NRP/B was increased in neuroblastoma cells in response to retinoic acid (Kim et al., 1998
) and in adipocytes treated with the phosphodiesterase inhibitor methylisobutylxanthine (Zhao et al., 2000
).
The BTB domain is found primarily in several zinc-finger-containing transcription factors and defines a newly characterized protein-protein interaction interface (Bardwell and Treisman, 1994; Collins et al., 2001
; Godt et al., 1993
; Zollman et al., 1994
). The BTB/POZ domain mediates both homodimerization and heterodimerization in vitro (Bardwell and Treisman, 1994
; Chen et al., 1995
; Soltysik-Espanola et al., 1999
). Several evolutionarily conserved residues within the BTB/POZ domain are critical for its dimerization. It was reported that dimerization of this domain was essential for the proper folding of the entire protein. In addition, the biological functions of the BTB/POZ domain in development, homeostasis, neoplasia and transcriptional repression were reported. Study of the BTB domain in PLZF, Bcl-6 and PLZF-RAR
indicated that this domain was essential for dimerization, transcriptional repression and nuclear localization (Dhordain et al., 1995
; Dong et al., 1996
).
Members of the kelch family are important for cytoskeletal organization and function (Varkey et al., 1995). Based on our studies (Kim et al., 1998
), NRP/B is a nuclear matrix protein as defined by biochemical and confocal analyses as well as by electron-microscopy analysis. Our previous work demonstrated that NRP/B is expressed specifically in neurons and contains two isoforms: NRP/B p57 (soluble) and p67 (less soluble) (Kim et al., 1998
). We found that the p57 form of NRP/B associates with p110RB and is involved in neuronal differentiation (Kim et al., 1998
). Furthermore, our recent report showed that NRP/B expression is upregulated in human brain tumors including glioblastomas and astrocytomas, whereas under normal conditions NRP/B expression is restricted to the nuclei of neurons (Liang et al., 2004
).
In the present study, we examined the structural and functional aspects of the BTB domain of NRP/B using three-dimensional structural analysis. This structural analysis allows for the examination of spatial, electrostatic, and hydrophilic/hydrophobic potential bindings, and of the relationships of the substitute residue with neighboring residues on the same or separate proteins. It provides an avenue to examine numerous potential changes, with the intention of selecting one or two critical sites for the actual cloning experiments. We therefore used three-dimensional modeling of the BTB domain using known crystal structures such as the PLZF BTB/POZ domain (Protein DataBank code 1BUO and 1CS3A), to elucidate the biological function of BTB in the brain. Coupling of sequence homology analysis with the three-dimensional model indicated several potential protein-protein interaction sites for NRP/B. Both targeted mutagenesis studies and crosslinking experiments showed that the N-terminus BTB domain of NRP/B is important for dimerization. To assign functions to the BTB domain of NRP/B, we expressed epitope-tagged NRP/B in neuronal cells, examined its ability to modulate neurite outgrowth, and also analyzed the subcellular distribution of mutant and wild-type NRP/B. In addition, we used Tet on/off as well as siRNA and single-cell microinjection approaches to determine the role of NRP/B in PC12 cells. These studies provide insights into the molecular structure of the NRP/B-BTB domain and its role in neuronal differentiation and neurite outgrowth.
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Materials and Methods |
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Three-dimensional modeling of NRP/B
Both the BLAST and AlignMaster programs use the known PLZF BTB/POZ domain crystal structure (that was previously analyzed in detail) as a three-dimensional template. The structure of the human PLZF BTB/POZ domain was obtained from the Protein DataBank (Protein DataBank codes 1BUO and 1CS3A). Superimposition, model building, construction of insertion regions, structure validation and calculation of structural properties were carried out using the subprograms ProMod v3.5, SPDBV v3.5, Loop v2.60, LoopDB v2.60, Parameters v3.5 and Topologies v3.5, which are available in the Automated SwissModel Package Program (www.expasy.ch/swissmod). RasWin v3.2b2a and Cn3D v4.0 (www.ncbl.nlm.nih.gov/Structure/CN3D/cn3d.html) were used to display the molecular structures.
