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Developmental and biochemical characterization of activators of the RB and p53
tumor suppressor pathways
den Besten, W.
Publication date 2007
Document Version Final published version
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Citation for published version (APA):
den Besten, W. (2007). Developmental and biochemical characterization of activators of the RB and p53 tumor suppressor pathways.
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The studies described in this thesis were performed in the Department of Genetics and Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, U.S.A., supported by NIH Grant CA071907, Howard Hughes Medical Institute and by the American Lebanese Syrian Associated Charities (ALSAC).
activators of the RB and p53 tumor suppressor pathways
ACADEMISCH PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus
prof. dr. J.W. Zwemmer
ten overstaan van een door het college van promoties ingestelde commissie,
in het openbaar te ververdedigen in de Aula der Universiteit op
donderdag 14 juni 2007, te 14:00 uur door
WILLEM DEN BESTEN
Promotor: Prof. dr. A. J. M. Berns Co-promotores: Dr. M. F. Roussel
Dr. C. J. Sherr
Overige leden: Prof. dr. F. Baas
Prof. dr. P. Borst
Prof. dr. R. Versteeg
Dr. R. Agami
Dr. D. S. Peeper
Page
Chapter 1
Introduction 9
Chapter 2
Control of spermatogenesis in mice by the cyclin D-dependent 43 kinase inhibitors p18Ink4c and p19Ink4d
Chapter 3
N-terminal polyubiquitination and degradation of the Arf tumor 57 suppressor
Chapter 4
Myeloid leukemia-associated nucleophosmin mutants perturb 81 p53-dependent and independent activities of the Arf tumor
suppressor protein
Chapter 5
Ubiquitination of, and Sumoylation by, the Arf Tumor Suppressor 89
Chapter 6
Summary and discussion 95
Samenvatting 103
Acknowledgements 109
Curriculum vitae 110
Introduction
In order to divide, a cell needs to faithfully replicate its DNA and distribute the duplicated genetic information equally between two daughter cells. The eukaryotic cell division cycle can be separated into several stages that include a DNA synthesis (S) phase and mitosis (M phase), each divided from one another by Gap (G) phases, G1 (between M and S) and G2 (between S and M). After a previously completed division, cells entering the ensuing G1 phase advance toward another S phase in response to extracellular stimulatory signals or withdraw from the cell cycle into a quiescent state (G0) in their absence.
Sustained growth factor stimulation during G1 phase is required for cells to pass a restriction point late in G1, after which they become committed to complete the cell cycle even in the absence of mitogens (Pardee, 1974; Pardee, 1989). Mitogen withdrawal or exposure to anti-proliferative cytokines before passing the restriction point prevents G1 progression and arrests proliferation. Cancer cells frequently lose these controls and tend to remain in cycle, dividing even in the absence of growth factors and losing their responsiveness to extracellular inhibitory signals (Sherr, 1996).
Regulation of G1 progression, S phase entry and exit
Transition through the different phases of the cell cycle is controlled by cyclin dependent protein kinases (CDKs). These holoenzymes consist of both regulatory (cyclin) and catalytic (CDK) subunits, and diverse CDK-cyclin complexes differentially govern distinct cell cycle transitions. Regulation of the mammalian G1-S transition can be distilled into two processes: the activation of a transcriptional program that initiates S phase and the inactivation of CDK inhibitors.
When quiescent cells are stimulated with growth factors to enter the G1 phase of the cell division cycle, D-type cyclins are induced and assemble with their catalytic partners CDK4 or CDK6 (Matsushime et al., 1992; Bates et al., 1994; Matsushime et al., 1994; Meyerson and Harlow, 1994) (Figure 1A and 1B). Assembled cyclin D-CDK complexes then enter the nucleus where they must be activated through phosphorylation by a CDK-activating kinase (CAK) to be able to phosphorylate protein substrates (Kato et al., 1994). Mitogen-activated signaling pathways promote the transcription of cyclin D genes and are also required for the assembly of newly synthesized D cyclins with CDK4 or CDK6. A distinct mitogen-induced signaling pathway negatively regulates the phosphorylation of cyclin D by glycogen synthase kinase-3β (GSK3β) (Diehl et al., 1998). Growth factor withdrawal leads to activation of GSK3β, cyclin D phosphorylation, its enhanced nuclear export, and accelerated ubiquitin-dependent proteasomal degradation of the protein in the cytoplasm (Diehl et al., 1997; Diehl et al., 1998). Therefore because their synthesis, assembly, nuclear
localization and stability strongly depend on continuous mitogenic stimulation, D-type cyclins act as growth factor sensors.
Figure 1. Regulation of the G1/S transition.
(A) Relative abundance of cyclins and p27Kip1 during the cell cycle. p27 levels are high in
quiescent cells, fall in response to mitogenic stimulation, remain at lower threshold levels in proliferating cells, and increase again when mitogens are withdrawn. D-type cyclins are expressed throughout the cell cycle in response to mitogen stimulation whereas the expression of cyclin E, A, and B is periodic (modified from (Sherr, 1996)).
(B) Restriction point control. Mitogen regulated induction of cyclin D expression leads to the
titration of p27 into active cyclin D/CDK4 kinase complexes which phosphorylate the retinoblastoma family of proteins. Phosphorylation of RB family of proteins results in the release of E2F transcriptional regulators and the induction of cyclin E and cyclin A transcription. Cyclin E and cyclin A assembly with CDK2 further phosphorylate RB family proteins. This creates a positive feedback loop and shift cells from mitogen dependence to mitogen independence as they pass the restriction point before entering S phase.
Mitogen stimulation in G1 phase leads to a build up of cyclin-CDK activity required to initiate transcription of genes essential for S phase entry and DNA synthesis. Once DNA synthesis begins, the activity of these G1-specific kinases is no longer necessary until cells complete division and re-enter the G1 phase of the next division cycle. Cyclin D-CDK complexes are quite specific in their substrate selection and, after localization to the nucleus, they phosphorylate proteins of the retinoblastoma family of transcriptional repressors (pRB, p107, p130) (Matsushime et al., 1992; Ewen et al., 1993; Kato et al., 1993) (Figure 1B). Rb-family members negatively control gene expression mediated by the E2F family of heterodimeric transcription factors. In quiescent cells, pRB binds to the “activating E2Fs “(E2F1, E2F2 and E2F3) to block their transcriptional activity. At the same time, other E2Fs (E2F4, and E2F5) recruit mostly p130 and associated corepressors to E2F-responsive promoters. As cells progress through the G1 phase of the cell cycle, phosphorylation of Rb family members by cyclin D-CDKs helps both to liberate the activating E2Fs and to antagonize the E2F repressor
complexes. This results in the replacement of E2F4/p130 and E2F5/p130 by activating E2Fs and their associated coactivators, which leads to the expression of E2F target genes (Takahashi et al., 2000).
