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J. Biol. Chem., Vol. 275, Issue 40, 31145-31154, October 6, 2000

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TOK-1, a Novel p21^Cip1-binding Protein That Cooperatively Enhances p21-dependent Inhibitory Activity toward CDK2 Kinase^*

Takashi Ono, Hirotake Kitaura, Hideyo Ugai^§, Takehide Murata^§, Kazunari K. Yokoyama^§, Sanae M. M. Iguchi-Ariga^¶, and Hiroyoshi Ariga^**

From the  Graduate School of Pharmaceutical Sciences, ^¶ College of Medical Technology, Hokkaido University, Kita-ku, Sapporo 060, Japan, ^§ RIKEN, Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, Japan, and  CREST, Japan Science and Technology Corp., 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Received for publication, April 10, 2000, and in revised form, June 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A p21^{Cip1/Waf1/Sdi1} is known to act as a negative cell-cycle regulator by inhibiting kinase activity of a variety of cyclin-dependent kinases. In addition to binding of the cyclin-dependent kinase to the N-terminal region of p21, p21 is also bound at its C-terminal region by proliferating cell nuclear antigen (PCNA), SET/TAF1, and calmodulin, indicating the versatile function of p21. In this study, we cloned cDNA encoding a novel protein named TOK-1 as a p21 C-terminal-binding protein by a two-hybrid system. Two splicing isoforms of TOK-1, TOK-1 and TOK-1, comprising 322 and 314 amino acids, respectively, were co-localized with p21 in nuclei and showed a similar expression profile to that of p21 in human tissues. TOK-1, but not TOK-1, directly bound to the C-terminal proximal region of p21, and both were expressed at the G₁/S boundary of the cell cycle. TOK-1 also preferentially bound to an active form of cyclin-dependent kinase 2 (CDK2) via p21, and these made a ternary complex in human cells. Furthermore, the results of three different types of experiments showed that TOK-1 enhanced the inhibitory activity of p21 toward histone H1 kinase activity of CDK2. TOK-1 is thus thought to be a new type of CDK2 modulator.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell-cycle movement is known to be coordinately regulated by several combinations of cyclin-cyclin-dependent kinase (CDK)¹ complex and their inhibitors. INK4 family proteins, including p15, p16, p18, and p19, inhibit CDK4/CDK6, and Cip/Kip family proteins, including p21, p27, and p57, inhibit all of the CDKs (see recent reviews, Refs. 1-3 and references therein). A cDNA of p21^{Cip1/Waf1/Sdi1} was cloned independently by different three procedures: Cip1 (4), a cyclin-dependent kinase 2 (CDK2)-binding protein; Waf1, p53-inducible protein (5); and Sdi1, a senescent-inducible protein (6). The p21 gene is induced dependently or independently by p53 in cells after various stresses. p53-dependent p21 induction occurs by x-ray or UV irradiation, which causes DNA damage, and by heat shock or osmotic shock to cells (5). Nutrition starvation, contact inhibition, terminal differentiation, or aging of cells triggers p53-independent p21 induction that is brought about by transcription factors, including STAT family protein (7), C/EBP (8), MyoD (9, 10), and vitamin D3 receptor (11). In any case, p21 induces cell cycle arrest, thus inhibiting CDK activity necessary for Rb inactivation.

In addition to binding of CDK-cyclin to the N-terminal region of p21 (12-16), a variety of proteins were found to bind to the C-proximal region of p21. DNA replication activity of DNA polymerase  is inhibited by p21 by binding to a proliferating cell nuclear antigen (PCNA), indicating that p21 also has the function of stopping the cell cycle during the S phase (12, 13, 15, 17-20). Furthermore, SET/TAF1 (21), human papilloma virus E7 (22, 23), c-Myc (24), and calmodulin (25) are reported to bind to the C-terminal region of p21. The molecular mechanisms explaining the versatile functions of p21, however, remain to be determined.

In this study, we identified and characterized a novel protein, TOK-1, as a p21 C-terminal region-binding protein. TOK-1 comprises two splicing isoforms, TOK-1 and TOL-1. Only TOK-1 bound to p21 in a ternary complex with CDK2 in cells and enhanced the p21 function of inhibition of the kinase activity of CDK2. Thus, TOK-1 is a new modulator of p21.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells-- Human HeLa, 293 and 293T cells, and monkey COS1 cells were cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum.

Plasmids-- All of the plasmid DNAs were basically constructed by polymerase chain reaction-based methods, in which the amplified fragments were inserted into the respective sites of the desired vectors. Details of the construction procedures are available upon request.

