RASSF1A Interacts with Microtubule-Associated Proteins and Modulates Microtubule Dynamics

[CANCER RESEARCH 64, 4112–4116, June 15, 2004] RASSF1A Interacts with Microtubule-Associated Proteins and Modulates Microtubule Dynamics

Ashraf Dallol,1 Angelo Agathanggelou,1 Sarah L. Fenton,1 Jalal Ahmed-Choudhury,1 Luke Hesson,1 Michele D. Vos,2 Geoffrey J. Clark,2 Julian Downward,3 Eamonn R. Maher,1,4 and Farida Latif1,4 1Section of Medical and Molecular Genetics, Division of Reproductive and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham, United Kingdom; 2Department of Cell and Cancer Biology, National Cancer Institute, Rockville, Maryland; 3Signal Transduction Laboratory, Cancer Research UK, London Research Institute, London, United Kingdom; and 4Cancer Research UK, Renal Molecular Oncology Research Group, University of Birmingham, The Medical School, Birmingham, United Kingdom

ABSTRACT RASSF1A also interacts with p120E4F (10), a negative modulator of cyclin A expression (11), and more recently, Liu et al. (12) showed The candidate tumor suppressor gene RASSF1A is inactivated in many RASSF1A to be a microtubule-associated protein able to protect types of adult and childhood cancers. However, the mechanisms by which microtubules from the effects of depolymerizing drugs. RASSF1A exerts its tumor suppressive functions have yet to be elucidated. To this end, we performed a yeast two-hybrid screen to identify novel Loss of RASSF1A expression is largely attributed to promoter RASSF1A-interacting proteins in a human brain cDNA library. Seventy hypermethylation because an exhaustive search for mutations yielded percent of interacting clones had homology to microtubule-associated only rare missense substitutions. The significance of many of these proteins, including MAP1B and VCY2IP1/C19ORF5. RASSF1A associa- missense changes in tumorigenesis have yet to be determined. Re- tion with MAP1B and VCY2IP1/C19ORF5 was subsequently confirmed cently, Shivakumar et al. (13) showed that RASSF1A, but not in mammalian cell lines. This suggested that RASSF1A may exert its RASSF1A mutants A133S or S131F, induce cell cycle arrest by tumor-suppressive functions through interaction with the microtubules. blocking cyclin D1 accumulation. Hence, naturally occurring tumor- We demonstrate that RASSF1A associates with the microtubules, causing associated variants can provide useful tools for the understanding of them to exist as hyperstabilized circular bundles. We found that two gene function. naturally occurring tumor-associated missense substitutions in the In this study, we provide yeast two-hybrid and colocalization data RASSF1A coding region, C65R and R257Q, perturb the association of RASSF1A with the microtubules. The C65R and R257Q in addition to to support the role of RASSF1A as a microtubule-associated protein. VCY2IP1/C19ORF5 showed reduced ability to induce microtubule acety- We identify two tumor-associated RASSF1A mutants, which show lation and were unable to protect the microtubules against the depoly- reduced association with the microtubules. Using these reagents, we merizing action of nocodazole. In addition, wild-type RASSF1A but not provide data that highlight the importance of microtubule association the C65R or the R257Q is able to block DNA synthesis. Our data identify for RASSF1A function in regulating microtubule stability and cell a role for RASSF1A in the regulation of microtubules and cell cycle cycle progression. dynamics that could be part of the mechanism(s) by which RASSF1A exerts its growth inhibition on cancer cells. MATERIALS AND METHODS

