From the Division of Regulation of Macromolecular Functions, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565, Japan
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ABSTRACT |
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Necdin is a nuclear protein expressed in virtually all postmitotic neurons, and ectopic expression of this protein strongly suppresses the proliferation of NIH3T3 cells. Simian virus 40 large T antigen targets both p53 and the retinoblastoma protein (Rb) for cellular transformation. By analogy with the interactions of the large T antigen with these nuclear growth suppressors, we examined the ability of necdin to bind to the large T antigen. Necdin was co-immunoprecipitated with the large T antigen from the nuclear extract of necdin cDNA-transfected COS-1 cells. Yeast two-hybrid and in vitro binding analyses revealed that necdin bound to an amino-terminal region of the large T antigen, which encompasses the Rb-binding domain. Moreover, necdin bound to adenovirus E1A, another viral oncoprotein that forms a specific complex with Rb. We then examined the ability of necdin to bind to the transcription factor E2F1, a cellular Rb-binding factor involved in cell-cycle progression. Intriguingly, necdin, like Rb, bound to a carboxyl-terminal domain of E2F1, and repressed E2F-dependent transactivation in vivo. In addition, necdin suppressed the colony formation of Rb-deficient SAOS-2 osteosarcoma cells. These results suggest that necdin is a postmitotic neuron-specific growth suppressor that is functionally similar to Rb.
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INTRODUCTION |
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In the vertebrate central nervous system, neurons withdraw from the cell cycle immediately after differentiation from their proliferative precursors, and remain in the postmitotic state all of their lives. Differentiated neurons are absolutely incapable of dividing even in the presence of chemical and physical stimuli that promote cell-cycle progression of proliferative cells. However, little is known about molecular mechanisms underlying the permanent quiescence displayed by all neurons. Several previous studies have suggested that the retinoblastoma protein (Rb),1 a well characterized growth suppressor protein, is involved in neuronal differentiation-associated growth arrest. In the brain of Rb-deficient mouse embryos, aberrant mitotic figures accompanied by massive neuronal death are observed particularly in the hindbrain, spinal cord, and sensory ganglia (1-3). In cultured murine embryonal carcinoma cells, Rb is markedly induced during neural differentiation (4). Expression of adenovirus E1A, an oncoprotein that suppresses Rb functions, impairs neuronal differentiation and induces cell death (5). These findings raise the possibility that Rb plays a critical role in cell-cycle arrest of certain types of neurons during differentiation. However, the fact that many neurons still differentiate properly in Rb-deficient mice (1-3) suggests that other growth-suppressive proteins compensate the loss of Rb functions in neurogenesis.
We have previously isolated a novel cDNA sequence encoding a 325-amino acid residue protein, termed necdin, from a subtraction cDNA library of neurally differentiated murine embryonal carcinoma cells (6). Necdin is a nuclear protein, whose gene is expressed in virtually all postmitotic neurons in the central and peripheral nervous systems of mice (7, 8). The necdin gene is expressed in postmitotic neurons derived from embryonal carcinoma cells, but not in transformed cell lines originating from neuroblastomas and pheochromocytomas even after they are induced to differentiate (7). In developing mouse brain, the necdin gene is constitutively expressed in neurons from early embryonal stages (e.g. embryonic day 10 at the forebrain) until adulthood, whereas necdin mRNA is undetectable in neuronal precursor cells (i.e. neuroepithelial stem cells) in the neural tube (8). These observations suggest that necdin is expressed in postmitotic neurons that are differentiated from their precursor cells in an irreversible manner. Furthermore, the fact that ectopic expression of necdin strongly suppresses the growth of proliferative NIH3T3 cells (9) leads to the speculation that necdin acts as a growth suppressor in postmitotic neurons.
The tumor suppressor gene products Rb and p53 are nuclear proteins that interact with transforming proteins encoded by small DNA tumor viruses such as simian virus 40 (SV40), adenovirus, and human papillomavirus (10). For example, SV40 large T antigen binds to both Rb and p53, whereas adenovirus E1A and E1B form specific complexes with Rb and p53, respectively. These viral transforming proteins target cellular growth suppressors that are operative in normal cells. Here we demonstrate that necdin binds to SV40 large T antigen and adenovirus E1A, both of which interact with Rb. Moreover, we found that necdin, like Rb, interacts with the transcription factor E2F1, which promotes cell-cycle progression. Necdin functionally replaces Rb as a growth suppressor in Rb-deficient SAOS-2 cells, leading to the suggestion that necdin is a neuron-specific growth suppressor that is functionally similar to Rb.
