Taken together, these results argue that Arf-induced sumoylation depends on a direct physical interaction of p19Arf with either Mdm2 or NPM, and that this process may preferentially occur in the nucleolus where the major pool of p19Arf resides

Taken together, these results argue that Arf-induced sumoylation depends on a direct physical interaction of p19Arf with either Mdm2 or NPM, and that this process may preferentially occur in the nucleolus where the major pool of p19Arf resides. Expression of Arf proteins protects p53 from Mdm2-mediated ubiquitination, leading to p53 stabilization and induction of p53-responsive genes. Mdm2 SGI-1776 (free base) was blocked. Reduction of Ubc9 levels with short hairpin RNAs rendered similar results. We suggest that Arf’s p53-independent effects SGI-1776 (free base) on gene expression and tumor suppression might depend on Arf-induced sumoylation. locus encodes two tumor-suppressor proteins, p16Ink4a and p19Arf (p14ARF in humans) that increase the growth-suppressive activities of the retinoblastomafamily proteins (Rb, p107, and p130) and the p53 transcription factor, respectively (1C3). By inhibiting Cdk4 and Cdk6, p16Ink4a helps to maintain Rb-family proteins in their active hypophosphorylated state, thereby inhibiting an E2F-dependent transcriptional program SGI-1776 (free base) that is required for cellular DNA replication. P2RY5 In contrast, p19Arf antagonizes the p53 negative regulator Mdm2 (Hdm2 in humans) SGI-1776 (free base) to trigger a p53 transcriptional response that can lead to proliferative arrest or apoptosis (4). Expression of and 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 gets its name) (2). The two genes are induced by different stress signals and can be separately mutated or silenced in tumor cells (4). 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, 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 mutation. By activating p53, serves to eliminate cells sustaining such mutations. However, is not directly induced by DNA damage signals that trigger p53 activity through other signal-transduction pathways (3), although its loss can modify the damage response (5). In this sense, serves as a fuse that gates mitogenic current, preventing abnormal cell proliferation in response to oncogene activation. Although it is generally accepted that much of also has p53-independent functions. Enforced expression of p19Arf can arrest the proliferation of and grow faster in culture than do cells lacking only one of the two genes (3, 7), and mice lacking are much more prone to developing cancer than are mice lacking and (8). Arf inhibits ribosomal RNA processing (9), interacts with topoisomerase I (10), and mediates p53-independent effects on gene expression by negatively regulating other transcription factors such as Btk coactivators (11), E2Fs (12C15), Myc (16, 17), and NFB (18). These p53-independent activities of Arf are relatively poorly understood. Enforced expression of p14ARF in human cells promotes sumoylation of several cotransfected cellular proteins, including Hdm2 (19, 20), the Werner helicase (WRN) (21), and the transcription factors E2F-1 and HIF-1 (22). Covalent addition of the small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) to the -amino groups of certain lysine residues in protein substrates is carried out by a single E1 enzyme (the SUMO-1-activating enzyme 1/2 heterodimer also known as Aos1/Uba2) and a single E2 enzyme (Ubc9) in a process analogous to ubiquitination (23C25). Before conjugation, SUMO isoforms must be cleaved by a SUMO-specific protease to generate a C-terminal diglycine motif. Processed SUMO can then be activated in an ATP-dependent manner to form a thioester bond between SUMO and the catalytic cysteine of the E1. The E1 mediates SUMO transfer to a cysteine residue in Ubc9, after which one of several E3s can interact with both Ubc9 and protein substrates to accelerate the rate and magnitude of their sumoylation (26C28). Mammalian protein inhibitor of activated signal transducer and activator of transcription.

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