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[Cancer Research 64, 8334-8340, November 15, 2004]
© 2004 American Association for Cancer Research

Regular Articles

Nuclear ßII-Tubulin Associates with the Activated Notch Receptor to Modulate Notch Signaling

Tien-Shun Yeh1, Rong-Hong Hsieh2, Shing-Chuan Shen3, Shwu-Huey Wang1, Min-Jen Tseng1,4, Chwen-Ming Shih5 and Jing-Jer Lin6

1 Graduate Institute of Cell and Molecular Biology and 2 Graduate Institute of Nutrition and Health Sciences, Taipei Medical University, Taipei, Taiwan; 3 Department of Dermatology, Taipei Municipal Wan-Fang Hospital-Affiliated with Taipei Medical University, Taipei, Taiwan; 4 Department of Life Science, National Chung Cheng University, Chia-Yi, Taiwan; 5 Department of Biochemistry, Taipei Medical University, Taipei, Taiwan; and 6 Institute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei, Taiwan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Notch signal pathway plays important roles in proliferation, apoptosis, and differentiation. Abnormalities in Notch signaling are linked to many human diseases. After ligand binding, Notch signaling is activated through the cleavage of Notch receptors to release and translocate the Notch intracellular domain into the nucleus. The Notch1 receptor intracellular domain (N1IC), the activated form of the Notch1 receptor, can modulate downstream target genes via C promoter-binding factor 1–dependent and -independent pathways. To further dissect the Notch1 signaling pathway, we screened the N1IC-associated proteins using a yeast two-hybrid system and identified nuclear ßII-tubulin as a candidate for the N1IC-associated proteins. It was suggested that the presence of ßII-tubulin in nuclei might be correlated with the cancerous state of cells. However, the function of ßII-tubulin locating in the nucleus still is unknown. Herein, we show that the complex of {alpha}- and ßII-tubulin is associated with N1IC in cancer cells by a coimmunoprecipitation analysis. The ankyrin domain of the Notch1 receptor alone was sufficient to associate with ßII-tubulin. Furthermore, {alpha}- and ßII-tubulin were localized in the nucleus and formed a complex with N1IC. Treatment with Taxol increased the amounts of nuclear {alpha}- and ßII-tubulin in K562 and HeLa cells and promoted the C promoter-binding factor 1–dependent transactivation activity of N1IC. We also show that nuclear ßII-tubulin was bound on the C promoter-binding factor 1 response elements via the association with N1IC. These results suggest that nuclear ßII-tubulin can modulate Notch signaling through interaction with N1IC in cancer cells.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Genes of Notch receptors encode evolutionarily conserved transmembrane receptors to regulate cell fate decisions during development (1) . Many reports have documented that the Notch signal pathway modulates proliferation, apoptosis, and differentiation (1, 2, 3) . Notch signaling also has been implicated in human cancers and in cancers induced by retroviral insertions in mice (4 , 5) . The Notch intracellular domain, the activated form of the Notch receptor, may function as an oncogene or tumor suppressor to promote or suppress tumorigenesis (for a review, see ref. 6 ). The effects of Notch signaling are cell type dependent, and these pathways that mediate growth and transformation may proceed through cross-talk with other signal pathways, such as transforming growth factor ß (7) and epidermal growth factor receptor–mitogen-activated protein kinase signaling pathways (8) .

After ligand binding, Notch signaling is activated through cleavage of Notch receptors, which allows the release and translocation of the Notch intracellular domain into the nucleus. The Notch intracellular domain modulates downstream target genes via C promoter-binding factor 1 (CBF1)–dependent and -independent pathways (9) .

A few Notch1 intracellular domain (N1IC)–associated cellular factors have been found, and we recently also reported the association of transcription factor Yin Yang 1 (YY1) with N1IC (10) . Only few downstream target genes of N1IC have been identified, such as the HES family (11) , Nrarp (12) , HERP2 (13) , cyclin D1 (14) , activator protein (15) , the pre–T-cell receptor {alpha} (pT{alpha}) gene (16) , and acid {alpha}-glucosidase (17) . However, the mechanisms controlling Notch1 signaling remain poorly understood.

