Sumoylation induced by the Arf tumor suppressor: A p53-independent function

Abstract

The mouse p19 Arf protein has both p53-dependent and p53-independent tumor-suppressive activities. Arf triggers sumoylation of many cellular proteins, including Mdm2 and nucleophosmin (NPM͞ B23), with which p19 Arf physically interacts in vivo, and this occurs equally well in cells expressing or lacking functional p53. In an Arf-null NIH 3T3 cell derivative (MT-Arf cells) engineered to reexpress an Arf transgene driven by a zinc-inducible metallothionein promoter, sumoylation of endogenous Mdm2 and NPM proteins was initiated as p19 Arf was induced and was observed before p53-dependent cell cycle arrest. Predominately nucleoplasmic molecules visualized by immunofluorescence with antibodies to small ubiquitin-like modifier (SUMO) 1 localized to nucleoli as p19 Arf accumulated there. Two Arf mutants, one of which binds to Mdm2 and NPM but is excluded from nucleoli and the other of which enters nucleoli but is handicapped in binding to Mdm2 and NPM, were defective in inducing sumoylation of these two target proteins and did not localize bulk sumoylated molecules to nucleoli. The CELO adenovirus protein, Gam1, which inhibits the SUMO activating enzyme (E1) and leads to down-regulation of the SUMO conjugating enzyme (E2͞Ubc9), had no overt effect on the ability of p19 Arf to activate p53 or the p53-responsive genes encoding Mdm2 and p21 Cip1 , despite the fact that Arf-induced sumoylation of Mdm2 was blocked. Reduction of Ubc9 levels with short hairpin RNAs rendered similar results. We suggest that Arf's p53-independent effects on gene expression and tumor suppression might depend on Arf-induced sumoylation. Gam1 ͉ Ubc9T he mouse Ink4a-Arf locus encodes two tumor-suppressor proteins, p16 Ink4a and p19 Arf (p14 ARF in humans) that increase the growth-suppressive activities of the retinoblastomafamily proteins (Rb, p107, and p130) and the p53 transcription factor, respectively (1-3). By inhibiting Cdk4 and Cdk6, p16 Ink4a helps to maintain Rb-family proteins in their active hypophosphorylated state, thereby inhibiting an E2F-dependent transcriptional program that is required for cellular DNA replication. In contrast, p19 Arf antagonizes the p53 negative regulator Mdm2 (Hdm2 in humans) to trigger a p53 transcriptional response that can lead to proliferative arrest or apoptosis (4). Expression of Ink4a and Arf is regulated by distinct promoters upstream of alternative first exons whose products are spliced to a common second exon translated in alternative reading frames (from which Arf gets its name) (2). The two genes are induced by different stress signals and can be separately mutated or silenced in tumor cells (4).Arf is activated by abnormally elevated and sustained mitogenic signals triggered by oncogenes but not by physiologic signaling thresholds conveyed by their appropriately regulated protooncogenic counterparts (4). For example, Arf is not induced by Myc or Ras during normal cell-cycle progression, but it is transcribed when such signals are constitutively enforced through Myc translocation or oncogenic Ras mu...

Nucleolar NF-κB/RelA mediates apoptosis by causing cytoplasmic relocalization of nucleophosmin

“…In addition to protein binding, NPM localization is regulated by posttranslational modifications such as ubiquitinylation and sumoylation. 22,36 Therefore, another possible mechanism by which RelA may cause its relocalization is by facilitating modulation of its post-transcriptional modification state. In support of this suggestion, we found that aspirin and GFP-NoLS RelA expression induced modified (high molecular weight) forms of NPM in the cytoplasm (data not shown).…”

Sumoylation of nucleophosmin/B23 regulates its subcellular localization, mediating cell proliferation and survival

“…Npm1 heterozygosity accelerates oncogenesis both in vitro and in vivo, supporting the fact that it has tumorsuppressive activity (16). Moreover, accumulating evidence indicates that B23 contributes to the growth-suppressing pathways through its interaction with Arf (8,17).…”

“…Recently, it has been shown that B23 is sumoylated at only one site in Arf-null NIH 3T3 cells. Its sumoylation is increased on Arf induction, and multisumoylated species are detected (17). However, the sumoylation residues are unknown, and the physiological functions of this event remain elusive as well.…”

ARF antagonizes the ability of Miz-1 to inhibit p53-mediated transactivation

“…In the Mdm2-null cell, ARF can activate the p53 through binding and inhibiting ARF-BP1/Mule, a newly identified E3 ubiquitin ligase for p53 (Chen et al, 2005). Moreover, increasing number of studies are providing evidence that ARF has p53-independent functions (Weber et al, 2000;Tago et al, 2005;Ha et al, 2007).…”