Cell Lines and Transfections. The non-small cell lung cancer cell lines INTRODUCTION NCI-H1299 and NCI-H1437, the green monkey kidney line COS-7, and the Loss of RASSF1A expression is one of the most common events in neuroblastoma cell line SK-N-AS were routinely maintained in DMEM sup- human cancers. Frequent epigenetic inactivation of RASSF1A pro- plemented with 10% fetal bovine serum (Invitrogen). Transfection was performed using Fugene 6 (Roche) according to the manufacturer’s instructions. moter region has been detected in many common human tumors, Plasmids and Antibodies. The bait plasmid pGBKT7-RASSF1A and including small cell lung cancer, non-small cell lung cancer, and pGEX-4T-1-RASSF1A were constructed by restriction digest of full-length breast and kidney cancers (Refs. 1–5; reviewed in Ref. 6). In addition RASSF1A from pcDNA3.1 using EcoRI and cloned in frame into yeast vector to its common inactivation, overexpression of RASSF1A in lung (1, 3), pGBKT7 (BD Clontech) and the glutathione S-transferase expression vector kidney (4), and prostate (7) cancer cell lines causes drastic reduction pGEX-4T-1, respectively (Amersham Pharmacia Biotech). pcDNA3HA/ of tumorigenicity both in vitro and in vivo. RASSF1A is the major and RASSF1A contains the complete coding region of RASSF1A as a BamHI-NotI longest splice variant coded for by the RASSF1 locus at 3p21.3. The fragment in pcDNA3HA. The RASSF1A mutant variants K21Q, C65R, RASSF1A protein contains two putative functional domains. A dia- S131F, A133F, R201H, V211A, R257Q, Y325C, and A336T were created by PCR-based site-directed mutagenesis using pcDNA3HA/RASSF1A as a tem- cylglycerol binding domain exists at the NH2 terminus, whereas a ras-association (RA) domain is found at the COOH terminus. plate. pEGFP-C2/RASSF1A contains the coding region of RASSF1A as an EcoRI fragment. The complete coding region of VCY2IP1/C19ORF5 was RASSF1A interacts only weakly with Ras despite containing a RAS- cloned in frame of the HA- and GFP- in pcDNA3 and pEGFP-C2, respectively. association domain (8). However, RASSF1A is brought into the RAS Rabbit polyclonal anti-hemagglutinin (HA) antibody (Y-11) was from Santa signaling pathway by heterodimerization with NORE1 (8). Both Cruz Biotechnology. Mouse monoclonal Anti-myc antibody was from Clon- NORE1 and RASSF1A were shown to interact with the proapoptotic tech. Goat polyclonal anti-MAP1B (N-10) antibody was from Santa Cruz serine/threonine kinase MST1 (9). We have recently shown that Biotechnology. Mouse monoclonal antibodies against ␣-tubulin (DM1A), ␤-tubulin (TUB 2.1), and acetyl-␣-tubulin (6-11B-1) and anti-HA (HA-7) were Received 1/27/04; revised 3/9/04; accepted 3/10/04. from Sigma. Mouse monoclonal anti-RASSF1A antibody (eb114) was from Grant support: Association for International Cancer Research, Sport Aiding Medical eBiosciences. HRP-conjugated Rabbit antimouse IgG and HRP-conjugated Research for Kids, Breast Cancer Campaign, and The Wellcome Trust and Cancer goat antirabbit IgG secondary antibodies were purchased from DAKOCyto- Research United Kingdom. mation. Alexafluor-488-conjugated goat antirabbit IgG and Alexafluor-594- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with conjugated rabbit antimouse IgG were purchased from Molecular Probes. 18 U.S.C. Section 1734 solely to indicate this fact. Yeast Two-Hybrid Screen. Yeast two-hybrid screening was performed as Note: A. Dallol and A. Agathanggelou contributed equally. described previously (10) Briefly, the bait vector pGBKT7-RASSF1A was Requests for reprints: Farida Latif, Section of Medical and Molecular Genetics, transformed in yeast strain Saccharomyces cerevisiae AH109 (RASSF1A-BD) Division of Reproductive and Child Health, University of Birmingham, The Medical School, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44-0-121-627-2741; using the Lithium Acetate/Polyethylene glycol method and selected on Trp Fax: 44-0-121-627-2618; E-mail: [email protected]. plates. Strain AH109 includes four reporter genes ADE2, HIS3, lacZ, and 4112

