Capable of inducing cell cycle arrest in G1 and G2 phases. Acts as a tumor suppressor. Binds to MDM2 and blocks its nucleocytoplasmic shuttling by sequestering it in the nucleolus. This inhibits the oncogenic action of MDM2 by blocking MDM2-induced degradation of p53 and enhancing p53-dependent transactivation and apoptosis. Also induces G2 arrest and apoptosis in a p53-independent manner by preventing the activation of cyclin B1/CDC2 complexes. Binds to BCL6 and down-regulates BCL6-induced transcriptional repression. Binds to E2F1 and MYC and blocks their transcriptional activator activity but has no effect on MYC transcriptional repression. Binds to TOP1/TOPOI and stimulates its activity. This complex binds to rRNA gene promoters and may play a role in rRNA transcription and/or maturation. Interacts with NPM1/B23 and promotes its polyubiquitination and degradation, thus inhibiting rRNA processing. Interacts with COMMD1 and promotes its 'Lys63'-linked polyubiquitination. Interacts with UBE2I/UBC9 and enhances sumoylation of a number of its binding partners including MDM2 and E2F1. Binds to HUWE1 and represses its ubiquitin ligase activity. May play a role in controlling cell proliferation and apoptosis during mammary gland development.
The ARF tumor suppressor gene antagonizes generation of various tumors. ARF-mediated tumor suppression occurs in a p53-independent manner as well as in a p53-dependent manner. We here demonstrate that BCL6 is a target of the ARF tumor suppressor. Either mouse p19(ARF) or human p14(ARF) binds to BCL6 and downregulates BCL6-induced transcriptional repression. ARF-mediated downregulation of the BCL6 activity may account in part for ARF-mediated tumor suppression.
Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.
The ARF gene (p19(ARF) in mouse and p14(ARF) in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogenously these proteins. Preparations of a GST-ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no effect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I.
The INK4a/ARF locus which is frequently inactivated in human tumours encodes two different tumour suppressive proteins, p16(INK4a) and ARF. p16(INK4a) is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a final effector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identified. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance.
Although the tumor suppressor ARF is generally accepted for its essential role in activating the p53 pathway, its p53-independent function has also been proposed. Here, we report that ARF associates with COMMD1 and promotes Lys(63)-mediated polyubiquitination of COMMD1 in a p53-independent manner. We found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggested that the N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, we found that endogenous ARF redistributes from the nucleolus to the nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, we found that ARF promotes the polyubiquitination of COMMD1 through Lys(63) of ubiquitin but not the polyubiquitination of Lys(48), which does not target COMMD1 for proteasome-dependent proteolysis. Moreover, ARF mutants lacking the domain interacting with COMMD1 did not promote COMMD1 polyubiquitination, indicating that physical association is a prerequisite condition for the polyubiquitination process. Together, these data suggest that the ability to promote Lys(63)-mediated polyubiquitination of COMMD1 is a novel property of ARF independent of p53.
The tumor suppressor Arf (Alternative Reading Frame) protein (p14ARF in human and p19ARF in mouse) is mainly located in the nucleolus consistent with its subcellular localization, the protein has been shown to specifically interact with 5.8S rRNA and with B23/Nucleophosmin and to regulate ribosome biogenesis. Here, we show that the p14ARF protein interacts with chromatin and is recovered by chromatin immunoprecipitation (ChIP) in a fraction that contains a DNA sequence of the rRNA gene promoter. In addition, topoisomerase I (Topo I) that has been shown to interact with p14ARF coprecipitates with p14ARF containing chromatin. These data, in view of the function for Topo I in rRNA transcription, are consistent with a role for the p14ARF-Topo I complex in rRNA transcription and/or maturation.
The p14ARF tumour suppressor regulates a series of cell cycle regulatory proteins to promote cell cycle arrest in response to abnormal hyperproliferative growth stimuli. p14ARF alterations are common in human cancers and, when inherited, confer susceptibility to cutaneous melanoma. We now propose that the mechanism of p14ARF action may involve the covalent modification of its binding partners with the small ubiquitin-related protein SUMO-1. In particular, we demonstrate that p14ARF interacts with the SUMO E2 conjugating enzyme, Ubc9 and enhances the sumoylation of its binding partners, hdm2, E2F-1, HIF-1alpha, TBP-1 and p120E4F. Furthermore, p14ARF-induced sumoylation is abrogated by a subset of melanoma-associated p14ARF mutations. These results provide a mechanism for p14ARF action through a common modification of diverse binding partners.
The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.