To further dissect the Notch1 signaling pathway, we used a yeast two-hybrid system to search the N1IC-associated proteins and found nuclear ßII-tubulin as one of the candidates. ß-Tubulin is one of the structural subunits of microtubules, which consists of the heterodimer of {alpha}- and ß-tubulin. In most normal cells, tubulin resides only in the cytosol and not in the nucleus. Although the existence of nuclear ßII-tubulin could not be detected in biopsy samples of normal human tissues (18) , the ßII isotype of tubulin recently was found in the nuclei of several tumor cells (19, 20, 21) . These observations suggest that the presence of nuclear ßII-tubulin may be correlated with the cancerous state of cells (20) .

Taxol, an antitumor drug, exhibits higher specificity for ßII-tubulin than for other isotypes. In a concentration-caused cellular apoptosis, Taxol could irreversibly deplete the nuclear ßII-tubulin content in rat C6 glioma cells (20) . Nuclear ßII-tubulin was found to exist as {alpha}ßII dimers instead of assembled microtubules (19) . However, the biological function of ßII-tubulin locating in nuclei still is unknown. In this study, we show that the association of nuclear ßII-tubulin with N1IC can modulate CBF1-dependent gene expression.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Plasmid Construction.
The expression construct of pcDNA-HA-N1IC contains cDNA encoding the amino acid residues 1764 to 2444 of the intracellular domain of the human Notch1 receptor with an NH2-terminal hemagglutinin tag (10) . The fusion protein plasmids pGST-ANK{Delta}EP and pGST-ANK express glutathione S-transferase (GST) fusion proteins with amino acid residues 1821 to 2095 and 1821 to 2205 of the human Notch1 receptor, respectively. Reporter plasmids 4xwtCBF1Luc and 4xmtCBF1Luc were described previously (10 , 22) .

Yeast Two-Hybrid Screening.
Plasmid pBTM116-N1-ANK{Delta}EP, which encodes the ankyrin (ANK) domain of the Notch1 receptor (amino acid residues 1821 to 2095), was used as a bait in two-hybrid screening of human testis cDNA library (Clontech, Palo Alto, CA) according to the Matchmaker two-hybrid system protocol (Clontech). Owing to the ability of this plasmid construct to autonomously activate the LacZ reporter gene, positive yeast clones were only selected by histidine prototrophy from 5.6 x 106 transformed colonies in the presence of 20 mmol/L 3-amino-1,2,4-triazole.

Cell Culture and Transfection.
All of the cell lines, including human erythroleukemia K562 cells, acute T-cell lymphoblastic leukemia SUP-T1 cells, and cervical carcinoma HeLa cells, were cultured in Roswell Park Memorial Institute 1640 and Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum. The stable K562 cell lines expressing HA-N1IC (K562/HA-N1IC) and their control cells (K562/pcDNA3) were established previously (10) . Taxol (Sigma-Aldrich, St. Louis, MO) at indicated concentrations in dimethyl sulfoxide or an equal volume of dimethyl sulfoxide was added for 24 hours, followed by washing with PBS three times and further incubated in the absence of Taxol for 24 hours or 48 hours. Cycloheximide (Sigma-Aldrich) was used at 25 µg/mL. For transient transfection of the luciferase reporter assay, K562 cells or HeLa cells (1 x 106) were seeded onto six-well plates and transfected using the SuperFect transfection reagent (Qiagen, Valencia, CA), and luciferase activities were measured as described previously (10) .

For chromatin immunoprecipitation (ChIP) experiments, the K562/HA-N1IC cells (5 x 106) were transfected with 5 µg of reporter plasmids 4xwtCBF1Luc; cells were harvested 24 hours after transfection.