DNA constructs, site-directed mutagenesis and sequencing
NRP/B-GFP constructs were generated by adding GFP tags to the NRP/B cDNA constructs including: NRP/B full length, NRP/B-BTB domain (amino acids 1-128) and NRP/B-kelch domain (a.a. 296-589) as shown in Fig. 2. Polymerase chain reactions were used for the site-directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene, La Jolla, CA). All mutants were sequenced and confirmed by automated PCR sequencing (BIDMC, Sequencing Core Facility).
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Expression and purification of NRP/B-(His)6 in SF-9 insect cells
Wild-type and mutant NRP/B-(His)6 baculovirus constructs were generated as described (Kim et al., 1998). The baculovirus construct was used to infect SF-9 cells grown in Grace's media. Infected cells were harvested 72 hours post-infection. Recombinant protein was purified on a Cobalt-Sepharose column. About 2 mg purified protein can be obtained from a 1-liter culture with >90% purity.
Association analysis of the BTB/POZ domain
293T cells were transfected with mutated and wild-type NRP/B cDNA constructs. Similar expression levels of wild-type and mutated NRP/B were observed. The cells were lysed in PBS containing 8 M urea. 200 µg total cell lysates were incubated in Ni-NTA HisSorb 96-well plates (Qiagen) which had bound 1 µg purified NRP/B-(His)6. The association mixture was diluted ten times with PBS for refolding, and for 10 minutes to form a dimer complex of purified NRP/B-(His)6 and mutant NRP/B-GFP. The wells were washed six times with PBS containing 0.05% Tween-20. The associated mutant NRP/B-GFP was solubilized in 40 µl of 2x Laemmli SDS sample buffer and analyzed by 10% SDS-PAGE. The blots were probed with anti-GFP polyclonal antibody (Clontech, 1 µg/ml final concentration) and visualized by enhanced chemiluminescence (ECL) reagent.
Immunolocalization by light and confocal microscopy
Two days after transient transfection, COS7 cells were fixed in 4% paraformaldehyde solution, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature and treated with blocking buffer (1.0% goat serum and 0.1% BSA in PBS). The Vectastain Elite ABC kit (Vector Laboratory, Burlingame, CA) was used with an anti-FLAG monoclonal antibody (M5, Kodak) or with specific anti-NRP/B monoclonal antibody generated in our laboratory as described (Kim et al., 1998). The staining by peroxidase reaction was monitored under a Nikon Diaphot 300 inverted microscope.
Expression of the N-terminal portion of NRP/B containing the BTB domain in Escherichia coli fused to glutathione-S-transferase (GST)
A GST-N-terminal NRP/B (GST-N-NRP/B) construct was generated by PCR using NRP/B-specific oligonucleotides containing restriction site linkers, and then subcloned into the pGEX 4T-2 vector (Amersham Pharmacia). This construct was used to transform E. coli DH5 and was analyzed by sequencing. GST-N-terminal NRP/B fusion protein expression was induced using 10 mM isopropyl ß-thiogalactopyranoside. Recombinant protein was affinity-purified using glutathione-Sepharose 4B beads, according to a protocol by Pharmacia (GST Gene Fusion System, Second Edition, 1996) with the exception of elution steps, which were performed on ice instead of at room temperature. Purified protein solubilized in glutathione elution buffer (Pharmacia) was dialyzed against cold PBS and stored in 50% ultrapure glycerol (United States Biochemical, Cleveland, OH) in 1x PBS at -20°C.
Crosslinking of GST-N-NRP/B fusion protein
The crosslinking experiment was performed similarly as in our previous report on Mayven (Soltysik-Espanola et al., 1999). The GST peptide was removed from the GST-N-NRP/B fusion protein by adding
2 U thrombin directly to
300 mg of fusion protein after elution from glutathione-agarose beads. The protein was then incubated in PBS (Pierce) with 10 mM dithiobis succinimidyl propionate (DSP), a bifunctional protein crosslinking reagent, freshly prepared as a 450 mM stock in DMSO (Sigma). Crosslinking was carried out at room temperature for 30 minutes and was terminated by adding either 2x Laemmli buffer, or 2x Laemmli buffer with DTT (an S-S reducing agent). Proteins were boiled for 5 minutes, resolved on a 4-12% gradient gel (BioRad, Hercules, CA), and visualized by staining with Coomassie Blue.
