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Originally published In Press as doi:10.1074/jbc.M412929200 on April 19, 2005

J. Biol. Chem., Vol. 280, Issue 24, 23273-23279, June 17, 2005
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Mutation of Cys105 Inhibits Dimerization of p12CDK2-AP1 and Its Growth Suppressor Effect*

Yong Kim{ddagger}, Hiroe Ohyama§, Vipel Patel§, Marxa Figueiredo{ddagger}, and David T. Wong{ddagger}¶||**{ddagger}{ddagger}§§

From the {ddagger}School of Dentistry and Dental Research Institute, Jonsson Comprehensive Cancer Center, ||Molecular Biology Institute, **Division of Head and Neck Surgery/Otolaryngology, and {ddagger}{ddagger}Henry Samueli School of Engineering and Applied Sciences, UCLA, Los Angeles, California 90095 and §Harvard School of Dental Medicine, Boston, Massachusetts 02115

Received for publication, November 16, 2004 , and in revised form, March 25, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
p12CDK2-AP1 (p12) is a CDK2-associated protein that negatively regulates its kinase activity. Growth arrest of normal diploid cells by contact inhibition resulted in an induction of p27kip1 and reduction of CDK2 levels. Interestingly, we observed concomitantly in growth-arrested cells, there was a reduction of nuclear p12 and the appearance of a nuclear 25-kDa molecule (p25) recognized by anti-p12 polyclonal antibody. Biochemical analysis showed that bacterial His-tagged p12 could be converted into a dimeric p25 in a reducing agent-dependent manner, and mutating the only cysteine residue of p12 (Cys105 -> Ala105) abolished the dimerization. Transient transfection of wild type p12 into U2OS cells showed a reducing agent-sensitive dimerization that was also abolished by the C105A mutation. Furthermore, reduction of p12 expression by a short interfering RNA resulted in a parallel reduction of p25. These data supports the possibility that p25 is a homodimeric form of p12 through the cysteine residue. More interestingly, transient transfection of p12 (C105A) into the normal diploid lung fibroblast CCD18LU cells resulted in a reduction of the growth-inhibitory effect of p12 and abolished the inhibitory effect of p12 on CDK2 kinase activity. In addition, we found that the C105A mutation did not alter nuclear localization of p12, but it prevented association with CDK2. Taken together, our data suggest that p12 forms a nuclear homodimers in contact inhibited normal diploid cells and dimerization of p12 is a necessary process for the growth inhibition effect by p12.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular proliferation is governed by elaborate interplay between mitogens and inhibitors. Growth arrest signals, such as serum reduction, medium depletion, contact inhibition, and other growth inhibiting agents can elicit cellular signaling pathways that target common cell cycle-regulating molecules, but it also seems that they might follow their own distinct pathways (1). Cellular growth arrest in normal cells is followed by cell cycle exit or apoptosis, which are frequently disrupted in neoplastic cells; however, the mechanisms that control these processes are not completely understood (2–4).

A CDK2-associated protein, p12 CDK2-associating protein 1 (p12CDK2-AP1, or p12),1 previously known as DOC-1 (deleted in oral cancer-1), is a 115-amino acid nuclear polypeptide that is down-regulated in ~70% of oral cancers (5). p12 is a highly conserved, ubiquitously expressed gene located on chromosome 12q24 and has a closely related sequence, DOC-1R, located on chromosome 11q13 (6, 7). Recently, p12 has been shown to be an S-phase regulator through two important cellular partners: CDK2 and DNA polymerase-{alpha}-primase (8, 9). Like other cell cycle regulators, p12 also has been implicated in apoptosis both in vitro and in vivo, although the mechanisms are less well defined (10, 11).