MEL18 whose expression is regulated by GAL4 responsive upstream activat- PBS/1% BSA for 30 min at room temperature. Incubation with the indicated ing sequences and promoter elements. The bait RASSF1A-BD was then used antibody was done at room temperature for 1 h followed by extensive washes to screen a pretransformed MATCHMAKER brain library cloned in pACT2 in PBS. This was followed by incubation for 30 min at room temperature with and introduced into yeast strain S. cerevisiae Y187. A total of 6 ϫ 106 clones the appropriate secondary antibody and several washes in PBS. The cover- were screened, of which 103 were positive for ␣-galactosidase expression and slips were mounted on glass slides using Vectashield with 4Ј,6-diamidino-2- turned blue. Clones were rescued and retransformed into yeast to reconfirm phenylindole mounting medium. Images were visualized with a Photometrics positive interactions. Positive clones were sequenced using automated DNA SenSys KAF 1400-G2 charged coupled device fitted to a Zeiss Axioplan sequence analysis (ABI) and homologies identified using National Center for fluorescence microscope and captured using the SmartCapture software from Biotechnology Information BLASTN/BLASTX. Digital Scientific. Immunoprecipitation and Western Blot Analysis. Generation of the Bromodeoxyuridine (BrdU) Incorporation Analysis. For BrdU incorpo- glutathione S-transferase-RASSF1A and the in vitro-translated HA-VCY2IP1/ ration assay, exponentially growing cells were plated on glass slides. Cells C19ORF5 and myc-RASSF1A was performed as described previously (10). were transfected with the indicated construct. The next day, 30 ␮M BrdU were The translated/tagged protein products were mixed and immunoprecipitated added to the transiently transfected cells. Twenty-four h later, the cells were using anti-HA antibody and protein G/A agarose beads. The protein complexes fixed in 4% paraformaldehyde, permeabilized in acetone at Ϫ20°C for 5 min, were then solubilized by addition of 15 ␮lof2ϫ SDS sample buffer and and then treated with 2 M HCl for 10 min. BrdU incorporation was visualized denatured before loading onto a 7.5–14% SDS-PAGE minigel. Western blot- with mouse monoclonal anti-BrdU antibody and alexafluor-594 secondary ting was performed according to standard procedures. Membranes were then antibody. HA-RASSF1A-expressing cells were visualized with anti-HA anti- probed with the antibodies indicated. body and alexafluor-488-conjugated secondary antibody. Microtubule Cosedimentation Assay. The microtubules cosedimentation Statistical Analysis. Comparisons were made by Fisher’s exact test or t assay was done as described previously (14). Cells were washed with ice-cold test when appropriate. PsofϽ0.05 were taken as statistically significant. PBS, scraped into 1 ml of PBS, and collected by centrifugation. The cells were solubilized in PTN buffer (100 mM PIPES, 30 mM Tris, 50 mM sodium chloride, RESULTS 1mM EGTA, 1.25 mM EDTA, 1 mM DTT, 1% Triton X-100, 10 mM phenyl- ␮ methylsulfonyl fluoride, and 1 g/ml aprotinin at pH 6.3). Supernatants were RASSF1A Interacts with Microtubules-Associated Proteins cleared by centrifugation for 30 min at 20,000 ϫ g at 25°C. Tubulin (5 mg/ml; Both in Vitro and in Vivo. We performed yeast two-hybrid screen to ICN Biochemicals) was diluted in buffer A (100 mM 4-morpholineethanesulfonic identify proteins that interact with RASSF1A. Approximately 6 ϫ 106 acid, 1 mM EGTA, 0.1 mM EDTA, 0.5 mM magnesium chloride, 100 ␮g/ml sucrose, 1 mM DTT, 0.1 mM GTP, 1 ␮g/ml leupeptin, and 1 ␮g/ml aprotinin at pH independent transformations were screened, of which, 103 clones were ␣ 6.8) and polymerized with and without 50 ␮M Taxol in the presence of 2.5 mM positive for -galactosidase expression. After excluding clones that con- GTP for 30 min at 37°C. Fifty ␮g of polymerized microtubules/assay were tained genomic repeats, we found 58 independent