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1Section of Medical and Molecular Genetics, Department of Reproductive and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham, United Kingdom; 2Signal Transduction Laboratory, Cancer Research United Kingdom, London Research Institute, London, United Kingdom; 3Institut de Ginitique Moliculaire, Uniti Mixte de Recherche 5535, Centre National de la Recherche Scientifique, Montpellier, France; 4AG Tumorgenetik der Medizinischen Fakultät, Martin-Luther Universität Halle Wittenberg, Halle (Saale), Germany; 5Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, Texas; and 6Cancer Research United Kingdom, Renal Molecular Oncology Research Group, University of Birmingham, The Medical School, Birmingham, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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RASSF1A is a Mr 39,000 (340 amino acids) protein containing two putative functional domains including a diacylglycerol binding domain (50–101 amino acids) at the NH2 terminus (absent in isoform C) and a RAS association domain (194–288 amino acids) in the COOH terminus. The presence of a RAS association domain suggests that RASSF1 proteins may function as RAS-effectors. Many known Ras effectors are oncoproteins in their own right; however, less is known about Ras effectors possessing tumor suppressor properties Consequently, the characterization of proteins encoded by tumor suppressor genes such as RASSF1A, their binding partners, and their location is crucial to understanding their function and role in tumor development.
Recently, several additional RASSF homologues have been reported, namely NORE1, RASSF2, and RASSF3. The biological functions of these polypeptides are not known. Murine Nore1 binds to RAS and several RAS-like GTPases in a GTP-dependent fashion; however, RASSF1A and C are not able to bind RAS directly. RASSF1A, however, can heterodimerize with NoreI and thereby associate with Ras-like GTPases (15) . The dimerization of RASSF1A requires the NH2-terminal 119 amino acids, which are not found in the C isoform. Human Nore1 interacts with the proapoptotic protein kinase MST1 to mediate a novel RAS-regulated apoptotic pathway. Interestingly, RASSF1A also interacts with MST1 suggesting a network of interacting proteins in the RAS-mediated apoptosis pathway (16) . More recently, we and others have demonstrated that RASSF1A blocks cell cycle progression and inhibits cyclin D1 accumulation (17 , 18) . Furthermore, our microarray analysis identified RASSF1A gene targets involved in many other cellular processes important in tumorigenesis including apoptosis, cell adhesion, transcription, protein synthesis, signaling, and cell migration (18) . Clearly, RASSF1A has a profound effect on gene expression and, hence, tumor cell biology. However, how these effects are exerted is not yet known. To establish a clearer picture of what the functions might be, and to identify additional cellular proteins that might bind to RASSF1A and modulate its biological activity we used a yeast two-hybrid approach to screen a human brain cDNA library for RASSF1A binding proteins. In this study, we isolated the ubiquitously expressed E1A-regulated transcription factor p120E4F a GLI-kruppel-related zinc finger phosphoprotein involved in cell cycle progression as a RASSF1A interacting partner and show that when coexpressed cell cycle arrest is enhanced.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All of the deletion constructs were generated using PFU turbo (Promega) and checked by sequence analysis. In vitro translated proteins were generated using a TNT-coupled reticulocyte lysate system (Promega) according to the manufacturer’s instructions. pEGFP-F was generated by inserting the farnesylation signal for H-Ras in frame into pEGFP-C2 (Clontech).
Yeast Two-Hybrid Screen.
Yeast two-hybrid screening was performed using MATCHMAKER system 3 (BD-Clontech). The bait vector pGBKT7-RASSF1A was transformed in yeast strain Saccharomyces cerevisiae AH109 (RASSF1A-BD) using the LiAc/PEG method and selected on Trp-plates. Strain AH109 includes four reporter genes, ADE2, HIS3, lacZ, and MEL1, of which the expression is regulated by GAL4-responsive upstream activating sequences and promoter elements. The bait RASSF1A-BD was then used to screen a pretransformed MATCHMAKER brain library cloned in pACT2 and introduced into yeast strain S. cerevisiae Y187. A total of 6 x 106clones were screened, of which 103 were positive for 
-galactosidase expression and turned blue. Clones were rescued and retransformed into yeast to reconfirm positive interactions. Positive clones were sequenced using automated DNA sequence analysis (ABI) and homologies identified using National Center for Biotechnology Information BLASTN/BLASTX.
Coimmunoprecipitation and Western Blot Analysis.