Protein dimerization, either cotranslational or posttranslational, is not a rare event occurring in the cell. Mostly DNA binding molecules, such as several known important transcription factors, are shown to form either homo- or heterodimers upon activation (12). It is well established that cytoplasmic domains of certain receptors are dimerized upon activation by ligand binding and autophosphorylation (13, 14). Furthermore, protein molecules localized in the cytoplasm can form homodimers by self-association or heterodimers by binding to other molecules and for transport into the nucleus (15). In acute myelogenous leukemia cells, STAT1 and -3 factors form stable heterodimers during constitutive STAT3 activation by hematopoietic growth factors, which induce proliferation and differentiation (16). It is known that E2F and DP subunits function as a heterodimer in the E2F-dependent pathway and DIP interacts with the DP subunit during growth arrest (17). During cellular growth arrest, several protein molecules are known to undergo either homo- or heterodimerization. It has been shown that activation of three distinct retinoid X receptor/retinoic acid receptor heterodimers induces growth arrest and differentiation of neuroblastoma cells (18). All-trans-retinoic acid induction leads to activation of STAT1 by the phosphorylation of tyrosine 701, dimerization, and subsequent nuclear translocation, resulting in cell cycle arrest and differentiation of human monoblastic U-937 cells (15).

In this report, we have identified a 25-kDa molecule that is recognized by anti-p12 polyclonal antibody in contact inhibited diploid cells. By using biochemical and molecular biological approaches, we have shown that p25 is a homodimer of p12, and its level is increased in the nucleus of contact-inhibited cells. Also, we have observed that there is an inverse correlation between the levels of p25 and active CDK2. We further have shown that the dimerization is mediated by the Cys105 residue of p12. More interestingly, eliminating the ability to form dimer by mutating Cys105 residue resulted in a suppression of the growth-inhibitory effect exerted by p12 along with a sustained CDK2 kinase activity. These findings suggest that p12 dimerizes in the nucleus of diploid cells upon growth arrest induced by contact inhibition and that dimerization may be a cellular process to form an active growth-inhibitory p12 targeting CDK2.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Normal diploid cell lines were cultured for p12 study. HaCaT (spontaneously immortalized human normal skin keratinocytes), CCD18Lu (human lung fibroblasts), Mv1Lu (mink lung epithelial cells), and C3HA (mouse liver fibroblast cells) were cultured. The cells were harvested at log phase as well as contact-inhibited conditions. At log phase, the cells were 50% confluent, whereas at contact-inhibited conditions, the cells were cultured for another 48–96 h after they were 100% confluent.

Western Blotting—Cells were lysed in ice-cold RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS) containing 1 µg/ml aprotinin, 2.5 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0.2 µg/ml lactacystin, and 1 mM Na3VO4. Proteins (30 µg) from whole cell lysates were loaded onto 12 or 15% SDS-polyacrylamide gel, blotted onto polyvinylidene difluoride membranes. The anti-p12 polyclonal antibody 86, anti-CDK2 antibody (C18520 [GenBank] Clone 55 from Transduction Laboratories, San Diego, CA), anti-p27 (C-19) from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), anti-actin (A-2066) from Sigma, and anti-Oct-1 (C-21) from Santa Cruz Biotechnology were used for immunodetection. The blots were visualized by the ECL system (Amersham Biosciences).

Gel Elution—Total cell lysate from CCD18Lu was fractionated by SDS-PAGE in duplicate, and one set of fractions was stained with Coomassie Blue staining (GELCODE® Blue Stain Reagent; Pierce), and the other was subjected to Western analysis. The relative location of p12 and p25 bands was measured from Western blot by using Quantity One imaging software (Bio-Rad), and the bands corresponding to the estimated locations were excised from the blue-stained gel. After the gel slices were cut out, the elution was performed using an Electro-Eluter (Bio-Rad) according to the manufacturer's protocol.

Construction of p12 Cysteine Mutants—To generate the bacterial fusion protein, an N-terminal His-tagged p12 DNA fragment was generated by PCR amplification using a specific primer set containing NdeI or EcoRI. The amplified DNA was digested with NdeI and EcoRI and subsequently subcloned into pET-17b vector (Novagen, San Diego, CA). Cysteine mutant of His-tagged p12 was generated by using the QuikChange site-directed mutagenesis system (Stratagene, San Diego, CA) according to the manufacturer's instructions. To generate the mammalian fusion construct, the p12 coding region was amplified by PCR and subcloned into pFLAG-CMV2 vector (Sigma). FLAG-tagged p12 was used as a template to generate a cysteine mutant by using the QuikChange site-directed mutagenesis system (Stratagene). A specific primer set (primer 1, 5'-CTGGTTCGGGAGGCCTTGGCAGAAACG-3'; primer 2, 5'-CGTTTCTGCCAAGGCCTCCCGAACCAG-3') was used to replace cysteine with alanine. The final clones were verified by DNA sequencing analysis.