Cloning of TOK-1 by a Two-hybrid System-- Saccharomyces cerevisiae L40 cells containing the lacZ gene driven by the GAL1 promoter were transformed first with pLex-p21, which did not activate lacZ transcription by itself. The transformant cells were subsequently transformed with human brain MATCHMAKER cDNA (CLONTECH), a cDNA library expressing the GAL4 activation domain fused to the cDNAs from human brain cells. Approximately 5 × 10⁶ colonies were screened for lacZ expression, which indicate the association of a GAL4 activation domain-fused protein with the LexA DNA binding domain (LexABD)-fused p21. The plasmid DNAs in the lacZ-positive cells were extracted by the procedure described in the protocols from CLONTECH. The plasmid derived from a positive colony was named pACT-TOK-1 and characterized.

Antibodies-- A fusion protein of GST to TOK-1 was expressed in Escherichia coli BL21(DE3) and purified as described previously (26). GST-TOK-1 was released from GST-TOK-1 by the PreScission protease (Amersham Pharmacia Biotech). Rats were immunized by GST-free TOK-1. The serum of the rats was used as a polyclonal anti-TOK-1 antibody after purification through a glutathione-Sepharose 4B column containing GST-TOK-1.

In Vitro Binding Assay-- GST-TOK-1, GST-TOK-1, GST-p21, and GST were purified from a 1-liter culture of E. coli BL21(DE3) transformed with pGEX-GST-TOK-1, pGEX-GST-TOK-1, GST-p21, and pGEX-6P-1, respectively, as described previously (26). GST-free TOK-1 and TOK-1 were released from the GST-TOK-1 and GST-TOK-1, respectively, by digestion with PreScission protease (Amersham Pharmacia Biotech). Three µg of GST-p21 or GST was first incubated with 3 µg of GST-free TOK-1 or TOK-1 for 2 h in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.25% gelatin, 1 mM EDTA, 0.1% Nonidet P-40, and 0.02% NaN₃ and then applied to a glutathione-Sepharose 4B (Amersham Pharmacia Biotech). After extensive washing of the column, the proteins recovered from the resin were separated in a 10% polyacrylamide gel containing SDS, blotted onto a nitrocellulose filter, and reacted with a rat polyclonal anti-TOK-1 antibody.

In Vivo Binding Assay-- One µg of pCMV-FLAG-TOK-1 or TOK-1 together with 1 µg of pCMV-p21 was transfected to human 293T cells 60% confluent in a 10-cm dish by the calcium phosphate precipitation technique. Forty-eight hr after transfection, the whole cell extract was prepared by the published procedure (27). Approximately 500 µg of the 293T cell proteins was first immunoprecipitated with 1 µg of a mouse anti-FLAG antibody (M2, Sigma) or with nonspecific mouse IgG in a buffer containing 50 mM Tris-HCL (pH 8.0), 150 mM NaCl, 0.25% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml bovine serum albumin, 2 µg/ml leupeptin, 10 µg/ml aprotinin, 5 µg/ml pepstatin A, 1 mM Na₃VO₄, and 10 mM -glycerophosphate. After washing with the same buffer except for 0.05% Nonidet P-40 instead of 0.25%, the precipitates were separated in a 10% of polyacrylamide gel containing SDS, blotted onto a nitrocellulose filter, and reacted with a mouse anti-p21 monoclonal antibody (Transduction Laboratories), a mouse anti-CDK2 monoclonal antibody (M2, Santa Cruz), or the rat anti-TOK-1 antibody.

Indirect Immunofluorescence-- COS1 cells were transfected with pCMV-FLAG-TOK-1 or TOK-1 together with pCMV-p21-HA by the calcium phosphate precipitation technique (28). Forty-eight hr after transfection, the cells were fixed with a solution containing acetone-methanol (3:7) and reacted with a mouse anti-FLAG monoclonal antibody (M2, Sigma) and a rabbit anti-HA polyclonal antibody (Santa Cruz). The cells were then reacted with an fluorescein isothiocyanate- or rhodamine-conjugated anti-mouse or anti-rabbit IgG, respectively, and observed under a confocal razor fluorescent microscopy.

Construction of a Recombinant Adenovirus Expressing TOK-1-- An adenovirus expressing TOK-1 was constructed according to the published procedure for the method by a homologous recombination (29). Basically, a blunt-ended cDNA containing TOK-1 was inserted into SwaI site of pAxCAwt followed by transfection together with adenovirus DNA containing a terminal protein to human 293 cells. After plaque purification of the desired adenovirus, a recombinant adenovirus for TOK-1 (AxCATOK-1) was selected.