The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the alpha transcript, p16(INK4a), is a recognized tumour suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A beta transcript, p14(ARF), activates a p53 response manifest in elevated levels of MDM2 and p21(CIP1) and cell cycle arrest in both G1 and G2/M. As a consequence, p14(ARF)-induced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14(ARF) acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14(ARF) expression and there is an inverse correlation between p14(ARF) expression and p53 function in human tumour cell lines. However, p14(ARF) expression is not involved in the response to DNA damage. These results place p14(ARF) in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.
Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.
Isoform
Iso 6
May be involved in regulation of autophagy and caspase-independent cell death; the short-lived mitochondrial isoform is stabilized by C1QBP.
The INK4a/ARF locus which is frequently inactivated in human tumours encodes two different tumour suppressive proteins, p16(INK4a) and ARF. p16(INK4a) is a major component of the RB pathway. ARF is part of an ARF-mdm2-p53 network that exerts a negative control on hyperproliferative signals emanating from oncogenic stimuli. Among these is the transcription factor E2F1, a final effector of the RB pathway, that induces ARF expression. Recent data suggest that ARF function is not restricted to the p53 pathway. However, ARF target(s) implicated in this p53-independent function remains to be identified. We show that ARF is able to inhibit the proliferation of human cell lines independently of their p53 status. In this context, we demonstrate that ARF interacts physically with E2F1 and inhibits its transcriptional activity. Moreover, we show that mdm2 is required for the modulation of E2F1 activity by ARF. Beside the well-known p53 and mdm2 partners, these results identify E2F1 as a new ARF target. Thus, ARF can be viewed as a dual-acting tumour suppressor protein in both the p53 and RB pathways, further emphasizing its role in tumour surveillance.
The ARF tumor suppressor gene antagonizes generation of various tumors. ARF-mediated tumor suppression occurs in a p53-independent manner as well as in a p53-dependent manner. We here demonstrate that BCL6 is a target of the ARF tumor suppressor. Either mouse p19(ARF) or human p14(ARF) binds to BCL6 and downregulates BCL6-induced transcriptional repression. ARF-mediated downregulation of the BCL6 activity may account in part for ARF-mediated tumor suppression.
Until recently, the ability of ARF (human p14(ARF), murine p19(ARF)) tumour-suppressor protein, encoded by the INK4A/ARF locus, to inhibit cell growth in response to various stimuli was related to its ability to stabilize p53 through the so-called ARF/MDM2/p53 pathway. However, recent data have demonstrated that ARF is not implicated in this unique p53-dependent pathway. By use of transient and stable expression, we show here that human p14(ARF) inhibits the growth of human tumoral cells lacking functional p53 by inducing a transient G(2) arrest and subsequently apoptosis. This p14(ARF)-induced G(2) arrest was correlated with inhibition of CDC2 activity, inactivation of CDC25C phosphatase and induction of the CDK inhibitor p21(WAFI). Apoptosis was demonstrated using Hoechst 33352 staining, proteolytic activation of caspase-3 and PARP cleavage. Similar results were obtained in experiments with cells synchronized by hydroxyurea block. Importantly, we were able to reproduce these effects 'in vivo' by showing that p14(ARF) inhibits the growth of p53 nullizygous human tumours in nude mice and induces the regression of p53 -/- established tumours. In these experiments, tumoral regression was associated with inhibition of cell proliferation as well as induction of apoptosis confirming the data obtained in cell lines.
The tumor suppressor ARF induces a p53-dependent and -independent cell cycle arrest. Unlike the nucleoplasmic MDM2 and p53, ARF localizes in the nucleolus. The role of ARF in the nucleolus, the molecular target, and the mechanism of its p53-independent function remains unclear. Here we show that ARF interacts with B23, a multifunctional nucleolar protein involved in ribosome biogenesis, and promotes its polyubiquitination and degradation. Overexpression of B23 induces a cell cycle arrest in normal fibroblasts, whereas in cells lacking p53 it promotes S phase entry. Conversely, knocking down B23 inhibits the processing of preribosomal RNA and induces cell death. Further, oncogenic Ras induces B23 only in ARF null cells, but not in cells that retain wild-type ARF. Together, our results reveal a molecular mechanism of ARF in regulating ribosome biogenesis and cell proliferation via inhibiting B23, and suggest a nucleolar role of ARF in surveillance of oncogenic insults.