Expression and Purification of Prokaryotic p12 Fusion Protein—pET-His-p12 was transformed into BL21 competent cells (Novagen), and recombinant clones were analyzed for the induction of His-tagged protein after isopropyl 1-thio-{beta}-D-galactopyranoside treatment. For purification of His-tagged p12 WT or C105A protein, clones were grown in 500 ml of LBAmp medium and induced with 0.1 mM isopropyl 1-thio-{beta}-D-galactopyranoside when the A650 was about 0.3. After a 3-h induction, the cells were pelleted, and total protein was exacted by using RIPA buffer containing protease inhibitors. After centrifugation at 10,000 x g for 30 min, the supernatant was applied to a nickel column equilibrated with RIPA buffer. After washing with 2 column volumes of RIPA buffer, the bound protein was eluted with a step gradient of immidazole and analyzed by Western blot analysis. Finally, the eluted protein was dialyzed against RIPA buffer overnight at 4 °C.

Nuclear and Cytoplasmic Fractionation—CCD18Lu cellular homogenate was produced by Dounce homogenization in 50 mM Tris buffer (pH 7.4), and the homogenate was centrifuged at 800 x g for 10 min to separate the nuclei and the F1 fraction. The nuclei were washed with homogenization buffer and resuspended in RIPA buffer to obtain the nuclear fraction (N). The F1 fraction from 800 x g centrifugation was further separated by differential centrifugation steps. After spinning at 10,000 x g for 30 min, the pellet was resuspended in RIPA to obtain Fraction 2 (F2). The supernatant was further centrifuged at 100,000 x g for 60 min, and the pellet was resuspended in RIPA buffer to obtain Fraction 3 (F3). The final supernatant was saved as the cytosolic fraction (Cy).

Transient Transfection and Proliferation Analysis—CCD18Lu cells were seeded on a 60-mm dish and transiently transfected with pFLAG-CMV2, pFLAG-p12 WT, or pFLAG-p12 C105A plasmid by using Lipofectamine Plus reagent according to the manufacturer's instructions (Invitrogen). After a 24-h transfection, cells were collected, and 5,000 cells were seeded per well on a 96-well plate. The next day, the proliferation of the cells was measured by using the CCK-8 assay reagent (Alexis Biochemical Corp., San Diego, CA) for 4 days following the manufacturer's instructions. CCK-8 assay reagent of 10% of the cell culture medium was added to each well and mixed gently. After a 1-h incubation at 37 °C, 1% SDS of a one-tenth volume of the culture medium was added, and the absorbance at 450 nm was measured. All of the assays were done in 16 replicates, and the growth rate was presented as -fold increase over the day 1 value.