Microinjection of NRP/B antibodies
The micromanipulator and microinjector were purchased from Eppendorf (Madison, WI). For the microinjection studies, PC12 cells were plated at 105 cells/ml in 60 mm dishes precoated with poly-D-lysine (50 µg/ml). Microinjection of each antibody into the nucleus was performed with prepulled microcapillaries (Eppendorf). The micropipette was controlled using an Eppendorf micromanipulator attached to a Nikon Diaphot inverted microscope. Injection pressure was controlled using the Eppendorf Microinjector model, 5242. Affinity-purified mouse specific monoclonal anti-Nrp/b antibodies and pEGFP-N2 vector (1:1, v/v) were diluted to a final concentration of 100 µg/ml in microinjection buffer (75 mM KCl, 10 mM potassium phosphate, pH 7.2). Injection volume was 10-12 µl/cell. After microinjection, fresh DMEM supplemented with 0.1% horse serum with or without NGF (50 ng/ml) was added to the PC12 cells, followed by 48 hours of incubation. Microinjected purified mouse IgG or non-relevant purified monoclonal antibody was used as a control. The pEGFP-N2 vector was used as an indicator for the injection of NRP/B antibody. The number of differentiated cells was counted based on the length of neurites that were more than twofold the length of their cell body, as previously described (Kim et al., 1998
).
Cell cycle analysis
PC12 cells were transfected with pEGFP vector harboring wild-type NRP/B and/or NRP/B mutants. Green fluorescent protein (GFP)-positive cells were sorted and the cell cycle was analyzed by FACS.
Preparation of siRNA and Transfection
The siRNA gene-specific SMARTpool for NRP/B was purchased from Dharmacon. Transfection of siRNA was performed with Lipofectamine 2000 (Life Technologies) in 24- and six-well plates. Briefly, Lipofectamine diluted in Opti-MEM (3 µl/50 µl) was incubated for 5 minutes. Empty pCMS-EGFP vector (1 µg) and siRNA (80 pmol) were diluted in DMEM (50 µl) and added to the Lipofectamine mixture. The incubation was continued for an additional 20 minutes before addition to cultures and transfection. For the neuritogenesis assays, PC12 cells were treated with NGF (50 ng/ml) 2 days after transfection and then monitored at various times thereafter for the proportion of cells with processes longer than two cell bodies in diameter (20 µm). For the neurite stability assays, PC12 cells were pretreated with NGF for 5 days, transfected, and scored at various times thereafter for the proportion of neurite-bearing cells.
Semi-quantitative RT-PCR
To investigate the effect of NRP/B siRNA on NRP/B levels, cells were transfected with siRNA at a final concentration of 20 µM. Total RNA extraction was carried out after 48 hours using Trizol Reagent (Life Technologies). One Step RT-PCR was performed as per the manufacturer's instructions using a set of specific primers (5'-GCCCTCTTCCTTCTGGGAG-3' and 5'-AGAAGGCAGATGTTTCCAG-3'). The reaction proceeded for 60 minutes at 50°C and then 5 minutes at 94°C. This was followed by 30 cycles with a 94°C denaturation for 30 seconds, 65°C annealing for 30 seconds and 68°C extension for 1 minute. The amplification generates a product of 885 bp of NRP/B.
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Results |
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The sites for the point mutations are as follows: D47A, H60A and R61D. The proposed interactions of these mutation sites are electrostatic, hydrophilic and electrostatic, respectively. The D47A mutation is aimed at neutralizing charge, the H60A mutation is aimed to reduce van der Waals and the R61D mutation is aimed at reducing charge reversal.
The three-dimensional structure of the BTB domain forms potential pocket-like structures that can act as a docking site for the binding partners (Fig. 1B). Moreover, these pocket sites contain highly conserved amino acids that imply the importance of these sites for NRP/B function.
As several BTB-related proteins have been shown to form homo- and/or hetero-dimers (Soltysik-Espanola et al., 1999), the mutated BTB domains of NRP/B were investigated for their dimerization potential. The dimerization property of the NRP/B-BTB domain was examined by (His)6 pull-down assays using wild-type and mutant NRP/B (Fig. 1C). The full-length NRP/B formed homodimers of NRP/B. Moreover, significant inhibition of dimerization was shown with the NRP/B-BTB mutant A. These results indicate that D47A, H60A and R61D within the BTB domain are important sites for protein-protein interactions that may facilitate dimerization of NRP/B.