The p14ARF tumour suppressor regulates a series of cell cycle regulatory proteins to promote cell cycle arrest in response to abnormal hyperproliferative growth stimuli. p14ARF alterations are common in human cancers and, when inherited, confer susceptibility to cutaneous melanoma. We now propose that the mechanism of p14ARF action may involve the covalent modification of its binding partners with the small ubiquitin-related protein SUMO-1. In particular, we demonstrate that p14ARF interacts with the SUMO E2 conjugating enzyme, Ubc9 and enhances the sumoylation of its binding partners, hdm2, E2F-1, HIF-1alpha, TBP-1 and p120E4F. Furthermore, p14ARF-induced sumoylation is abrogated by a subset of melanoma-associated p14ARF mutations. These results provide a mechanism for p14ARF action through a common modification of diverse binding partners.
The tumor suppressor Arf (Alternative Reading Frame) protein (p14ARF in human and p19ARF in mouse) is mainly located in the nucleolus consistent with its subcellular localization, the protein has been shown to specifically interact with 5.8S rRNA and with B23/Nucleophosmin and to regulate ribosome biogenesis. Here, we show that the p14ARF protein interacts with chromatin and is recovered by chromatin immunoprecipitation (ChIP) in a fraction that contains a DNA sequence of the rRNA gene promoter. In addition, topoisomerase I (Topo I) that has been shown to interact with p14ARF coprecipitates with p14ARF containing chromatin. These data, in view of the function for Topo I in rRNA transcription, are consistent with a role for the p14ARF-Topo I complex in rRNA transcription and/or maturation.
Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.
Although the tumor suppressor ARF is generally accepted for its essential role in activating the p53 pathway, its p53-independent function has also been proposed. Here, we report that ARF associates with COMMD1 and promotes Lys(63)-mediated polyubiquitination of COMMD1 in a p53-independent manner. We found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggested that the N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, we found that endogenous ARF redistributes from the nucleolus to the nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, we found that ARF promotes the polyubiquitination of COMMD1 through Lys(63) of ubiquitin but not the polyubiquitination of Lys(48), which does not target COMMD1 for proteasome-dependent proteolysis. Moreover, ARF mutants lacking the domain interacting with COMMD1 did not promote COMMD1 polyubiquitination, indicating that physical association is a prerequisite condition for the polyubiquitination process. Together, these data suggest that the ability to promote Lys(63)-mediated polyubiquitination of COMMD1 is a novel property of ARF independent of p53.
The two distinct proteins encoded by the CDKN2A locus are specified by translating the common second exon in alternative reading frames. The product of the alpha transcript, p16(INK4a), is a recognized tumour suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of the retinoblastoma protein by the cyclin-dependent kinases, CDK4 and CDK6. In contrast, the product of the human CDKN2A beta transcript, p14(ARF), activates a p53 response manifest in elevated levels of MDM2 and p21(CIP1) and cell cycle arrest in both G1 and G2/M. As a consequence, p14(ARF)-induced cell cycle arrest is p53 dependent and can be abrogated by the co-expression of human papilloma virus E6 protein. p14(ARF) acts by binding directly to MDM2, resulting in the stabilization of both p53 and MDM2. Conversely, p53 negatively regulates p14(ARF) expression and there is an inverse correlation between p14(ARF) expression and p53 function in human tumour cell lines. However, p14(ARF) expression is not involved in the response to DNA damage. These results place p14(ARF) in an independent pathway upstream of p53 and imply that CDKN2A encodes two proteins that are involved in tumour suppression.
The ARF gene (p19(ARF) in mouse and p14(ARF) in man) has become a central actor of the cell cycle regulation process as it participates to the ARF-MDM2-p53 pathway and the Rb-E2F-1 pathway. By use of immunoprecipitation and Western blotting (IP/WB), we now show that ARF physically associates with topoisomerase I (Topo I). ARF-Topo I immune complexes were detected in SF9 insect cells infected with recombinant baculoviruses encoding the two genes as well as in 293 cells that express endogenously these proteins. Preparations of a GST-ARF recombinant protein stimulated the DNA relaxation activity of Topo I but, in contrast, had no effect on the decatenation activity of Topo II. The Topo I stimulation was also detected in cell extracts of SF9 cells expressing both proteins. A confocal microscopy study indicated that part of ARF and Topo I colocalized in the granular component structure of the nucleolus. As a whole, our data indicate that Topo I is a new partner of ARF and suggest that ARF is involved in cell reactions that require Topo I.
The INK4a-ARF locus encodes two unrelated proteins that both function in tumor suppression. p16INK4 binds to and inhibits the activity of CDK4 and CDK6, and ARF arrests the cell cycle in a p53-dependent manner. We show here that ARF binds to MDM2 and promotes the rapid degradation of MDM2. This interaction is mediated by the exon 1beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, deletion of the ARF-INK4a locus simultaneously impairs both the INK4a-cyclin D/CDK4-RB and the ARF-MDM2-p53 pathways.