In vitro translated E4F and RASSF1A was generated using the TNT-coupled reticulocyte lysate system (Promega). The T7 promoter was incorporated into the sequence of library cDNAs using the following commercially available primers: AD HA forward Co-IP primer and AD Reverse Co-IP primer (BD-Clontech). PCR products were cleaned using a PCR clean up kit (Qiagen) according to the manufacturer’s instructions. For coimmunoprecipitation studies, 10 µl of in vitro translated (Transcend tRNA labeled) bait and library protein were mixed and incubated for 1 h at room temperature before the addition of either 1 µg of c-Myc monoclonal antibody or HA-polyclonal antibody, in the presence of Protein G/A agarose beads (Santa Cruz Biotechnology) and coimmunoprecipitation buffer [freshly prepared 20 mM Tris-HCL (pH7.5), 150 mM NaCl, 1 mM DTT, 5 µg/ml aprotinin, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1% Tween 20 (v/v)]. Samples were then incubated for an additional 2 h at 4°C on a rotating wheel. The beads were then washed three times in 500 µl of TBST (Tris buffered saline and 0.1% Tween 20), and the bound protein was then solubilized by addition of 15 µl of 2x SDS sample buffer. Once eluted the samples were then denatured before loading onto a 7.5–14% SDS-PAGE minigel. Western blotting was performed according to standard procedures. Membranes were then probed with c-Myc monoclonal antibody, HA polyclonal antibody (Clontech), RASSF1A polyclonal antibody N15 (Santa Cruz Biotechnology), RASSF1A monoclonal antibody (eB114 eBiosciences), ß- actin monoclonal (Sigma), or E4F polyclonal antibody-88.2 (generated using a NH2-terminal peptide of p120E4F corresponding to amino acids 50–64 EEDEDDVHRCGRCQA; Ref. 19
) and the appropriate secondary antibodies. The bands were then visualized by enhanced chemiluminescence (Amersham). When no antibody was appropriate Coomassie blue staining was applied (according to standard procedures).
Fusion Protein Expression and GST Pull-Down Assay.
To prepare GST fusion proteins, Escheria coli strain BL29 was transformed with a pGEX expression vector (pGEX-4T-1) containing the appropriate insert. Briefly, overnight cultures were diluted 1:10 and grown to A600 = 0.7, then the generation of fusion proteins was induced by the addition of 0.4 mM isopropyl-D-thiogalactoside at 30°C overnight. Pelleted cells were lysed by mild sonication (3 x 30 s blasts) in ice-cold PBS (1 x PBS) containing 1% Triton X-100 and Complete protease inhibitor (Roche) before resuspension in Bugbuster protein extraction reagent. After centrifugation, supernatants were applied to glutathione Sepharose 4B beads (Amersham) at 4°C overnight, collected, and resuspended in 1x PBS and 10% glycerol before storage at -80°C. Immobilized GST protein was detected by SDS-PAGE and Western blotting using Coomassie blue staining and probing with a GST specific antibody (Amersham).
For the pull-down assay 10 µl in vitro translated proteins (generated using the TNT-coupled reticulocyte lysate system) was incubated with 25 µl GST-RASSF1A bound beads in a final volume of 440 µl of PBS and incubated for 1 h at 30°C, followed by an additional incubation for 2 h at 4°C with rotation. The beads were then pelleted, washed three times in 1 x PBS, before being resuspended in 2 x SDS sample buffer and analyzed by SDS-PAGE (10–13% gels). Western blotting was performed according to standard procedures. Membranes were then probed with the appropriate primary and secondary antibodies. The bands were then visualized by enhanced chemiluminescence.
Immunoprecipitation Assays.
After transfection (48 h; except for endogenous immunoprecipitations, which were harvested when confluent) cells were harvested by trypzination and washed with ice-cold 1 x PBS. Pelleted cells were lysed with modified radioimmunoprecipitation assay buffer (Mammalian Cell Lysis kit; Sigma) containing Complete protease inhibitor (Roche) and mild sonication (3 x 10 s). Protein content was standardized as determined by the Bradford assay using Coomassie Plus Protein Assay Reagent (Bio-Rad). Fifty-µg lysates were precleared with protein A/G PLUS agarose beads, and then incubated with appropriate antibody and beads overnight at 4°C on a rotating wheel. The beads were then pelleted, washed three times in modified radioimmunoprecipitation assay buffer, before resuspension in 2x SDS sample buffer and analyzed by SDS-PAGE (10–13% gels). Western blotting was preformed according to standard procedures. Membranes were then probed with the appropriate primary and secondary antibodies. The bands were then visualized by enhanced chemiluminescence.
Cell Lines Used in This Study.
All of the cell lines were maintained in DMEM containing 10% fetal bovine serum in a 37°C incubator and 5% CO2. For cell cycle distribution and protein analysis, cultured cells (2 x 105) were seeded in 25-cm2 flasks and cotransfected with the expression plasmids (2 µg). Cells were harvested using trypsin 48 h after transfection.