The recombinant BTB domain of NRP/B dimerizes in solution
It has been shown that the BTB domain of several proteins forms homodimers (Dhordain et al., 1995). Other BTB/POZ-containing transcription factors can form both homodimers and heterodimers, which have been demonstrated to regulate the transcriptional activity of zinc-finger proteins (Bardwell and Treisman, 1994
). To determine whether NRP/B can form dimers in solution, in a similar manner as we demonstrated previously in Mayven (Soltysik-Espanola et al., 1999
), the NRP/B-BTB domain was cleaved away from the GST sequence by thrombin and then crosslinked by DSP, a bifunctional protein crosslinking agent. DSP forms stable linkages between free amine groups of interacting proteins, and these linkages are cleavable with S-S reducing reagents. We observed dimerization of the NRP/B-BTB domain in the non-reduced gel as compared with the reduced gel of the crosslinked NRP/B and with the non-crosslinked NRP/B run under non-reducing conditions (Fig. 1D). Thus, the BTB domain of the NRP/B protein dimerized in solution.
The NRP/B-BTB domain is essential for nuclear localization
Our previous work showed the nuclear localization of endogenous NRP/B in primary hippocampal neurons and human neuroblastoma cells (SH-SY5Y) (Kim et al., 1998). To investigate which domain of NRP/B is important for its subcellular localization, plasmids expressing the BTB domain and the kelch motif were constructed and used for the transfection studies. Transfection studies in PC12 cells (which express endogenous NRP/B) revealed that NRP/B wild-type (WT) and the NRP/B-BTB domain were expressed in the nucleus in these cells (Fig. 2). However, when NRP/B-kelch constructs were transiently transfected, PC12 cells showed cytoplasmic perinuclear expression of NRP/B (Fig. 2). Of note, we assessed the survival of PC12 cells transfected with various NRP/B constructs and observed similar levels of cell survival under the different conditions (93-97%). In addition, we did not observe any cytotoxic effects of these constructs following transfection in PC12 cells. These results indicate that the BTB domain of NRP/B is necessary and might be crucial for its translocation to the nucleus.
Interaction of the NRP/B-BTB domain with p110RB
We have shown previously the in vivo and in vitro interactions of NRP/B with p110RB (Kim et al., 1998). As the association of NRP/B with p110RB is proposed to be essential in the regulation of neuronal differentiation, we therefore examined which domain of NRP/B directly interacts with p110RB. Both the full-length NRP/B and the NRP/B-BTB strongly interacted with wild-type p110RB (Fig. 3A). The BTB domain of NRP/B specifically interacted with the TR subdomain within the B pocket of p110RB (Fig. 3B). This TR subdomain is known to play a role in the protein association of p110RB with transcriptional activators, such as MyoD (Gu et al., 1993
), which is involved in myogenesis. No interaction was observed between NRP/B-kelch and p110RB. These results confirm our initial studies showing the in vivo and in vitro interaction of NRP/B with p110RB. Furthermore, as shown here, this interaction is mediated through the BTB domain of NRP/B and the B pocket of p110RB, indicating the significance of the NRP/B-BTB domain in neuronal differentiation.
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NRP/B-BTB mutants inhibit neurite outgrowth
To investigate the functional effect of the NRP/B-BTB domain on neurite outgrowth, PC12 cells were transfected with NRP/B-BTB mutants. Although GFP-transfected cells had no effect on neurite outgrowth, NRP/B-BTB had significant induction of neurite outgrowth similar to that obtained with the wild-type NRP/B-transfected cells. Further morphological analysis showed that BTB mutant A significantly reduced neurite outgrowth (by about 70-80%) compared to PC12 cells stimulated with NGF (Fig. 6A) and to the wild-type NRP/B which induced neurite outgrowth (Fig. 6B and 6C). Interestingly, PC12 cells stimulated with NGF also showed low expression of NRP/B in the neurite extensions (T.-A.K. et al., unpublished data). Similar results were obtained with primary hippocampal neurons transfected with NRP/B constructs (Table 1). We did not observe any cytotoxic effects in PC12 cells transfected with the various constructs of NRP/B, and the survival of the cells transfected with the different NRP/B constructs was similar (92-96%). Thus, dimerization and protein-protein interactions (such as NRP/B-p110RB) within the BTB domain of NRP/B, specifically mediated by the amino acids D47A, H60A and R61D, indicate that the BTB domain is essential for neurite outgrowth.