The INK4a-ARF locus encodes two unrelated proteins that both function in tumor suppression. p16INK4 binds to and inhibits the activity of CDK4 and CDK6, and ARF arrests the cell cycle in a p53-dependent manner. We show here that ARF binds to MDM2 and promotes the rapid degradation of MDM2. This interaction is mediated by the exon 1beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, deletion of the ARF-INK4a locus simultaneously impairs both the INK4a-cyclin D/CDK4-RB and the ARF-MDM2-p53 pathways.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionBHF-UCL
We performed a yeast interactive screen in search of p19(ARF)-binding partners and have isolated a novel serine rich protein that is assigned to human chromosome 4q35 and mouse chromosome 8. The human and mouse proteins showed 84.2% homology. It is named CARF for its role as a putative Collaborator of ARF. CARF binds to both mouse and human ARF proteins. Its expression was detected in a variety of human tissues. The cDNA was expressed in bacteria and mammalian cells as a soluble and predominantly nucleoplasmic protein, respectively. CARF is a novel binding partner of ARF and might be involved in its p53-dependent or -independent tumor suppressive functions.
Evidence
2:
Inferred from Physical InteractionUniProtKB
The tumour suppressor ARF is specifically required for p53 activation under oncogenic stress. Recent studies showed that p53 activation mediated by ARF, but not that induced by DNA damage, acts as a major protection against tumorigenesis in vivo under certain biological settings, suggesting that the ARF-p53 axis has more fundamental functions in tumour suppression than originally thought. Because ARF is a very stable protein in most human cell lines, it has been widely assumed that ARF induction is mediated mainly at the transcriptional level and that activation of the ARF-p53 pathway by oncogenes is a much slower and largely irreversible process by comparison with p53 activation after DNA damage. Here we report that ARF is very unstable in normal human cells but that its degradation is inhibited in cancerous cells. Through biochemical purification, we identified a specific ubiquitin ligase for ARF and named it ULF. ULF interacts with ARF both in vitro and in vivo and promotes the lysine-independent ubiquitylation and degradation of ARF. ULF knockdown stabilizes ARF in normal human cells, triggering ARF-dependent, p53-mediated growth arrest. Moreover, nucleophosmin (NPM) and c-Myc, both of which are commonly overexpressed in cancer cells, are capable of abrogating ULF-mediated ARF ubiquitylation through distinct mechanisms, and thereby promote ARF stabilization in cancer cells. These findings reveal the dynamic feature of the ARF-p53 pathway and suggest that transcription-independent mechanisms are critically involved in ARF regulation during responses to oncogenic stress.
Evidence
3:
Inferred from Physical InteractionIntAct
The alternative reading frame (ARF) mRNA encodes two pro-death proteins, the nucleolar p19ARF and a shorter mitochondrial isoform, named smARF (hsmARF in human). While p19ARF can inhibit cell growth by causing cell cycle arrest or type I apoptotic cell death, smARF is able to induce type II autophagic cell death. Inappropriate proliferative signals generated by proto-oncogenes, such as c-Myc and E2F1, can elevate both p19ARF and smARF proteins. Here, we reveal a novel means of regulation of smARF protein steady state levels through its interactions with the mitochondrial p32. The p32 protein physically interacts with both human and murine smARF, and colocalizes with these short isoforms to the mitochondria. Remarkably, knocking down p32 protein levels significantly reduced the steady state levels of smARF by increasing its turn over. As a consequence, the ability of ectopically expressed smARF to induce autophagy and to cause mitochondrial membrane dissipation was significantly reduced. In contrast, the protein levels of full-length p19ARF, which mainly resides in the nucleolus, were not influenced by p32 depletion, suggesting that p32 exclusively stabilizes the mitochondrial smARF protein. Thus the interaction with p32 provides a means of specifically regulating the expression of the recently identified autophagic inducer, smARF, and adds yet another layer of complexity to the multifaceted regulation of the ARF gene.
Evidence
4:
Inferred from Physical InteractionUniProtKB
Although the tumor suppressor ARF is generally accepted for its essential role in activating the p53 pathway, its p53-independent function has also been proposed. Here, we report that ARF associates with COMMD1 and promotes Lys(63)-mediated polyubiquitination of COMMD1 in a p53-independent manner. We found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggested that the N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, we found that endogenous ARF redistributes from the nucleolus to the nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, we found that ARF promotes the polyubiquitination of COMMD1 through Lys(63) of ubiquitin but not the polyubiquitination of Lys(48), which does not target COMMD1 for proteasome-dependent proteolysis. Moreover, ARF mutants lacking the domain interacting with COMMD1 did not promote COMMD1 polyubiquitination, indicating that physical association is a prerequisite condition for the polyubiquitination process. Together, these data suggest that the ability to promote Lys(63)-mediated polyubiquitination of COMMD1 is a novel property of ARF independent of p53.