Coimmunoprecipitation.
To prepare whole-cell lysates, cells were lysed in NETN buffer [50 mmol/L Tris-HCl (pH 7.9), 150 mmol/L NaCl, 0.5 mmol/L EDTA, and 0.5% NP40] containing protease and phosphatase inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 100 mmol/L sodium fluoride). Two alternative buffers, buffer A [20 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 2 mmol/L MgSO4, and 0.5% NP40] and buffer B (PBS containing 0.5% NP40), containing protease and phosphatase inhibitors also were used as indicated. Cell lysates briefly were centrifuged to remove cell debris and then immunoprecipitated with protein A–Sepharose-bound anti–ßII-tubulin antibody (Sigma-Aldrich) as described previously (10) . Western blot analysis was performed with anti-Notch1 COOH-terminal (Santa Cruz Biotechnology, Santa Cruz, CA), anti–ßII-tubulin, and anti–{alpha}-tubulin antibodies (Santa Cruz Biotechnology).

Glutathione S-Transferase Pull-Down Assay.
N1IC proteins expressed as GST-ANK{Delta}EP and GST-ANK fusion proteins from the expression constructs of pGST-ANK{Delta}EP and pGST-ANK were induced and purified as described previously (10) . Whole-cell extracts of K562/HA-N1IC cells were prepared in NETN buffer as described previously. A 50% (v/v) slurry of glutathione-agarose resin prebound with 0.5 µg of GST or GST fusion proteins was incubated with 500 µg of whole-cell extracts for the pull-down assay at 4°C for 2 hours as described elsewhere (10) .

Subcellular Fractionation and Sucrose Gradient Analysis.
To prepare the nuclear extracts, cell pellets were suspended and lysed in a hypotonic buffer; after centrifugation, the nuclear pellets were resuspended in a high-salt buffer as described previously (10) . Nuclear extracts of K562/HA-N1IC or K562/pcDNA3 cells were loaded on the top of a 10% to 60% (w/v) sucrose gradient for centrifugation (10) . The gradients were fractionated into 0.5-mL fractions each from the top, and aliquots of each fraction were subjected to Western blot analysis to detect ßII-tubulin and N1IC proteins. We also prepared the protein standards (catalase, 11.3 S, Mr 232,000; thyroglobulin, 19.4 S, Mr 669,000) to be run on a sucrose gradient.

Oligoprecipitation and Chromatin Immunoprecipitation.
The 5'-biotinylated oligonucleotides and the protocol for oligoprecipitation were as described by Yeh et al. (10) . The procedure for ChIP of K562/HA-N1IC cells transfected with luciferase reporter plasmids using protein A–Sepharose-bound anti–ßII-tubulin antibody and the specific primers for PCR amplification also was described previously (10) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ßII-Tubulin Is a Candidate of Notch1 Receptor Intracellular Domain–Associated Proteins.
To date, molecular mechanisms regulating the Notch signal pathway remain obscure. To understand the Notch1 signaling pathway, we used the yeast two-hybrid system to search the protein(s) that interact with the activated Notch1 receptor (N1IC). The truncated fragment of N1IC, ANK{Delta}EP (23) , was expressed to screen N1IC-associated cellular factors from a human testis cDNA library in the presence of 3-amino-1,2,4-triazole. The cytoskeletal protein ßII-tubulin was one of the candidates for the N1IC-associated proteins. The COOH-terminal region of ßII-tubulin (accession no. BC019829) encompassing amino acid residues from 197 to 445 was identified as the region associated with the ANK domain of the Notch1 receptor (ANK{Delta}EP) in the yeast two-hybrid system (data not shown).

The Notch1 Receptor Intracellular Domain Associates with ßII-Tubulin in Cancer Cells.
To confirm the association of N1IC with ßII-tubulin in cancer cells, whole-cell extracts of K562 cells (K562/pcDNA3), HA-N1IC protein-expressing K562 cells (K562/HA-N1IC), and SUP-T1 cells were used for coimmunoprecipitation by antimouse IgG or anti–ßII-tubulin antibodies (Fig. 1A and B)Citation . The HA-N1IC fusion protein was coimmunoprecipitated with ßII-tubulin in K562/HA-N1IC and SUP-T1 cells by anti–ßII-tubulin antibody. This interaction between ßII-tubulin and N1IC also was observed in Jurkat cells using anti-Notch1 COOH-terminal antibody for immunoprecipitation (data not shown). {alpha}-Tubulin also was coimmunoprecipitated with ßII-tubulin; this might be because of the nuclear ßII-tubulin existing as {alpha}ßII dimers (19) .