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Microinjection of specific NRP/B antibodies inhibited the neurite outgrowth of PC12 cells
To elucidate the role of NRP/B protein on neurite outgrowth, specific NRP/B antibodies (both monoclonal and polyclonal) or anti-mouse IgG were microinjected into the nuclei of PC12 cells. There were no effects observed in PC12 cells co-microinjected with control IgG and GFP vector in the absence or presence of NGF (Fig. 7A, panels a-c) or in cells microinjected with monoclonal anti-NRP/B antibody in the absence of NGF (Fig. 7A, panel d). However, NRP/B-specific antibodies blocked the neurite outgrowth of PC12 cells upon NGF stimulation (Fig. 7A, Panels e-f) and (Fig. 7C). Importantly, we observed identical morphological changes to those found using NRP/B antisense oligodeoxynucleotides (Kim et al., 1998). When control antibody (mouse IgG or Csk kinase antibody) was microinjected into PC12 cells followed by NGF stimulation, these cells became differentiated as demonstrated by the appearance of prominent neurite outgrowth similar to that of the PC12 cells treated with NGF alone (Fig. 7B, panels a and b). Thus, these results together with our previously published data (Kim et al., 1998
) strongly suggest that NRP/B expression is involved in the neurite outgrowth of PC12 cells.
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Effects of NRP/B siRNA on neurite outgrowth
To investigate further the function of NRP/B in neuronal differentiation, we used a siRNA construct designed to specifically reduce the cellular levels of the NRP/B transcripts. PC12 cells transfected with siRNA in the presence or absence of NGF showed little or no neurites as compared to control PC12 cells treated with NGF (Fig. 8A,B). This effect was evident 24 hours after transfection and persisted for 3-5 days. To control for the specificity of the siRNA, transfected cells were also immunostained for the closely related family member Mayven (Soltysik-Espanola et al., 1999). The NRP/B siRNA had no detectable effect on the expression of Mayven (Fig. 8C). Such selectivity is consistent with previous reports regarding the specificity of siRNA constructs in mammalian cells (Elbashir et al., 2001
). When the PC12 cells transfected with the NRP/B siRNA construct were assessed for their response to NGF, it was observed that the rate of neurite genesis was greatly diminished in comparison with the untransfected NGF-stimulated cells (Fig. 8A,B). In contrast, a control siRNA construct (which shows no homology with known rat sequences) did not reduce neurite outgrowth in the presence of NGF (Fig. 8A,B), indicating that the actions of the GFP-directed siRNA do not have effects on neurite outgrowth. Of note, a significant decrease in NRP/B mRNA expression was observed in PC12 cells transfected with NRP/B siRNA (Fig. 8C), whereas the levels of GAPDH remained relatively constant under these experimental conditions (Fig. 8C), confirming again the specificity of the effects of NRP/B siRNA.
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Effects of NRP/B siRNA on p110RB phosphorylation
We have shown previously that the association of NRP/B with p110RB was required for neurite outgrowth (Kim et al., 1998). To examine if p110RB is a target of NRP/B, PC12 cells were seeded in six-well plates overnight and transfected with GFP siRNA or NRP/B siRNA. After 24 hours, cells were treated with NGF (50 ng/ml), as indicated. After 48 hours, total cell lysates were prepared and analyzed by western blot analysis using anti-p110RB (RB), anti-phosphoRB (pRB Ser795) or anti-NRP/B (VD2) antibodies. Cells transfected with NRP/B siRNA showed a decrease in p110RB phosphorylation at Ser795 at the G1-S stage of the cell cycle (Fig. 8D), whereas no effect on p110RB protein expression was observed (Fig. 8D). A similar outcome was obtained when the blot was probed for Ser801/811 with anti-phosphoRB (data not shown). These results indicate that NRP/B siRNA leads to a reduction in p110RB phosphorylation.