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EXPERIMENTAL PROCEDURES |
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Western Blotting and Immunoprecipitation--
Necdin
cDNA-carrying p94BFL was transfected into COS-1 cells by
DEAE-dextran method as described (6). Nuclear extracts were prepared
from COS-1 cells 48 h after transfection (11), separated by 10%
SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to
Immobilon membrane (Millipore) by electroblotting. The membrane was
incubated with the antibody against necdin (antibody C2) (1:500) (6) or
anti-SV40 large T antigen monoclonal antibody (1:500) (a gift from Dr.
N. Yamaguchi, University of Tokyo). The proteins were detected by the
avidin-biotin-peroxidase complex technique using a kit (Vector Labs).
For immunoprecipitation of necdin-large T antigen complex, the nuclear
extract was dialyzed against buffer N (20 mM Tris-HCl (pH
7.5), 100 mM NaCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride), and incubated with
antibody C2 for 1 h at room temperature. After adding the equal
volume of 50% protein A-Sepharose (Pharmacia) slurry suspended in
buffer N, the mixture was incubated for 1 h at 20 °C. Immune
complexes were separated by 10% SDS-PAGE, and analyzed by immunoblot
with the anti-large T antigen antibody. For immunoprecipitation of necdin-E2F1 complex, cDNAs encoding FLAG-tagged E2F1 (amino acids 55-430), necdin (amino acids 1-325), and necdinN (amino acids 110-325) were inserted into pRc/CMV expression vector (Invitrogen) to
make pRc-E2F1*, pRc-necdin, and pRc-necdin
N, respectively. Mouse
E2F1 cDNA used was prepared from mRNA of P19 cells by reverse transcription-polymerase chain reaction, sequenced, and confirmed to be
identical with the reported sequence (12). Sets of these expression
vectors and the expression vector for SV40 large T antigen (pEF321-T, a
gift of Dr. S. Sugano, University of Tokyo) were transfected into
~70% confluent SAOS-2 cells in a 60-mm dish by the calcium phosphate
method (13). Nuclear extracts and their immunoprecipitates of
cDNA-transfected SAOS-2 cells were prepared, and the proteins were
detected by antibodies C2 and anti-FLAG M2 (Kodak) as described
above.
Two-hybrid Assay--
GAL4 DNA-binding domain vector (pGBT9),
GAL4 activation domain vector (pGAD424), pTD1 encoding SV40 large T
antigen, and pVA3 encoding mouse p53 were purchased from
CLONTECH. Rb cDNA and adenovirus type 5 E1A
gene were provided by Dr. T. Akiyama (Osaka University) and Dr. K. Shiroki (University of Tokyo), respectively. DNA fragments for hybrid
proteins were generated by polymerase chain reaction using synthetic
oligonucleotide primers with restriction sequences at both ends. After
treatment with respective restriction enzymes, the fragments were
directionally inserted in pGBT9 and pGAD424, and introduced into
Saccharomyces cerevisiae SFY 526. Transformants were spread
onto a 100-mm dish, and selected for both leucine and tryptophan
requirements. The above procedure and colony lift filter assay for
-galactosidase activity were carried out as recommended by CLONTECH.
The reaction was evaluated 4 ranks with the time for the appearance of
blue colonies at 30 °C: +++, less than 2 h; ++, 2-6 h; +,
6-12 h;
, remaining white over 12 h.
In Vitro Binding Assay-- EcoRI-BamHI fragments were excised from inserted cDNAs in pGAD424, and subcloned directionally into pMALC2 (New England Biolabs) to make maltose-binding protein (MBP) fusion proteins, which were purified as recommended by New England Biolabs. RNA was synthesized in vitro by transcribing linearized Bluescript II (Stratagene) carrying cDNA for necdin (amino acids 1-325) or Rb (amino acids 379-928) with T7 RNA polymerase (New England Biolabs), and translated in the rabbit reticulocyte lysate system (Promega) supplemented with [35S]methionine (Amersham). For the large T antigen binding, 10 µl of the translation reaction mixture was incubated for 2 h at 4 °C with 150 µl of binding buffer A (20 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 1 mM EDTA, 2% bovine serum albumin, 0.2 mM phenylmethylsulfonyl fluoride) containing MBP fusion proteins bound to amylose resin (5 µg). For E1A binding, 10 µl of the translation reaction mixture was incubated for 30 min at 4 °C with 150 µl of binding buffer B (50 mM Hepes (pH 7.0), 500 mM NaCl, 0.1% Nonidet P-40, 0.2 mM phenylmethylsulfonyl fluoride) containing MBP fusion proteins bound to amylose resin (5 µg). After washing three times with binding buffer A, bound 35S-labeled proteins were eluted with 20 mM maltose, separated by 10% SDS-PAGE, and visualized by fluorography.