Evidence
5:
Inferred from Physical InteractionIntAct
Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.
Evidence
6:
Inferred from Physical InteractionBHF-UCL
The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.
Evidence
7:
Inferred from Physical InteractionIntAct
Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull-down assay. Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation.
Evidence
8:
Inferred from Physical InteractionIntAct
The p14(ARF) (ARF) tumour suppressor plays an important role in the cellular response to oncogene activation. In this report, we demonstrate an interaction between ARF and DAXX, a highly conserved protein with identified roles in the regulation of gene expression. HDM2 was shown to interact with each of ARF and DAXX upon upregulation of expression as well as at lower expression levels following transfection of ARF and DAXX. Through immunofluorescence analysis, we observed that endogenous ARF and DAXX colocalize both to nucleoli and to nuclear bodies in cell lines that co-express both proteins. Similar results were obtained upon co-transfection of ARF and DAXX. Co-expression of ARF and DAXX was further found to inhibit ARF-mediated HDM2 sumoylation and to induce sumoylation and ubiquitination of DAXX itself, implicating DAXX as a substrate of ARF-mediated post-translational events. We also observed induction of p53 sumoylation in the presence of ARF and DAXX, an effect that was inhibited by upregulation of HDM2 expression. In summary, we have identified DAXX as a novel ARF binding partner and substrate of ARF-mediated sumoylation and suggest that DAXX acts as a modifier of both p53-dependent and p53-independent ARF function.
Evidence
9:
Inferred from Physical InteractionIntAct
The CDKN2A locus encodes two distinct proteins, p16INK4a and p14ARF, both of which are implicated in replicative senescence and tumor suppression in different contexts. Here, we describe the characterization of a novel strain of human diploid fibroblasts (designated Milan HDFs) from an individual who is homozygous for the R24P mutation in p16INK4a. As this mutation occurs in the first exon of INK4a (exon 1alpha), it has no effect on the primary sequence of p14(ARF). Based on both in vitro and in vivo analyses, the R24P variant is specifically defective for binding to CDK4 but remains able to associate with CDK6. Nevertheless, Milan HDFs behave as if they are p16INK4a deficient, in terms of sensitivity to spontaneous and oncogene-induced senescence, and the R24P variant has little effect on proliferation when ectopically expressed in normal fibroblasts. It can, however, impair the proliferation of U20S cells, presumably because they express more CDK6 than primary fibroblasts. These observations suggest that CDK4 and CDK6 are not functionally redundant and underscore the importance of CDK4 in the development of melanoma.
The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.
The cleavage of DNA during apoptosis, which usually occurs in two stages: cleavage into fragments of about 50 kbp followed by cleavage between nucleosomes to yield 200 bp fragments.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.
A cell aging process stimulated in response to cellular stress, whereby normal cells lose the ability to divide through irreversible cell cycle arrest.
Inactivation of the ARF-p53 tumor suppressor pathway leads to immortalization of murine fibroblasts. The role of this pathway in immortalization of human epithelial cells is not clear. We analyzed the functionality of the p14(ARF)-p53 pathway in human mammary epithelial cells (MEC) immortalized by human papillomavirus 16 (HPV16) E6, the p53 degradation-defective E6 mutant Y54D, or hTERT. E6-MEC or E6Y54D-MEC maintains high-level expression of p14(ARF). Late-passage hTERT-immortalized MEC express p53 but down-regulate p14(ARF). Enforced expression of p14(ARF) induces p53-dependent senescence in hTERT-MEC, while both E6-MEC and E6Y54D-MEC are resistant. We show that E6Y54D inhibits p14(ARF)-induced activation of p53 without inactivation of the p53-dependent DNA damage response. Hence, p53 degradation and inhibition of p14(ARF) signaling to p53 are independent functions of HPV16 E6. Our observations imply that long-term proliferation of MEC requires inactivation of the p14(ARF)-p53 pathway.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.
