CARF Is a Novel Protein That Cooperates with Mouse p19 ARF (Human p14 ARF ) in Activating p53*

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16 INK4a and the alternative reading frame protein, ARF (p19 ARF in mouse and p14 ARF in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19 ARF activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14 ARF occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novelcollaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16 INK4a and the alternative reading frame protein, ARF (p19 ARF in mouse and p14 ARF in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19 ARF activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14 ARF occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.
The INK4a locus on chromosome 9p21 is frequently affected in human tumors. It encodes two structurally distinct tumor suppressor proteins, p16 INK4a and the alternative reading frame protein, ARF (p14 ARF in human and p19 ARF in mouse) (1)(2)(3)(4)(5). These act upstream of pRB and p53 tumor suppressor proteins and regulate their activities and thus the progression of cell cycle (6 -8). p16 INK4a prevents phosphorylation and functional inactivation of the retinoblastoma protein (pRB) by cyclindependent kinases (9). ARF negatively regulates MDM2-mediated degradation of p53 (10 -16). ARF expression is regulated by pRB through the family of E2F transcription factors; Rb represses E2F function and ARF expression. ARF therefore provides a mechanism whereby inactivation of Rb and release of E2F lead to the stabilization and functional activation of p53, linking the Rb and p53 pathways (6,17). Therefore, functional regulation of ARF is critical for cell cycle control in response to a variety of cellular and environmental signals. The current prevailing model proposes that ARF functions by sequestering MDM2 in the nucleolus and thereby preventing the degradation of p53 that requires nuclear-cytoplasmic shuttling of p53-MDM2 complexes. It was, however, recently shown that stabilization of p53 by p14 ARF occurs independent of the relocalization of MDM2 to the nucleolus (11). This indicated the possibility that the function of ARF may be accomplished by interacting partners other than MDM2. We recently reported that p19 ARF binds to Pex19p that sequesters it in the cytoplasm and negatively regulate its p53 activation function (18). However, the cellular factors that regulate ARF activity remain poorly defined. In the present study, we report a novel collaborator of ARF, CARF. It is a novel ARF-interacting nuclear protein that co-localizes, cooperates, and is co-regulated with ARF and thus acts as a novel component of the ARF-MDM2-p53 pathway.

MATERIALS AND METHODS
Yeast Two-hybrid Screen-cDNA encoding full-length p19 ARF in the yeast expression vector pODB8 (a kind gift from O. Louvet) was used for screening of the human testis cDNA library in yeast two-hybrid vector pACT2 (Clontech) as described (18). Interactions of two proteins were examined by growth of the yeast transformed by two plasmids on nutrient-deficient (His Ϫ /Leu Ϫ /Trp/Ade Ϫ ) plates and ␤-galactosidase reporter activity. The isolated cDNA-derived plasmids were recovered from yeast and sequenced using an ABI sequencer (PerkinElmer Life Sciences).
Anti-CARF Antigen and Antibody-His-tagged CARF protein was expressed in Escherichia coli and purified by its binding to nickelnitrilotriacetic acid-agarose (Qiagen). Purity of the protein preparations was analyzed by SDS-PAGE and silver staining and was used for rabbit immunizations. An anti-CARF antibody thus obtained was analyzed for its reactivity to CARF by immunoprecipitation and Western blotting.
Immunostaining-Cells grown on glass coverslips placed in 35-mm plastic dishes were washed with cold PBS and fixed with 4% formalde-* This work was supported in part by a Hougateki research grant from the National Institute of Advanced Industrial Science and Technology (to S. C. K.). 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. ‡ ‡ Supported by a Carcinogenesis Fellowship from the New South Wales Cancer Council.
§ § To whom correspondence should be addressed. Fax: 81-298-61-6692; E-mail: s-kaul@aist.go.jp. . Cells transfected with an expression plasmid encoding CARF-V5 showed the presence of endogenous CARF and a slightly higher sized hyde for 10 min at room temperature. Fixed cells were washed with PBS and permeabilized with 0.2% bovine serum albumin in PBS for 20 min. Cells were then incubated in blocking buffer containing the primary antibody (either tag-specific or protein-specific as indicated in the figure legends) for 1 h and washed extensively in PBS before incubation with the appropriate fluorochrome-conjugated secondary antibody for a further 30 min. Various secondary antibodies were used, including Alexa-488-conjugated goat anti-rabbit and anti-mouse or Alexa-594conjugated goat anti-rabbit and anti-mouse (Molecular Probes). After six washes in PBS with 0.1% Triton X-100, cells were overlaid with a coverslip with Fluoromount (Difco). The cells were examined on a Zeiss microscope with epifluorescence optics or a Fluoview confocal laserscanning microscope (Olympus, Tokyo, Japan). The extent to which the two proteins co-localized was assessed by combining the two images using Fluoview software.
