ARF (known as p14ARF in humans and p19 ARF in mouse) was originally identified as an alternative transcript of the lnk4a/ARF tumor suppressor locus. Numerous studies indicate that ARF suppresses aberrant cell growth in response to oncogene activation, at least in part, by inducing the p53 pathway. The ARF induction of p53 appears to be mediated through Mdm2, since over-expressed ARF interacts directly with Mdm2 and inhibit its ability to promote p53 degradation. Interestingly, ARF also has tumor suppressor functions that do not depend on p53 or Mdm2. For example, ARF can induce cell growth arrest in tumor cells that lack a functional p53 gene. Moreover, it was recently shown that ARF suppresses the growth, progression, and metastasis of mouse skin carcinomas through both p53-dependent and p53-independent pathways. Nevertheless, the identity of these factors and the mechanisms by which they mediate p53/Mdm2- independent tumor suppression by ARF remain unclear. To understand the mechanism of ARF-mediated growth suppression, we isolated ARF-containing nuclear complexes from human cells and identified a novel ubiquitin ligase ARF-BP1, as a major component of the complexes. ARF-BP1 harbors a signature HECT motif and its ubiquitin ligase activity is inhibited in the presence of 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-wildtype cells, ARF-BP1 inactivation serves to stabilize the p53 polypeptide and induce p53-dependent apoptosis. We further show that ARF-BP1 directly binds and ubiquitinates p53 and that inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization in Mdm2-null cells. 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. However, it also raises more general questions regarding the role of ARF-BP1 in ARF-mediated tumor suppression function. For example, 1) what are additional targets that mediate p53-independent cell growth arrest and 2) how are the ARF/ARF-BP1 and ARF-BP1/p53 interactions regulated? Further analysis of this process should clarify the precise role of ARF-BP1 and yield broader insights into the mechanisms of ARF-mediated tumor suppression. The first specific Aim is to elucidate regulation of the ARF-BP1/p53 and ARF/ARF-BP1 interactions. We will test whether the ARF-BP1/p53 and ARF/ARF-BP1 interactions are regulated upon stress and also examine whether ARF-BP1 is functionally regulated by phosphorylation. The second specific aim is to identify novel cellular factors which mediate p53-independent functions of ARF-BP1. By biochemical purification and mass spectrometry, we will identify novel cellular factors that specifically interact with ARF-BP1 and investigate the roles of these factors in its mediating p53-independent function. ? ? ?
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