MEG3 is a maternally expressed imprinted gene suggested to function as a non-coding RNA. Our previous studies suggest that MEG3 has a function of tumor suppression. The tumor suppressor p53 plays a central role in tumor suppression and mediates the functions of many other tumor suppressors. Therefore, we hypothesized that MEG3 functions through activation of p53. We found that transfection of expression constructs for MEG3 and its isoforms results in a significant increase in p53 protein levels and dramatically stimulates p53-dependent transcription from a p53-responsive promoter. Using this as the functional assay, we demonstrated that the open reading frames encoded by MEG3 transcripts are not required for MEG3 function, and the folding of MEG3 RNA is critical to its function, supporting the concept that MEG3 functions as a non-coding RNA. We further found that MEG3 stimulates expression of the growth differentiation factor 15 (GDF15) by enhancing p53 binding to the GDF15 gene promoter. Interestingly, MEG3 does not stimulate p21(CIP1) expression, suggesting that MEG3 can regulate the specificity of p53 transcriptional activation. p53 degradation is mainly mediated by the mouse double minute 2 homolog (MDM2). We found that MDM2 levels were down-regulated in cells transfected with MEG3, suggesting that MDM2 suppression contributes at least in part to p53 accumulation induced by MEG3. Finally, we found that MEG3 is able to inhibit cell proliferation in the absence of p53. These data suggest that MEG3 non-coding RNA may function as a tumor suppressor, whose action is mediated by both p53-dependent and p53-independent pathways.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.
Any process that stops, prevents, or reduces the frequency, rate or extent of ubiquitin ligase activity, the catalysis of the reaction: ATP + ubiquitin + protein lysine = AMP + diphosphate + protein N-ubiquityllysine.
Any process that increases the rate, frequency, or extent of cell cycle arrest, the process in which the cell cycle is halted during one of the normal phases.
The INK4a-ARF locus encodes two unrelated proteins that both function in tumor suppression. p16INK4 binds to and inhibits the activity of CDK4 and CDK6, and ARF arrests the cell cycle in a p53-dependent manner. We show here that ARF binds to MDM2 and promotes the rapid degradation of MDM2. This interaction is mediated by the exon 1beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, deletion of the ARF-INK4a locus simultaneously impairs both the INK4a-cyclin D/CDK4-RB and the ARF-MDM2-p53 pathways.
Isoform
Iso 4
Positive regulation of DNA damage response, signal transduction by p53 class mediatordefinition[GO:0043517]
Any process that activates, maintains or increases the rate of the cascade of processes induced by the cell cycle regulator phosphoprotein p53, or an equivalent protein, in response to the detection of DNA damage.
The INK4a-ARF locus encodes two unrelated proteins that both function in tumor suppression. p16INK4 binds to and inhibits the activity of CDK4 and CDK6, and ARF arrests the cell cycle in a p53-dependent manner. We show here that ARF binds to MDM2 and promotes the rapid degradation of MDM2. This interaction is mediated by the exon 1beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, deletion of the ARF-INK4a locus simultaneously impairs both the INK4a-cyclin D/CDK4-RB and the ARF-MDM2-p53 pathways.
The PTEN tumour suppressor gene is induced by the early growth response 1 (EGR1) transcription factor, which also transactivates p53, p73, and p300/CBP as well as other proapoptotic and anti-cancer genes. Here, we describe a novel Akt-EGR1-alternate reading frame (ARF)-PTEN axis, in which PTEN activation in vivo requires p14ARF-mediated sumoylation of EGR1. This modification is dependent on the phosphorylation of EGR1 at S350 and T309 by Akt, which promotes interaction of EGR1 with ARF at K272 in its repressor domain by the ARF/Ubc9/SUMO system. EGR1 sumoylation is decreased by ARF reduction, and no EGR1 sumoylation is detected in ARF(-/-) mice, which also exhibit reduced amounts of PTEN. Our model predicts that perturbation of any of the clinically important tumour suppressors, PTEN, EGR1, and ARF, will cause some degree of dysfunction of the others. These results also explain the known negative feedback regulation by PTEN on its own synthesis through PI3 kinase inhibition.