One of the E2F target genes induced as cells approach the G1/S transition is cyclin E which assembles with CDK2 and further phosphorylates Rb on residues distinct from the ones targeted by cyclin D-CDK4 or CDK6 kinases (Figure 1B). In addition to genes whose products are required for DNA synthesis, such as enzymes involved in nucleotide biosynthesis (thymidylate synthetase, thymidine kinase and dihydrofolate reductase) and the main components of the DNA-replication machinery (Cdc6, ORC1 and the minichromosome maintenance (MCM) proteins), E2F1 also stimulates its own transcription. Thus, the mitogen-regulated abundance and association of D-type cyclins with CDK4 and CDK6 triggers an initial burst of transcriptional activation, which is then accelerated by cyclin E-CDK2 through a positive feedback loop as cells approach the G1-S boundary (Figure 1B). Hence, Rb inactivation shifts cells from a mitogen-dependent (cyclin D driven) to mitogen-independent (cyclin E driven) state, and this coincides with the irreversible commitment of cells to enter S-phase (Figure 1B) (Sherr and Roberts, 1999).
Once cells pass the restriction point and enter S-phase, the levels of cyclin D and cyclin E fall as their phosphorylation signals them for ubiquitin-dependent destruction (Figure 1A). Phosphorylation of cyclin D by nuclear GSK3β enhances its cytoplasmic export, ubiquitination, and proteasomal degradation, but its continued synthesis and increased stability later in the cell cycle allow cyclin D to reaccumulate during G2 (Diehl et al., 1998; Sherr, 2002) (Figure 1A). Cyclin E is phosphorylated on multiple residues by its own catalytic partner, CDK2 (Won and Reed, 1996; Clurman et al., 1996), as well as by GSK3β and another yet unknown kinase (Welcker et al., 2003). These processes similarly target cyclin E for destruction.
Following degradation of cyclin E, CDK2 associates with cyclin A, another cyclin induced by E2F during late G1. CDK2-cyclin A and, later, CDK1-cyclin B complexes take over part of cyclin E’s function and keep Rb hyperphosphorylated throughout the rest of the cell cycle until dephosphorylation reactivates its function at the end of mitosis. In addition, CDK2-cyclin A also phosphorylates DP1, one of the heterodimeric components of the E2F complex, thereby inhibiting its DNA binding. This results in decreased E2F transactivation as cells approach the S-G2 boundary. Of the two mitotic cyclins (cyclin A and B), cyclin A is the first to be degraded in pro-metaphase by a multiprotein E3 ubiquitin ligase complex, APC/CCdc20. Later in metaphase and only after spindle assembly has been completed is cyclin B proteolysis initiated by APC/CCdc20 and later continued by APC/CCdh1. The latter complex remains active in the ensuing G1 phase and prevents the reaccumulation of cyclins A and B until cells again enter S phase. Fully activated CDK1-cyclin B is required for cells to enter mitosis, whereas the destruction of cyclin B and the resulting inactivation of CDK1 kinase activity are necessary for mitotic exit. These events, together with the earlier decrease in E2F transactivation, help to reset the system to the ground state and
reinstitute a period of mitogen-dependence by reestablishing the requirement for cyclin D in the subsequent G1 phase of the next cell cycle.
The Cip/Kip family of CDK inhibitors
In addition to transcriptional activation of genes required for S phase, cells
also need to inactivate the Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) in order to pass the restriction point. This family of CKIs contains three members (p21Cip1, p27Kip1 and p57Kip2) which are able to bind and inhibit CDK2 kinase activity by changing the shape of the catalytic cleft and by inserting a helix inside the ATP binding site (Russo et al., 1996; Pavletich, 1999). Paradoxically, cyclin D-CDK complexes are resistant to Cip/Kip inhibition, and their activation and assembly is actually facilitated by their interactions with these CKIs (LaBaer et al., 1997; Cheng et al., 1999). This sequestration of Cip/Kip proteins highlights a second, noncatalytic role for the cyclin D-dependent kinases in G1 phase progression. Quiescent cells express relatively high levels of nuclear p27Kip1 which, in response to mitogenic stimulation, delocalizes to the cytoplasm where part of it is targeted for degradation (Pagano et al., 1995; Shirane et al., 1999) (Figure 1A). Once in the cytoplasm, p27Kip1 promotes the activation of cyclin D-CDK complexes. Cip/Kip molecules contact both the cyclin D and D-CDK4/D-CDK6 subunits to stabilize them in an active higher order complex with an estimated molecular weight of ~150 kDa. Once assembled, these heteromeric complexes are directed to the nucleus through the nuclear localization signal in the Cip/Kip proteins, allowing the kinases to phosphorylate Rb-family proteins. The titration of unbound p27Kip1 and p21Cip1 proteins into higher order complexes with assembling cyclin D-dependent kinases relieves cyclin E-CDK2 from Cip/Kip inhibition. In addition, cyclin E-CDK2 can facilitate its own activation by phosphorylating p27Kip1 to trigger its degradation and thereby further reducing the p27Kip1 levels (Sheaff et al., 1997; Vlach et al., 1997). The tethered Cip/Kip proteins remain bound to cyclin D-dependent kinases throughout the rest of the cell cycle, but are released when mitogens are withdrawn and cyclin D levels decrease. As a result, Cip/Kip proteins redistribute to cyclin E-CDK2 complexes and induce G1 arrest.
The INK4 family of CDK inhibitors
A second class of cyclin-dependent kinase inhibitors are the INhibitors of CDK4, or INK4 proteins, whose members (p16INK4a, p15INK4b, p18INK4c and p19INK4d) exclusively bind to and inhibit the D-type cyclin-dependent kinases. The INK4 proteins, p16INK4a, p18INK4c and p19INK4d were identified as interactors in
yeast two-hybrid screens that employed CDK4 or CDK6 as baits (Serrano et al., 1993; Guan et al., 1994; Hirai et al., 1995). In contrast, p15INK4b was cloned as an INK4a cDNA homolog using a low stringency hybridization procedure
(Hannon and Beach, 1994). The INK4 proteins are highly conserved among species and share roughly 40% homology with one another. All four members are structurally very similar and primarily consist of either four (p16INK4a and p15INK4b) or five (p18INK4c and p19INK4d) ankyrin-like repeats that mediate binding to both the N-terminal and the C-terminal parts of CDK4 or CDK6. Binding distorts the ATP binding site of CDK4/6 and results in a reduced ATP affinity or a disorientation of already bound ATP. This allosteric change alters the cyclin D binding site and therefore noncompetitively blocks the interaction between these CDKs and cyclin D (Pavletich, 1999).
