Regulation of the tumor suppressor p53 by sumoylation and identification of two independent sumo-interacting motifs in Daxx

Posttranslational modification with small ubiquitin related modifier (SUMO) is an important regulatory mechanism of protein function. SUMO is an evolutionary conserved protein with a molecular weight of 10kDa in humans. It is covalently linked to certain lysine residues of target proteins. SUMO modification impacts subcellular localization of a modified protein, protein-protein interactions, and transcriptional regulation. One target protein of sumoylation is p53, a tumor suppressor protein. Research highlighting the role of SUMO modification of this protein is controversial. There have been conflicting reports regarding the effects of SUMO modification on p53 activities, particularly how it impacts transcriptional regulation of some p53 target genes and if SUMO modification affects subcellular localization of p53. We performed a comprehensive molecular biological study employing two unique approaches: (1) fusion of SUMO to the p53's C-terminus and (2) chemical induced dimerization of SUMO and p53. Our results have shown that when p53 is modified by SUMO, its biochemical functions are attenuated. It can no longer arrest clonogenic growth and activate the expression of its target genes involved in cell-cycle arrest or apoptosis. Furthermore, it appears that p53 is exported from the nucleus to the cytoplasm upon chemical induced sumoylation. This nuclear export may involve the CRM1-mediated pathway. Surprisingly, our results suggest that CRM1 interacts directly with the p53 tetramer. Thus, sumoylation of p53 may have an important role in promoting the nuclear export of p53 and that dissociation of the p53 tetramer required for the exposure of the nuclear export signal (NES) might not be necessary for CRM1-dependent nuclear export of p53, challenging the prevalent paradigm regarding the nuclear export of p53.;Another protein that SUMO interacts with is Daxx. The death-associated protein, Daxx, is essential for embryonic development and implicated in apoptosis and transcriptional regulation. Here we show that in addition to a conserved core of about 200 residues, Daxx possesses several conserved domains and two essentially invariable short SUMO-interacting motifs (SIMs) both of which can independently interact with SUMO. Daxx is known to interact with the SUMO E2 conjugating enzyme, Ubc9, responsible for SUMO conjugation to substrates via the Ubc9-SUMO complex. This interaction strictly requires at least one SIM. Interestingly, the Ubc9 H20D mutation that abolishes non-covalent Ubc9-SUMO interaction also interrupts Daxx-Ubc9 interaction. Thus, SUMO serves as the intermediate for Daxx-Ubc9 interaction. Remarkably, Daxx strongly stimulates c-Jun-mediated transcription and both SIMs are required for this stimulation. These results suggest that the conserved SIMs are involved in mediating protein-protein interactions that underlie Daxx's diverse cellular functions.;Overall, these studies provide important knowledge about cellular functions of sumoylation with respect to p53 and Daxx and may have implications in our understanding of tumorigenesis and anticancer therapy.