CDK2 Kinase Activity Assay—Total cell lysate was prepared with 1x lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM {beta}-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 1 µg/ml leupeptin, 2.5 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). An in vitro CDK2 kinase assay was done as described previously (20). Briefly, 1 mg of total cell lysate was incubated with 2 µg of anti-CDK2 antibody (Clone 55; BD Biosciences) overnight at 4 °C. After incubating with 40 µl of protein A/G-agarose (Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h at 4 °C, the beads were washed twice with 1x cell lysis buffer and then twice with 1x kinase buffer (25 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, and 10 mM MgCl2). The final pellet was resuspended in 15 µl of 2x kinase buffer, and a kinase activity assay was done by adding 1 µg of Rb-C substrate (Cell Signaling Technology, Beverly, MA), 1 µl of [{gamma}-32P]ATP, and 50 µM ATP. After a 30-min incubation at 30 °C, the reaction was stopped by adding 10 µl of 4x SDS-PAGE sample buffer and boiled for 5 min before loading onto a 10% SDS-polyacrylamide gel. The gel was stained with Coomassie Blue and dried before autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased Expression of p25 Related to p12 in Contact-inhibited Diploid Cells—p12 is ubiquitously expressed in most of the human tissues, and a polyclonal antibody raised against a GST fusion of p12 recognizes a 12-kDa molecule (6, 8, 19). Its cellular expression has been further confirmed by immunoprecipitation, partial chromatographic purifications, and peptide sequencing analysis on proteins from mouse and human cell lysates (19). The most definitive evidence that the detected 12-kDa molecule is the protein product of p12 was that, when both alleles of p12 were disrupted in murine embryonic stem cells, the anti-p12 antibody (polyclonal antibody 86) no longer detected the 12-kDa protein (20). Although previous studies suggest that p12 may function as a growth suppressor by inhibiting CDK2 and that it also can interact with the DNA polymerase-{alpha}-primase complex, its cellular significance has not yet been elucidated (9). In order to examine the role of p12 during growth arrest induction in cells, we used several normal diploid cells (rodent C3HA and Mv1Lu; human HaCaT and CCD18Lu) that exhibit growth arrest induced by cell-cell contact inhibition. After the cells reached full confluence, the levels of p12 and CDK2 were monitored at different time points (24, 48, 72, and 96 h) along with the level of the known growth arrest marker p27kip1 (21). The experiment was repeated three times independently to avoid any experimental biases. As expected, as the cells went into the growth arrest phase, the level of p27kip1 increased substantially (Fig. 1A). Based on our previous data, which show that ectopic overexpression of p12 resulted in inhibition of CDK2 and growth suppression, we expected to see increased levels of p12 in growth-arrested cells (8). Interestingly, as shown in Fig. 1A, contact-inhibited normal diploid cells showed a reduced level of p12 compared with the log phase cells. In addition to the expected 12-kDa molecule, the polyclonal anti-p12 antibody also recognized a higher molecular weight species with an estimated size of 25 kDa. Interestingly, we observed an increased level of p25 in contact-inhibited cells compared with the log phase cells as the cells went into the growth-arrested state. It was further noted that the cellular level of p25 was inversely correlated with the level of p12 in contact-inhibited cells (Fig. 1B). The level of p25 consistently paralleled the level of p27kip1, whereas the level of CDK2 was reduced as the cells went into the growth-arrested stage. Therefore, the cellular level of CDK2 parallels that of p12 and is inversely correlated with p25.



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  		FIG. 1.
Growth arrest induced the formation of p25 recognized by anti-p12 antibody. Human (HaCaT and CCD18Lu) and rodent (C3HA and Mv1Lu) diploid cells were forced into a growth arrest stage by contact inhibition (from 24 to 96 h after reaching 100% confluence), and an equal amount of total protein was analyzed by Western analysis. A, immunoblot analysis with anti-p12 (polyclonal antibody 86) revealed the presence of a cross-reacting 25-kDa molecule. The levels of CDK2, p27kip1, p12, p25, and {beta}-actin were monitored as the cells were driven into the growth arrest stage. B, the relative level of CDK2, p27kip1, p12, and p25 was measured against {beta}-actin in HaCaT and C3HA. As the cells were driven into the contact-inhibited stage, there was a decrease in the level of p12 and an increase in the level of p25.

 
The Formation of Dimerized p25 Is Increased in the Nucleus upon Contact-inhibited Growth Arrest—It has been reported that dimerization of certain proteins can be related to either molecular binding to form an active complex or be involved in subcellular trafficking of a molecule to a different cellular compartment (15). As a way of understanding the physiological significance of dimerization of p12 in the cell, we first tested whether two different forms of molecules are localized in the same or different cellular compartments. We fractionated CCD18Lu cellular proteins by the differential centrifugation method. After fractionation, an equal amount of protein was analyzed by Western blotting analysis with anti-p12 polyclonal antibody that recognizes both 12- and 25-kDa forms. The efficiency of fractionation was confirmed by Western analysis of the fractions with antibody against nuclear Oct-1 antibody. As shown in Fig. 2, p12 was predominantly localized in the nucleus of the log phase cells, whereas p25 was exclusively present in the microsomal compartment (F2 fraction). Nuclear localization of p12 assessed by anti-p12 polyclonal antibody was consistent with the previously reported immunohistochemical data in human oral tissues, showing a predominant nuclear staining (5). However, when the cells were forced into a contact-inhibited growth arrest state, there was a significant increase of p25 and concomitant decrease of p12 only in the nucleus. Interestingly, there was no change at the level of microsomal p25. This finding suggests that the appearance of p25 in the nucleus of contact-inhibited cells is from the dimerization of nuclear p12 but not from the nuclear import of the microsomal pool of p25.