Proper folding of CDK4, before its association with D-type cyclins, is catalyzed in a 450 kDa cytoplasmic complex that contains the chaperone proteins Hsp90 and p50/Cdc37 (Dai et al., 1996; Stepanova et al., 1996) (Figure 2A). Cdc37 binds CDK4 and then acts to recruit dimeric Hsp90 to the complex. Chaperone-bound CDK4 is stabilized and, when properly folded, is released and assembled into complexes with a D-type cyclin and a Cip/Kip protein. Overexpression of INK4 proteins disrupts much of the 450-kDa CDK4-containing chaperone complex and shifts CDK4 into a binary 50-kDa complex with one INK4 protein (McConnell et al., 1999). Furthermore, INK4 proteins also bind and disrupt cyclin D-Cip/Kip-CDK4 complexes. Unbound cyclin D gets rapidly degraded, and the INK4-induced release of tethered Cip/Kip proteins coordinates cyclin E-CDK2 inhibition and promotes arrest in the G1-phase of the cell cycle (Figure 2B). This indirect negative effect that INK4 proteins have on cyclin E-CDK2 activity and cell cycle progression underscores the functional collaboration between Cip/Kip and INK4 proteins in vivo (Sherr and Roberts, 1995).
Figure 2. Function of INK4 proteins.
(A) Proper folding of CDK4 (and CDK6, not shown) is catalyzed by the chaperones Hsp90
and Cdc37. Mitogenic signals induce the expression of D-type cyclins and promote their assembly, together with Cip/Kip proteins and chaperone bound CDK4, into active kinase complexes. INK4 proteins bind and inactivate CDK4 redirecting it into small ~50kDa complexes (modified from (Sherr and Roberts, 1999)).
(B) INK4 proteins collaborate with Cip/Kip family proteins to induce cell cycle arrest.
Increased INK4 protein expression results in the formation of INK4-CDK complexes and the destabilization of cyclin D. Released Cip/Kip proteins bind and inhibit cyclin E (and A)-dependent CDK2 (modified from (Sherr and Roberts, 1999)).
Ink4 proteins and mouse development
Despite their functional redundancy, Ink4 proteins are differentially expressed during mouse development and in adulthood (Zindy et al., 1997). Transcripts for both Ink4a and Ink4b are not detected during embryonic mouse development, whereas Ink4c and Ink4d mRNAs are readily detected by Northern blotting as early as mouse embryonic day 7. This suggests that Ink4c and Ink4d contribute to cell cycle regulation during mouse development and differentiation, whereas Ink4a and Ink4b do not. To address the in vivo function of the Ink4 proteins, several laboratories generated mice lacking the individual Ink4 genes (Franklin et al., 1998; Latres et al., 2000; Sharpless et al., 2001) or in the case of Ink4a, a knock-in allele expressing a disabled, mutant form of the protein (Krimpenfort et al., 2001). None of these mice display severe developmental defects, although mice lacking Ink4d exhibit testicular atrophy and progressive hearing loss (Zindy et al., 2000; Chen et al., 2003).
Given that p18Ink4c and p19Ink4d are each highly expressed during mouse
development, one might naively expect a more striking phenotype in mice carrying targeted deletions of both genes. It is possible that p18Ink4c and p19Ink4d play redundant roles in certain tissues, and even that their elimination might activate other Ink4 genes as part of a compensation mechanism. One way to address these possibilities is to generate mice doubly-deficient for Ink4c and Ink4d or mice lacking all the Ink4 proteins. Results obtained with doubly-deficient Ink4c/Ink4d-null mice are described in Chapter 2 of this thesis and underscore contributions of these CKIs to male germ cell development and fertility.
A second possibility is that the aforementioned collaboration between Ink4 and Cip/Kip proteins might reveal phenotypes when particular combinations of these CKI genes are disabled. If Ink4 proteins require Cip/Kip proteins to inhibit cell proliferation, deletion of one inhibitor might not necessarily lead to a defect but co-deletion will. This is dramatically demonstrated in mice doubly deficient for p19Ink4d and p27Kip1. Both p19Ink4d and p27Kip1 are expressed in post-mitotic neurons, and mice lacking both genes develop severe bradykinesia, proprioceptive abnormalities and seizures, and die shortly after birth, a phenotype absent in mice lacking only one of the genes (Zindy et al., 1999). These in vivo experiments prove that, although seemingly restricted in inhibiting D-type cyclin-dependent kinases, Ink4 proteins can collaborate with Cip/Kip family proteins in a cell lineage-specific manner to inhibit cyclin E-CDK2 and impose cell cycle arrest.
RB pathway and cancer
Genetic and biochemical analyses of human tumor samples provide compelling evidence that the proteins that control the G1-S phase transition act as tumor suppressors or proto-oncogenes. The high frequency with which mutations in these particular components are found in human neoplasia suggests
that an essential step in the transformation of a normal cell into a cancer cell is the disabling of the so-called RB-pathway (Sherr and McCormick, 2002). Tumor-associated alterations include increased expression of positive cell cycle regulators, such as cyclins (mainly cyclin D and E) and CDKs (CDK4 and CDK6), as well as loss of negative cell cycle regulators, such as CKIs (mainly INK4a, INK4b, INK4c and Kip1) and RB.
Depending on the tumor type, there are a variety of ways in which the RB pathway can be affected by genetic changes. For example, in mantle cell lymphoma with the t(11;14) translocation, the cyclin D1 gene is fused to the immunoglobin heavy chain promoter, which leads to ectopic expression of cyclin D1 in B lymphocytes. In squamous cell carcinoma of the head and neck, the chromosomal region encoding cyclin D1 gene is amplified (Hall and Peters, 1996). CDK4 and CDK6 overexpression is frequently found in glioblastoma multiforme (Hall and Peters, 1996), whereas miscoding mutations discovered in melanoma can render CDK4 and CDK6 insensitive to INK4 proteins (Wolfel et al., 1995; Zuo et al., 1996). Because of the seeming linearity of the INK4-Cyclin D/CDK4-RB axis, mutations affecting the RB pathway generally occur in a mutually exclusive fashion with only a single hit in one of these components (Sherr, 1996). For example, small cell lung cancers that have either lost RB (80%) or INK4a (15%) express normal levels of cyclin D, whereas those overexpressing cyclin D (5%) retain wild-type RB and INK4a. In addition, the frequency of particular genetic events varies among tumor types. In adenocarcinoma of the lung, INK4a loss predominates, whereas the majority of small cell lung carcinomas lose RB. The basis for these cell type-specific alterations remains unclear.
The p53 pathway
Metazoan cells have evolved protective responses to potentially harmful stimuli that induce uncontrolled cell division by activating compensatory mechanisms that either induce cell cycle arrest or induce programmed cell death (apoptosis). Cancer cells lose these defenses by acquiring additional mutations that inactivate key components of these checkpoint pathways. The most frequently disrupted archetypal checkpoint regulator is the p53 gene.