Reporter Assay for E2F Site-dependent
Transcription--
Three wild-type E2F motifs (14) were linked to the
minimal L23 gene promoter (15), and inserted in the luciferase reporter plasmid PGV-B (Toyo Ink). SV40 early promoter and human polypeptide chain elongation factor 1 (EF-1
) promoter were excised from PGV-C
(Toyo Ink) and pEF-BOS (a gift from Dr. S. Nagata, Osaka University),
respectively, inserted in PGV-B, and used as controls. cDNAs
encoding Rb (amino acids 379-928) and E2F1 (amino acids 55-430) were
inserted in pRc/CMV to make pRc-Rb and pRc-E2F1, respectively. Sets of
plasmids were co-transfected into ~70% confluent SAOS-2 cells in a
35-mm dish by the calcium phosphate method (13). Luciferase activities
were measured using a luminometer (Lumat LB9501, Berthold). A LacZ
reporter plasmid (pRc-LacZ) was constructed by inserting pCH110-derived
LacZ gene in pRc/CMV, and co-transfected (1 µg/assay) with the
reporter genes for normalizing the activities.
Colony Formation Assay--
The assay was carried out as
described previously (16): SAOS-2 cells were grown to ~70%
confluence, and transfected with pRc/CMV, pRc-necdin, pRc-necdinN,
or pRc-Rb (10 µg each per 60-mm dish) by the calcium phosphate method
(13). G418 (500 µg/ml) was added to the culture medium 48 h
after transfection. The cells were incubated for 14 days, fixed with
10% acetate, 10% methanol for 15 min, and stained with 0.4% crystal
violet in 20% ethanol for 15 min for visualizing the colonies. For
immunocytochemistry, SAOS-2 cells were fixed with 0.5%
paraformaldehyde solution for 15 min on ice, permeabilized with
methanol at 25 °C, stained with antibody C2 by the
avidin-biotin-peroxidase complex method (Vector Labs), and photographed
with a phase-contrast microscope.
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RESULTS |
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Necdin Interacts with SV40 Large T Antigen-- COS-1 cells, a monkey kidney cell line transformed by SV40, constitutively express the large T antigen, which forms stable complexes with Rb and p53 (17). We have previously found that ectopic necdin is accumulated in the nucleus of cDNA-transfected COS-1 cells (6). To examine whether necdin interacts with SV40 large T antigen in the nucleus in vivo, necdin cDNA was transiently transfected into COS-1 cells. By Western blot analysis, similar levels of the large T antigen were present in untransfected and necdin cDNA-transfected COS-1 cells (Fig. 1A, lanes 1 and 2), and a ~45-kDa band of necdin was detected in the cDNA-transfected cells (Fig. 1A, lane 4). The large T antigen was co-immunoprecipitated with necdin from the nuclear extract of the cDNA-transfected cells (Fig. 1B, lane 4, compared with negative controls in lanes 2 and 3), suggesting that ectopic necdin forms a specific complex with the large T antigen in the nucleus in vivo.
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Necdin Interacts with Adenovirus E1A--
Rb forms a specific
complex with adenovirus oncoproteins E1A, whereas p53 interacts with
E1B (10). Rb-binding sites of the large T antigen and E1A contain the
LXCXE motif (X = any amino acid)
(19). Our data that necdin and Rb share the ability to bind to the
NH2 terminus of the large T antigen led us to examine whether necdin interacts with adenovirus E1A. We used part of the
E1A gene (amino acids 1-185) including three functional domains designated conserved regions (CR) 1-3, of which CR1 and CR2 possess transforming activities (20). Both necdin and Rb bound to E1A (amino
acids 1-185), with which p53 failed to interact (Fig.
4A). We then tested whether
necdin binds to three types of deletion mutants; E1ACR2 (lacking
CR2), E1A
CR2/3 (lacking CR2 and CR3), and E1A-CR3 (containing only
CR3). Necdin bound weakly but significantly to both E1A
CR2 and
E1A
CR2/3, but failed to bind to E1A-CR3. On the other hand, Rb
failed to bind to the three deletion mutants. It was confirmed, by
in vitro binding assay, that both necdin and Rb bound to E1A
(Fig. 4B). Necdin bound, albeit weakly, to E1A
CR2 (Fig.