Establishment of TOK-1- and p21-expressing Cell Lines-- HeLa-Tet-on cells were purchased from CLONTECH. HeLa-Tet-on cells in a 10-cm dish were transfected with 5 µg of pUHD-TOK-1 or pUHD-p21 together with 0.5 µg of pSV-bsr, a blasticidin S expression vector, by the calcium phosphate precipitation technique and cultured in the presence of 0.5 µg/ml of blasticidin and 1 µg/ml of tetracycline. About 2 weeks after transfection, blasticidin-resistant colonies were selected and used as TOK-1 or p21-expressing cell lines.

Kinase Assay-- HeLa cells expressing TOK-1 or p21 under the control of doxcycline were cultured in the presence or absence of 1 µg/ml of doxcycline for 2 days, and the cell extracts were prepared by sonication of cells in an H1-kinase buffer containing 50 mM Tris-HCL (pH 8.0), 200 mM NaCl, 0.5% Nonidet P-40, 0.3 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 0.1 mM Na₃VO₄, and 20 mM NaF. One hundred µg of proteins in the extracts was first incubated with 250 ng of an anti-CDK2 antibody for 2 h at 4 °C and further incubated with protein A/G-agarose for 1 h. After extensive washing of the mixture with buffer A containing 20 mM Tris-HCl (pH 8.0), 250 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 2 µg/ml leupeptin, 5 µg/ml pepstatin A, 0.1 mM Na₃VO₄, 1 mM NaF, and 10 mM -glycerophosphate, then with buffer A containing 100 mM NaCl and, finally, with H1-kinase buffer, the mixture was reacted with 5 µCi (6000 mmol/ml) of -ATP and 1 µM ATP and 5 µg of histones H1 for 20 min at 30 °C. The mixture was boiled in Laemmli buffer, and the proteins in the mixture were separated on polyacrylamide gel and autoradiographed.

Fifty µg of H9 cell extract prepared as described above was first reacted with a recombinant TOK-1 with or without a recombinant p21 extracted from E. coli for 2 h at 4 °C. After the anti-CDK2 antibody was added to the mixture, the kinase assay was carried out as described above.

HeLa cells expressing TOK-1 or p21 under the control of doxcycline were infected with a recombinant adenovirus of TOK-1 at multiplicity of infection 10 and cultured in the presence or absence of doxcycline for 2 days. Three hundred µg of cell extract prepared as described above was added to 250 µg of anti-CDK2 antibody, and the kinase assay was carried out as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of cDNAs Encoding p21-binding Proteins-- To screen cDNAs encoding p21-associated proteins, a segment spanning amino acid number 87 to 164 of human p21 cDNA was fused to the LexA DNA-binding domain (LexABD) in pGLex (30), and S. cerevisiae L40 cells were transformed with the plasmid. A library of human brain cDNAs in pACT was then introduced to the transformants, and the colonies resistant to the His marker were selected. Among the 5 × 10⁶ cells transformed in total, 87 colonies were His-positive. After the -galactosidase assay of the colonies, 5 positive clones were obtained and identified as the same clones by DNA sequencing analysis. A clone was termed TOK-1 (ptwenty-one and CDK-associated protein-1) and characterized in this study.

The TOK-1 cDNA isolated contained 1,272 nucleotides and encoded 320 amino acids without the putative first methionine. After an EST data base search, two types of cDNA homologous to the cloned TOK-1 cDNA were found. One clone was TOK-1 cDNA with an extra 18 bases at the 5' end encoding 322 amino acids, and the other, encoding 314 amino acids, was a splicing isoform of TOK-1, changing after amino acid 259. The former of the original isolate was named TOK-1, and the splicing form was named TOK-1 (Fig. 1). TOK-1 cDNA was cloned by polymerase chain reaction with desired primers from the TOK-1 DNA sequence using human brain cDNAs as a template. Although there was no in-frame stop codon upstream of the first methionine of cDNA clones obtained, the nucleotide sequences around the putative first methionine were well matched with the Kozak consensus sequence (31), suggesting that these cDNAs encode full-size TOK-1 and TOK-1. There were no unique structural motifs therein.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   Nucleotide and amino acid sequences of TOK-1. A, nucleotide and amino acid sequences of TOK-1 and TOK-1 are shown. The plausible initiation and stop codons are boxed. A putative nuclear localization signal is underlined. The polyadenylation signal are indicated by a hatched square. B, structures of TOK-1 and TOK-1. Open boxes indicate regions of TOK-1 and TOK-1 that contain the same amino acids (aa), and the hatched boxes represent the regions containing unique amino acids.