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  		FIG. 2.
Nuclear dimerization of p12 into p25 during growth arrest. Total cell lysate from either contact-inhibited or log phase CCD18Lu cells was fractionated by the differential centrifugation method as described under "Materials and Methods," and equal amounts of protein from each fraction (L, total lysate; N, nuclear; Cy, cytosolic; F2 (microsomal fraction), pellet from 10,000 x g; and F3 (membranous fraction), pellet from 100,000 x g) were analyzed by Western analysis to examine the level of p12 and p25. The result showed the induction of nuclear p25 in contact-inhibited cells with a concurrent reduction of p12. The levels of actin and nuclear Oct-1 were also monitored.

 
p25 Is a Homodimeric Form of p12 through the Cysteine Residue—In order to assure that p25 is related to p12 and that it is a homodimer of p12, we performed biochemical characterization of p25. First, we performed an immunodepletion analysis by blocking the polyclonal antibody with either purified GST or GST-p12 recombinant proteins. As shown in Fig. 3A, the detection of both 12- and 25-kDa bands was completely abolished by preincubating the antibody with GST-p12 but not with GST alone. Analysis of the deduced p12 protein sequence indicates that there is only one cysteine residue at position 105 that may mediate the dimerization of p12 into p25. If the dimerization is solely through this cysteine residue, the dimer formation should be at least partly reversible by reducing agent. In order to see if p25 formation was sensitive to reducing reagent treatments, we isolated endogenous cellular p12 and p25 by gel elution as described under "Materials and Methods." As shown in Fig. 3B, the gel-eluted p12 could also form a dimer in solution when analyzed by Western blotting, and the association was decreased by treatment with {beta}-mercaptoethanol (about 50%), which suggests that dimerization is sensitive to reducing conditions. In parallel, the gel-eluted p25 in solution dissociated into the monomeric p12, and the extent of dissociation was significantly higher in the presence of reducing agent. The possible reason for incomplete association or dissociation could be that the endogenous p12 or p25 might have been irreversibly denatured during the gel elution process and did not maintain the proper physicochemical property to permit reversible intermolecular conversion. It is also possible that an additional irreversible cellular modification process exists in the formation of p25. Nonetheless, these results confirm that p12 produced in mammalian cells can form a dimer in a reducing agent-sensitive manner, firmly implicating the involvement of the cysteine residue in the dimerization process. To further validate whether p25 is a dimeric form of p12, we generated a bacterial recombinant His-tagged p12 and examined whether the purified protein could form a dimer. As shown in Fig. 3C, the purified His-tagged p12 formed a dimer recognized by Western analysis with anti-p12 antibody (lane 1). The dimerization was also sensitive to {beta}-mercaptoethanol treatment (lane 2), which suggests that the dimerization involves the cysteine residue of the p12. We explored whether the dimerization was actually through the cysteine residue at 105 present in p12 protein by site-directed mutagenesis. We replaced the cysteine residue of His-tagged p12 WT with an alanine to produce the C105A mutant and purified it through a nickel column. Compared with the p12 WT, the mutated p12 C105A did not form any dimer as shown in Fig. 3C either in the presence or absence of {beta}-mercaptoethanol (lanes 3 and 4). This finding convincingly indicates that intermolecular association through the cysteine residue mediates the homodimerization of p12. Dimerization of p12 mediated by cysteine 105 was further confirmed in mammalian cells. U2OS cells were transiently transfected with either FLAG-p12 WT or mutated FLAG-p12 C105A (Fig. 4). To visualize specifically the FLAG-tagged recombinant molecule, the blot was probed with anti-FLAG antibody, and the result clearly showed dimer formation only in FLAG-p12 WT (lane 2) and not in the C105A mutant (lane 3). Interestingly, there was a doublet of the dimer, suggesting posttranslational modification. It should be noted that we also observed the formation of a doublet with the monomer (data not shown). Furthermore, in the presence of the reducing agents ({beta}-mercaptoethanol or dithiothreitol), the dimer formation was distinctively reduced (Fig. 4, lanes 5 and 8).



