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ARF Promotes Accumulation of Retinoblastoma Protein Through Inhibition of MDM2

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ARF Promotes Accumulation of Retinoblastoma Protein Through Inhibition of MDM2 Oncogene (2007) 26, 4627–4634 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE ARF promotes accumulation of retinoblastoma protein through inhibition of MDM2 DLF Chang1, W Qiu1, H Ying2, Y Zhang3, C-Y Chen4 and Z-XJ Xiao1,2 1Graduate Program in Molecular Medicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA; 2Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA; 3The Pulmonary Center, Boston University School of Medicine, Boston, MA, USA and 4Department of Pathology, Boston University School of Medicine, Boston, MA, USA The INK4a/ARF locus,encoding two tumor suppressor binds to and inhibits cyclin D-associated kinases proteins,p16 INK4a and p14ARF (ARF),plays key roles in resulting in hypophosphorylated retinoblastoma protein many cellular processes including cell proliferation, (Rb), a biologically active Rb species in tumor suppres- apoptosis,cellular senescence and differentiation. Inacti- sion (Weinberg, 1995). It was later discovered that the vation of INK4a/ARF is one of the most frequent events same locus encodes a second protein through the usage during human cancer development. Although p16INK4a is of a separate promoter with a distinct first exon (exon a critical component in retinoblastoma protein (Rb)- 1b), which results in translation from an alternate mediated growth regulatory pathway,p14 ARF plays a reading frame (ARF) encoding a basic, nucleolar pivotal role in the activation of p53 upon oncogenic stress protein named p14ARF in human (p19ARF in mouse). signals. A body of evidence indicates that ARF also ARF shares no amino acid homology with p16INK4a possesses growth suppression functions independent of protein and is activated by oncogenic stress signals, such p53,the mechanism of which is not well understood. We as Ras, c-Myc, E1A and E2F (Serrano, 2000; Sherr, have recently shown that MDM2 interacts with Rb and 2001). promotes proteasome-dependent Rb degradation. In this Mice lacking both INK4a and ARF alleles (INK4aÀ/À/ study,we show that ARF disrupts MDM2–Rb interaction ARFÀ/À) are prone to spontaneous and carcinogen- resulting in Rb accumulation. Wild-type ARF,but not induced tumor formation (Serrano et al., 1996). ARF mutant defective in MDM2 interaction,stabilizes Selective disruption of ARF alone recapitulates the Rb and inhibits colony foci formation independent of p53. phenotypic consequences seen in the INK4aÀ/À/ARFÀ/À In addition,ablation of Rb impairs ARF function in double-null mice (Kamijo et al., 1997; Sharpless et al., growth suppression. Thus,this study demonstrates that 2004). The ARFÀ/À cells are highly susceptible to Ras ARF plays a direct role in regulation of Rb and suggests transformation and are resistant to both Ras- and that inactivation of ARF may lead to defects in both p53 culture-induced senescence (Groth et al., 2000). In and Rb pathways in human cancer development. addition, exon 1b is frequently targeted in human Oncogene (2007) 26, 4627–4634; doi:10.1038/sj.onc.1210254; cancers (Kumar et al., 1998; Iida et al., 2000; Randerson- published online 5 February 2007 Moor et al., 2001; Rizos et al., 2001; Viswanathan et al., 2001). The N-terminal portion (amino acids (aa) 1–64) Keywords: ARF; retinoblastoma protein; MDM2; cell of p14ARF is necessary and sufficient for growth arrest cycle; cancer (Quelle et al., 1997; Zhang et al., 1998). ARF functions to activate p53 through inhibition of MDM2 (Pomerantz et al., 1998; Zhang et al., 1998). ARF interacts with MDM2 in the central acidic domain (CAD) (Weber et al., 2000b; Bothner et al., 2001) and Introduction antagonizes MDM2 E3 ligase function, resulting in p53 stabilization (Kamijo et al., 1998; Pomerantz et al., The INK4a/ARF locus, located on chromosome 9p21, is 1998; Stott et al., 1998; Zhang et al., 1998). In fact, ARF one of the most frequently targeted loci for inactivation inhibits several known functions of MDM2 toward p53, in human cancer (Hall and Peters, 1996; Hainaut et al., including MDM2’s ability to block p53 transcription 1997). The INK4a/ARF gene encodes the p16INK4a and (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., p14ARF tumor suppressor proteins. p16INK4a specifically 1998), and to ubiquitinate p53 (Honda and Yasuda, 1999; Midgley et al., 2000; Llanos et al., 2001). In addition, ARF sequesters MDM2 in the nucleolus to Correspondence: Dr Z-XJ Xiao, Department of Biochemistry, Boston prevent nucleo-cytoplasmic shuttling (Tao and Levine, University School of Medicine, 715 Albany St., K-423, Boston, MA 1999; Weber et al., 1999, 2000b; Zhang and Xiong, 02118, USA. 1999). E-mail: [email protected] Received 5 June 2006; revised 30 November 2006; accepted 4 December A growing body of evidence suggests that ARF 