4B, left panel, lane 3) and E1A
CR2/3 (data not shown).
Since CR2 contains the LXCXE motif (19), it is
suggested that this motif is less requisite for binding to necdin.
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Necdin Interacts with E2F1--
Since necdin and Rb show similar,
if not identical, binding characteristics toward the large T antigen
and E1A, we then examined whether necdin interacts with cellular
Rb-binding factor E2F, which directly regulates the transcription of a
diverse set of genes involved in DNA replication and cell growth
control (19). We first analyzed the binding of necdin to E2F1 by the
two-hybrid assay (Fig. 5A).
Both necdin and Rb bound to F2F1 (amino acids 55-430), which covers
the domains for cyclin A binding, DNA binding, and transcriptional
activation that comprises the Rb-binding domain (19). Necdin bound
weakly to a carboxyl (COOH)-terminally truncated form lacking the
Rb-binding domain (21) (E2F1RB), but failed to interact with a form
devoid of the entire transactivation domain (22) (E2F1
TA). These
results suggest that necdin binds to the transactivation domain of E2F1
to modulate E2F-driven transcription.
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Necdin Represses E2F Site-dependent
Transcription--
We then examined whether necdin represses
E2F-driven transcription in vivo using SAOS-2 cells (Fig.
7A). Necdin and Rb reduced the
basal (intrinsic) E2F site-dependent transcription to 57 and 40%, respectively. E2F1 increased 13-fold the basal
transcriptional activity, and necdin and Rb repressed the activation to
41 and 52%, respectively. Necdin had no effect on SV40 early promoter activity. In this analysis, the suppression of E2F
site-dependent transcription by the truncated form of Rb
(amino acids 379-928) was weaker than the previous report in which
full-length Rb was used (23). We analyzed the expression of Rb in
transfected SAOS-2 cells by Western blotting, and found that a
considerable amount of Rb immunoreactivity underwent degradation (data
not shown), inferring that the weak suppression by Rb is attributable,
at least in part, to its metabolic instability. The inhibition of E2F1-driven transcriptional activity by necdin was
concentration-dependent, and the maximum suppression was
~30% of the control value (Fig. 7B, left panel). On the
other hand, the NH2-terminally truncated form of necdin
(necdinN, amino acids 110-325), which lacks E2F1 binding activity,
exhibited no inhibition. Full-length necdin showed no inhibition of
EF-1
promoter activities at the concentrations that repressed the
E2F site-dependent transactivation (Fig. 7B, right
panel). These results suggest that necdin specifically suppresses E2F1-driven transcription in vivo by interacting with the
transactivation domain of E2F1.
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Necdin Suppresses the Growth of Rb-deficient Cells--
SAOS-2
cells are Rb-deficient osteosarcoma cells, whose growth is inhibited by
reintroduction of wild-type Rb (16). Expression of the functional
domain of Rb (amino acids 379-928) suppresses the formation of
macroscopic colonies of SAOS-2 cells (16). Thus, we examined the growth
suppressive effect of necdin in this assay system. Ectopic necdin
markedly suppressed the colony formation of transfected SAOS-2 cells
(Fig. 8A). On the other hand,
the NH2-terminally truncated necdin (necdinN, amino
acids 110-325) exerted little or no growth suppression of SAOS-2
cells. To rule out the possibility that the repressed colony formation
by necdin is attributable to its cytotoxicity, we analyzed the levels
of necdin expressed in these transfectants. Similar amounts of necdin were detected in cell lysates from the 4- and 18-day cultures (Fig.
8B), suggesting that the suppression by necdin is due to its
constitutive expression and not to the cytotoxic effect. Immunoreactive necdin was localized to the nuclei of transfected SAOS-2 cells (Fig.
8C). Moreover, the necdin-positive cells exhibited a
severalfold increase in size, a typical phenotype displayed by Rb
cDNA-transfected SAOS-2 cells (16). These results suggest that
necdin serves as a substitute for Rb in these Rb-deficient cells.