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  		Fig. 1. Human N1IC associates with ßII-tubulin in cancer cells. A and B. Whole-cell extracts of K562 cells (K562/pcDNA3), HA-N1IC protein-expressing K562 cells (K562/HA-N1IC) (A), and SUP-T1 cells (B) were immunoprecipitated (IP) with antimouse IgG or anti–ßII-tubulin antibodies. The precipitated proteins were resolved by SDS-PAGE and analyzed by Western blot analysis using the anti-Notch1 COOH-terminal (C-ter) antibody (top), anti–ßII-tubulin antibody (middle), or anti–{alpha}-tubulin antibody (bottom). C. Whole-cell extracts of HA-N1IC protein-expressing K562 cells (K562/HA-N1IC) prepared by buffer A or buffer B were immunoprecipitated with anti-His or anti–ßII-tubulin antibodies. The precipitated proteins were analyzed by Western blot analysis using the anti-Notch1 C-ter antibody (top) and anti–ßII-tubulin antibody (bottom).

 
To avoid the binding artifacts generated by the denaturation of tubulins, either HEPES buffer (buffer A) or PBS (buffer B) also was used for the coimmunoprecipitation instead of NETN buffer. The interaction between ßII-tubulin and N1IC also was found in K562/HA-N1IC cells (Fig. 1C)Citation . Therefore, these results suggest that the Notch1 receptor intracellular domain associated with the heterodimer form of {alpha}- and ßII-tubulin in cells.

The ANK Domain of the Notch1 Receptor Is Sufficient to Associate with the ßII-Tubulin.
The in vitro GST fusion protein pull-down assay was used to dissect the region of N1IC required for the association with ßII-tubulin. Partially purified GST and GST fusion proteins of N1IC were analyzed by SDS-PAGE and Coomassie Blue staining (Fig. 2)Citation . Whole-cell extracts of K562/pcDNA3 cells were prepared for the pull-down assay by GST and GST fusion proteins. Both fusion proteins of GST-ANK{Delta}EP and GST-ANK were shown to associate with endogenous ßII-tubulin of K562/pcDNA3 cells. Therefore, the ANK domain of the Notch1 receptor alone was sufficient to associate with ßII-tubulin. This is consistent with the result of two-hybrid screening, in which the COOH-terminal half of ßII-tubulin was identified as the prey of ANK{Delta}EP protein.



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  		Fig. 2. ANK domain of the Notch1 receptor is sufficient to associate with ßII-tubulin. Whole-cell extracts of K562/pcDNA3 cells were used for the pull-down assay with purified GST, GST-ANK{Delta}EP, and GST-ANK fusion proteins. The pull-down pellets were resolved by SDS-PAGE and analyzed by Western blot analysis using anti–ßII-tubulin antibody (bottom). The same amounts of inputs of various GST fusion proteins were resolved by SDS-PAGE and analyzed by Coomassie Blue (CB) staining (top).

 
Nuclear ßII-Tubulin Exists in Cancer Cells.
The ßII isotype of tubulin was found in the nuclei of several tumor cells, including HeLa cells, and the presence of nuclear ßII-tubulin might be correlated with the cancerous state of cells (20) . To determine the existence of nuclear ßII-tubulin in the hematopoietic tumor cells, the nuclear and cytosolic extracts of K562 and SUP-T1 cells were used for Western blot analysis (Fig. 3)Citation . ßII-Tubulin was present in the nuclei and the cytoplasm of K562 and SUP-T1 cells and HeLa cells. In the same blotting membranes, nucleolar B23 only was detected in the nuclear fractions, whereas glyceraldehyde-3-phosphate dehydrogenase only was observed in cytosolic fractions. Therefore, the existence of nuclear ßII-tubulin from the possibility of contamination of cytosolic proteins in the nuclear extracts of these cells could be excluded.



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  		Fig. 3. Nuclear ßII-tubulin is present in cancer cells. Cytosolic extracts (C) and nuclear extracts (N) of K562 cells (left), HeLa cells (middle), and SUP-T1 cells (right) were prepared for Western blot analysis using the anti–ßII-tubulin antibody. The same membranes were stripped and reprobed with anti-B23 (a nuclear marker) or anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies as indicated.