Expression of TOK-1 in Various Tissues and Cell Cycle-- The expression of TOK-1 mRNA was examined by Northern blot analysis in various human and mouse tissues using a common or specific probe to TOK-1 and TOK-1. The common probe to TOK-1 and TOK-1 gave 1.5- and weak 3.0-kilobase mRNA (Fig. 2A, TOK-1, ). By using a specific probe to TOK-1 and TOK-1, a TOK-1 mRNA of 1.5 kilobases was expressed highly in skeletal muscle, and TOK-1 mRNA of 1.5 kilobase was expressed highly in skeletal muscle and the heart, moderately in the placenta and pancreas, and weakly in the brain, kidney, and liver, the patterns of which were similar to those of p21 (Fig. 2A, TOK-1, TOK-1, and p21, respectively). A polyclonal antibody against TOK-1 was prepared by using recombinant TOK-1 expressed in E. coli and purified as an immunogen to the rat. In Western blot analyses using the polyclonal anti-TOK-1 antibody, two distinct proteins of 50 and 45 kDa were detected in human 293T cells (Fig. 2B, lane 1). When an excess amount of the recombinant TOK-1 was added to the reaction with the antibody, both the 50- and 45-kDa proteins disappeared (Fig. 2B, lane 2), and both proteins were thus identified as endogenous TOK-1 or TOK-1. Expression vectors for TOK-1 and TOK-1 were transfected to 293T cells, and the cell extract was blotted with the anti-TOK-1 antibody (Fig. 2A, lanes 3 and 4). Proteins of 50 and 45 kDa were detected in TOK-1- and TOK-1-transfected cells, respectively. The results suggest that the TOK-1 and TOK-1 cDNAs cloned correspond to the 1.5-kilobase mRNAs encoding the 50 and 45 kDa proteins, respectively.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   Expression of TOK-1 and TOK-1 mRNA and the proteins in human tissues and cells. A, Northern blot analyses were carried out using three multiple Northern blot sheets of human tissues with different probes. The probes used were the region spanning amino acids 1-322 of TOK-1 cDNA (TOK-1, ), 259-322 of TOK-1, 259-314 of TOK-1, and the respective full-size cDNAs of p21 and -actin (p21 and -actin). B, cell extracts were prepared from non-transfected human 293 cells (lanes 1 and 2) and 293 cells transfected with an expression vector for TOK-1 (lane 3) or TOK-1 (lane 4), and their proteins were separated on 10% polyacrylamide gel. After blotting onto a nitrocellulose filter, the proteins on the filter were reacted with an anti-TOK-1 polyclonal antibody and visualized by an ECL system (lanes 1-3) (Amersham Pharmacia Biotech). To see the specificity of the proteins that reacted with the anti-TOK-1 polyclonal antibody, the anti-TOK-1 antibody absorbed with 1 µg of GST-TOK-1 was used (lane 2).

To see the expression of TOK-1 during the cell cycle, HeLa cells were treated with mimosine to restore the cells at the G₁/S boundary. Then the total RNAs and proteins were extracted from the cells at various times after mimosine release, and Northern and Western blotting analyses were carried out (Fig. 3, A and B, respectively). To confirm that the cells were synchronized by this procedure, DNA from an aliquot of cells was stained with propidium iodide and analyzed by flow cytometry (Fig. 3C). Cells entered the S phase at 6 h after mimosine release, then entered the G₂/M phase from 12 to 18 h and reentered the G₀/G₁ phase after 30 h. Northern blot analysis showed that TOK-1 mRNA was expressed after the G₂/M phase and peaked before the S phase of the cell cycle. TOK-1 mRNA, on the other hand, was expressed during the cell cycle with a tendency to the pattern of TOK-1. The p21 mRNA was expressed throughout the cell cycle. At the protein level, however, distinct expression patterns among TOK-1, TOK-1, and p21 were observed. Both TOK-1 and p21 were prominently expressed at the boundary of G₁/S of the cell cycle (Fig. 3B, lane 8), whereas TOK-1 was expressed throughout the cell cycle (Fig. 3B). The different patterns between RNA and protein levels of TOK-1 and p21 may be due to the modification of proteins to affect their stabilization.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Expression of TOK-1 in the cell cycle. HeLa cells were synchronized to the G1/S boundary in a medium containing 500 µM mimosine for 24 h. After extensive washing of the cells with the medium, the cells started to enter the S phase of the cell cycle by the addition of a new medium without mimosine. The time for adding the new medium was set to 0. RNAs and proteins were extracted from the cells at various times after the addition of the new medium, blotted onto filters, and Northern (A) and Western blotting (B) analyses were carried out as in Fig. 2. C, to confirm cell synchronization, an aliquot of the above cells was stained with propidium iodide and analyzed by flow cytometry (Fax Sort, Becton Dickinson). G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