Small Interfering RNA (siRNA) Treatment of Cells.
The double-stranded siRNA oligonucleotide targeting RASSF1A was synthesized by MWG Biotech United Kingdom Ltd. (Milton Keynes, United Kingdom), and the sequences used were those published previously (17)
: sense 5'-GACCUCUGUGGCGACUUCATT-3' and antisense 5'-UGAAGUCGCCACAGAGGUCTT-3'.
A negative control duplex (Ambion Europe Ltd., Huntingdon, United Kingdom) was used to demonstrate that transfection did not induce nonspecific effects on gene expression. The day before transfection, cells were plated onto six-well cell culture plates in 2 ml of growth medium without antibiotics and grown to 30–50% confluence. On the day of transfection, for each transfection sample, the duplexes were diluted to give a final concentration of 20 nM in Opti-Mem I (Invitrogen Life Technologies, Inc., San Diego, CA). Sixty µl of Oligofectamine reagent (Invitrogen Life Technologies, Inc.; diluted 1:4 with Opti-Mem I) were added to the diluted duplex, and the mixture was incubated at room temperature for 20 min to allow the siRNA:Oligofectamine complexes to form. This mixture was then added to the transfection well and incubated for 72 h at 37°C before whole cell lysates were taken.
Cell Cycle Analysis.
All of the transfected cells were cotransfected with a 1:5 ratio of pEGFP-F, a GFP vector, and appropriate vector. This was used as a measure of transfection efficiency and allowed accurate gating (20)
. Forty-eight h after transfection, cells were trypsinized and washed with PBS before fixing in 50% ethanol in 1% BSA at 4°C for 30 min. Cells were stained with propidium iodide (50 µg/ml) containing RNase A (1 mg/ml). DNA content from at least 25,000 events was analyzed by flow cytometry using a Coulter Epics Xl-MCL flow cytometer running system software. Data were collated using WINMDI software and then analyzed using Cylchred software. S phase inhibition was calculated from four independent experiments using the following formula: [(percentage of cells transfected with vector alone cells in S phase) - (percentage of transfected cells in S- phase in RASSF1A, p120E4F, or RASSF1A/p120E4F)]/(percentage of cells transfected with vector alone cells in S phase) x 100.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-galactosidase expression. Nucleotide sequence analysis revealed that two separate clones (designated E4F
) contained a 1-Kb insert with an open reading frame of 1113 bp encoding a protein fragment of 324 amino acids, which corresponds to the COOH-terminus half of the E1A regulated transcription factor p120E4F (NP 004415). We also identified a number of other interesting targets including the proapoptotic protein kinases MST1 and MST2 (10 clones) already known to interact with RASSF1A (16)
. The remaining novel binding partners are under additional investigation.
In the absence of either RASSF1A or E4F, or the replacement of RASSF1A by the unrelated protein lamin, diploid cells generated no -galactosidase signal. To determine the region of RASSF1A essential for interaction with E4F, RASSF1C (the shorter variant missing amino acids 1–119) was expressed as a fusion protein with the yeast two-hybrid vector pGBKT7 and tested for interaction with E4F. No -galactosidase production was observed as an indicator of a direct interaction (data not shown). p120E4F contains 10 putative zinc finger domains, which are clustered in two separate regions (Fig. 1A)
. Four motifs are contained within an NH2-terminal domain that is also present in p50E4F (cleaved product), whereas the remaining 6 motifs are grouped within a central region (amino acids 277–437) found only in the full-length protein (1)
. The isolated clone E4F (3)
encodes the last 5 zinc finger domains and all of the additional COOH-terminal residues that are specific to p120E4F (Fig. 1A)
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-HA were tested in a coimmunoprecipitation before resolving on a 10% SDS-PAGE gel and Western blotting. In agreement with the data obtained in the yeast two-hybrid system an interaction was observed generating specific bands close to the predicated sizes of Mr 41,000 and Mr 60,000. These bands were absent in the presence of empty vector. Therefore, these data demonstrate that RASSF1A can directly associate with p120E4F in yeast (Fig. 1B)
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GST-RASSF1A Interacts with E4F and p120E4F in Vitro.
To confirm that RASSF1A interacted with E4F and full-length p120E4F in vitro, a GST pull down assay was performed. A GST fusion protein corresponding to full-length RASSF1A (1–340 amino acids) was created and incubated with in vitro translated E4F and p120E4F protein. E4F and wild-type p120E4F specifically bound to the GST-RASSF1A (1–340) fusion protein but not to the GST alone (Fig. 1C)
, indicating a physical association between these two proteins in vitro.