 
The Intrinsic Activated Notch1 Receptor Associates with Endogenous ßII-Tubulin in Nuclear Complexes.
The Notch1 receptor intracellular domain previously had been shown to form a high molecular weight complex with cellular factors in the nuclei (10 , 24) . To gain further insights into the physiology of the interaction between N1IC and ßII-tubulin, their distributions in nuclei were investigated by sucrose gradient analysis. The nuclear extracts of K562/pcDNA3- and HA-N1IC–expressing K562/HA-N1IC cells were prepared for sucrose gradient ultracentrifugation. The collected fractions were analyzed by Western blot analysis using anti-Notch1 COOH-terminal and anti–ßII-tubulin antibodies sequentially. The sucrose gradient analysis profile showed that N1IC existed in both lower molecular weight fractions (fractions 3 to 7) and higher molecular weight fractions (fractions 14 to 16; Fig. 4ACitation ), whereas ßII-tubulin was detected in fractions 2 to 15. In the absence of the activated Notch1 receptor (i.e., K562/pcDNA3 cells), ßII-tubulin showed the same sucrose gradient profile as that of K562/HA-N1IC cells.



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  		Fig. 4. N1IC associates with endogenous ßII-tubulin in nuclei of cancer cells. A. Nuclear extracts from K562/pcDNA3 and K562/HA-N1IC cells were subjected to sucrose gradient centrifugation. N1IC and ßII-tubulin were visualized by Western blot analysis with anti-Notch1 COOH-terminal (C-ter) and anti–ßII-tubulin antibodies, respectively. Arrows indicate the native molecular masses of known standards. B. Nuclear extracts of K562/pcDNA3 cells, K562/HA-N1IC cells (left), and SUP-T1 cells (right) were prepared for immunoprecipitation by antibodies against mouse IgG or ßII-tubulin. The immunoprecipitates were resolved by SDS-PAGE and analyzed by Western blot analysis using anti-Notch1 C-ter, anti–ßII-tubulin, or anti–{alpha}-tubulin antibodies.

 
To confirm the association between N1IC and nuclear ßII-tubulin, coimmunoprecipitation was applied to the nuclear extracts of K562/pcDNA3 and stable N1IC-expressing K562/HA-N1IC cells. ßII-tubulin was immunoprecipitated in the nuclear fractions of these two cells using anti–ßII-tubulin antibody (Fig. 4B)Citation . After stripping and reprobing this immunoprecipitated blot, N1IC, but not transcription factor YY1, also was detected in the nuclear fraction of K562/HA-N1IC cells. Furthermore, {alpha}-tubulin also was coimmunoprecipitated in this analysis, presumably through association with ßII-tubulin. These results showed that ectopically expressed N1IC was associated with endogenous ßII-tubulin in nuclei by the analysis of coimmunoprecipitation.

To check whether intrinsic N1IC interacts with endogenous ßII-tubulin in the nuclei of cancer cells, nuclear extracts of SUP-T1 cells were prepared for coimmunoprecipitation using the anti–ßII-tubulin antibody. Fig. 4BCitation shows that N1IC could be coimmunoprecipitated with ßII-tubulin in the nuclei of SUP-T1 cells. This N1IC–ßII-tubulin association also was found in nuclei of HeLa cells (data not shown).

Contents of Nuclear {alpha}- and ßII-Tubulin Were Elevated by Nonapoptotic Concentrations of Taxol in Cancer Cells.
To evaluate the effect of Taxol on ßII-tubulin, nuclear and cytosolic extracts of K562 and HeLa cells were prepared for Western blot analysis after treatment with Taxol for 24 hours. The K562 cell, a human chronic myelogenous leukemia cell line, expresses Bcr-Abl, which mediated high resistance to Taxol-induced apoptosis as showed higher concentrations of Taxol (µmol/L range) were used (25) . Nevertheless, Taxol at low concentrations (10 nmol/L for 20 hours) already induced mitotic block in HeLa cells by suppressing the dynamics of spindle microtubules (26) . To confirm whether Taxol inhibited the cell proliferation in these conditions, HeLa cells were treated with various concentrations of Taxol for 24 hours. As described previously (27) , cell numbers of HeLa cells were not affected in the presence of Taxol at lower concentrations (5 and 10 nmol/L; Fig. 5ACitation ). However, the numbers of survival cells were decreased after treatment with higher concentrations of Taxol (25 and 50 nmol/L) in HeLa cells.