The p53 protein is a homotetrameric transcription factor, which, in normal cells, is kept at a low concentration through a negative feedback loop that depends upon the activity of one of its direct transcriptional targets, Mdm2. Mdm2 not only inhibits p53 transcriptional activity by binding to its N-terminal transactivation domain (Momand et al., 1992; Oliner et al., 1993), but it also catalyzes the ubiquitination of p53, leading to its delocalization from the nucleus to the cytoplasm and its proteasomal degradation (Haupt et al., 1997; Kubbutat et al., 1997; Zhang and Xiong, 2001). The levels and transcriptional activity of p53 increase in response to several cellular stress signals, such as DNA damage induced by ionizing or ultraviolet irradiation, reduction in oxygen levels (hypoxia),
expression of activated oncogenes, changes in cell adhesion and rNTP depletion (Giaccia and Kastan, 1998). This increased p53 activity triggers the transcriptional activation of target genes such as the CDK2 kinase inhibitor, p21Cip1, as well as a host of pro-apoptotic gene products (e.g. Bax, PUMA) and, depending on the cellular context, leads to cell cycle arrest or apoptosis.
p53 mutations, and most commonly missense mutations that affect its DNA binding, are found in about 50 % of all human cancers. In addition, p53 can be functionally inactivated through overproduction of Mdm2 or through binding to viral oncoproteins. Both SV40 Large T antigen and Adenoviral protein E1B-55Kd block p53-mediated transcriptional activation, whereas E6 proteins of oncogenic strains of human papillomaviruses promote the degradation of p53 independently of Mdm2.
In mice, the complete inactivation of p53 promotes spontaneous tumor development (Donehower et al., 1992; Jacks et al., 1994; Purdie et al., 1994); moreover, loss of only one p53 allele increases the probability that the second will be somatically mutated, thereby leading to a more indolent but still recognizable tumor-prone phenotype (Donehower et al., 1992; Harvey et al., 1993). Similarly, familial inheritance of a defective p53 allele in the germ line in the human Li-Fraumeni syndrome predisposes to tumors in which the second p53 allele is somatically mutated (Malkin et al., 1990; Srivastava et al., 1990; Iavarone et al., 1992). The “two-hit” inactivation of both alleles underscores the recessive nature of many canonical tumor suppressor genes, Rb also among them (Knudson, Jr., 1971). In the case of p53, a complication is that the dominant-negative activity of many p53 mutants endows them with additional oncogenic properties. On the one hand, because they tetramerize with wild type p53 protein subunits, some mutant forms of p53 inhibit the resulting complexes from binding to DNA and activating transcription. But, mutant forms of p53 can also oligomerize with other p53 family members (p63 and p73), and in so doing, can again act in a dominant manner to inactivate their functions as well (Strano et al., 2000; Olive et al., 2004; Lang et al., 2004). Therefore, mutation and deletion of p53 need not connote identical phenotypic consequences, because those p53 mutations that abrogate its transcriptional activity but do not affect its tetramerization are likely to convey a more potent oncogenic activity than mutants that can no longer oligomerize and therefore act as true nulls.
INK4A/ARF locus: Discovery of p19Arf
Genes encoding the cyclin-dependent kinase inhibitors p15Ink4b and p16Ink4a are closely linked on the short arm of human chromosome 9p21 (and the corresponding locus on chromosome 4 in mice), are separated by only 60 kb, and are located 100-150 kb centromeric to the methylthioadenosine phosphorylase gene (MTAP) (Figure 3A) (Quelle et al., 1995a). Experiments designed to isolate the Ink4a cDNA paralog from a mouse erythroleukemia (Mel) cDNA library (Quelle et al., 1995b) revealed an additional transcript that
corresponded to the previously described INK4a beta cDNA (Mao et al., 1995; Stone et al., 1995). The beta transcript originates from an alternative first exon, exon 1β (E1β), which is located 13 kb upstream of exon 1α (E1α) of the Ink4a gene (Figure 3A). Transcription initiation for Exon 1α and Exon 1β is regulated by distinct promoters, and each exon 1-coded RNA segment is joined through the identical splice acceptor site to sequences specified by exon 2. However exon 2 in the Exon 1β-containing transcript is translated in an alternate reading frame and encodes a protein with a molecular weight of 19 kDa which was subsequently named p19Arf for Alternative Reading Frame protein (Quelle et al., 1995b).
Mouse p19Arf (p14ARF in human) bears no sequence homology to any other protein in the genome, and its enforced expression in wild type cells results in efficient arrest in both the G1 and G2 phases of the cell division cycle (Quelle et al., 1995b). This type of p19Arf- induced arrest requires the presence of a functional p53 protein (Kamijo et al., 1997). Arf associates directly with Mdm2 (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998; Zhang et al., 1998) and inhibits its E3 ubiquitin protein ligase activity (Honda and Yasuda, 1999) (Figure 3B). This leads to p53 stabilization and, depending on the biological context, results in either cell cycle arrest or apoptosis. Thus, the Ink4a/Arf locus encodes two unrelated proteins which function as upstream activators of two of the most frequently targeted tumor suppressor pathway in human cancer (Figure 3B).
Figure 3. INK4a/Arf locus.
(A) Schematic representations (not to scale) of the Ink4b, Arf, Ink4b and MTAP genes in mouse, puffer fish, and chickens. Arf is encoded by Exon 1β (E1β), Exon 2 (E2) and Exon 3 (E3) (in red) and Ink4a is encoded by Exon 1α (E1α), E2 and E3 (in yellow). Dotted lines denote the splicing of the Arf and Ink4a transcripts. Ink4b exons are shown as black and MTAP as gray boxes. The arrows on top of the figure indicate the location of centromere and telomere whereas arrows above the exons indicate the direction of transcription (modified from (Kim et al., 2003)).
(B) The products of the Ink4a/Arf locus link the RB and p53 pathways. p16Ink4a binds to and
inhibits cyclin D-dependent kinases CDK4 and CDK6 thereby preventing RB phosphorylation. p19Arf prevents p53 degradation by binding to and thereby inhibiting the p53 ubiquitin E3 ligase, Mdm2.
Evolutionary history of the Ink4a/Arf locus
The observation that a single locus, through the dual utilization of coding sequences, encodes two unrelated proteins was completely unexpected. This then raises questions of how evolutionarily conserved this unusual genome architecture is; how two regulators of the Rb pathway (p15Ink4b and p16Ink4a) became linked to an activator of the p53 pathway (p19Arf); and what selective pressures have evolutionarily maintained both open reading frames in Ink4a/Arf exon 2. Arf transcripts have been identified in placental mammals such as human, non human primates, mouse, rat, pig, golden hamster, and in marsupials, such as the opossum. In contrast, the puffer fish (Fugu rubripes) has only one identifiable Ink4 gene and lacks any Arf encoding potential in this genomic region (Figure 3A) (Gilley and Fried, 2001). On the other hand, the chicken genome has, in addition to an Ink4b gene, an exon 1β-like transcript that terminates in a second exon that has high sequence homology with exon 2 of chicken Ink4b (Figure 3A) and contains the capability to encode two distinct open reading frames if splicing occurs in the correct register. Surprisingly, no exon 1α was identified. Instead, duplicated genomic sequences from a region near the putative exon 1β of chicken Arf are present in the region where exon 1α of Ink4a is expected to be located.