RASSF1A and p120E4F Interact in Vivo.
Full-length RASSF1A and p120E4F-HA were overexpressed transiently in mouse NIH 3T3 fibroblast cells and human A549 non-small cell lung cancer line. Approximately 48 h after transfection, cell lysates were prepared and protein expression confirmed (Fig. 2A)
. Cell lysates were then subject to immunoprecipitation with RASSF1A polyclonal antibody and control IgG antibody before running on a 10% SDS-PAGE gel and Western blot analysis with E4F (88.2) antibody. As shown in Fig. 2A
, p120E4F-HA was efficiently coprecipitated in both NIH 3T3 and A549 cells with RASSF1A but not control IgG. In addition the specificity of the Co-IP was confirmed by Western blotting with anti-HA antibody also (data not shown). These data show that RASSF1A physically interacts with p120E4F in vivo when both proteins are exogenously overexpressed.
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endogenous levels of RASSF1A interact with p120E4F in vivo. In addition NCI-H1792 cells were treated with RASSF1A-siRNA before cell lysates were subject to immunoprecipitation (Fig. 2C)
. In the absence of endogenous RASSF1A no specific interaction was seen with p120E4F in NCI-H1792 cells.
NH2 Terminal of RASSF1A Interacts with p120E4F.
To additionally define the region of RASSF1A involved in the interaction with p120E4F, various GST-fusion constructs of RASSF1A deletions were created and tested for their ability to bind to in vitro translated p120E4F (see Fig. 3
). As summarized in Fig. 3
A, full-length GST-RASSF1A protein (1–340 amino acids) bound strongly to p120E4F (construct 1). Likewise, p120E4F bound strongly to the smaller COOH-terminal truncated GST construct 1–119 but failed to bind with the NH2-terminal truncated construct GST 143–340. We observed a very weak band in GST-RASSF1C (Fig. 3A
, Lane 4). Together this data suggests that the residues 1–119 specific to RASSF1A are important for the interaction with p120E4F.
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. As demonstrated previously, wild-type p120E4F bound strongly to GST-RASSF1A, as did E4F, which is missing the first zinc finger domain. Deletion of the COOH-terminal 243 amino acids had no effect on the ability of p120E4F to bind GST-RASSF1A (construct 3). Removal of residues 299–683 that includes the central zinc finger domain (constructs 4–8) completely abolished the interaction indicating that the site of interaction lies within this region. Sequential deletion of each zinc finger within the second region indicated that the site of interaction lies within the COOH-terminal zinc finger. Only construct 9, which contained the last zinc finger motif, might be involved in mediating the interaction. Indeed removal of the last intact zinc finger motif (construct 10: 445–683 amino acids) resulted in no interaction. In addition construct 11 containing only the last zinc finger motif (417–560 amino acids) demonstrated interaction with RASSF1A. These data indicate that p120E4F contacts RASSF1A through a COOH-terminal region located between amino acids 411 and 445.
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. The percentage of cells in G1 was increased by 10%, G2 was reduced by 
6%, whereas significant inhibition of S phase was evident 15.5 ± 3% (P < 0.05). RASSF1A overexpression had a small effect (>5%) on the percentage of cells in G1 compared with vector alone. In addition a small reduction in G2 was evident (5%), whereas no significant difference was seen on the percentage of cells in S phase (0.5 ± 1%; Fig. 5B
). Coexpression of RASSF1A and p120E4F, however, caused S phase inhibition to increase to 40 ± 4%, whereas increasing the number of cells in G1 by 18%. Little additive effect was seen in the G2 peak. Thus, coexpression of p120E4F and RASSF1A induced enhanced cell cycle arrest.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In mice and humans p120E4F is a GLI-kruppel-related zinc finger phosphoprotein that is ubiquitously expressed. p120E4F is synthesized as a Mr 120,000 protein that upon proteolytical cleavage gives rise to p50E4F, a Mr 50,000 NH2-terminal fragment. Full-length p120E4F (murine homologue 
AP3) is one of two related zinc finger proteins that differentially regulate the adenovirus E4F gene in an E1A-dependent manner (21)
. The full-length protein, p120E4F, represses this promoter in the absence, but not in the presence of E1A. In response to adenovirus E1A or treatment with phorphol ester, p120E4F becomes hyperphosphorylated and undergoes a reduction in both DNA binding and transcriptional repressor activities (22
, 23)
. The other E4F protein, p50E4F is a proteolytically derived Mr 50,000 NH2-terminal fragment of p120E4F that has been shown to stimulate the E4 promoter when coexpressed with E1A. Interestingly, although p50E4F and p120E4F can recognize the same DNA motif (RTGACGTC/AAY) in vitro, the additional residues specific to p120E4F confer functional and regulatory properties distinct from p50E4F. Expression of p120E4F significantly inhibits cellular growth and cell cycle progression. p50E4F has no such effect. p120E4F, but not p50E4F, blocked cell proliferation when expressed in mouse fibroblasts by inducing cell cycle arrest near the G1-S transition (19
, 21, 22, 23, 24, 25, 26, 27)
.