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  		FIG. 3.
p25 is a dimerized product of p12. A, immunocompetition with GST-p12 abolished the cross-reactivity of antibody to p25 compared with GST control, suggesting that p25 is related to p12. B, p12 and p25 were partially purified from CCD18Lu total protein by gel elution as described under "Materials and Methods." Immunoblot analysis showed that the gel eluted p12 could form a dimer, and also p25 could be dissociated into a monomeric 12-kDa molecule in vitro. The conversion was also shown to be sensitive to a reducing agent ({beta}-mercaptoethanol (ME)) and reducing condition affected the conversion by up to 50% as measured by densitometric scanning of the intensities. C, bacterial His-tagged p12 WT showed a formation of dimer that is sensitive to reducing agent. However, mutation of cysteine into alanine abolished a dimer formation.

 



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  		FIG. 4.
Dimerization of p12 in U2OS cells. Dimer formation of p12CDK-AP1 was confirmed in mammalian cells. U2OS cells transfected with FLAG-tagged p12CDK2-AP1 wild type (lane 2) showed a formation of dimer recognized by anti-FLAG antibody (Sigma). Treatment of cell lysate with either {beta}-mercaptoethanol ({beta}-ME) or dithiothreitol (DTT) abolished dimerization. Transfection with either pFLAG-CMV2 null vector (lane 1) or C105A mutant construct (lane 3) did not show dimer formation. WB, Western blot.

 
In addition to the validation of dimer formation in mammalian cells by transient transfection, we further examined the consequence of down-regulating endogenous p12 in the formation of p25. We generated stable gene silencing clones of p12 from human normal oral keratinocytes (OKF6tert1) by using short interfering RNA (siRNA) (20). As shown in Fig. 5A, stable p12 siRNA clones showed a reduced level of p12 protein expression (up to 80%) as compared with control. In parallel, there was a reduced level of p25 in proportion to the level of p12 reduction as determined by densitometric analysis (Fig. 5B). This result further supports that the p25 is a dimeric form of p12. We also performed peptide-sequencing analysis of purified cellular p12 and p25 by using an anion exchange column, reverse phase high pressure liquid chromatography, and mass spectrum analyses (data not shown). The results showed that both the p12 fraction and the p25 fractions contained the actual peptide sequences deduced from the p12 gene. This series of biochemical and molecular characterizations of p12 and p25 confirmed that p25 is a dimeric form of p12 and that this dimer can form through the cysteine under physiological conditions.

Inhibition of Cellular Proliferation by Wild Type p12 but Not by the C105A Mutant—Our previous work has shown that transfection of wild type FLAG-p12 resulted in an inhibition of cellular proliferation (8). To evaluate a functional consequence of the dimerization of p12, the effect of the mutation of the cysteine residue on cellular proliferation was examined. Human normal diploid lung fibroblast CCD18LU cells were transiently transfected with either wild type or the C105A mutant construct of FLAG-p12, and cellular proliferation was measured (Fig. 6A). Interestingly, substitution of alanine for the cysteine residue that we have shown to be involved in dimerization of p12 in this report resulted in a significant reduction of the growth-inhibitory effect (p < 0.001). In fact, cellular proliferation of transfectant with FLAG-p12 C105A mutant was not significantly distinguishable from a null vector control transfectant. It has been previously demonstrated that the growth-inhibitory function of p12 is accounted for through modulation of CDK2 activity. In order to examine whether the dimerization of p12 has any effect on CDK2 activity, we have measured CDK2 kinase activity after transiently transfecting either WT or the C105A mutant form of FLAG-p12 into CCD18Lu cells. As shown in Fig. 6B, ectopic overexpression of WT p12 resulted in a reduction of CDK2 kinase activity against Rb-C substrate. In contrast, the C105A mutant showed a sustained CDK2 kinase activity. Although we observed relatively moderate inhibition of CDK2 activity, probably due to low transfection efficiency (about 30% as monitored by control green fluorescent protein expression), the result showed significant differences between WT and the C105A mutant (p = 0.01705 between vector and WT; p = 0.001451 between WT and C105A). These results suggest that dimerization of p12 may be a prerequisite event for p12 to play a role as a growth suppressor through modulation of CDK2 activity. It also suggests that nuclear dimerization of p12 in growth-inhibited diploid cells by contact inhibition may mediate this important cellular process.