In mammals, the splicing of the exon 1β transcript to that from exon 2 enables Arf translation to continue in the -1 reading frame relative to that of p16Ink4a, whereas the matching splice in chickens puts exon 1β in the third possible register. As this +1 reading frame encodes a stop codon at the beginning of exon 2, the chicken Arf protein (p7Arf) is encoded entirely by exon 1β; yet, despite its small molecular mass, p7Arf is still able to stabilize chicken p53
(Kim et al., 2003). This is consistent with results obtained with truncation mutants in which the p53 stabilizing function was also mapped to the exon 1β encoded portion of the mouse p19Arf and human p14ARF proteins (Quelle et al., 1997; Stott et al., 1998). Overall, it appears that a single genomic duplication generated the tandemly linked Ink4b and Ink4a genes. This occurred before the insertion of an Arf-like gene, and in avian species, exon 1α was subsequently lost. The origin of the Arf gene itself, however, remains enigmatic.
It may be that the unusual structure of the Ink4b-Arf-Ink4a locus connotes a requirement for co-transcriptional en bloc regulation of the three genes. For example, the three genes are not expressed during embryonic and fetal development (Zindy et al., 1997), presumably because their activation at these times might interfere with proper gestational processes. On the other hand, explantation of mid-gestation mouse embryo fibroblasts (MEFs) into tissue culture provides a sufficient environmental stress to activate all three genes simultaneously (Zindy et al., 1997; Zindy et al., 1998), thereby coordinating the Rb and p53 checkpoint response. Importantly, an evolutionary requirement for co-regulation of the entire locus would not preclude the possibility that, once so organized, individual genes in the locus could acquire the ability to be activated by distinct stress signals, as is indeed the case in other circumstances (Zindy et al., 1998; Williams et al., 2006; Gil and Peters, 2006).
The Arf tumor suppressor pathway
Although the three genes encoded by the INK4/ARF locus affect both the RB and the p53 tumor suppressor pathways, the frequent occurrence of 9p21 deletions that inactivate p15INK4b, p16INK4a and p14ARF in many human cancers, while emphasizing the importance of the locus, makes it hard to assess which of the tumor suppressor pathways is compromised, and which proteins (p15INK4b,
p16INK4a, p14ARF) confer tumor suppressor activities under different
circumstances. If we consider that both RB and p53 are inactivated as part of the life history of many cancer cells, it is reasonable to assume that co-inactivation of the ARF and two INK4 genes would mimic these effects, and that loss of each would independently contribute.
Apart from deletion, there are reports of selective inactivation of one gene in the INK4/ARF locus leaving the other genes intact. For example, some germline mutations found in kindreds with familial melanoma map to exon 1α without affecting p14ARF at all (Hussussian et al., 1994; Kamb et al., 1994; Goldstein et al., 1995), whereas a recent study identified mutations in exon 1β that leave p16INK4a unaffected (Randerson-Moor et al., 2001; Rizos et al., 2001; Harland et al., 2005). Also, promoter methylation of p14ARF, in the absence of p15INK4b and p16INK4a silencing, has been observed in colon cancer (Esteller et al., 2000; Esteller et al., 2001; Sato et al., 2002), whereas p15INK4b promoter methylation in myeloid and lymphoid cells occurs without p16INK4a promoter
methylation (Herman et al., 1996). These reports firmly establish a role for all three genes as tumor suppressors with their relative importance perhaps dictated by cell type-specific factors.
The first effort to disable the Ink4a/Arf locus in mice (without knowledge of the Arf gene) was undertaken by Serrano and coworkers who disrupted both exon 2 and exon 3 of the Ink4a gene in the mouse germ line (Serrano et al., 1996). Homozygous exon 2/3 mutant mice are highly tumor prone and develop spontaneous tumors early in life, the appearance of which could be accelerated by treatment with the chemical carcinogens 9, 10-dimethyl-1, 2-benzanthracene (DMBA) either alone or in combination with ultraviolet B (UVB) irradiation. All known functions of p19Arf map to exon 1β, which is intact in exon 2/3 mutant mice. However, removal of exon 2 and exon 3 likely results in destabilization of the Arf mRNA transcript, so that exon 2/3 mutant mice are functionally deficient for both p16Ink4a and p19Arf. Subsequent studies designed to specifically disrupt either Arf, by deleting exon 1β (Kamijo et al., 1997), or Ink4a, by deleting exon 1α or by a knock-in mutation in exon 2 (Sharpless et al., 2001; Krimpenfort et al., 2001), showed that many of the key phenotypes of the exon 2/3 mutant mice, while present in the p19Arf-null mice, are mostly absent in the p16Ink4a-null
animals. Therefore, it appears that, at least in mice, p19Arf and not p16Ink4a is the dominant contributing tumor suppressor of the Ink4a/Arf locus. This situation is likely to be different in humans, where a wealth of evidence implicates INK4a mutation and silencing in a wide variety of cancers (Ruas and Peters, 1998).
Ink4a/Arf and replicative senescence
When isolated at embryonic day 13.5 and explanted into culture, primary mouse embryo fibroblasts (MEFs) grow rapidly, but their proliferation does not continue indefinitely. Fixed numbers of plated cells (3 X 105) when allowed to proliferate, enumerated, and diluted to the same starting number every three days (the so-called 3T3 protocol) progressively lose their proliferative capacity and eventually stop dividing (Todaro and Green, 1963). This seemingly irreversible cell cycle arrest, also termed replicative senescence, was suggested by some to be the result of a mitotic clock that measured generation number. We now appreciate, however, that senescence is likely caused by a stress response resulting from nonphysiologic cell culture conditions, such as disruption of cell-cell contact, lack of heterotypic interactions between different cell types, the medium-to-cell ratio, persistent Ras activation by mitogens, absence of survival factors, hyperoxia, and plating on plastic (Sherr and DePinho, 2000). In fact, cells grown under more defined culture conditions or in physiological (3%) oxygen levels can sometimes proliferate much longer (Loo et al., 1987; Parrinello et al., 2003).
With each passage on a 3T3 or similar protocol, MEFs express increasing levels of negative cell cycle regulators, including p16Ink4a and p19Arf. The elevation of p19Arf levels results in the stabilization of p53 and transcriptional activation of p21Cip1 which, together with p16Ink4a, prevents cell cycle progression. Two key observations indicate that activation of the Arf/p53 pathway is central in the establishment and maintenance of replicative senescence in MEFs. First, MEFs derived from p53-null or Arf-null animals maintain their proliferative potential when put into culture, do not senesce, and can seemingly be propagated indefinitely. Second, when wild type MEFs are continually passaged on a 3T3 protocol, rare spontaneously immortalized variants emerge which invariably are either tetraploid with mutant p53 (~80%) or remain pseudodiploid and exhibit bi-allelic Ink4a/Arf deletions (Kamijo et al., 1997).
Recent observations, also implicate the Rb pathway in the establishment of senescence in MEFs. Similar to p53 or Arf-null MEFs, fibroblasts lacking all Rb family members are immortal when explanted into culture. Because such cells are resistant to arrest induced by p19Arf and p53, these data argue that the Arf-p53 and Rb pathways influence one another’s activities and are parts of an integral signaling network that responds to various forms of cellular stress (Dannenberg et al., 2000; Sage et al., 2000). Furthermore, the acute inactivation of Rb in senescent MEFs can reverse the cellular senescence program (Sage et al., 2003). Therefore, both Rb and E2F act downstream of the p19Arf-p53 axis and are required for the execution of the senescence program. Disruption of Rb family members, p53 function, or perturbing p53 regulators such as Arf and Mdm2 allow many mouse cell types to bypass senescence and proliferate as immortal established cell lines.