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  		Fig. 5. Contents of nuclear {alpha}- and ßII-tubulin were elevated by Taxol in cancer cells. A. HeLa cells (1 x 105) were seeded in the presence of Taxol for 24 hours, and the cell number then was counted by trypan blue exclusion. B and C. After treatment with various concentrations of Taxol, the cytosolic and nuclear fractions of K562 cells (B) and HeLa cells (C) were prepared for Western blot analysis using anti–ßII-tubulin antibody. The same membranes were stripped and reprobed with anti–{alpha}-tubulin, anti–glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or anti-B23 antibodies as indicated. Cycloheximide (CHX; 25 µg/mL) was added 30 minutes before Taxol treatment to block de novo protein synthesis. D. After treatment with 5 nmol/L Taxol for 24 hours, HeLa cells were washed by PBS three times (24) and further incubated in the absence of Taxol for 24 hours (24 + 24) or 48 hours (24 + 48). The cytosolic and nuclear fractions were prepared for Western blot analysis.

 
The Taxol-treated K562 cells showed dramatic quantity increment of {alpha}- and ßII-tubulin in the nuclei without affecting both contents in the cytosol (Fig. 5B)Citation . Furthermore, the amounts of nuclear {alpha}- and ßII-tubulin also were elevated without affecting cytosolic counterparts apparently in Taxol-treated HeLa cells (Fig. 5C)Citation . Previous treatment of HeLa cells with cycloheximide did not affect the increment of nuclear tubulins by Taxol. Therefore, this phenomenon was not because of de novo protein synthesis. This increase of nuclear ßII-tubulin induced by Taxol was sustained within 2 days after the removal of Taxol (Fig. 5D)Citation . However, the effect on the augmentation of nuclear tubulins was not observed in the presence of colchicine, an antimicrotubule agent (data not shown). Analyzing using confocal microscopy, the nuclear ßII-tubulin was increased in K562/HA-N1IC cells and colocalized with N1IC in nucleus after treatment with 3.5 µmol/L Taxol for 24 hours (data not shown). These results suggest that Taxol may increase the nuclear import of {alpha}- and ßII-tubulin in K562 and HeLa cells.

Enhancement of Luciferase Reporter Activity Transactivated by the Notch1 Receptor Intracellular Domain after Treatment with Taxol.
To elucidate the biological function of the association between N1IC and nuclear ßII-tubulin in the Notch signaling pathway, a luciferase reporter assay was performed. K562 cells were cotransfected with a luciferase reporter plasmid containing four copies of wild-type CBF1 response elements (4xwtCBF1Luc) and an N1IC-expressing construct, pcDNA3-HA-N1IC, or their control vectors in the presence of various concentrations of Taxol. The transfected cells were harvested and assayed for luciferase activity 24 hours after transfection. Although Taxol had been shown to alter gene expression (28, 29, 30) , it did not affect the activity of the luciferase reporter gene containing CBF1 response elements (Fig. 6A)Citation . In the absence of Taxol, N1IC enhanced the expression of the reporter gene containing CBF1 response elements by ~12-fold in K562 cells. This promotion of luciferase activity was further elevated ~1.5- and 4-fold in the presence of 3.5 and 7 µmol/L Taxol, respectively. Enhancement of luciferase activity of approximately twofold to threefold by Taxol also was observed in HeLa cells (Fig. 6B)Citation . These effects of Taxol were not detected in the luciferase reporter plasmid containing four copies of the mutant CBF1 response elements (4xmtCBF1Luc) in either cell line (data not shown). Moreover, the activation of CBF1-dependent luciferase reporter activity in K562 cells and HeLa cells was not observed in the presence of colchicine (data not shown). These results imply that Taxol augments the CBF1-dependent transactivation activity of N1IC.