MEG3 is a maternally expressed imprinted gene suggested to function as a non-coding RNA. Our previous studies suggest that MEG3 has a function of tumor suppression. The tumor suppressor p53 plays a central role in tumor suppression and mediates the functions of many other tumor suppressors. Therefore, we hypothesized that MEG3 functions through activation of p53. We found that transfection of expression constructs for MEG3 and its isoforms results in a significant increase in p53 protein levels and dramatically stimulates p53-dependent transcription from a p53-responsive promoter. Using this as the functional assay, we demonstrated that the open reading frames encoded by MEG3 transcripts are not required for MEG3 function, and the folding of MEG3 RNA is critical to its function, supporting the concept that MEG3 functions as a non-coding RNA. We further found that MEG3 stimulates expression of the growth differentiation factor 15 (GDF15) by enhancing p53 binding to the GDF15 gene promoter. Interestingly, MEG3 does not stimulate p21(CIP1) expression, suggesting that MEG3 can regulate the specificity of p53 transcriptional activation. p53 degradation is mainly mediated by the mouse double minute 2 homolog (MDM2). We found that MDM2 levels were down-regulated in cells transfected with MEG3, suggesting that MDM2 suppression contributes at least in part to p53 accumulation induced by MEG3. Finally, we found that MEG3 is able to inhibit cell proliferation in the absence of p53. These data suggest that MEG3 non-coding RNA may function as a tumor suppressor, whose action is mediated by both p53-dependent and p53-independent pathways.
MEG3 is a maternally expressed imprinted gene suggested to function as a non-coding RNA. Our previous studies suggest that MEG3 has a function of tumor suppression. The tumor suppressor p53 plays a central role in tumor suppression and mediates the functions of many other tumor suppressors. Therefore, we hypothesized that MEG3 functions through activation of p53. We found that transfection of expression constructs for MEG3 and its isoforms results in a significant increase in p53 protein levels and dramatically stimulates p53-dependent transcription from a p53-responsive promoter. Using this as the functional assay, we demonstrated that the open reading frames encoded by MEG3 transcripts are not required for MEG3 function, and the folding of MEG3 RNA is critical to its function, supporting the concept that MEG3 functions as a non-coding RNA. We further found that MEG3 stimulates expression of the growth differentiation factor 15 (GDF15) by enhancing p53 binding to the GDF15 gene promoter. Interestingly, MEG3 does not stimulate p21(CIP1) expression, suggesting that MEG3 can regulate the specificity of p53 transcriptional activation. p53 degradation is mainly mediated by the mouse double minute 2 homolog (MDM2). We found that MDM2 levels were down-regulated in cells transfected with MEG3, suggesting that MDM2 suppression contributes at least in part to p53 accumulation induced by MEG3. Finally, we found that MEG3 is able to inhibit cell proliferation in the absence of p53. These data suggest that MEG3 non-coding RNA may function as a tumor suppressor, whose action is mediated by both p53-dependent and p53-independent pathways.
The INK4a-ARF locus encodes two unrelated proteins that both function in tumor suppression. p16INK4 binds to and inhibits the activity of CDK4 and CDK6, and ARF arrests the cell cycle in a p53-dependent manner. We show here that ARF binds to MDM2 and promotes the rapid degradation of MDM2. This interaction is mediated by the exon 1beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, deletion of the ARF-INK4a locus simultaneously impairs both the INK4a-cyclin D/CDK4-RB and the ARF-MDM2-p53 pathways.
A protein ubiquitination process in which a polymer of ubiquitin, formed by linkages between lysine residues at position 63 of the ubiquitin monomers, is added to a protein. K63-linked ubiquitination does not target the substrate protein for degradation, but is involved in several pathways, notably as a signal to promote error-free DNA postreplication repair.
Although the tumor suppressor ARF is generally accepted for its essential role in activating the p53 pathway, its p53-independent function has also been proposed. Here, we report that ARF associates with COMMD1 and promotes Lys(63)-mediated polyubiquitination of COMMD1 in a p53-independent manner. We found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggested that the N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, we found that endogenous ARF redistributes from the nucleolus to the nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, we found that ARF promotes the polyubiquitination of COMMD1 through Lys(63) of ubiquitin but not the polyubiquitination of Lys(48), which does not target COMMD1 for proteasome-dependent proteolysis. Moreover, ARF mutants lacking the domain interacting with COMMD1 did not promote COMMD1 polyubiquitination, indicating that physical association is a prerequisite condition for the polyubiquitination process. Together, these data suggest that the ability to promote Lys(63)-mediated polyubiquitination of COMMD1 is a novel property of ARF independent of p53.
Although the tumor suppressor ARF is generally accepted for its essential role in activating the p53 pathway, its p53-independent function has also been proposed. Here, we report that ARF associates with COMMD1 and promotes Lys(63)-mediated polyubiquitination of COMMD1 in a p53-independent manner. We found that ARF interacts with COMMD1 in vivo. Deletion analysis of ARF suggested that the N-terminal amino acids 15-45 are important for its interaction with COMMD1. In addition, we found that endogenous ARF redistributes from the nucleolus to the nucleoplasm and interacts with COMMD1 when DNA is damaged by actinomycin D. Interestingly, we found that ARF promotes the polyubiquitination of COMMD1 through Lys(63) of ubiquitin but not the polyubiquitination of Lys(48), which does not target COMMD1 for proteasome-dependent proteolysis. Moreover, ARF mutants lacking the domain interacting with COMMD1 did not promote COMMD1 polyubiquitination, indicating that physical association is a prerequisite condition for the polyubiquitination process. Together, these data suggest that the ability to promote Lys(63)-mediated polyubiquitination of COMMD1 is a novel property of ARF independent of p53.