Cultured primary human fibroblasts seem to behave differently. Initially these cells have a greater proliferative capacity than MEFs and undergo 60-80 population doublings (versus 15-30 in MEFs) before reaching the “Hayflick limit”
and undergoing senescence (Hayflick and Moorhead, 1961). Dual inactivation of RB and p53 function with, for instance, SV40 large T antigen prolongs the life span of human cells and allows them to bypass senescence, but this is insufficient for their immortalization (Wright and Shay, 1992). Because human cells have shorter telomeres than mice (~12 kb versus ~60 kb) and do not normally express telomerase, continuous cell division is accompanied by telomere erosion. Telomere shortening may initiate DNA damage responses and activate RB and p53 dependent checkpoints that contribute to senescence. Inactivation of these functions by viral oncoproteins or mutations allows cells to continue to divide. This leads to a further reduction in telomere length, and cells eventually enter a “crisis” stage in which end-to-end chromosomal fusion is a hallmark. This promotes a state of massive chromosome instability (via so-called breakage-fusion-bridge cycles) in which most cells undergo apoptosis, and only few escape. Either through the reactivation of telomerase or by employing an alternative (ALT) recombinational mechanism to maintain telomeres, rare surviving cells restabilize their chromosomes, and this allows them to proliferate indefinitely as established cell lines. The fact that human cancer cells not only lack proper RB and p53 functions but have reactivated telomerase or the ALT pathway explains why virtually all established human cell lines have been derived from tumors and not from primary cells.
In summary, primary human cells require, in addition to the inactivation of both p53 and RB function, the presence of telomerase or ALT activity to become immortalized. On the other hand, MEFs can sustain proliferation without the requirement to solve “the telomere problem”.
Arf is induced by abnormal oncogenic signals
Transformation of primary MEFs can be achieved when an oncogene such as Myc or adenovirus E1A is co-introduced with activated Ras (Land et al., 1983; Ruley, 1983). This leads to the hypothesis that separate establishment (immortalization) and transforming functions collaborate in the tumorigenic conversion of rodent cells in culture. Expression of Myc or E1A leads to immortalization of primary rodent cells, whereas Ras can only transform cells when they are first immortalized. In fact, expression of oncogenic Ras in cells which are not yet established leads to accelerated activation of the senescence program (premature senescence), and this is accompanied by the rapid activation of the p19Arf-p53 pathway. In contrast, Ras efficiently transforms established MEFs lacking p53 or Arf (Kamijo et al., 1997; Serrano et al., 1997).
Like Ras, overexpression of Myc or E1A induces p19Arf expression in MEFs (Zindy et al., 1998; de Stanchina et al., 1998). Although Myc and E1A have immortalizing functions, both are also potent inducers of apoptosis, and this is potentiated by depriving cells of extracellular survival factors (Askew et al., 1991; White et al., 1991; Rao et al., 1992; Evan et al., 1992). Overexpression of Myc in cells grown in low serum conditions selects for loss of either p53 or Arf
function (Zindy et al., 1998). In agreement, Arf- and p53-null MEFs are highly resistant to Myc-induced apoptosis (Zindy et al., 1998).
Similar results are obtained in Myc-induced B-cell tumors in mice. Here, Myc expression driven by the immunoglobulin heavy chain promoter-enhancer (Eµ-Myc) within the B lymphocyte lineage induces lymphomas that kill mice within one year (Adams et al., 1985). Early in the course of disease, the increase in the numbers of B cells is restrained, at least in part, by the Arf-p53 checkpoint. Still, lymphomas ultimately emerge that eventually kill the mice. Interestingly, more than half of the lymphomas exhibit inactivating mutations in either p53 or Arf (Eischen et al., 1999). Therefore, inappropriate Myc expression provides a strong selective pressure for events that dismantle Arf or p53 function and thereby select for immortalized cells (Eischen et al., 1999; Jacobs et al., 1999b; Schmitt et al., 1999).
Such studies helped to further elucidate the tumor suppressive function of p19Arf. In primary cells, p19Arf expression is negligible but is highly induced when cells are exposed to cellular stress signals such as activated oncogenes. The p19Arf tumor surveillance pathway senses these hyperproliferative signals and, depending on the context, responds with either induction of cell cycle arrest or apoptosis, thereby protecting the organism from tumor development. This concept was validated with the use of an engineered reporter mouse strain (Arf-GFP mouse) in which exon 1β was substituted by a green fluorescent protein (GFP) cassette placed under the control of the intact Arf promoter (Zindy et al., 2003). Since these mice are deficient for Arf, they rapidly develop lymphoma when crossed with Eµ-Myc mice and, strikingly, the tumors are brightly fluorescent green. In addition, MEFs derived from these Arf-GFP mice turn green when passaged in culture or when they are exposed to oncogenic Ras. These data confirm that oncogenic signals impinge on the Arf locus to induce p19Arf and activate the p53 pathway.
Arf transcriptional regulation
In addition to Myc, Ras and E1A, expression of p19Arf is induced by
activated signaling molecules such as β-catenin (Damalas et al., 2001) and v-Abl (Radfar et al., 1998) and by transcription factors like E2F1 (Bates et al., 1998) and Dmp1 (Inoue et al., 1999). The discovery of E2F binding sites in the proximal Arf promoter and the induction of Arf transcription after overexpression of E2F1 could provide a possible mechanism by which Arf senses proliferative signals. However, unlike classic E2F-responsive genes, Arf is not appreciably induced as cells enter S phase. Thus, if Arf is a genuine E2F target, it must be activated by a mechanism that distinguishes between normal and abnormal proliferative thresholds.
Cells deficient for the E2F3 splice forms, E2F3a and E2F3b, have a reduced rate of proliferation which is accompanied by elevated p19Arf levels (Aslanian et al., 2004). Chromatin immunoprecipitation (ChIP) experiments
revealed that in unstimulated cells, E2F3, and most probably E2F3b, was present on the Arf promoter, whereas the activator E2Fs, E2F1 and E2F3a, were only recruited after oncogenic stimulation by E1A overexpression, for example. Surprisingly, suppression of Arf mediated by E2F3b does not depend on Rb family proteins, which might explain why p19Arf fails to be periodically induced
during the normal cell cycle. One possibility is that under conditions where activator E2Fs (E2F1, 2 and 3a) can replace E2F4/p130 repressor complexes during the normal cell cycle, they cannot displace the E2F3b repressor complex on the Arf promoter. A greater affinity of E2F3b for its binding site, possibly determined by other components in the repressor complex, might account for this phenomenon. This type of threshold control ensures that Arf is activated only during abnormal and uncontrolled proliferation when Rb family proteins are deregulated and a certain level of free activating E2Fs is reached.