In Vivo Co-immunoprecipitation-Cell lysates (400 g of protein) in 400 l of Nonidet P-40 lysis buffer were incubated at 4°C for 1-2 h with an antibody used for immunoprecipitation. Immunocomplexes were separated by incubation with Protein-A/G-Sepharose, and Western blotting was performed with the indicated antibodies using procedures described above.
Colony-forming Assays-U2OS cells stably transfected for IPTGinducible expression of ARF were secondarily transfected with expression plasmid for metal-inducible CARF and pPur (for puromycin selection) in the ratio of 20:1, respectively. Transfected cells were selected in puromycin (2.5 g/ml)-supplemented medium for 4 days and were plated (1000 cells per 10-cm dish) in duplicate. Cells were treated with IPTG (1 mM) for ARF expression and ZnSO 4 (100 M) for CARF expression. Cells were maintained until the appearance of colonies with a regular change of medium. Colonies were fixed in methanol, stained with 10% Giemsa solution, photographed, and counted.  4) and induced for expression of ARF by addition of 100 M ZnSO 4 to the medium. The level of p53 protein and that of its downstream protein, p21 WAF1 , was enhanced by more than 10-fold by CARF expression in the presence of p19 ARF (lanes 2 and 4 of b and c). A mild increase (about 3-fold) in p53 and p21 WAF1 was detected in the absence of p19 ARF (lanes 1 and 3 of b and c). Expression of p19 ARF stabilizes CARF (a, compare lanes 3 and 4), and vice versa expression of CARF stabilized p19 ARF (d, compare lanes 2 and 4). B, quantitation of the Western blot; increase (relative units) in the level of proteins (p53 and p21 WAF1 , p19 ARF , and CARF) normalized against actin is plotted for the presence of ARF, CARF, or ARF and CARF. C, co-existence of ARF and CARF. Three different cell types were stained for endogenous ARF and CARF. In an unsynchronized culture, the cells (marked with white arrows) lacking CARF were also deficient in ARF staining, suggesting the companionship of the two proteins.
CARF-V5 protein (lane 2). The small size proteins in lanes 1 and 2 may be the degraded protein products. CARF-V5 protein was immunoprecipitated with CARF serum but not preserum (compare lanes 3 and 4). The serum IgG (used for immunoprecipitation) cross-reacted to the anti-V5 antibody (smear extending from 50 -65 kDa in lanes 3 and 4). However, the CARF serum specifically precipitated CARF protein. C, coimmunoprecipitation of p14 ARF with endogenous CARF from NARF cells that were induced for a low level of expression of p14 ARF (lane 1, 20 g of lysate). p14 ARF was co-immunoprecipitated with CARF but not with preserum control (400 g of lysates used for immunoprecipitation). Anti-p14ARF antibody cross-reacted to a protein of ϳ70 kDa. D, visualization of CARF-green fluorescent protein (GFP) (a) and p19 ARF -Myc (b) in HeLa cells by green fluorescent protein fluorescence and staining with anti-Myc antibody, respectively. Whereas p19 ARF localizes predominantly in the nucleolus, CARF was seen in the nucleoplasm as well as in the nucleolus. CARF and p19 ARF co-localized only in the periphery (granular region) of nucleoli (seen as a yellow ring in c). NARF (U2OS cells with an inducible expression of p14 ARF ) cells were transfected with CARF-V5 and were induced for p14 ARF expression by 1 mM IPTG overnight. These were doubly stained for CARF (anti-V5 monoclonal antibody) and p14 ARF (anti-p14 ARF polyclonal serum 8 (d-f). Co-localization of CARF and ARF was seen in the granular region of the nucleolus of NARF cells. Saos-2 cells were doubly stained for CARF (endogenous) and p14 ARF -Myc (exogenous) with a polyclonal CARF antiserum and a monoclonal anti-Myc antibody, respectively. Note the co-localization of the proteins in the granular region of the nucleolus (g-i). HeLa cells stained for endogenous CARF and endogenous ARF also showed co-localization of the proteins in the granular region of the nucleolus (j-l). Taken together, CARF and ARF proteins (tagged exogenous or endogenous) co-localize in the granular region of the nucleolus (a-l). E, a higher magnification image of CARF and ARF showing co-localization of the two proteins in the nucleolus in its periphery (granular region).