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Discussion |
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The BTB domain of bric-a-brac, ZID, LAZ/BCL6 and the kelch-motif-containing proteins, can mediate homo- or hetero-dimerization, suggesting that this domain is an important conserved motif for protein-protein interactions (Bardwell and Treisman, 1994; Robinson and Cooley, 1997
). In the bab protein, the first 51 amino acids of the BTB domain and in the Mayven protein, the N-terminus containing the BTB domain were found to be sufficient for dimerization (Soltysik-Espanola et al., 1999
). Interestingly, we have also observed dimerization of NRP/B by both crosslinking experiments (Fig. 1D) and (His)6 pull-down assays using wild-type and mutant NRP/B (Fig. 1C). The BTB domains are found in several actin-binding proteins and in nuclear-DNA-binding proteins (Bomont et al., 2000
). These domains have been proposed to function in DNA-protein and/or protein-protein interactions that organize higher order structures of the cytoskeleton or chromatin (Geisert et al., 1990
). Another proposed function of this domain is protein targeting to specific nuclear domains, as the appearance of ZID, LAZ/BCL6 and hZF5 proteins in nuclear dots depends on the integrity of their BTB domain (Chen et al., 1995
; Sugiura et al., 1997
). The BTB domain of NRP/B, but not the kelch motif, was sufficient for NRP/B translocation to the nucleus (Fig. 2). Although there is no complete classical nuclear localization signal (NLS) in the NRP/B protein sequence, we proposed that its translocation to the nucleus might be either by dimerization of two existing halves of the NLS within the BTB domain or by cotransportation by another nuclear carrier protein (Kim et al., 1998
).
Based on the findings presented here, the N-terminus of NRP/B, within the first 115 amino acid residues (BTB domain) of the protein, was found to contain a potential novel nuclear localization signal and a p110RB binding motif. In tetracycline-inducible stable PC12 cell lines, upregulation of NRP/B corresponded with a significant induction of neurite outgrowth and hypophosphorylation of p110RB (Fig. 4). The mechanism of induction of p110RB hypophosphorylation by NRP/B will be addressed in future studies. Significant changes in neurite length were observed upon overexpression of NRP/B mutants in PC12 cells compared to wild-type NRP/B (Fig. 5). The high level of expression of NRP/B in fetal and adult brains (Kim et al., 1998) and its localization pattern in PC12 cells upon NGF differentiation, imply that NRP/B may play an important role in the differentiation of neuronal cells.
The BTB domain of NRP/B is necessary for its interaction with p110RB (Fig. 3). The amino acids (Asp 47, His 60 and Arg 61) within the NRP/B-BTB domain were found to be important for the NRP/B-p110RB association (Fig. 5). NRP/B specifically interacts with the TR subdomain within the B pocket of p110RB (Fig. 3). This subdomain of p110RB has an important function in protein-protein interactions. It is known for its interaction with transcriptional activators such as MyoD, which is involved in the differentiation of myoblasts (Gu et al., 1993). The TR subdomain is conserved among cyclins, is present in the basal transcription factor TFIIB, and defines a superfamily of nuclear regulatory proteins (Gibson et al., 1994
). p110RB is also a nuclear-matrix-associated protein and has dual roles in cell cycle regulation and neuronal differentiation (Lee et al., 1994
; Mancini et al., 1994
; Slack et al., 1998
). Thus, the NRP/B association with p110RB is through the specific protein-protein interactions of their BTB domain and TR subdomain, respectively. Among the NRP/B-BTB mutants, BTB mutant A abolished the effect of the wild-type NRP/B and significantly affected dimerization and neurite outgrowth (Figs 1 and 5, respectively). It has been shown that DIP (DP-interacting protein) is involved in growth control mediated through its BTB domain. Interestingly, cell cycle analysis, based on sorting the GFP-transfected cells and non-transfected cells using two different excitation emissions, indicated that wild-type NRP/B-GFP increased the G0-G1 population significantly (
23%) compared to levels in the non-transfected cells and mock-transfected PC12 cells (Fig. 5A).