Binding of TOK-1 with p21 in Vitro-- To determine the TOK-1/p21 binding domains on both of the proteins, GST fusion proteins of various deletion mutants of TOK-1, TOK-1, and p21 were constructed to be expressed in E. coli and purified by a GST affinity column. GST- TOK-1 or TOK-1 was subsequently treated with PreScission protease (Amersham Pharmacia Biotech) to release GST-free TOK-1 or TOK-1. GST-p21 or its deletion mutants were incubated with GST-free TOK-1 or TOK-1 and then trapped on the glutathione-Sepharose resin. After extensive washing, the proteins bound to the column were recovered and analyzed by Western blotting using an anti-TOK-1 antibody. The presence of GST-p21 and GST-p21 deletion mutants was first confirmed by blotting against an anti-GST antibody (Fig. 4 middle). TOK-1 bound to the wild type and the C-proximal region of GST-p21 but not to the N-terminal half of p21, which includes the regions bound by cyclin and CDK (Fig. 4). Fine deletion mutants of the C-proximal region of GST-p21 indicated that TOK-1 bound to a region spanning amino acids 149 to 164 of p21, which included the region bound by PCNA and cyclin.

View larger version (36K): [in this window] [in a new window]   Fig. 4.   Determination of the p21 region bound by TOK-1. The wild type (p21 wt) or various deletion mutants of TOK-1 were fused to GST and used for the pull-down assay using recombinant TOK-1 that was extracted as GST-TOK-1 from E. coli and then released from GST. After the reaction, the bound proteins were blotted with an anti-TOK-1 antibody (upper panel). To see the GST-21 and its deletion mutants applied to the reaction, an aliquot of the samples was also blotted with the anti-GST antibody (lower panel). In the schematic drawing of the TOK-1 construct, the domains bound by cyclin, CDK, and PCNA are indicated by hatched or black boxes.

To see the interaction domain of TOK-1 with p21, the wild type or deletion mutants of FLAG-tagged TOK-1 or TOK-1 was incubated with GST wild type p21 (GST-p21 wt) or the GST-N-terminal half of p21 (GST-p21 1-158) and then applied to glutathione-Sepharose resin. The proteins bound to the resin were analyzed by Western blotting using an anti-FLAG antibody (Fig. 5). The wild type and the mutants containing a region spanning amino acids 161 to 259 of TOK-1 were recovered from the resin bound by the GST-p21 wt but not GST-p21 1-158 (Fig. 5, lanes 1-10). Contrary to TOK-1, neither the wild type nor the deletion mutants of TOK-1, including even the mutant C containing the region spanning amino acids 161 to 258 common to TOK-1 and TOK-1, bound to GST-p21 (Fig. 5, lanes 11-16). These results suggest that p21 directly binds to the region spanning amino acids 161 to 259 of TOK-1 and that the C-proximal region of TOK-1 spanning amino acids 259 to 322 interferes with the binding between p21 and TOK-1.

View larger version (47K): [in this window] [in a new window]   Fig. 5.   Determination of the binding region of TOK-1 with p21. The GST fusion proteins of wt and deletion mutants spanning amino acids 1-158 () of p21 were prepared in E. coli and incubated with glutathione beads coupled with FLAG-TOK-1 (lanes 1-10) or FLAG-TOK-1 (lanes 1-16). Proteins bound to the beads were then separated by SDS-polyacrylamide gel electrophoresis (10%) and blotted using an anti-FLAG antibody (upper panel). The 1/200 volumes of the proteins used for binding reaction were applied to the same gel (lanes 17-24).

Co-localization of p21 with TOK-1 in the Cells-- To determine the cellular localization of TOK-1 or TOK-1, an expression vector for FLAG-tagged TOK-1 or TOK-1 was transfected to monkey COS1 cells. Two days after transfection, the cells were stained with an anti-FLAG antibody, and the proteins were visualized under a confocal razor microscopy. Cell nuclei were identified by Hoechst staining. Both FLAG-TOK-1 and -TOK-1 were observed in the nuclei but not in the cytoplasm (Fig. 6A). When both p21 fused to HA and FLAG-TOK-1 or -TOK-1 were cotransfected to COS1 cells and detected by the rhodamine- and fluorescein isothiocyanate-conjugated second antibodies, respectively, the TOK-1 or TOK-1 (green) and p21 (red) were co-localized in nuclei as shown by the yellow color (Fig. 6B). These results suggest that although TOK-1 does not directly bind to p21, both exist in the same region or near regions in cells.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Cellular localization of PAP-1. A, an expression vector for FLAG-TOK-1 or FLAG-TOK-1 was transfected to monkey COS1 cells by the calcium phosphate precipitation technique. Two days after transfection, the cells were fixed, reacted with an anti-FLAG monoclonal antibody (M2, Sigma), and visualized with an fluorescein isothiocyanate-conjugated anti-mouse antibody. The same slides as in A were also stained with Hoechst 33258. B, the expression vector for FLAG-TOK-1 or FLAG-TOK-1 was cotransfected with an expression vector for p21-HA to COS1 cells as in A and visualized with a rhodamine-conjugated anti-mouse IgG and the fluorescein isothiocyanate-conjugated anti-rabbit antibody, respectively (-FLAG and -HA, respectively). Both figures were merged (merge).