2006; published online 5 February 2007 possesses growth suppressive properties independent of ARF upregulates Rb through inhibition of MDM2 DLF Chang et al 4628 p53. ARFÀ/À mice develop carcinomas and neurogenic (Figure 1a). Transfection efficiency was comparable as tumors rarely seen in p53À/À mice. In addition, c-Myc- shown by a comparable expression of co-transfected induced lymphomas are more aggressive in ARFÀ/À/ GFP. In addition, expression of p19ARF by retroviral p53À/À mice compared to either p53À/À or ARFÀ/À mice infection in H1299 cells also led to Rb accumulation. alone (Eischen et al., 1999). Triple knock mice Importantly, ARF led to a significant increase in nullizygous for ARF, p53 and Mdm2 develop multiple hypophosphorylated Rb (Figure 1b). tumors at higher frequency than that of mice lacking both p53 and Mdm2 or p53 alone (Weber et al., 2000a). Recently, it has been shown that tumors emerged p14ARF upregulates Rb in an MDM2-dependent manner significantly earlier in mice lacking both ARF and p53 Rb consists of the N-terminus, the central portion (A–B than in the mice lacking p53 alone (Christophorou et al., domain) and the C-pocket (Figure 2a). The A–B 2006). Notably, ARF can upregulate hypophosphory- domain, referred to as the Rb small pocket (RbSP), lated Rb and inhibits cell cycle progression in p21À/À binds to several viral oncoproteins and a subset of cells (Modestou et al., 2001). These observations cellular proteins. The Rb large pocket (RbLP), contain- indicate that ARF growth suppression function can be ing the RbSP and C-pocket, is required for Rb growth achieved independent of the p53–p21 pathway. suppressive function. As the Rb C-pocket binds to We have shown previously that MDM2 interacts with MDM2 (Xiao et al., 1995; Sdek et al., 2004), we Rb and inhibits its growth suppressive function (Xiao examined the role of Rb C-pocket in ARF-mediated Rb et al., 1995). MDM2 binds to Rb C-pocket, blocks Rb- upregulation. H1299 cells were co-transfected with E2F interaction and promotes cell cycle progression RbLP or RbSP and p14ARF expression plasmids. (Sdek et al., 2004). Recently, we demonstrated that Western-blot analysis showed that co-expression of MDM2 facilitates Rb interaction with the 20S protea- ARF led to a significant increase in RbLP, but not some resulting in accelerated Rb degradation (Sdek RbSP proteins (Figure 2b), suggesting that the Rb C- et al., 2005). pocket is critical for ARF effect on Rb. We next In this study, we investigated the role of ARF tumor examined whether ARF could directly inhibit MDM2- suppressor protein in the regulation of Rb. Our results mediated Rb degradation. As shown in Figure 2c, show that ARF inhibits MDM2-mediated Rb degrada- expression of ARF-stabilized MDM2, consistent with tion by disruption of MDM2–Rb interaction. In previous reports (Stott et al., 1998; Honda and Yasuda, addition, we show that lack of ARF led to reduced Rb 1999; Llanos et al., 2001). Importantly, whereas MDM2 protein levels independent of p53, and that ARF growth destabilized Rb, as expected, co-expression of ARF suppression function is impaired in the absence of Rb. led to concomitant MDM2 and Rb stabilization (Figure 2c), suggesting that ARF upregulates Rb through inhibition of MDM2. We then examined the effect of ARF on Rb in p53À/À murine embryonic Results fibroblast (MEF) and p53À/À/MDM2À/À cells. As shown in Figure 2d, ARF markedly induced Rb in p53À/À MEF ARF Expression of p14 increases Rb levels independent cells but failed to do so in p53À/À/MDM2À/ÀÀ/À cells. of p53 Taken together, these data suggest that ARF inhibits MDM2 has been shown to facilitate Rb degradation MDM2 and upregulates Rb. (Sdek et al., 2005; Uchida et al., 2005). Given the critical role of ARF in regulation of MDM2, we investigated whether ARF can modulate MDM2-mediated Rb ARF inhibits colony foci formation independent of p53 ARF degradation. Co-expression of p14 and Rb in p53- The N-terminus of ARF (ARF-N; aa 1–64) is critical for null H1299 cells led to a marked increase in Rb levels ARF-mediated growth suppressive function. Notably, both p14ARF and p19ARF N-terminal domains contain MDM2-binding modules (Zhang et al., 1998; Weber et al., 2000b). Thus, we examined the effects of wild type, ARF-N and ARF-C (aa 64–132) on the expression of MDM2 and Rb. Our results showed that whereas wild-type ARF and ARF-N promoted MDM2 expres- sion, keeping with its reported ability to block MDM2 E3 ligase activity (Figure 3a), they also led to the accumulation of Rb protein (Figure 3b). In contrast, ARF-C was unable to affect either MDM2 or Rb (Figure 3a and b). We next examined the effect of ARF Figure 1 ARF increases Rb protein levels independent of p53. and ARF mutants on colony foci formation. As shown (a) H1299 cells were co-transfected with p14ARF, myc-tagged Rb in Figure 3c, wild-type ARF as well as ARF-N (aa 1–64) (myc-Rb) and GFP expression plasmids.
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