The mammalian ARF-INK4a locus uniquely encodes two cell cycle inhibitors by using separate promoters and alternative reading frames. p16INK4a maintains the retinoblastoma protein in its growth suppressive state while ARF stabilizes p53. We report that human ARF protein predominantly localizes to the nucleolus via a sequence within the exon 2-encoded C-terminal domain and is induced to leave the nucleolus by MDM2. ARF forms nuclear bodies with MDM2 and p53 and blocks p53 and MDM2 nuclear export. Tumor-associated mutations in ARF exon 2 disrupt ARF's nucleolus localization and reduce ARF's ability to block p53 nuclear export and to stabilize p53. Our results suggest an ARF-regulated MDM2-dependent p53 stabilization and link the human tumor-associated mutations in ARF with a functional alteration.
The INK4a-ARF locus encodes two unrelated proteins that both function in tumor suppression. p16INK4 binds to and inhibits the activity of CDK4 and CDK6, and ARF arrests the cell cycle in a p53-dependent manner. We show here that ARF binds to MDM2 and promotes the rapid degradation of MDM2. This interaction is mediated by the exon 1beta-encoded N-terminal domain of ARF and a C-terminal region of MDM2. ARF-promoted MDM2 degradation is associated with MDM2 modification and concurrent p53 stabilization and accumulation. The functional consequence of ARF-regulated p53 levels via MDM2 proteolysis is evidenced by the ability of ectopically expressed ARF to restore a p53-imposed G1 cell cycle arrest that is otherwise abrogated by MDM2. Thus, deletion of the ARF-INK4a locus simultaneously impairs both the INK4a-cyclin D/CDK4-RB and the ARF-MDM2-p53 pathways.
The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.
The mammalian ARF-INK4a locus uniquely encodes two cell cycle inhibitors by using separate promoters and alternative reading frames. p16INK4a maintains the retinoblastoma protein in its growth suppressive state while ARF stabilizes p53. We report that human ARF protein predominantly localizes to the nucleolus via a sequence within the exon 2-encoded C-terminal domain and is induced to leave the nucleolus by MDM2. ARF forms nuclear bodies with MDM2 and p53 and blocks p53 and MDM2 nuclear export. Tumor-associated mutations in ARF exon 2 disrupt ARF's nucleolus localization and reduce ARF's ability to block p53 nuclear export and to stabilize p53. Our results suggest an ARF-regulated MDM2-dependent p53 stabilization and link the human tumor-associated mutations in ARF with a functional alteration.
Protein involved in apoptotic programmed cell death. Apoptosis is characterized by cell morphological changes, including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation, and eventually death. Unlike necrosis, apoptosis produces cell fragments, called apoptotic bodies, that phagocytic cells are able to engulf and quickly remove before the contents of the cell can spill out onto surrounding cells and cause damage. In general, apoptosis confers advantages during an organism's life cycle.
Protein involved in the complex series of events by which the cell duplicates its contents and divides into two. The eukaryotic cell cycle can be divided in four phases termed G1 (first gap period), S (synthesis, phase during which the DNA is replicated), G2 (second gap period) and M (mitosis). The prokaryotic cell cycle typically involves a period of growth followed by DNA replication, partition of chromosomes, formation of septum and division into two similar or identical daughter cells.
Protein involved in the transfer of genetic information from DNA to messenger RNA (mRNA) by DNA-directed RNA polymerase. In the case of some RNA viruses, protein involved in the transfer of genetic information from RNA to messenger RNA (mRNA) by RNA-directed RNA polymerase.
Protein involved in ubiquitin-like modifier processing, activation, conjugation or deconjugation such as Ubl-activating enzymes (E1s), Ubl-conjugating enzymes (E2s), Ubl-protein ligases (E3s), some thiol proteases (Ubiquitin carboxyl-terminal hydrolases (UCH), Ubiquitin- specific processing proteases (UBP) and ubiquitin-like proteases) and the ubiquitin-like modifier proteins. Besides signaling proteolysis, ubiquitination for example can be a signal for trafficking, kinase activation and other nonproteolytic fates.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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