Binding of TOK-1 to p21 in Vivo and a Ternary Complex among TOK-1, p21, and CDK2-- Expression vectors driven by the CMV promoter for FLAG-TOK-1 and various amounts of p21 or its deletion mutant containing amino acids 1 to 152 (p21 ()) were transfected to human 293T cells. Forty-eight hours after transfection, cell extracts were prepared, and the proteins in the extract were first precipitated with an anti-FLAG antibody. The precipitates were subjected to Western blot analyses using an anti-p21 or CDK2 antibody (Fig. 7). Since the immunogen to the anti-p21 antibody used here (Transduction Laboratories C24420) was the peptide spanning amino acids 1-150 of p21, the expressions of both wild-type p21 and p21 () in transfected cells were confirmed by Western blotting with this antibody (Fig. 7, second autoradiogram from the top, lanes 8-10, and data not shown). Relatively constant amounts of the introduced FLAG-TOK-1 were detected in the precipitate with the anti-FLAG antibody (Fig. 7, lanes 1-5). Furthermore, p21, but not p21 (), and the endogenous CDK2 were detected in a dose-dependent manner in the precipitate with the anti-FLAG antibody in FLAG-TOK-1-transfected cells but not in non-transfected cells (Fig. 7, lanes 2-5 and 6, respectively). It was noted that an active phosphorylated form of CDK2 was precipitated much more than was an inactive unphosphorylated CDK2 in Flag-TOK-1-transfected cells (Fig. 7, lanes 2-4), whereas the inactive unphosphorylated CDK2 was more precipitated in cells transfected with p21 alone (Fig. 7, lane 11). By using recombinant CDK2 and TOK-1 expressed in and purified from E. coli, no direct binding between the two proteins was observed (data not shown). The results suggested that TOK-1 makes a ternary complex with p21 and CDK2 via p21 in cells.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Association of TOK-1 with p21 and CDK2 in human 293T cells. The expression vectors for FLAG-TOK-1 and p21 or its deletion mutant spanning amino acids 1-152 were introduced into human 293T cells. Two days after transfection, cell extracts were prepared, and the proteins in the extracts were first immunoprecipitated (IP) with an anti-FLAG antibody (lanes 1-6). The proteins in the precipitates were separated in a 10% polyacrylamide gel and blotted with an anti-p21 antibody (first autoradiogram), anti-CDK2 antibody (second autoradiogram), or anti-FLAG antibody (third autoradiogram). The 1/200 volumes of the extract used for binding reaction were applied in the same gel (lanes 7-10, input). The amounts of the p21 expression vector used were 0.01, 0.1, and 1 µg in lanes 2, 3, and 4, respectively. The expression vector for p21 was introduced into human 293T cells, and the proteins in the cells were immunoprecipitated with the anti-p21 antibody (lane 11). p21 and CDK2 were detected by Western blotting as in lanes 1-10.

Stimulation of p21-dependent Inhibitory Activity to CDK2 by TOK-1-- Human HeLa-Tet-on cells that harbor tetracycline/doxcycline-dependent reverse-repressor (CLONTECH) were transfected with a expression vector for TOK-1 or p21 under the control of a doxcycline-dependent CMV promoter (32, 33), together with pSV-bsr, an expression vector for blasticidin S-resistant gene, and were cultured in the presence of blasticidin S. Then, blasticidin S-resistant HeLa cell lines that express TOK-1 or p21 in the presence of doxcycline were established. Inducibility of the desired proteins in the cells was tested in the presence or absence of 2 µg/ml of doxcycline by Western blotting (Fig. 8). Cells harboring the vector alone, HV9, expressed neither TOK-1 nor p21 irrespective of the presence of doxcycline (Fig. 8, A and B, lanes 1 and 2). Two cell lines, HT39 and HT52, expressed TOK-1 only in the presence of doxcycline (Fig. 8A, lanes 3-6). HP28 and HP8 expressed p21 only and highly in the presence of doxcycline (Fig. 8B, lanes 3-6). All these cell lines, however, equally expressed both inactive and active forms of CDK2 under both conditions of doxcycline (Fig. 8C).