clones that included pelleted by centrifugation for 30 min at 20,000 ϫ g at 25°C. The microtubules known and novel genes that were able to interact with RASSF1A in the were resuspended in 60 ␮l of precleared cell supernatant and incubated for 30 min yeast two-hybrid system. Thirty-two clones were homologous to the at 37°C. Samples were then centrifuged for 30 min at 20,000 ϫ g at 25°C, and VCY2IP1 (also known as C19ORF5, NP_060644) gene, which has high supernatants and pellets were analyzed by SDS-PAGE and immunoblotting. overall similarity with MAP1A and MAP1B. The NH2 terminus of Immunofluorescence. Exponentially growing NCI-H1299 cells were VCY2IP1/C19ORF5 (residues 3–564 aa, NP_060644) has 58% similar- plated on coverslips and transfected the next day with indicated constructs ity with the NH terminus of MAP1B (residues 16–650 aa, NP_005900). using Fugene 6 as described by the manufacturer. After 48 h of transfection, 2 cells were either fixed with 4% paraformaldehyde for 10 min at room tem- The COOH terminus of VCY2IP1/C19ORF5 (residues 649–848 aa, perature or treated with 10 ␮M nocodazole (microtubule stability assay) or with NP_060644) has a 42% similarity with the COOH terminus of MAP1B carrier only for 1 h followed by fixation with 4% paraformaldehyde. After (residues 2009–2225 aa, NP_005900). Similarly, the NH2 terminus of fixation, cells were permeabilized with 0.1% Triton X-100 in PBS/1% BSA for VCY2IP1/C19ORF5 (residues 205–544 aa, NP_060644) has 57% simi-

2 min. Nonspecific binding was blocked by incubating the coverslips with larity with the NH2 terminus of MAP1A (residues 2–390 aa,

Fig. 1. RASSF1A interacts with microtubule-associated proteins. A, endogenously expressed MAP1B was pulled down by glutathione S-transferase-RASSF1A (GST-RASSF1A)-coated- Sepharose beads from SK-N-AS cell lysates. Extracts were resolved by SDS-PAGE, and MAP1B was visualized using anti-MAP1B (N-10) antibody after Western blotting (WB). B, RASSF1A is associated with MAP1B in SK-N-AS cells. pcDNA3-HA and pcDNA3-HA-RASSF1A were transiently transfected into SK-N-AS cells. Recombinant proteins were immunoprecipitated (IP) using anti-hemagglutinin (HA) antibody (Y-11), and bound MAP1B was visualized as above. C, Myc-RASSF1A interacts with HA-VCY2IP1/C19ORF5 clone37D in vitro. Anti-Myc antibody (Clontech) was used to IP Myc-RASSF1A from a mixture of in vitro-generated Myc-RASSF1A and HA-VCY2IP1/C19ORF5 clone37D. The IP was resolved by SDS-PAGE, and bound HA-VCY2IP1/C19ORF5 clone37D was visualized with anti-HA antibody. Input proteins are shown and Myc-Lamda (Lam) was used for control. D, exogenously overexpressed RASSF1A and HA-VCY2IP1/C19ORF5 associate in COS-7 cells. HA-VCY2IP1/C19ORF5 was immunoprecipitated with anti-HA antibody from extracts of COS-7 cells cotransfected with RASSF1A and HA-VCY2IP1/C19ORF5. RASSF1A was detected using the eb114 antibody (eBiosciences). E, HA-RASSF1A expressed in COS-7 cells cosedimented with in vitro-polymerized tubulin. Protein extracts from COS-7 cells transfected with HA-RASSF1A were incubated with 50 ␮g of tubulin polymerized in vitro in the presence or absence of 50 ␮M Taxol. The mixture was spun down, fractionated into a supernatant (S) and pellet (P), and HA-RASSF1A content was analyzed by SDS-PAGE (L ϭ 10 ␮l of lysate). 4113