Additional transcriptional activators most likely collaborate with E2Fs, and this combined action could further determine the threshold required to trigger gene expression and fine tune Arf induction in response to specific signals or in certain tissues. For example, the Dmp1 transcription factor, following its induction by the Ras-MAP kinase signaling pathway, binds directly to a DNA element in the proximal Arf promoter adjacent to the E2F consensus site (Sreeramaneni et al., 2005). Dmp1 may well play a role in determining the magnitude of the Arf response, since its genetic disruption in mice blunts Arf activity, facilitates Ras-induced transformation, and results in spontaneous tumor development and increased carcinogen sensitivity (Inoue et al., 2001).
Transcriptional repression of the Inka /Arf locus
Despite being “sensors” of hyperproliferative signals, the products of the Ink4a/Arf locus are not expressed during mouse embryonic development. Instead, the locus is insulated from activation during this period of massive proliferation by a large multi-protein complex containing the two closely related polycomb group (PcG) proteins, Bmi-1 and Mel18 (Jacobs et al., 1999a) and possibly a second complex containing CBX7 (Gil et al., 2004). PcG complexes exhibit histone methyltransferase and ubiquitin ligase activities that modify lysine residues on histone tails, thereby leaving PcG-specific methyl and ubiquitin marks. These nucleosome imprints serve as an epigenetic memory and maintain specific targets, such as homeobox-cluster genes, in a repressed state through each round of DNA replication. MEFs derived from mice deficient in Bmi-1 undergo premature replicative senescence and express elevated levels of p16Ink4a, p19Arf and p15Ink4d (Jacobs et al., 1999a). In mice, loss of Bmi-1 expression results in neurological and hematopoietic defects (Jacobs et al., 1999a). This is caused by the postnatal depletion of blood and neural stem cells (Molofsky et al., 2003; Park et al., 2003) and can be partially rescued on an Ink4a/Arf- deficient background (Jacobs et al., 1999a; Bruggeman et al., 2005; Molofsky et al., 2005). How the Bmi-1-containing complex represses the
Ink4a/Arf locus remains unclear. PcG proteins lack a DNA binding domain and must be tethered to Polycomb responsive elements via interactions with chromatin-bound factors. Interactions between PcG and DNA-associated factors such as E2F6 and YY1 have been reported (Garcia et al., 1999; Satijn et al., 2001; Trimarchi et al., 2001); however it is not known whether these proteins play a role in the repression of the Ink4a/Arf locus.
The identification of specific regulators that maintain the Ink4a/Arf locus in a repressed state during development has important implications for human cancer. For example, Bmi-1 levels are reported to be elevated in some breast carcinomas (Kim et al., 2004), non-small cell lung cancers (Vonlanthen et al., 2001), squamous cell carcinomas (Breuer et al., 2005), and mantle cell lymphomas (Bea et al., 2001), and this correlates with low to undetectable levels of p16INK4a and/or p14ARF. In addition, Twist and TBX2 also act as ARF repressors, and they are overexpressed in rhabdomyosarcomas (Maestro et al., 1999) and breast cancers, respectively (Jacobs et al., 2000). Therefore, the inappropriate suppression of the INK4a/ARF locus could provide yet another mechanism by which the p53 and RB pathways are functionally compromised in human cancers. Extending this concept, it might well be that most, if not all, cancers have defects in RB and p53 function resulting from mutations in these genes themselves or in the many genes that modify their functions.
p53- and Mdm2-independent functions of the Arf tumor suppressor
The growth suppressive functions of p19Arf described above are thought to depend heavily on functional p53. However, two key observations suggest that the Arf-Mdm2-p53 pathway is not strictly linear, and that in certain settings, p19Arf has activities that are independent of Mdm2 and/or p53. First, mice lacking Arf, Mdm2 and p53 develop a much broader spectrum of tumors than animals lacking Arf or p53 alone, or both Mdm2 and p53. Tumors in these triple knock-out (TKO) mice frequently develop simultaneously at independent sites and originate from different cell types (Weber et al., 2000). MEFs derived from TKO mice can be arrested by Arf overexpression, albeit with slower kinetics than those that retain p53.
Second, Arf-null mice become blind soon after birth, a phenotype not seen in mice lacking p53 (McKeller et al., 2002). In the eye, Arf expression is found in mural cells that surround the hyaloid vascular system within the vitreous (McKeller et al., 2002). These perivascular, pericyte-like cells stabilize vessel formation and provide angiogenic factors that maintain endothelial cells and vessel integrity. p19Arf blocks mural cell proliferation driven by platelet-derived growth factor (Pdgf-β), which is elaborated, in turn, by endothelial cells (Silva et al., 2005). The absence of Arf expression causes mural cells to accumulate, and this prevents hyaloid vessels from undergoing regression during early postnatal development. Indeed, in Arf-GFP homozygotes that lack a functional Arf gene, the cells that accumulate in the vitreous exhibit vivid green fluorescence (Zindy et
al., 2003). The ultimate result of Arf inactivation is the formation of a retrolental fibrous mass that destroys the retina and leads to blindness. In cultured pericyte-like cells, p19Arf expression can decrease transcription of the Pdgf-β receptor
gene and blocks Pdgf-β-driven proliferation. Reinforcing the biological importance of this interaction, crossing Arf-null mice with animals lacking the PdgfR-β gene ameliorates the embryonic Arf-null eye phenotype (Silva et al., 2005).
The tumor incidence in TKO mice and blindness in Arf null mice offer the best evidence to date for p53-independent activities of p19Arf. How p19Arf exerts its anti-proliferative activity in the absence of Mdm2 and p53 or affects development of the mouse eye is not known. Some in vitro observations hint at a possible mechanism.
Most of the Arf protein in the cell localizes to the nucleolus, a sub-nuclear compartment where 47S ribosomal RNA (rRNA) transcribed by polymerase I is processed into 28S, 18S and 5.8S rRNA and assembled into pre-ribosomal particles. Arf suppresses rRNA transcription and attenuates rRNA processing, the latter even in the absence of p53 and Mdm2 (Sugimoto et al., 2003). p19Arf is able to bind to 5.8 S rRNA (Sugimoto et al., 2003) and in a separate study was detected on the rDNA promoter following ChIP (Ayrault et al., 2004). Besides regulating RNA polymerase II and III activity, some Myc protein also localizes to the nucleolus at sites of active ribosomal DNA (rDNA) transcription where it stimulates RNA pol I activity (Grandori et al., 2005). A recent study shows that Arf can inhibit the transcriptional activation of several target genes through a direct interaction with both N and C terminal domains of the Myc protein (Qi et al., 2004). Hence, p19Arf might directly interact with Myc on the rDNA promoter
but whether this, in combination with inhibition of rRNA processing, is the mechanism of p19Arf-induced cell cycle arrest and possibly tumor suppression in the absence of Mdm2 and p53 remains to be determined.