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Establishment of cell line expression TOK-1 or p21 under the control of a doxcycline-dependent promoter. HeLa TeT-on cells (CLONTECH) were transfected with a Tet-inducible expression vector for TOK-1 or p21 and cultured in the presence of 2 µg/ml doxcycline. Two weeks after transfection, doxcycline-resistant clones were selected. Cell extracts were prepared from cell lines expressing TOK-1 (HT39 and HT52), p21 (HP28 and HP8), and the vector alone (HV9), and the expressions of TOK-1 (A), p21 (B), and CDK2 (C) were examined by Western blotting with the respective antibodies.

To determine the biological functions of TOK-1, the effect of TOK-1 on the inhibitory effect of p21 on CDK2 kinase activity was examined by three procedures. First, HV9, HT39, and HP28 cell extracts were prepared from cells cultured in the presence or absence of doxcycline, and they were immunoprecipitated with an anti-CDK2 antibody. The precipitates were reacted with [-³²P]ATP and histone H1 as a substrate (Fig. 9A). Autoradiogram and its quantitation result are shown. HV9 cells harboring the vector alone did not change H1 kinase activity irrespective of the presence or absence of 1 µg/ml doxcycline, and HP28 cells, p21-expressing cells, inhibited H1 kinase activity of CDK2 (Fig. 9A, lanes, 1, 2, 5, and 6). HT39 cells, on the other hand, also inhibited H1 kinase activity in the presence of 1 µg/ml doxcycline less than did p21 (Fig. 9A, lanes 3 and 4). We then tested the activity of TOK-1 toward CDK2 activity by the ectopic expression system. HV9, HP28, and HP8 cells were infected with a recombinant adenovirus to express TOK-1 (Ad-TOK-1) or with an adenovirus harboring the vector alone (Ad-V), and they were cultured in either absence or presence of low amounts (0.02 and 0.2 µg/ml) of doxcycline, where the inhibition of H1 kinase activity by induced p21 was approximately 30% that of 1 µg/ml doxcycline. Cell extracts were then prepared and used for the H1 kinase assay of CDK2 (Fig. 9B). HV9 cells infected with Ad-TOK-1a showed kinase activities that were decreased to a similar extent in all of the concentrations of doxcycline used, whereas Ad-V-infected cells had no effect (Fig. 9B, lanes 1-6). As shown in Fig. 9A, the H1 kinase activity was inhibited in a dose-dependent manner in HP28 and HP8 cells infected with Ad-V, and infection of Ad-TOK-1 further inhibited H1 kinase activity (Fig. 9B, lanes 7-18). These results suggest that TOK-1 alone moderately inhibits CDK2 activity and/or enhances the inhibitory activity of endogenous p21 toward CDK2. To assess this possibility more precisely, a reconstitution experiment was carried out. Cell extracts from HV9 cells were reacted with the purified recombinant TOK-1 with or without the purified recombinant p21, then immunoprecipitated with the anti-CDK2 antibody, and their H1 kinase activities were examined (Fig. 9C). The results clearly showed that TOK-1 alone did not inhibit H1 kinase activity of CDK2 over the range of TOK-1 used (Fig. 9C, lanes 1-4). The inhibitory activity of p21 toward CDK2 was, however, further enhanced by the recombinant TOK-1 in a dose-dependent manner (Fig. 9, lanes 5-8). This result suggests that TOK-1 enhances the p21 inhibitory activity of CDK2s.