RESULTS AND DISCUSSION
To isolate p19 ARF -interacting proteins, a Gal4 binding domain (BD)-p19 ARF fusion protein was used as a bait to screen a human cDNA library cloned into a Gal4 activation domain (AD) yeast two-hybrid plasmid. Two clones, A-1 (Pex19p) (18) and A-10, were strongly positive by His prototrophy and induction of ␤-galactosidase expression. The nucleotide sequence of A-10 clone (accession number AF 246705, assigned to chromosome 4) matched with the sequence from a cDNA clone (accession number NM-017612; NEDO human cDNA sequencing project). We isolated a full-length cDNA encoding a novel serine-rich (21%) protein consisting of 580 amino acids and named it CARF for a novel protein that collaborates/cooperates with ARF as demonstrated in this study. The amino acid sequence of CARF did not match significantly with any of the protein entries in the data base and did not reveal the presence of any known protein motifs. Therefore no hints to its putative function could be obtained. To detect p19 ARF (ARF) and CARF interactions in mammalian cells, we transfected HeLa cells with expression plasmids encoding CARF-V5 and p19 ARF -Myc proteins and performed in vivo co-immunoprecipitation assays (Fig. 1A). CARF was precipitated with p19 ARF , and vice versa, the control immunoprecipitations performed from untransfected cells and control IgG were negative. The results revealed that p19 ARF (Fig. 1A) interacts with CARF. Similar co-immunoprecipitation of p14 ARF was obtained with CARF from cell lysates transfected with CARF-V5 and p14ARF-Myc (data not shown). To rule out the possibility that the interactions are because of overexpression of the two proteins, we next immunoprecipitated endogenous CARF from NARF cells with a mild level of expression of ARF (U2OS cells that were induced for ARF expression by 0.01 mM IPTG for 12 h). An antibody raised against a full-length recombinant CARF protein and demonstrated to be reactive to CARF by Western blotting and immunoprecipitation (Fig. 1B) was used for immunoprecipitation of endogenous CARF. Immunoprecipitation of endogenous CARF pulled down p14 ARF (Fig. 1C, level of p14 ARF protein in onetwentieth of the lysate used for immunoprecipitation is seen in lane 1). These data supported CARF-ARF interactions in in vivo. Taken together, it was concluded that CARF interacts with both p19 ARF and p14 ARF .
To determine whether ARF and CARF proteins co-localize within intact cells, we performed co-immunolocalization studies for exogenous and endogenous proteins (Fig. 1D). In all cell types (HeLa, NIH 3T3, and COS 7) used, p19 ARF localized mainly in the nucleolus with some diffuse staining in the nu-FIG. 3. CARF targeting by RNA interference decreased ARF function. A, U2OS cells were transfected with target or control siRNA. CARF expression was analyzed either by immunostaining or Western blotting with anti-CARF antibody. Note that 60 -70% of cells showed negligible CARF expression in target siRNA-transfected cells. B, U2OS cells transfected with CARF-siRNA were induced for ARF expression by IPTG addition into the medium. ARF induction resulted in an increase in p53 and p21 WAF1 (compare lanes 1, 3, and 5 with 2, 4, and 6, respectively). Note that ARF induced an increase in p53, and p21 WAF1 expression was reduced when CARF was targeted with siRNA (compare lane 2 with 4 and 6). Targ., target; Cont., control; Unt., untransfected. C, quantitation of Western blot; relative protein units (for each of the protein analyzed) normalized against actin are plotted for control and target siRNA-treated cells. D, U2OS cells transfected with target or control siRNA were induced for ARF expression by IPTG addition into the medium. Targeting of CARF also resulted in reduced expression of ARF. cleoplasm and the cytoplasm. CARF localized in the nucleoplasm and appeared to be excluded from the core of the nucleolus (Fig. 1D). The two proteins co-localized in the periphery (granular region) of the nucleolus (Fig. 1D, c). Similarly, p14 ARF (exogenous or endogenous) and CARF (exogenous or endogenous) co-localized in the periphery of the nucleolus (Fig.  1D, d-l, and E). From these data, it was concluded that exogenous and endogenous CARF proteins have identical subcellular localization and that CARF co-localizes with ARF (p19 ARF or p14 ARF ) in the periphery (granular region) of the nucleolus.
