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UBE1L Represses PML/RARA by Targeting the PML Domain for Isg15ylation

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UBE1L Represses PML/RARA by Targeting the PML Domain for Isg15ylation 905 UBE1L represses PML/RARA by targeting the PML domain for ISG15ylation Sumit J. Shah,1 Steven Blumen,1 Ian Pitha-Rowe,1 PML/RARA degradation through different domains and Sutisak Kitareewan,1 Sarah J. Freemantle,1 distinct mechanisms. Taken together, these findings Qing Feng,1 and Ethan Dmitrovsky1,2,3 advance prior work by establishing two pathways con- verge on the same oncogenic protein to cause its degra- Departments of 1Pharmacology and Toxicology and 2Medicine dation and thereby promote antineoplastic effects. The 3 and Norris Cotton Cancer Center, Dartmouth Medical School, molecular pharmacologic implications of these findings Hanover, New Hampshire and Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire are discussed. [Mol Cancer Ther 2008;7(4):905–14] Abstract Introduction Acute promyelocytic leukemia (APL) is characterized by Acute promyelocytic leukemia (APL) is a distinct subtype expression of promyelocytic leukemia (PML)/retinoic acid of acute myeloid leukemia (FAB, M3; ref. 1). APL is (RA) receptor A (RARA) protein and all-trans-RA-mediated characterized by accumulation of abnormal promyelocytes clinical remissions. RA treatment can confer PML/RARA expressing the oncogenic product of the balanced t(15;17) degradation, overcoming dominant-negative effects of rearrangement between the promyelocytic leukemia (PML) this oncogenic protein. The present study uncovered and retinoic acid (RA) receptor a (RARa) genes (2, 3). independent retinoid degradation mechanisms, targeting PML/RARa is a dominant-negative transcriptional factor different domains of PML/RARA. RA treatment is known (3). All-trans-RA treatment is clinically successful in APL to repress PML/RARA and augment ubiquitin-activating by triggering leukemic cell differentiation (4). RA treatment enzyme-E1-like (UBE1L) protein expression in NB4-S1 APL can cause degradation of PML/RARa protein, thereby cells. We previously reported RA-induced UBE1L and the reversing its dominant-negative effects (5). IFN-stimulated gene, 15-kDa protein ISG15ylation in APL Protein degradation is regulated by the ubiquitin- cells. Whether the ubiquitin-like protein ISG15 directly proteasome pathway (6). Ubiquitin, a 76–amino acid conjugates with PML/RARA was not explored previously polypeptide, is conjugated to target proteins through and is examined in this study. Transient transfection isopeptide bonds, resulting in monoubiquitinated and experiments with different PML/RARA domains revealed polyubiquitinated substrates (6). Polyubiquitination that RA treatment preferentially down-regulated the RARA marks the substrate protein for proteolysis by the protea- domain, whereas UBE1L targeted the PML domain for some (6). Protein ubiquitination is tightly regulated by a repression. As expected, ubiquitin-specific protease 18 ubiquitin-conjugating multienzyme cascade, including the (UBP43/USP18), the ISG15 deconjugase, opposed UBE1L ubiquitin-activating enzyme (E1), ubiquitin conjugases but not RA-dependent PML/RARA degradation. In con- (E2), ubiquitin ligases (E3), and deubiquitinating (DUB) trast, the proteasomal inhibitor, N-acetyl-leucinyl-leucinyl- enzymes, which are deconjugases that remove ubiquitin norleucinal, inhibited both UBE1L- and RA-mediated from substrate proteins (6, 7). PML/RARA degradation. Notably, UBE1L induced ISG15y- The IFN-stimulated gene, 15-kDa protein (ISG15), is a lation of the PML domain of PML/RARA, causing its ubiquitin-like protein (7, 8). Ubiquitin-like proteins conju- repression. These findings confirmed that RA triggers gate intracellular proteins via an isopeptide linkage (7, 8). ISG15ylation is reversible and regulated by a similar set of enzymes as in the ubiquitin system (7, 8). The ubiquitin- activating enzyme-E1-like protein (UBE1L; E1), ubiquitin- conjugating enzyme 8 (E2), estrogen-responsive finger Received 7/31/07; revised 1/18/08; accepted 2/21/08. protein (E3), and ubiquitin-specific protease 18 (UBP43 or Grant support: NIH, National Cancer Institute grants R01-CA087546, USP18) have each been reported as enzymes involved in R01-CA062275, and R01-CA111422 (E. Dmitrovsky); Samuel Waxman Foundation Cancer Research Award (E. Dmitrovsky); American Cancer ISG15ylation (9–12). Biochemical steps involved in ubiq- Society Institutional Grant (S.J. Freemantle); and NIH grant T32-CA00959 uitination have been examined previously, but consequen- (S. Blumen). ces of ISG15ylation and fate of ISG15ylated substrates are The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked still being determined (6–8). advertisement in accordance with 18 U.S.C. Section 1734 solely