NP_002364). The COOH terminus of VCY2IP1/C19ORF5 (residues precipitation of HA-VCY2IP1/C19ORF5 and probing with anti- 566–860 aa, NP_060644) has a 36% similarity with the COOH terminus RASSF1A antibody (eb114) from cotransfected COS-7 cells (Fig. of MAP1A (residues 2234–2597 aa, NP_002364). We also identified 9 1D). This data are supported by immunofluorescence staining that independent clones that contained the MAP1B gene. In total, there were showed HA-RASSF1A and GFP-VCY2IP1/C19ORF5 colocalize in 41 RASSF1A-interacting clones that were microtubule-associated pro- NCI-H1299 cells (Fig. 2A). teins. In contrast, we identified only 10 clones for MST1 and MST2 that RASSF1A Colocalizes with Microtubules and Promote Their already have been shown to interact with RASSF1A. The interaction Stability. The high frequency with which microtubule-associated pro- between RASSF1A and MAP1B was confirmed in vitro, using glutathi- teins were identified by the yeast two-hybrid screen suggested RASSF1A one S-transferase-RASSF1A fusion protein and extracts from the neuro- localized to the microtubules. This was confirmed by immunofluores- blastoma cell line SK-N-AS. As shown in Fig. 1A, glutathione S-trans- cence staining in several cell lines transfected with HA-RASSF1A (NCI- ferase-RASSF1A bound efficiently to MAP1B from SK-N-AS cell H1299, A549, MCF7, and COS-7). Fig. 2, B and D, shows that HA- lysates. The interaction was additionally confirmed in SK-N-AS cells RASSF1A colocalized with ␤-tubulin and ␣-tubulin, respectively, in transiently transfected with HA-RASSF1A; Fig. 1B shows that exog- NCI-H1299 cells and all other cell lines studied (data not shown). enously overexpressed HA-RASSF1A immunoprecipitated with endog- Furthermore, overexpression of RASSF1A appeared to rearrange the enously expressed MAP1B. microtubules into either circular or bundled perinuclear rings, whereas the We confirmed the interaction of VCY2IP1/C19ORF5 with untransfected cells show the microtubules radiating from a microtubule- RASSF1A in vitro using sequences from one of the positively inter- organizing center located at the centrosome. This phenotype is consistent acting clones, clone 37D, that contained the COOH terminus 2.5 kb of with microtubule staining in cell lines with or without endogenous the 3.183 kb of the complete coding region of VCY2IP1/C19ORF5 RASSF1A expression (Fig. 2C). RASSF1A-negative cell lines (NCI- (Fig. 1C). The interaction was additionally confirmed by immuno- H1299, A549, and MCF7) displayed unbundled microtubules radiating

Fig. 2. RASSF1A associates with tubulin and VCY2IP1/C19ORF5 and promotes microtubule stabilization. A, RASSF1A (HA-RASSF1A) colocalized with VCY2IP1/C19ORF5 (HA-C19ORF5). NCI-H1299 cells were transfected with green fluorescent protein-VCY2IP1/C19ORF5 (GFP-C19ORF5) and HA-RASSF1A. HA-RASSF1A was visualized using anti-HA (HA7; Sigma) antibody (red), GFP-VCY2IP1/C19ORF5 (green), and 4Ј,6-diamidino-2-phenylindole (DAPI) counterstained (blue). B, RASSF1A colocalized with ␤-tubulin. NCI-H1299 non-small cell lung cancer cells were transfected with HA-RASSF1A, which was visualized using anti-HA (Y11) antibody (green), and ␤-tubulin was stained with TUB 2.1 antibody (Sigma). C, NCI-H1437 cell line with endogenous RASSF1A expression and NCI-H1299 (RASSF1AϪ/Ϫ) were grown on coverslips and stained for ␤-tubulin as above. The magnified images shows the differences in the microtubules orientation and structure in both lines. D, wild-type RASSF1A protects microtubules from drug-induced depolymerization. NCI-H1299 cells were transfected with the indicated constructs (see “Materials and Methods”). Forty-eight h after transfection, cells were treated with 10 ␮M nocodazole or vehicle for 1 h. ␣-Tubulin was visualized with DM1A antibody (red), and transfected proteins were stained green. E, wild-type RASSF1A is associated with acetylated ␣-tubulin. NCI-H1299 cells were transfected with the indicated constructs and stained as before. Acetylated ␣-tubulin was visualized using 6-11B-1 antibody (red). 4114