It has been shown that ARF acts upstream of p53 and activates its transcriptional activation functions (6,12,13,20,21). We investigated how CARF affects this function of ARF. NIH 3T3 cells that lack endogenous p19 ARF (because of biallelic loss of the INK4a locus) but were stably transfected for heavy metal-inducible expression of hemagglutinin-tagged p19 ARF were employed (18,19). Induction of p19 ARF by addition of 100 M ZnSO 4 to the culture medium ( Fig. 2A (d, lanes 1 and 2)) caused an increase in the p53 level (Fig. 2, A (b, lanes 1 and 2) and B) and activity as detected by the level of expression of its downstream regulator gene p21 WAF1 (Fig. 2, A (c, lanes 1 and 2) and B). Expression of CARF along with ARF further enhanced p53 and p21 WAF1 at least by 2-3-fold (Fig. 2, A, compare lanes  2 and 4, and B). Noticeably, cells induced for ARF showed a higher level of stable CARF expression suggesting that ARF stabilizes the CARF protein and vice versa (Fig. 2, A (a, com-pare lanes 3 and 4, and d, compare lanes 2 and 4) and B). The data were further supported by CARF and ARF staining of unsynchronized cells. In three different cell lines, cells lacking ARF were also deficient in CARF staining (Fig. 2C), suggesting that CARF and ARF are co-regulated. Such co-absence of ARF and CARF was seen in ϳ70% of three different types of cells examined. Llanos et al. (11) have shown that only a minor fraction of p14 ARF is bound to MDM2 and the nucleolar localization of ARF and MDM2 is not essential for the p53 activation function of ARF. We have shown here that CARF is a novel binding partner (collaborator) of ARF; it co-localizes with ARF in the nucleolus, co-regulates with it, and cooperates in its p53 activation function.
To further elucidate the role of CARF for ARF-mediated p53 activation function, we next employed RNA interference (RNAi) in NARF cells (U2OS with inducible expression of p14 ARF ). The transfection of target siRNA resulted in loss of CARF expression as observed by immunostaining (Fig. 3A, significant decrease in the intensity of CARF staining was observed in 60 -70% cells) and Western blotting (Fig. 3, B, compare lanes 1 and 2 with 3-6, and C). As expected, induction of p14 ARF caused increased levels of p53 and p21 WAF1 (Fig. 3, B, compare lanes 3 and 4 and lanes 5 and 6). In the presence of CARF-siRNA (Fig. 3B, lanes 1 and 2), the p14 ARF -induced increase in p53 and p21 WAF1 was reduced to about 55%. These data showed that CARF is required for efficient ARF function. Noticeably, ARF levels were also reduced when CARF was targeted (Fig. 3, B and D). These data strongly supported co-regulation of ARF and CARF (also shown in Fig. 2, B and C). Taken together, it was concluded that ARF and CARF are co-regulated and CARF cooperates with ARF in its p53 activation function.
We next examined the biological activity of CARF by the colony-forming assay of U20S cells (have stably integrated IPTG-inducible p14 ARF expression plasmid (12) and metal-inducible expression of CARF along with endogenous CARF) (Fig. 4A). As expected, induction of ARF expression resulted in reduced colony number and size (Fig. 4A, compare a and d with  b and e, respectively). Expression of CARF along with ARF caused further reduction (Fig. 4A, compare b and e with c and f, respectively). These results are consistent with the cooperative role of CARF in ARF function and thus assign CARF as a novel regulator of the p19 ARF -MDM2-p53 pathway. ARF is an important mediator of cellular senescence and regulates a variety of other p53-dependent or -independent cellular responses (2,5,6,(22)(23)(24). Intriguingly, CARF was seen to cause some reduction (up to 20 -30%) in colony-forming efficiency of cells in the absence of ARF (Fig. 4B). This can be explained, at least in part, by CARF-induced moderate stabilization and activation of p53 in the absence of p19 ARF (Fig. 2A) and suggests that CARF may partly function independent of ARF; thus studies on its relation to other members of the ARF-MDM2-p53-p21 WAF1 pathway are warranted. Elucidation of mechanism(s) of ARF action, its binding partners, and factors that regulate its activity are of critical importance in our understanding of tumor growth, progression, and therapeutics. We have identified CARF as a novel ARF binding partner. Besides their interactions and co-regulation in vivo, we have demonstrated that CARF cooperates with ARF in its p53 activation function.