to RA is a differentiation-inducing agent that has been used indicate this fact. clinically to treat APL and other diseases, as reviewed Requests for reprints: Ethan Dmitrovsky, Department of Pharmacology and (13, 14). Notably, RA treatment can induce complete Toxicology, Dartmouth Medical School, 7650 Remsen Building, Hanover, a New Hampshire 03755. Phone: 603-650-1667; Fax: 603-650-1129. clinical remissions in APL cases that express PML/RAR E-mail: [email protected] (4, 15). Prior work revealed pharmacologic treatment with Copyright C 2008 American Association for Cancer Research. RA overcomes dominant-negative PML/RARa effects (3). doi:10.1158/1535-7163.MCT-07-0515 RA response is accompanied by induced PML/RARa Mol Cancer Ther 2008;7(4). April 2008 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. 906 UBE1L Represses PML/RARa through ISG15ylation degradation through caspase- and proteasome-dependent independently cloned into the pCMV-HA vector using pathways (5, 16), which inhibits PML/RARa activity. RA EcoRI and BglII restriction endonuclease sites to engineer causes ubiquitination of RARa and PML/RARa, implicat- pCMV-HA-PML/RARa (S) and pCMV-HA-PML/RARa ing the RARa domain in mediating this degradation (5, 17). (L), respectively (3, 15). The pCMV-HA-PML expression Mutagenesis studies identified the DNA-binding domain vector was engineered using an amplified cloned fragment and the transcription-activation domain (AF-2) of RARa encoding residues 1 to 390 of PML/RARa. The pSG5-RARa as involved in proteasomal degradation (17). Involvement vector was engineered using an amplified cloned fragment of the PML domain in RA-mediated degradation of PML/ coding for amino acids 86 to 462 of RARa. The pcDNA-His- RARa has been less extensively studied. One study found UBP43 plasmid was provided by Dr. Bret Hassel (Univer- RA engaged caspase-3 activation, which targeted the PML sity of Maryland). The enhanced green fluorescent protein domain of PML/RARa for repression (16). (EGFP) plasmid (pEGFP-N2) and the h-galactosidase Prior work withmicroarray and biochemicalanalyses reporter plasmid (pCH111) were eachpurchased (Clontech). uncovered UBE1L as a direct RA target gene that repressed Transfection Procedures PML/RARa (18, 19). However, how UBE1L causes this was Transient transfection of BEAS-2B cells was accom- not discerned. RA treatment of APL cells induced ISG15 plished using Effectene transfection reagent (Qiagen) and and ISG15ylation and whether ISG15 physically conjugates methods described in previous work (21). Briefly, BEAS-2B withPML/RARa was not determined (19). The current (1 Â 106) cells were plated per treatment group and allowed study sought to examine how retinoid-mediated induction to attachovernight. Cells were then transfected with of UBE1L and ISG15ylation targets PML/RARa for Effectene reagent for 24 h. Following this, cells were repression. Findings reported here establish that, in exposed for 0 to 24 hto different treatments as described addition to RA effects on ubiquitination of RARa and in the figure legends. Transient transfection of NB4-S1 APL PML/RARa, RA engaged a distinct biochemical pathway cells was accomplished using the Nucleofector technology through induction of UBE1L and ISG15. This caused (Amaxa) that combines electroporation with cell-specific ISG15ylation of the PML domain of PML/RARa to confer optimized solutions to achieve entry of expression vectors its repression. Taken together, these findings revealed that into these cells. The EGFP or h-galactosidase reporter retinoid treatment engages two distinct pathways that plasmids were cotransfected to control for transfection converge on PML/RARa to trigger its degradation and efficiency in transfectants. In selected experiments, single- overcome its oncogenic effects in APL. cell expression assays for transfected EGFP determined the percentages of transfected cells using established methods (20). Coomassie blue staining of gels was also used to Materials and Methods confirm comparable loading of protein lysates in eachlane. Cells and Reagents Quantification of signal intensities was accomplished and The PML/RARa-expressing NB4-S1 APL cell line was displayed using previously optimized methods (22). cultured in Advanced RPMI 1640 (Invitrogen) supple- Immunoblot Analyses mented with2% fetal bovine serum (Gemini Bio-Products), Cells were treated as described in individual figure 4 mmol/L L-glutamine (Invitrogen), 100 units/mL penicil- legends. Whole-cell lysates were prepared using radio- lin (Invitrogen), and 100 Ag/mL streptomycin (Invitrogen) immunoprecipitation assay buffer [150 mmol/L NaCl, as described previously (18, 19). The BEAS-2B immortal- 50 mmol/L Tris-HCl (pH 7.4), 1 mmol/L EDTA, 1 mmol/L ized human bronchial epithelial cell line was cultured
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