ARF interacting proteins
Since the discovery of the p19Arf protein, many laboratories have tried to
identify Arf-binding proteins other than Mdm2. Numerous proteins involved in a wide range of cellular processes have been proposed to regulate, or be regulated by, the human or mouse Arf proteins. For example, Arf binds to transcriptional regulators such as the activating E2Fs, DP1, c-Myc, HIF1-α and p120E4F, and proteins such as topoisomerase I, MdmX, Rad6, TBP-1, Spinophilin, Pex19p, CARF, EBNA-5, Ubc9 and Werner’s helicase (WRN) (Gjerset and Bandyopadhyay, 2006). Despite the growing list of Arf interacting proteins, it remains unclear whether any of these associations actually take place in vivo and/or what the biological significance, if any, of the binding may be.
Arf proteins are highly basic (~22% arginine) with isoelectric points (pI) of ~12 and, because of their positive charge at physiological pH, can interact with acidic proteins and nucleic acids. In order to properly fold and remain soluble in
cells, Arf proteins must be “buffered” by binding to other molecular species (DNA, RNA and/or protein). Most of the p19Arf protein expressed in mouse fibroblasts is bound to nucleophosmin (NPM or B23) and contained in high molecular weight nucleolar complexes of 2-5-MDa (Bertwistle et al., 2004). When induced, only a small fraction of p19Arf associates with nucleoplasmic Mdm2 in separate
complexes of about 500-600 kDa (Llanos et al., 2001; Bertwistle et al., 2004). NPM is an abundant, mostly nucleolar protein which actively shuttles between the cytoplasm and the nucleus (Borer et al., 1989). It has been implicated in a variety of cellular processes such as rRNA processing and ribosome biogenesis, protein folding and disaggregation, and centrosome duplication (Okuda et al., 2000), and it has been reported to possess ribonuclease (Herrera et al., 1995) and molecular chaperone activities (Szebeni and Olson, 1999). Indeed, the fact that NPM has been implicated in numerous, unrelated biological processes argues for a “chaperone-like” function, but its actual biochemical role remains unclear.
Although four independent studies have identified NPM as an Arf binding protein (Itahana et al., 2003; Bertwistle et al., 2004; Brady et al., 2004; Korgaonkar et al., 2005), each interprets the interactions in a different way. Arf targets NPM for degradation and this, according to one report, provides a mechanistic explanation for Arf’s effect on rRNA processing (Itahana et al., 2003). However, NPM is induced in rapidly proliferating cells and diminished in cells that exit the division cycle (Feuerstein and Mond, 1987; Feuerstein et al., 1988), so Arf-induced arrest would be expected to lower the amount of NPM in cells. Moreover, cells derived from NPM-null mice have no overt rRNA processing defect (Colombo et al., 2005), and while overexpression of an NPM mutant lacking its carboxyl-terminal nucleic acid-binding domain can override the ability of p19Arf to retard rRNA processing, overexpression of wild type NPM does not (Bertwistle et al., 2004). Together these data strongly argue against NPM as the direct target for Arf mediated inhibition of rRNA processing.
Whereas NPM may not affect rRNA processing, it may still play a central role in ribosome biogenesis. NPM rapidly shuttles from the nucleolus/nucleus to the cytoplasm and was recently shown to interact with several ribosomal proteins (Brady et al., 2004; Yu et al., 2006). Mapping of the nuclear export signal (NES) and mutation of its leucyl residues creates a dominant negative form of NPM that blocks ribosomal protein and rRNA nuclear export (Yu et al., 2006). In the absence of p53 and Mdm2, Arf is able to inhibit NPM shuttling, trapping it within the nucleolus, and this may be important for the Mdm2/p53- independent growth suppressing function of p19Arf (Brady et al., 2004). This interpretation differs from
that of another group (Korgaonkar et al., 2005). These investigators reported that not only does NPM bind Arf through the same domains that mediate the Arf-Mdm2 interaction, but that NPM also increases Arf’s nucleolar localization thereby separating Arf from nucleoplasmic Mdm2 (Korgaonkar et al., 2005). This would suggest that in the presence of p53 and Mdm2, NPM inhibits Arf-mediated growth suppression and p53 activation.
In chapter 3 and 4 of this thesis, I provide further information concerning the Arf-NPM interaction. In chapter 3, I describe how the degradation of the Arf
protein is regulated and provide evidence that NPM is a critical factor in determining the stability of the Arf protein. In chapter 4, I discuss how an acute myeloid leukemia (AML)-associated NPM mutant (NPMc), which mislocalizes to the cytoplasm, affects Arf functions.
Ubiquitin conjugation pathway and Arf degradation
Selective degradation of most proteins is regulated by ubiquitin, a small 76 amino acid polypeptide which, through an isopeptide linkage with lysine residues, is conjugated to target proteins. Because ubiquitin itself contains seven lysines, a chain of ubiquitin molecules can be formed on each target protein and at least four ubiquitin molecules linked to one another are required to target a protein for proteasome-dependent destruction (Thrower et al., 2000). Ubiquitination of proteins requires at least three enzymes. The ubiquitin-activating enzyme (E1) adenylates ubiquitin and transfers it to a ubiquitin conjugating enzyme (E2) which, in turn, interacts with a ubiquitin ligase (E3) that selects substrates for ubiquitin modification. Although Arf binds two ubiquitin E3 ligases, Mdm2 (Kamijo et al., 1998; Pomerantz et al., 1998; Zhang et al., 1998) and the recently identified ARF-BP1 (Chen et al., 2005), little is known about the regulation of Arf protein turnover. In chapter 3 of this thesis, I describe kinetic measurements of Arf protein half-life and test the involvement of the ubiquitination machinery in Arf protein turnover.
Arf and the conjugation of the ubiquitin-like molecule, Sumo
In addition to ubiquitin there are ubiquitin-like molecules that are conjugated to proteins through mechanisms analogous to the ubiquitin enzyme cascade. One such ubiquitin-like molecule is the small ubiquitin-related modifier (Sumo). The consequences of sumoylation are diverse and can range from regulating subcellular protein localization to regulating histone assembly and transcription, but sumoylation plays no direct role in the regulation of protein degradation. Surprisingly several Arf interacting proteins are modified by Sumo. Both p19Arf and p14ARF promote the sumoylation of Hdm2 (Xirodimas et al., 2002), WRN (Woods et al., 2004), E2F1 (Rizos et al., 2005), HIF1α (Rizos et al., 2005) and NPM (Tago et al., 2005), and this requires a direct interaction of target proteins with Arf but is independent of p53 (Tago et al., 2005). Arf seems to function downstream of the E1 and E2 enzymes of the cascade, because sumoylation induced by Arf is sensitive to Gam1, an avian adenovirus protein which specifically interferes with coordinating Sumo E1 and E2 functions (Boggio et al., 2004; Tago et al., 2005). Given that Arf binding is a requirement for sumoylation, it is possible that Arf functions as a component of a Sumo E3 ligase that bridges the Sumo E2, Ubc9, with a substrate. Although Arf-induced
sumoylation is not involved with Arf’s ability to inhibit rRNA processing or induce cell cycle arrest, it does open up the possibility that Arf is able to affect diverse biological processes previously not connected to the Arf/p53 pathway.
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