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DISCUSSION |
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This study has shown that necdin interacts with SV40 large T antigen and adenovirus E1A, both of which possess cellular transforming activities. Necdin bound to the NH2-terminal region of the large T antigen (amino acids 84-120) (see Fig. 3). The NH2-terminal fragment of the large T antigen (amino acids 1-120) induces cellular transformation, and missense mutations and short deletions in this region are defective in transformation (17). Thus, necdin potentially inhibits the transforming activity of the large T antigen by binding to the NH2-terminal region. Moreover, necdin bound to the region encompassing two conserved regions, CR1 and CR2 of E1A (see Fig. 4), both of which are responsible for cellular transformation and induction of cellular DNA synthesis (20). We failed to obtain replication-defective recombinant adenovirus carrying necdin cDNA from 293 cells, a cell line constitutively expressing adenovirus oncoproteins,2 suggesting that ectopic necdin inactivates endogenous E1A that promotes the replication of adenovirus DNA. These findings raise the possibility that necdin modifies or neutralizes the transforming activities of these viral oncoproteins. Conversely, it seems likely that these viral oncoproteins target necdin for cellular transformation because this nuclear protein is normally involved in growth-suppressive mechanisms.
Necdin is functionally similar, but structurally dissimilar to Rb. Besides necdin and Rb, two Rb-related proteins, p107 and p130, interact with the large T antigen and E1A (24). These Rb family proteins are structurally similar, and comprise the binding region designated "pocket domain," with which various factors such as viral oncoproteins and cellular transcription factors interact (25). Necdin consists of 325 amino acid residues (6), much smaller than Rb family proteins, and no homologous sequences are found between necdin and Rb family proteins. We found recently that amino acid sequences of the core functional domain of necdin (amino acids 83-292, see Fig. 6) are highly conserved (91% identity) between human and mouse, whereas the sequences of the NH2-terminal region (amino acids 1-82) are less conserved (60% identity).3 Therefore, this region may be evolutionally conserved because of its functional importance.
The biological significance of the formation of necdin-E2F1 complex in postmitotic neurons remained to be elucidated: postmitotic neurons differentiated from P19 cells contain high levels of mRNAs for E2F1 (5), necdin (6), and Rb (4), leading to speculation that E2F1 activities in these neurons are regulated by Rb and necdin in combination. As shown presently, necdin binds to the transactivation domain of E2F1, and represses E2F1-induced transactivation in vivo. E2F-binding sites exist within the promoters of a number of cellular genes involved in cell-cycle progression (19). Thus, it is tempting to speculate that necdin, in cooperation with Rb, suppresses the expression of a battery of genes required for DNA replication by silencing E2F1 in postmitotic neurons. We are currently investigating whether necdin substantially controls E2F1 functions in postmitotic neurons by transferring cDNAs encoding necdin and E2F1 using recombinant adenovirus vectors.
Rb is expressed in both neuroepithelial stem cells and postmitotic neurons (26), and thus potentially induces growth arrest of the stem cells at the very beginning of neuronal differentiation. On the other hand, necdin is expressed only in postmitotic neurons (7, 8), suggesting that necdin has a function to keep differentiated neurons staying in the postmitotic state. Rb loses its growth inhibitory functions upon phosphorylation by D-type cyclins and cyclin-dependent kinases (27). Interestingly, these cyclins and kinases physiologically coexist with the Rb-E2F system in postmitotic neurons (5, 28). If Rb is the sole molecule that arrests the cell cycle of differentiated neurons, then pathological or accidental phosphorylation of Rb would lead quiescent neurons to undergo abortive mitosis and death. Thus, necdin might be involved in a "fail-safe" mechanism, complementing Rb to prevent postmitotic neurons from resuming cell division. Studies using necdin-defective animal models are currently under way in our laboratory to clarify the implications of necdin in neuronal postmitotic phenotype in vivo. Further information about interactions between necdin and cell-cycle regulatory machinery will lead to a better understanding of molecular mechanisms underlying permanent quiescence displayed by all nerve cells.
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ACKNOWLEDGEMENTS |
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We thank Drs. Y. Hayashi and K. Matsuyama for information about yeast two-hybrid assay, and Drs. T. Uetsuki and M. Niinobe for advice and discussions.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to K. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Div. of Regulation of
Macromolecular Functions, Institute for Protein Research, Osaka
University, Yamadaoka 3-2, Suita, Osaka 565, Japan. Tel.: 81-6879-8621;
Fax: 81-6879-8623; E-mail: yoshikaw{at}protein.osaka-u.ac.jp.
1
The abbreviations used are: Rb, retinoblastoma
protein; SV40, simian virus 40; PAGE, polyacrylamide gel
electrophoresis; MBP, maltose-binding protein; EF-1, polypeptide
chain elongation factor 1
; CR, conserved region.
3 Y. Nakada and K. Yoshikawa, manuscript in preparation.
2 T. Uetsuki and K. Yoshikawa, unpublished observations.
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REFERENCES |
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