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  		FIG. 5.
Knockdown of p12 by siRNA resulted in a reduction of p25. The level of p25 was examined after knocking down the expression of p12 by siRNA in human normal oral keratinocytes (OKF6tert1). A, Western analysis showed a reduction of p25 in p12 siRNA clones. The levels of p12 and p25 were compared with the vector control (V) and the negative siRNA clone (N). B, densitometric scanning of band intensities showed concurrent reduction of p25 in p12 siRNA clones.

 
C105A Mutation Inhibits Its Binding to CDK2—To address how the prevention of dimerization by C105A mutation resulted in a reduced inhibition of CDK2 activity by p12, the cellular localization of p12 C105A and its association with CDK2 were examined. CCD18Lu cells were transiently transfected with FLAG-tagged p12 constructs (WT, C105A, or A3 CDK2 binding mutant) and fractionated by using a NucBuster protein extraction kit (EMD Biosciences Inc., San Diego, CA). It has been previously reported that A3 (mutation of TER residues 109–111 to AAA) prevented p12 from binding to CDK2 (8). As shown in Fig. 7A, the C105A mutation did not affect the cellular localization of p12, and both expressed p12 mutants (C105A and A3) and WT were exclusively localized in the nuclear fraction. The integrity of cellular fractionation was confirmed by using an antibody against Oct-1 nuclear protein, and loading of protein was visualized by probing with anti-actin antibody. We then examined whether C105A mutation could affect the physical association with CDK2 by coimmunoprecipitation analysis. As shown in Fig. 7B, p12 WT formed a dimer, and there was a reduction of dimer formation from the p12 A3 mutant, whereas p12 C105A did not form a dimer. We were able to pull down CDK2 with p12 WT and a greatly reduced level of CDK2 with p12 A3 mutant. Interestingly, p12 C105A failed to associate with CDK2. This result suggests that an ability of p12 to form a dimer is correlated to its ability to associate with CDK2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The expression of p12CDK2-AP1 (p12), previously known as the DOC-1 gene has been shown to be down-regulated in oral cancers and microsatellite unstable colorectal cancers (5, 22). There is growing evidence that p12 can exert its cellular effect in cell cycle regulation through interaction with important cell cycle-regulatory molecules such as CDK2 and/or possible other unidentified molecules. Also, it has been found that the other related member of p12 exists in the genome and is expressed as a possible functional molecule. There has been significant evidence correlation between decreased or abolished p12 expression and oral cancer development (5). However, it has not been directly shown that the deletion or reduction of p12 expression causes cellular transformation and in turn tumorigenesis in vivo. Most scientifically proven data on the possible function of p12 is the interaction with an important cell cycle-regulatory molecule, CDK2, and down-regulation of its activity (5, 8). We have previously demonstrated that p12 can interact with CDK2 and DNA polymerase-{alpha}-primase; however, the mechanisms of these interactions have not been fully elucidated. Also, the physiological consequences of the interactions have not been completely understood. Based on the growth-inhibitory effect of p12, we hypothesized that p12 is present as an active molecule when the cells are in growth arrest or at the onset of the growth arrest phase.



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  		FIG. 6.
p12 C105A showed a reduction of growth-inhibitory effect with a sustained CDK2 activity. A, CCD18Lu cells were transiently transfected with a control vector (pFLAG), p12 WT, or C105A mutant, and cellular proliferation was determined by using a cell counting kit (CCK-8; Alexis Biochemicals, San Diego, CA). B, after transfection with pFLAG, pFLAG-p12 WT, or pFLAG-p12 C105A, CDK2 was immunoprecipitated, and in vitro kinase activity was measured against the Rb-C fragment as described under "Materials and Methods." The levels of CDK2, Rb-C fragment, and transfected genes were determined by Western analysis. The CDK2 kinase activity was normalized against Rb-C and depicted as a graph. The experiments were repeated three times independently, and data were analyzed by a paired t test (p = 0.01705 between vector and WT; p = 0.001451 between WT and C105A). IP, immunoprecipitation; IB, immunoblot.