View larger version (50K): [in this window] [in a new window]   Fig. 9.   Effect of TOK-1 on p21 inhibitory activity toward CDK2 kinase. A, HV9, HT39, and HP28 cells were cultured in the presence or absence of 1 µg/ml doxcycline for 48 h. Then cell extracts were prepared and immunoprecipitated with an anti-CDK2 antibody. H1 kinase activity was examined in a reaction mixture containing cell extract and histone H1 as described under "Experimental Procedures." B, HV9, HP28, and HP8 cells were infected with an adenovirus expressing TOK-1 (AxCATOK-1) or the vector alone (AxCA) and cultured in the presence of 0, 0.02, or 0.2 µg/ml doxcycline for 48 h. Then, H1 kinase activity was examined as in A. To easily realize the adenoviruses used in this experiment, AxCATOK-1 and AxCA were described as Ad-TOK-1 and Ad-V, respectively, in this figure. C, the H9 cell extract prepared above was first reacted with various amounts of a recombinant TOK-1 with or without a recombinant p21 extracted from E. coli for 2 h at 4 °C. After the anti-CDK2 antibody was added to the mixture, the kinase assay was carried out as described above.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have described the cloning and characterization of novel proteins, TOK-1 and TOK-1. TOK-1 is a splicing form of TOK-1 in which two-thirds amino acids from the N terminus are identical to those of TOK-1. These were found to be co-localized in cell nuclei. TOK-1, but not TOK-1, was expressed in the G₁/S boundary of the cell cycle at the same timing as that of p21 and bound directly to p21 both in vitro and in vivo. TOK-1 also preferentially bound to an active form of CDK2 (34) via p21 in a ternary complex in cells. Since p21 alone tended to bind to the inactive form of CDK2, TOK-1 was assumed to modulate a function or status of p21 when both were in a complex. TOK-1 enhanced p21 activity to inhibit H1 kinase activity of CDK2 in three types of experiments in which the precipitates of cell extracts with an anti-CDK2 antibody were used as the kinase source on an exogenously added histone H1 substrate, suggesting that TOK-1 helps p21 to absorb the active form of CDK2 in cells. TOK-1 expressed in doxcycline-induced or TOK-1 adenovirus-infected human cells moderately inhibited the H1 kinase activity of CDK2 by itself, whereas TOK-1 alone purified from E. coli did not show any inhibitory activity but still enhanced the inhibitory activity of p21 toward CDK2 in a reconstitution system. This discrepancy between TOK-1 in human cells and the recombinant TOK-1 from E. coli cells may be explained as follows: the modified form of TOK-1 due to phosphorylation or acetylation in human cells efficiently binds to p21 endogenously present in a low amount but sufficient to inhibit CDK2, whereas the unmodified form of TOK-1 in E. coli has no or only weak activity. Alternatively, another protein or other proteins associated with TOK-1 present in the immunoprecipitates might be necessary for TOK-1 to enhance p21 activity.

Although two-thirds of the N-terminal amino acids of TOK-1 were identical to those of TOK-1, p21 bound to this identical region of only TOK-1. This suggests that the different one-third C-terminal amino acids of TOK-1 from that of TOK-1 interfere with the binding activity of TOK-1 toward p21, probably due to the tertiary structure of the protein. TOK-1 was, however, co-localized with p21 in cell nuclei. It is plausible to speculate from these phenomena that TOK-1 affects or modulates the some functions of TOK-1 or p21.

Originally, p21 was reported to inhibit CDK activity (4), and it was later found that p21 stimulated the assembly of CDK4/cyclin D and translocation of CDK4-cyclin D to the nucleus (35). To do this, the CDK-cyclin complex binds to the N-proximal region of p21. In addition to these N-terminal functions of p21, p21 was assumed to have other functions due to the interaction of proteins with the C-proximal region of p21. These include PCNA (12, 13, 15, 17-20), SEN/TAF1 (21), c-Myc (24), and calmodulin (25). The DNA replication function of PCNA, a cofactor of DNA polymerase , was inhibited by p21 during the S phase of the cell cycle (17, 18). SEN/TAF1, a putative oncogene product related to non-lymphomatic acute lymphoma, bound to the region spanning amino acid 125 to 146 of p21, the same region as that of TOK-1 binding (21). SEN/TAF1 abrogates the inhibitory effect of p21 on CDK2-cyclinE (21). Calmodulin also binds to the same region of p21, and this interaction leads to translocation of CDK4-cyclinD to the nucleus (25). It would therefore be interesting to examine whether TOK-1 interacts with or competitively interferes with SEN/TAF1 or calmodulin on p21. Taken together, TOK-1 is a new type of modulator of p21.

	ACKNOWLEDGEMENTS

We thank Yoko Misawa and Kiyomi Takaya for technical assistance.

	FOOTNOTES

^* This work was supported by grants-in-aid from the Ministry of Education, Science, Culture, and Sports of Japan.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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB040450 and AB040451.

^** To whom correspondence should be addressed: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan. Tel.: 81-11-706-3745; Fax: 81-11-706-4988; E-mail: hiro@pharm.hokudai.ac.jp.

Published, JBC Papers in Press, June 30, 2000, DOI 10.1074/jbc.M003031200

	ABBREVIATIONS

The abbreviations used are: CDK2, cyclin-dependent kinase 2; TOK-1, p21-binding protein; HA, hemagglutinin antigen; GST, glutathione S-transferase; PCNA, proliferating cell nuclear antigen; wt, wild type; CMV, cytomegalovirus.

	REFERENCES

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