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  		Fig. 6. CBF1-mediated transactivation activity of N1IC is modulated by Taxol. A and B. Reporter plasmid containing wild-type CBF1 response elements was cotransfected with HA-N1IC protein-expressing plasmid or its control vector into K562 cells (A) or HeLa cells (B) in the presence of various concentrations of Taxol. After 24 hours, luciferase activity was determined from whole-cell extracts, and the basal promoter activity of the reporter construct was set to unity. Mean values and SDs from at least four independent experiments are shown. C. The ßII-tubulin–N1IC-associated complex binds on the wild-type CBF1 response element but not on the mutant one. The nuclear extract of K562/HA-N1IC cells was incubated with the 5'-biotinylated double-stranded oligonucleotides of wild-type (WT) or mutant (mt) CBF1 response elements and then precipitated with streptavidin-agarose beads. The precipitated proteins were resolved by SDS-PAGE and analyzed by Western blot analysis using anti-Notch1 COOH-terminal (C-ter) antibody (top) or anti–ßII-tubulin antibody (bottom). D. K562/HA-N1IC cells were transiently transfected with reporter plasmid containing wild-type CBF1 response elements. Twenty-four hours after transfection, transfected cells were harvested for ChIP assay using anti-His and anti–ßII-tubulin antibodies. The immunoprecipitated DNA was used to amplify a 470-bp PCR product by specific primers for the region of the CBF1 response element in the reporter plasmid. (+) PCR-positive control uses 4 ng of 4xwtCBF1Luc plasmid as the DNA template.

 
The ßII-Tubulin–N1IC-Associated Complex Binds on the Wild-Type CBF1 Response Element, but not on the Mutant One.
The aforementioned data show that Taxol treatment increases the quantity of nuclear ßII-tubulin and also turns on CBF1-dependent Notch signaling in K562 and HeLa cells. We surmised that nuclear ßII-tubulin might participate in modulation of the Notch signal pathway. Therefore, oligoprecipitation was performed to study the interaction between the ßII-tubulin–N1IC-associated complex and the CBF1 recognition DNA sequence. In K562/HA-N1IC cells, N1IC and ßII-tubulin could be precipitated, together with the 5'-biotinylated wild-type CBF1 response element, by streptavidin-agarose beads but not with the mutant one (Fig. 6C)Citation . Furthermore, the ChIP assay also was used to examine the specific association of ßII-tubulin and N1IC with targeted DNA sequence in the context of living cells. K562/HA-N1IC cells were transiently transfected with reporter plasmid 4xwtCBF1Luc. Twenty-four hours after transfection, the cells were treated with formaldehyde to cross-link DNA and protein and then harvested for the ChIP assay using anti-His and anti–ßII-tubulin antibodies. The amplified PCR product of 470 bp was only present in the sample immunoprecipitated by anti–ßII-tubulin antibody (Fig. 6D)Citation . These data imply that nuclear ßII-tubulin indirectly binds on the wild-type CBF1 response element via associating with N1IC, which directly interacts with CBF1, and this association may enhance the transactivation activity of N1IC.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role and regulation of Notch signal pathway are complicated and not yet fully understood. Notch signaling integrates with several other signal pathways during development. Many of these integrations are cell-type and -context dependent, suggesting that these are either directly or indirectly modulated by cell-specific cofactors. The activated Notch1 receptor recently was shown to form a high molecular weight complex containing Mastermind-like 1, CBF1, and transcription factor YY1 in the nucleus (10 , 24) . These associations modulate the transactivation activity of N1IC in regulating the Notch signal pathway.

In this study, we identified ßII-tubulin as an N1IC-associated protein, and this association modulates Notch signaling. The biological function of ßII-tubulin locating in nucleus still is unclear, although it was suggested that nuclear ßII-tubulin might be correlated with the cancerous state of cells (20) . The relationship between the intracellular domain of the human Notch1 receptor and ßII-tubulin was investigated. We show herein that N1IC associated with ßII-tubulin in the nuclei of cancer cells and that Taxol treatment induced the increment of nuclear tubulins and increased the CBF1-dependent transactivation activity of N1IC. This is the first report regarding the involvement of nuclear ßII-tubulin in CBF1-dependent Notch signaling. These data suggest that nuclear ßII-tubulin may be involved in the control of tumorigenesis through the interaction with activated Notch1 receptor, which has been implicated in cancers (5 , 6) .

