In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.
SUMO (small ubiquitin-like modifier) modification regulates many cellular processes, including transcription. Although sumoylation often occurs on specific lysines within the consensus tetrapeptide ⌿KxE, other modifications, such as phosphorylation, may regulate the sumoylation of a substrate. We have discovered PDSM (phosphorylation-dependent sumoylation motif), composed of a SUMO consensus site and an adjacent proline-directed phosphorylation site (⌿KxExxSP). The highly conserved motif regulates phosphorylation-dependent sumoylation of multiple substrates, such as heat-shock factors (HSFs), GATA-1, and myocyte enhancer factor 2. In fact, the majority of the PDSM-containing proteins are transcriptional regulators. Within the HSF family, PDSM is conserved between two functionally distinct members, HSF1 and HSF4b, whose transactivation capacities are repressed through the phosphorylation-dependent sumoylation. As the first recurrent sumoylation determinant beyond the consensus tetrapeptide, the PDSM provides a valuable tool in predicting new SUMO substrates.heat-shock factor ͉ heat-shock protein ͉ transcription S UMO (small ubiquitin-like modifier) is covalently linked by an isopeptide bond to lysine residues in their substrates (1). SUMO conjugation often requires a consensus sequence ⌿KxE (⌿, large hydrophobic residue; x, any amino acid) around the target lysine (2). Because not all available SUMO consensus sites are modified, other factors must also affect the target site specificity. For example, phosphorylation of the substrate has been shown to regulate the sumoylation both positively and negatively (3-5). However, apart from the ⌿KxE tetrapeptide, no common regulatory motifs involved in SUMO conjugation are known (1). The SUMO family consists of four members, SUMO-1, -2, -3, and -4, of which SUMO-1 is best studied; SUMO-4 has been so far detected only at the RNA level (1, 6). SUMO-1 and SUMO-2͞3 have, at least partially, distinct substrates and regulation. For example, the general pattern of conjugates and the subnuclear distribution are different between SUMO-1 and SUMO-2͞3, and the amount of conjugated SUMO-2͞3, but not SUMO-1, is increased upon protein-damaging stresses (7,8). Beyond this, the functional differences between the SUMO paralogs are largely unknown.The mammalian HSF family is composed of three members, HSF1, HSF2, and HSF4. HSF1 is the mammalian counterpart of the single invertebrate HSF and is indispensable for the heatshock response (9). Upon stress, HSF1 is activated by trimerization-induced DNA binding and hyperphosphorylation (10,11). Moreover, HSF1 is sumoylated on a conserved lysine, lysine-298 (4, 12). We have demonstrated that HSF1 sumoylation depends on phosphorylation of an adjacent site, serine-303 (4). Sumoylation was originally suggested to induce HSF1 DNA binding and to be needed for HSF1-mediated transcription (12). This hypothesis was, however, questioned by our previous study, showing that HSF1 could be fully activated also when the sumoylation was prevented (4). Therefore...
PIAS (protein inhibitor of activated STAT) proteins interact with and modulate the activities of various transcription factors. In this work, we demonstrate that PIAS proteins x␣, x, 1, and 3 interact with the small ubiquitin-related modifier SUMO-1 and its E2 conjugase, Ubc9, and that PIAS proteins themselves are covalently modified by SUMO-1 (sumoylated). PIAS proteins also tether other sumoylated proteins in a noncovalent fashion. Furthermore, recombinant PIASx␣ enhances Ubc9-mediated sumoylation of the androgen receptor and c-Jun in vitro. Importantly, PIAS proteins differ in their abilities to promote sumoylation in intact cells. The ability to stimulate protein sumoylation and the interaction with sumoylated proteins are dependent on the conserved PIAS RING finger-like domain. These functions are linked to the activity of PIASx␣ on androgen receptor-dependent transcription. Collectively, our results imply that PIAS proteins function as SUMO-1-tethering proteins and zinc finger-dependent E3 SUMO protein ligases, and these properties are likely to explain their ability to modulate the activities of various transcription factors.Members of the recently identified PIAS (protein inhibitor of activated STAT) protein family have been found to interact with several distinct nuclear proteins. PIAS1 and PIAS3 bind to STAT1 and STAT3, respectively, and inhibit their action (4, 28). PIASx␣/ARIP3 (androgen receptor [AR]-interacting protein 3) was first characterized as an AR-interacting protein, and it modulates the transcriptional activity of the receptor (34). Other PIAS proteins have recently been demonstrated to function as coregulators for AR and other steroid receptors (25, 57). PIASx/Miz1 (Msx-interacting zinc finger) associates with a homeodomain-containing Msx2 protein (61), and GBP (Gu/RNA helicase II-binding protein), which is nearly identical to PIAS1, interacts with Gu/RNA II-helicase (59). More recently, PIAS proteins have been identified in many yeast two-hybrid screens, including PIASx␣/ARIP3 from its interaction with mouse disabled 2 (mDab2) (3) and DJ-1 protein (54), PIAS1 from its interaction with p53 (9), and PIAS3 from its interaction with high-mobility-group protein HMGI-C (63) and zinc finger protein Gfi-1 (43). In addition to PIASx (PIASx␣/ARIP3 and PIASx/Miz1), SUMO-1 (small ubiquitinrelated modifier 1) was identified as a p73␣-interacting protein by Minty et al. (32), who suggested that PIASx was isolated via the interaction with Smt3p (yeast SUMO) covalently linked to p73␣. We have also detected SUMO-1 as a major PIASx␣/ ARIP3-interacting protein in yeast (unpublished results).Members of the SUMO protein family, also known as Sentrin, GMP1, PIC1, and Ubl1 (31, 37, 62), are present in protozoa, metazoa, plants, and fungi. SUMO proteins from metazoa can be divided into two families: the SUMO-1 family and the SUMO-2 and -3 family (31, 37, 62). SUMO-2 and SUMO-3 are very similar at the amino acid level (97% identity for the human proteins), but they are only ϳ50% identical to SUMO-1. SUMO-1 is...
Activation of androgen receptor (AR) is crucial for prostate cancer growth. Remarkably, also castration-resistant prostate cancer (CRPC) is dependent on functional AR, and several mechanisms have been proposed to explain the addiction. Known causes of CRPC include gene amplification and overexpression as well as point mutations of AR. We report here the pharmacological profile of ODM-201, a novel AR inhibitor that showed significant antitumor activity and a favorable safety profile in phase 1/2 studies in men with CRPC. ODM-201 is a full and high-affinity AR antagonist that, similar to second-generation antiandrogens enzalutamide and ARN-509, inhibits testosterone-induced nuclear translocation of AR. Importantly, ODM-201 also blocks the activity of the tested mutant ARs arising in response to antiandrogen therapies, including the F876L mutation that confers resistance to enzalutamide and ARN-509. In addition, ODM-201 reduces the growth of AR-overexpressing VCaP prostate cancer cells both in vitro and in a castration-resistant VCaP xenograft model. In contrast to other antiandrogens, ODM-201 shows negligible brain penetrance and does not increase serum testosterone levels in mice. In conclusion, ODM-201 is a potent AR inhibitor that overcomes resistance to AR-targeted therapies by antagonizing both overexpressed and mutated ARs. ODM-201 is currently in a phase 3 trial in CRPC.
Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a monogenic autosomal disease with recessive inheritance. It is characterized by multiple autoimmune endocrinopathies, chronic mucocutaneous candidiasis, and ectodermal dystrophies. The defective gene responsible for this disease was recently isolated, and several different mutations in the novel gene, AIRE, have been identified, by us and by others, in patients with APECED. We have shown that the APECED protein is mainly localized, both in vitro and in vivo, to the cell nucleus, where it forms distinct speckles. This accords with the predicted structural features of the protein, which suggest involvement of AIRE in the regulation of gene transcription. Here, we report the results of mutational analyses of a series of 112 patients with APECED who were from various ethnic backgrounds. A total of 16 different mutations, covering 91% of disease alleles, were observed; of these, 8 were novel. The mutations are spread throughout the coding region of AIRE, yet four evident mutational hotspots were observed. In vitro expression of four different naturally occurring nonsense and missense mutations revealed a dramatically altered subcellular location of the protein in cultured cells. Interestingly, the wild-type APECED protein tethered to the Gal4 DNA-binding domain acted as a strong transcriptional activator of reporter genes in mammalian cells, whereas most of the analyzed mutant polypeptides had lost this capacity.