The N-terminus containing the BTB domain of NRP/B (del C) had the same growth suppressive effect as the full-length NRP/B (wt), whereas the C-terminus kelch repeat domain of NRP/B (del N) had no such effects (Fig. 5). These results have been confirmed by transfections with the NRP/B-BTB mutant A. BTB mutant A abolished the effects of the wild-type NRP/B and significantly affected dimerization, neurite outgrowth and cell cycle progression. The negative-growth-regulating properties of DIP are analogous to those possessed by p110RB. It has been reported that the BTB domain directly interacts with the silencing mediator of retinoid and thyroid receptor (SMRT) corepressor to form a transcriptional repressor complex that includes another corepressor, mSIN3A and the HDAC-1 histone deacetylase (Dhordain et al., 1997; Dhordain et al., 1998
; Lin et al., 1998
; Wong and Privalsky, 1998
). Yeyati et al. (Yeyati et al., 1999
) reported that the promyelocytic leukemia zinc finger (PLZF) protein functions as a transcriptional repressor by the binding of its BTB domain to promoters of target genes involved in the regulation of the cell cycle, such as cyclin A. In hematopoietic cells, expression of PLZF leads to growth suppression, cell cycle arrest in G1-S phase, and a differentiation blockade (Reid et al., 1995
). The growth suppressive effect of NRP/B may be mediated through the interaction of its BTB domain with p110RB (Fig. 3) or possibly through other key cell cycle regulatory proteins such as E2F or the cdks. It is conceivable that the recruitment of transcriptional repression by the BTB domain of NRP/B can lead to growth suppression, resulting in neuronal differentiation.
The effect of the NRP/B-BTB mutant on growth suppression was related to its loss of ability to dimerize and to induce neurite outgrowth. Our structure-function studies indicated that amino acids (Asp 47, His 60 and Arg 61) within the highly conserved charged pocket of the NRP/B-BTB domain are important for the function of NRP/B in neuronal cells. Specifically, amino acids (D47 and R61) within the NRP/B-BTB domain are the most conserved charged residues as was also found in the PLZF BTB domain (D35 and R49). Point mutations of these amino acids in the PLZF BTB domain functionally inactivate BTB-dependent repression of transcription while maintaining the ability of these amino acids to dimerize (Melnick et al., 2002; Melnick et al., 2000b
). These data support the hypothesis that NRP/B functions as an important regulator of neuronal differentiation, through protein-protein interactions via the BTB domain. Individual components of NRP/B can create dynamic protein-protein interactions, such as the binding of the NRP/B-BTB domain with p110RB. The intervening sequence domain (IVS) (Fig. 1A) may allow flexible conformational changes by utilizing energy produced from protein-protein interactions or from dimerization of NRP/B. It has been reported that upon dimerization of a protein, potential
-helical IVS changes the conformation of the protein, possibly from an unconstructed state to a stable coiled-coil structure (Carr et al., 1997
). Mutations in the BTB domain of NRP/B led to a significant inhibition of NRP/B dimerization (Fig. 1C).
It has been shown previously that p110RB is important for neurite outgrowth (Kranenburg et al., 1995), and that NRP/B also plays a role in neurite outgrowth (Kim et al., 1998
). Here, we observed that overexpression of the BTB domain induced neurite outgrowth whereas the BTB mutant A abolished this effect. In contrast, downregulation of NRP/B by NRP/B siRNA but not control siRNA significantly reduced neurite production in PC12 cells. In addition, we demonstrated that p110RB is a target of NRP/B, as cells transfected with NRP/B siRNA showed a decrease in p110RB phosphorylation at Ser795 at the G1-S stage of the cell cycle (Fig. 8D) and at Ser 801/811 (data not shown). Thus, association of NRP/B with p110RB is important for the neurite outgrowth of PC12 cells in response to NGF stimulation, and p110RB is a downstream target of NRP/B during the neuronal differentiation of these cells. Our findings are consistent with the microinjection data and with our proposed function of NRP/B in neurite outgrowth.
Nuclear-matrix-anchored NRP/B may have a unique role in cellular functions through its interaction with insoluble cellular components such as actin and chromatin. As we have shown by the in-situ subfractionation of nuclei, insoluble NRP/B mainly was associated with nuclear actin through the kelch motif (T.-A.K. and S.A., unpublished data). The matrix-free soluble form of NRP/B may have other important biological functions such as homo- or hetero-dimerization and/or interaction with p110RB.
Thus, the studies presented here demonstrate that NRP/B, as a neuronal-specific nuclear matrix protein, is involved in neurite outgrowth, and that p110RB is a downstream target of NRP/B. We have further shown that the BTB domain of NRP/B is important for its dimerization and that this domain induced neurite outgrowth through its binding to the B pocket of p110RB. The BTB domain is also important for nuclear localization. The critical residues for these interactions were suggested by three-dimensional modeling and were confirmed by biochemical studies. Our findings, supported by three-dimensional modeling and structure-based targeting, provide further insight into the molecular structure-function of NRP/B and the involvement of its BTB domain in neuronal function, as determined by several methods including microinjection and siRNA approaches.
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