 



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  		FIG. 7.
C105A mutation did not alter the cellular localization of p12 but affected its association with CDK2. A, CCD18Lu cells were transiently transfected with FLAG-tagged p12 WT, C105A, or A3 mutant construct. After 48 h of transfection, cellular proteins were fractionated, and the localization of expressed genes was examined by Western analysis. The integrity of cellular fractionation was confirmed by probing for a nuclear Oct-1 protein. B, the effect of C105A mutation on the association with CDK2 was examined by coimmunoprecipitation analysis. 293 cells were transiently co-transfected with p12 construct and CDK2-HA. Total protein was subjected to co-immunoprecipitation with anti-FLAG antibody. The resulting complex was analyzed by Western analysis with anti-FLAG and anti-CDK2 antibody. IP, immunoprecipitation; IB, immunoblot.

 
In order to see the fate of p12 protein product in the growth arrest stage, we investigated the regulation of p12 in contact-inhibited growth-arrested cells. It is well known that normal diploid cells can be growth-arrested through contact inhibition, and several important cell cycle regulators are known to be involved in this process (2, 23–27). However, the exact mechanism of growth arrest induced by contact inhibition has not been established. It may share some common cellular mechanism governing growth arrest induced by other causes, such as serum starvation, medium depletion, or treatment with transforming growth factor-{beta}, but it may also follow its own distinctive cellular pathway (28, 29). Our previous results showed that p12 was a negative S phase regulator, and its expression peaked at the S phase. Deletion of p12 in murine embryonic stem cells by homologous recombination resulted in an increased cellular proliferation and a shift in cell cycle profile (20), which supports the growth-inhibitory role of p12. In this report, we have shown that p12 could form a dimer in contact-inhibited diploid cells, and the dimerization is through an intermolecular association via the cysteine residue. Furthermore, we have shown that the dimerization of p12 is a functionally significant event, since an abolishment of dimerization by mutating a cysteine residue abolished its growth-inhibitory effect in CCD18Lu cells. We have further shown that abolishing dimer formation by C105A mutation abrogated its CDK2 inhibitory function. These data suggest that dimerization of p12 is functionally significant in the modulation of CDK2 activity and cell growth inhibition. In addition, we have shown that C105A mutation did not alter nuclear localization of p12, but it prevented p12 from forming a complex with CDK2. However, it is still not clear whether the dimer is directly associated with CDK2. Considering that the A3 mutant that has an intact Cys105 showed a reduced dimerization, it is speculated that p12 forms a dimer through Cys105, but it is significantly affected by a neighboring molecular structure. We have observed that in asynchronous cell culture, the dimer is exclusively in the cytoplasm. It is still not clear how this cytoplasmic dimer is formed from its monomer or whether it has a different function compared with the nuclear dimer. Also, it needs to be addressed how the dimerization affects the association p12 with CDK2 and how mechanistically the nuclear dimer functions in the modulation of CDK2 activity. Based on our previous data showing an in vitro association of monomeric p12 and the inactive cytoplasmic form of CDK2, it was speculated that p12 might play a role in sequestering a pool of CDK2 and might also be involved in cellular trafficking of CDK2 in and out of the nucleus (8). Although it needs to be further validated, it is possible that dimerization of p12 might be a way of sequestering an active pool of CDK2 and involved in the cellular trafficking process. It will be intriguing to examine a cellular localization of p12 WT or C105A by immunocytochemistry and also its co-localization with CDK2 by confocal microscopy to determine whether a mutation of cysteine has any effect on this process. These future studies on the process of p12 in the formation of dimer will reveal the cellular and molecular mechanism of p12 in the regulation of CDK2 and its subsequent effect on cell cycle regulation.


   FOOTNOTES
 
* This work was supported by Public Health Service Grants T32 DE 007296-08 (to Y. K.) and RO1 DE 14857 (to D. T. W.). 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. Back

§§ To whom correspondence should be addressed: Dental Research Institute, School of Dentistry, UCLA, 10833 Le Conte Ave., 73-017 CHS, Los Angeles, CA 90095. Tel.: 310-206-3048; Fax: 310-794-7109; E-mail: dtww{at}ucla.edu.

1 The abbreviations used are: p12CDK2-AP1 or p12, p12 CDK2-associating protein 1; siRNA, short interfering RNA; STAT, signal transducers and activators of transcription; RIPA, radioimmune precipitation; WT, wild type; GST, glutathione S-transferase. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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