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. RASSF1A AND MAPs INTERACTION from the microtubule-organizing center. RASSF1A-positive cell lines Table 1 The effect of wild-type RASSF1A and mutant variants expression on BrdU a (NCI-H1437, NCI-H1792, and HeLa) display a more bundled and cir- incorporation BrdU incorporation Values are normalized to those of the vector control (set at 100%). Eighty to 90% of cular microtubules. Similar phenotype was observed when pEGFP- NCI-H1299 were BrdU positive. All cultures were adherent. Values represent RASSF1A is used instead of HA-RASSF1A (data not shown). The means Ϯ SEs from at least three independent experiments. interaction with the microtubules was confirmed in vitro using a micro- RASSF1A variant % BrdU incorporation tubule cosedimentation assay with extracts of HA-RASSF1A expressing RASSF1A wt 23.00 Ϯ 3.00 COS-7 cells. Fig. 1E shows RASSF1A cosedimented with in vitro C65R 45.34 Ϯ 2.08 polymerized microtubules. R257Q 70.34 Ϯ 4.04 Mutant Forms of RASSF1A Show Loss of Microtubule Asso- a BrdU, bromodeoxyuridine. ciation. We compared the subcellular localization of wild-type HA- RASSF1A with nine naturally occurring tumor-associated variant forms of the protein. All variants of RASSF1A showed similar levels fected with wild-type HA-RASSF1A expressed high levels of acety- of expression in COS-7 cells (Fig. 3A). However, with all mutants, lated ␣-tubulin (Fig. 2E). However, this phenotype was lost or greatly aberrant localization of RASSF1A was observed (Figs. 2D and 3B). reduced in cells expressing C65R and R257Q variants (Fig. 2E). Variants C65R and R257Q showed the most drastic change in the VCY2IP1/C19ORF5 Does Not Stabilize Microtubules. Because subcellular localization of RASSF1A. The majority of cells express- HA-VCY2IP1/C19ORF5 appeared to colocalize with RASSF1A, its ing HA-tagged C65R and R257Q variants showed atypical localiza- ability to stabilize microtubules was examined. However, unlike wild- tion of RASSF1A, where it existed in the nucleus or in other unde- type RASSF1A, VCY2IP1/C19ORF5 did not cause the microtubules fined cytoplasmic compartments. to exist in circular bundles and was not able to protect the microtu- Loss of Microtubule-Association Correlated with Loss of Mi- bules from nocodazole-induced depolymerization. In addition, crotubule Stability. Fig. 2D shows that the wild-type HA-RASSF1A VCY2IP1/C19ORF5 was not able to induce the acetylation of ␣- protected the microtubules against depolymerization induced by treat- tubulin (Fig. 2, D and E). ing NCI-H1299 cells with 10 ␮M nocodazole for 1 h. The same effect Loss of RASSF1A Association with the Microtubules Affects Its was seen in MCF7 or when pEGFP-RASSF1A was used (data not Ability to Delay the Cell Cycle. We used BrdU incorporation assay shown). Examination of the RASSF1A variants showed that the C65R to compare the ability of wild-type RASSF1A and variants C65R/ and R257Q were not able to protect the microtubules against depo- R257Q to stop DNA synthesis in NCI-H1299 cells (Table 1). Wild- lymerization when RASSF1A localized to the nucleus (Fig. 2D). In type RASSF1A caused a significant decrease in BrdU incorporation general, the microtubules were protected against nocodazole-induced compared with the mutant variants (P Ͻ 0.001). Wild-type RASSF1A depolymerization whenever wild-type RASSF1A or any of the vari- blocked BrdU incorporation in at least 77 Ϯ 3.00% of cells, whereas ants colocalized with the microtubules. variants C65R and R257Q blocked BrdU incorporation in only RASSF1A-Induced Acetylation of Microtubules. Acetylation of 54.66 Ϯ 2.08 and 29.66 Ϯ 4.04% of cells, respectively. ␣-tubulin is a marker for increased microtubules stability. Cells trans- DISCUSSION The data presented in this study support previous studies, suggest- ing that the RASSF1A tumor suppressor is a microtubule-associated protein. We used a yeast two-hybrid screen to show that RASSF1A interacts with microtubule-associated proteins, which comprised 70% of the interacting clones. Consistent with these findings, we show that RASSF1A colocalized with ␣- and ␤-tubulin in mammalian cell lines, protected microtubules from nocodazole-induced