p120E4F has been shown to be associated with a number of cell cycle regulating proteins including cyclin E, cyclin B, the post-transcriptional up-regulation of cyclin-dependent kinase inhibitors p21waf1 and p27kip1, reduced cdk2, cdk4/6, and cdc2 kinase activities, and the down-regulation of cyclin A gene expression (19 , 21, 22, 23, 24, 25, 26, 27) . Cyclin A is the only cellular target demonstrated to be regulated by p120E4F to date (25) . There are probably other genes regulated by p120E4F that are involved in cell cycle progression because p120E4F repression was not affected by a reduction in cyclin A protein and RNA levels. Cell cycle arrest by p120E4F is also enhanced by interacting with retinoblastoma, the p53 transcription factor (19 , 24) and the p14 ARF tumor suppressor (28) . Thus, although the precise cellular signals that regulate endogenous p120E4F have yet to be clearly defined the evidence to date suggests that p120E4F may regulate cell cycle progression in response to a number of different signals.
Here we demonstrate that p120E4F associates both in vitro and in vivo with RASSF1A protein in human and mouse cells to form a complex, which modulates cell cycle progression. The strong association with p120E4F occurs mainly through residues that are specific to RASSF1A and are required for its growth suppression activity, and amino acids 411–445 of p120E4F, a region that contains both p53 and retinoblastoma protein binding domains (19 , 24) .
p120E4F contacts RASSF1A in vivo and enhances RASSF1A- mediated growth arrest. At 48 h after transfection, RASSF1A alone induced a small increase in G1 phase cells, with no significant effect on S phase. Overexpression of p120E4F affected cell cycle consistent with previous published data (19 , 21, 22, 23, 24, 25, 26, 27) . Another study has demonstrated recently in the non-small cell lung cancer cell line NCI-H1299 that RASSF1A induced G1-S cell cycle arrest and blocked accumulation of cyclin D1 (17) . Although we did not observe a dramatic effect by RASSF1A alone in our experiments we cannot rule out contrasting results due to the cells lines used or culture conditions. More importantly, however, NIH-3T3 cells overexpressing both RASSF1A and p120E4F showed a much greater reduction in the percentage of S phase cells, with a consistent increase in the number of cells in G1 phase. Interestingly it has also been shown that p120E4F also affects G2 phase progression (27) , although the mechanisms underlying this effect are not clear. We also saw a small difference in G2 phase in the cell lines we studied.
Both proteins have been demonstrated to evoke changes in cell cycle regulatory proteins, including the post-transcriptional elevation of cyclin B1, p21Waf1, and p27Kip1 protein levels, reduced expression of cyclin A, and reduced levels of cdk2 and cdc kinase activities in the case of p120E4F, and down-regulation of cyclin D1 in the case of RASSF1A (17, 18) . It is conceivable that p120E4F may be involved in modulating the transcriptional function of RASSF1A. A better understanding of the role of the p120E4F/RASSF1A complex requires characterization and functional analysis of other factors that associate with the COOH half of p120E4F and the NH2 terminus of RASSF1A. Additional studies need to be focused on understanding the regulation of cellular RASSF1A, as well as the molecular mechanisms of cell cycle control and identification of cellular target genes. Ultimately control of gene expression generally requires the interaction of multiple factors, which make up the transcriptional complex, including several activators, coactivators, and repressors, which stabilize the transcriptional machinery. The evidence presented here that p120E4F can physically associate with RASSF1A suggests that p120E4F is a novel interacting partner in the signaling pathway(s) of RASSF1A.
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FOOTNOTES |
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Grant Support: Breast Cancer Campaign, AICR (Association for International Cancer Research), The Wellcome Trust, SPARKS (Sport Aiding Medical Research for Kids), and Cancer Research United Kingdom.
Requests for reprints: Farida Latif, Department of Reproductive and Child Health, The Medical School, Edgbaston, Birmingham, B15 2TT, United Kingdom. Fax: 44-0-121- 627-2618; E-mail: flatif{at}hgmp.mrc.ac.uk
Received 8/21/03. Revised 10/30/03. Accepted 10/31/03.
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