It was reported that the distribution of nuclear ßII was highly dependent on the type of cancer (21) . These findings and our results suggest that nuclear ßII-tubulin could be a useful diagnostic agent in cancers. In addition to clarifying the biological function of nuclear tubulin in cancer cells, this study also raises the possibility that regulation of tumor formation could be regulated by nuclear ßII-tubulin through Notch signaling. We will further dissect the roles of nuclear ßII-tubulin in tumorigenesis of various cancers.

An intriguing similar situation is that the cytoskeletal protein actin also has been detected in the cell nucleus, and it has been tentatively implicated in gene expression (31, 32, 33, 34, 35) . Percipalle et al. (36) recently also showed that the actin-ribonucleoprotein interaction is involved in transcription by RNA polymerase II.

Taxol not only binds to tubulin to promote microtubule assembly and to stabilize microtubules by bundle formation (37, 38, 39) but also to modulate gene expression (28, 29, 30) . Furthermore, Taxol markedly enhances the nuclear factor {kappa}B and activator protein transcription factors binding to their response elements in the interleukin-8 promoter, which in turn up-regulates the IL-8 gene in Taxol-responsive ovarian cancer cells (40) . To clarify whether elevation of nuclear ßII-tubulin by Taxol of nonapoptotic concentration was caused by activation of gene expression or nuclear translocation, cycloheximide was used to block the de novo protein synthesis. Elevation of nuclear ßII-tubulin by Taxol also was observed in the presence of this inhibitor (Fig. 5B and C)Citation . The amount of nuclear ßII-tubulin in the cytosolic fraction also showed no apparent variation with the various treatments. These results may exclude the possibility that Taxol activates ßII-tubulin gene expression and suggest that Taxol enhances the nuclear import of ßII-tubulin. This observation in K562 and HeLa cells is different from that of rat C6 glioma cells; Taxol in an apoptotic concentration has been shown to deplete nuclear ßII-tubulin in C6 glioma cells (20) .

How does ßII-tubulin enter nuclei of cancer cells? The COOH-terminal region of tubulin is less conserved among various isotypes (41) ; therefore, this isotype-specific region may be involved in the nuclear entrance of ßII-tubulin. The nuclear ßII-tubulin, most likely in the form of {alpha}ßII dimer, was suggested to bind with the ßII-interacting protein to remain itself in the nucleus (21) . In the experiment of yeast two-hybrid system, we found that the COOH-terminal region of ßII-tubulin (amino acid residues 197 to 445) is sufficient to associate with N1IC. It is possible that N1IC with the nuclear localization signal may be involved in the nuclear localization of ßII-tubulin through their association.

Alternatively, it also has been speculated that ßII-tubulin remains attached to chromatin after cell division and then is trapped in the nucleus during interphase (42) . However, cell cycle arrest induced by Taxol cannot enhance nuclear localization of ßII-tubulin through attachment to chromatin from the mitotic phase into interphase. This implies that there must be other mechanisms through which ßII-tubulin enters the nucleus.


   ACKNOWLEDGMENTS
 
We thank Dr. S. D. Hayward for the gift of reporter plasmids 4xwtCBF1Luc and 4xmtCBF1Luc.


   FOOTNOTES
 
Grant support: National Health Research Institutes (NHRI-EX93–9315BC) and National Science Council (NSC 92–2320-B-038–054) of the Republic of China.

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 18 U.S.C. Section 1734 solely to indicate this fact.

Requests for reprints: Tien-Shun Yeh, Graduate Institution of Cell & Molecular Biology, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan. Phone: 886-2-2930-7930 ext. 2541; E-mail: cmbtsyeh{at}tmu.edu.tw

Received 6/22/04. Revised 8/26/04. Accepted 9/12/04.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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