depolymerization, and induced acetylation of ␣-tubulin. We identified two tumor-associated RASSF1A variants (C65R/R257Q), which have drastically reduced ability to associate with the microtubules. C65R and R257Q variants failed to protect against drug-induced microtubule depolymerization and did not induce acetylation of ␣-tubulin. Furthermore, unlike wild-type RASSF1A, the mutant forms did not block DNA synthesis. Taken together, this data suggest that association of RASSF1A with the microtubules is central to its function and disrup- tion of this association may promote tumorigenesis. The interactions between RASSF1 and VCY2IP1/C19ORF5 was previously reported but not confirmed (15). The identification of MAP1B as an RASSF1A-interacting protein is a novel finding. We have confirmed the interaction between RASSF1A and VCY2IP1/ C19ORF5 and MAP1B using in vitro-generated proteins and with whole cell lysates. Consistent with these findings, immunofluorescence staining showed RASSF1A colocalized with VCY2IP1/ C19ORF5. However, unlike RASSF1A, overexpression of VCY2IP1/ Fig. 3. A, HA-tagged wild-type RASSF1A and mutant variants were transfected in C19ORF5 did not protect microtubules from drug-induced de- COS-7 cells, and Western blotting was used to asses the expression level of each construct 48 h after transfection. B, NCI-H1299 cells were grown on coverslips and transfected with polymerization or induced acetylation of ␣-tubulin. These findings are wild-type or RASSF1A mutant variants. Atypical RASSF1A localization was defined as consistent with the observed weak association of VCY2IP1/C19ORF5 any cell that exhibited HA-RASSF1A localization in a compartment other than the microtubules. One-hundred cells were scored/slide with n ϭ 2. Similar fractions were with the microtubules in the absence of RASSF1A. VCY2IP1/ obtained in MCF7. C19ORF5 is an interacting partner for VCY2, a testis-specific protein 4115

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2004 American Association for Cancer Research. RASSF1A AND MAPs INTERACTION located in the frequently deleted AZFc region on chromosome Yq RASSF1A to the microtubules. Our data suggest that through interaction (16). VCY2 in turn interacts with UBE3A, a ubiquitin-protein ligase with the microtubules and microtubule-associated proteins, RASSF1A E3A (17). Previously, VCY2IP1/C19ORF5 has also been shown to regulates cell cycle progression and may also modulate cell migration. interact with LRPPRC, the mitochondrial inner membrane NADH dehydrogenase I and cytochrome c oxidase I (15). MAP1B is the earliest microtubule-associated protein expressed in the developing REFERENCES nervous system (18) and is thought to have roles on axonal transport 1. Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP. Epigenetic inactivation and neurite outgrowth (19). 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Monoclonal antibodies specific for an acetylated form of wild-type RASSF1A have reduced motility compared with controls. alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol 1985;101:2085–94. Microarray expression profiling has shown that genes involved in cell 24. Wadsworth P. Regional regulation of microtubule dynamics in polarized, motile cells. adhesion and motility such as zyxin and CDH2 are significantly up- Cell Motil Cytoskeleton 1999;42:48–59. regulated after expression of RASSF1A in A549, which might indicate a 25. Hubbert C, Guardiola A, Shao R, et al. HDAC6 is a microtubule-associated deacety- lase. Nature (Lond.) 2002;417:455–8. role for RASSF1A in the control of cell migration and adhesion (27). 26. Palazzo A, Ackerman B, Gundersen GG. Cell biology: tubulin acetylation and cell Overall, the evidence presented here confirms the localization of motility. Nature (Lond.) 2003;421:230. 27. 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