The heat shock response, which is accompanied by a rapid and robust upregulation of heat shock proteins (Hsps), is a highly conserved protection mechanism against protein-damaging stress. Hsp induction is mainly regulated at transcriptional level by stress-inducible heat shock factor 1 (HSF1). Upon activation, HSF1 trimerizes, binds to DNA, concentrates in the nuclear stress granules, and undergoes a marked multisite phosphorylation, which correlates with its transcriptional activity. In this study, we show that HSF1 is modified by SUMO-1 and SUMO-2 in a stress-inducible manner. Sumoylation is rapidly and transiently enhanced on lysine 298, located in the regulatory domain of HSF1, adjacent to several critical phosphorylation sites. Sumoylation analyses of HSF1 phosphorylation site mutants reveal that specifically the phosphorylationdeficient S303 mutant remains devoid of SUMO modification in vivo and the mutant mimicking phosphorylation of S303 promotes HSF1 sumoylation in vitro, indicating that S303 phosphorylation is required for K298 sumoylation. This finding is further supported by phosphopeptide mapping and analysis with S303/7 phosphospecific antibodies, which demonstrate that serine 303 is a target for strong heat-inducible phosphorylation, corresponding to the inducible HSF1 sumoylation. A transient phosphorylation-dependent colocalization of HSF1 and SUMO-1 in nuclear stress granules provides evidence for a strictly regulated subnuclear interplay between HSF1 and SUMO.The heat shock response is an evolutionarily well-conserved cellular stress response mechanism that is characterized by the elevated synthesis and accumulation of heat shock proteins (Hsps). Hsps are molecular chaperones involved in protein folding and maintenance of protein homeostasis, and a robust increase in Hsp levels is essential for survival when cells are exposed to various protein-damaging stresses (for reviews see references 15 and 34). The stress-inducible expression of Hspencoding genes is regulated by a family of heat shock transcription factors (HSFs), which bind to the heat shock element (HSE) in the promoter of heat shock genes and stimulate their transcription (for reviews see references 37 and 62). Three members of the HSF family, HSF1, HSF2, and HSF4, have been identified in mammalian species (41,47,54,56). HSF1, the vertebrate homologue of the single HSF found in the yeast and the fly, is the major stress-responsive family member, since no other HSF is able to functionally substitute for HSF1 or to rescue the heat shock response in HSF1-deficient cells or mice (35,44,64).The elevated synthesis of Hsps upon heat shock is caused by a multistep activation of HSF1 (for a review see reference 45). Under normal growth conditions, inactive HSF1 is distributed throughout the cell as a monomer that is constitutively phosphorylated on certain serine residues. Upon activation, HSF1 undergoes several modifications, such as trimerization and localization to specific nuclear structures, called nuclear stress granules (for a review s...
Identical N-terminal deletions in the wild-type rat androgen receptor (rAR) and a constitutively active rAR (AR⌬641-902) devoid of the ligand-binding domain (LBD) resulted in dissimilar consequences in transcriptional activation: deletion of residues 149 -295 abolished wild-type AR activity, but did not influence that of AR⌬641-902. The activity of the N-terminal transactivation domain is thus controlled by the hormone-occupied LBD, suggesting that the N-and C-terminal regions of rAR communicate. Consistent with this idea, a strong androgen-dependent interaction between the N-terminal region and LBD was demonstrated in a mammalian two-hybrid system using GAL4 and VP16 fusion proteins. This interaction can be direct or indirect. Several nuclear receptor coactivators (CBP, F-SRC-1, SRC-1, and RIP140) that interact with other steroid receptors were tested as potential mediators of the Nand C-terminal interaction of rAR using the mammalian two-hybrid system. CBP or F-SRC-1 not only enhanced AR-mediated transactivation, but also facilitated the androgen-dependent interaction between the N-and Cterminal domains, implying that part of the coactivatordependent transcriptional activation occurs via this mechanism. In contrast, SRC-1, a coactivator for the progesterone receptor, inhibited both AR-mediated transactivation and interaction between the N and C termini. Recruitment of coregulators may involve AR domains other than the LBD, as F-SRC-1 and CBP enhanced, but SRC-1 repressed, the transcriptional activity of AR⌬641-902. Collectively, interplay between the N-terminal region and LBD of rAR results in the formation of a transactivation complex that includes coregulators and that is mandatory for optimal activation of androgen-induced promoters.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.