The ISG15 Isopeptidase UBP43 Is Regulated by Proteolysis via the SCFSkp2 Ubiquitin Ligase

Abstract

The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-β. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2–/–mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling. The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-β. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2–/–mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling. Modification of proteins by ubiquitin and ubiquitin-like (Ubl) 1The abbreviations used are: Ubl, ubiquitin-like; SCF, Skp1, cullin, F-box protein; LPS, lipopolysaccharide; IFN, interferon; MEF, mouse embryonic fibroblast; LRR, leucine-rich repeat; HEK, human embryonic kidney; HA, hemagglutinin; SCFSkp2, Skp1-Cul1-F-box protein Skp2. molecules, including SUMO, Nedd8, and ISG15, has emerged as a critical regulatory process in eukaryotes, controlling pathways such as the cell cycle, cellular stress response, intracellular signaling, development, and the immune response (1Hochstrasser M. Nat. Cell Biol. 2000; 2: E153-E157Crossref PubMed Scopus (368) Google Scholar, 2Kim K.I. Zhang D.E. Biochem. Biophys. Res. Commun. 2003; 307: 431-434Crossref PubMed Scopus (63) Google Scholar, 3Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6907) Google Scholar, 4Hicke L. Nat. Rev Mol. Cell. Biol. 2001; 2: 195-201Crossref PubMed Scopus (994) Google Scholar, 5Jentsch S. Pyrowolakis G. Trends Cell Biol. 2000; 10: 335-342Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Deregulation of ubiquitin or Ubl modification can cause autoimmune and neurodegenerative diseases, developmental abnormalities, and cancer. Conjugation of ubiquitin and Ubls involves a three-step mechanism initially demonstrated for ubiquitin as follows. A single E1 ubiquitin-activating enzyme activates the Ubl molecule via formation of a thioester bond. Activated Ubl is then transferred to one of a large family of E2 ubiquitin-conjugating enzymes. In most cases, E2 enzymes are targeted to appropriate substrates by a class of substrate receptor complexes termed E3 ubiquitin ligases. Together, the E2 and E3 enzymes catalyze the formation of isopeptide bonds between ubiquitin and lysine residues on the target proteins. In the case of multiubiquitination, additional ubiquitin molecules are added to form ubiquitin chains. The multiubiquitinated proteins are then recognized and rapidly degraded into short peptides by the 26 S proteasome (3Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6907) Google Scholar, 6Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar). Modification by ubiquitin and Ubl molecules is a reversible process mediated by a large family of isopeptidases that exist to remove these molecules from their substrates. Although the function and regulation of these isopeptidases is poorly defined, the few that have been studied indicate an important role in growth and development (7Gray D.A. Inazawa J. Gupta K. Wong A. Ueda R. Takahashi T. Oncogene. 1995; 10: 2179-2183PubMed Google Scholar, 8Huang Y. Baker R.T. Fischer V.J. Science. 1995; 270: 1828-1831Crossref PubMed Scopus (242) Google Scholar). Specificity of the ubiquitin and Ubl pathways is conferred by the nature and activity of the E3 complexes. One of the beststudied E3 ligase complexes is the SCF complex. SCF complexes are composed of four subunits: the three core components Skp1, a Cullin family member, the RING finger protein Rbx1/Roc1, and one variable component, the F-box protein, that acts as the substrate recognition factor. A large number of F-box proteins have been identified in organisms ranging from yeast to humans. The mammalian F-box protein Skp2 may play a pivotal role in oncogenesis and has been implicated in degradation of several key regulators of cell proliferation including the tumor suppressor proteins p27Kip1, p57Kip2, (9Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1335) Google Scholar) and p130 (10Tedesco D. Lukas J. Reed S.I. Genes. Dev. 2002; 16: 2946-2957Crossref PubMed Scopus (238) Google Scholar) and the replication factor, hORC1 (11Mendez J. Zou-Yang X.H. Kim S.Y. Hidaka M. Tansey W.P. Stillman B. Mol. Cell. 2002; 9: 481-491Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). We used the yeast two-hybrid technique to isolate potential Skp2-interacting proteins. We identified seven proteins, including UBP43, which will be described in this report. UBP43 belongs to the family of ubiquitin isopeptidases, one of the two classes of ubiquitin and Ubl deconjugating enzymes (12Schwer H. Liu L.Q. Zhou L. Little M.T. Pan Z. Hetherington C.J. Zhang D.E. Genomics. 2000; 65: 44-52Crossref PubMed Scopus (57) Google Scholar). UBP43 was identified as strongly expressed mRNA in hematopoietic tissue from an acute myeloid leukemia mouse model (13Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar). Thereafter, UBP43 was identified as an up-regulated gene in melanoma cells treated with IFN-β and the protein kinase C activator MEZ. This treatment causes cells to irreversibly lose proliferation potential and begin progression toward terminal differentiation (14Kang D. Jiang H. Wu Q. Pestka S. Fisher P.B. Gene. 2001; 267: 233-242Crossref PubMed Scopus (59) Google Scholar). UBP43 functions as an isopeptidase responsible for cleaving the ubiquitin-like protein ISG15 from substrates (15Malakhov M.P. Malakhova O.A. Kim K.I. Ritchie K.J. Zhang D.E. J. Biol. Chem. 2002; 277: 9976-9981Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Upon treatment of cells with type I IFN or lipopolysaccharide (LPS), ISG15, UBP43, and the ISG15 E1 enzyme, UBE1L, are rapidly up-regulated (16Malakhova O. Malakhov M. Hetherington C. Zhang D.E. J. Biol. Chem. 2002; 277: 14703-14711Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). It seems that tight regulation of both the ISG15 conjugation and deconjugation pathways is required to ensure proper response to cellular stress. In UBP43 knockout mouse embryonic fibroblasts (MEFs), ISG15 cleavage from cellular substrates is strongly reduced, suggesting that UBP43 is the major ISG15 isopeptidase (17Ritchie K.J. Malakhov M.P. Hetherington C.J. Zhou L. Little M.T. Malakhova O.A. Sipe J.C. Orkin S.H. Zhang D.E. Genes Dev. 2002; 16: 2207-2212Crossref PubMed Scopus (134) Google Scholar). UBP43–/–MEFs demonstrate prolonged STAT1 signaling and IFN hypersensitivity, which is in accordance with data showing that signal transducer and activator of transcription 1 is an ISG15-modified protein (18Malakhov M.P. Kim K.I. Malakhova O.A. Jacobs B.S. Borden E.C. Zhang D.E. J. Biol. Chem. 2003; 278: 16608-16613Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). These observations and the identification of UBP43 in the two tumor models suggest that UBP43 plays an important role in cellular proliferation and differentiation and that UBP43 levels need to be carefully controlled in cells. Indeed, UBP43 protein levels are regulated at the level of transcription (16Malakhova O. Malakhov M. Hetherington C. Zhang D.E. J. Biol. Chem. 2002; 277: 14703-14711Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and post-translationally, as we show here, via the SCFSkp2-mediated ubiquitin pathway. DNA Constructs—Skp1, Skp2, and UBP43 were PCR-amplified using a human lymphocyte cDNA library (HL4006AE; BD Biosciences Clontech). Skp1 and Skp2 were cloned into MatchMaker (BD Biosciences Clontech) yeast two-hybrid vectors pGADT7 and pGBKT, respectively. All other plasmids were created with the GATEWAY cloning system using vectors from Invitrogen following manufacturer's protocols. The C-terminally V5-tagged UBP43 adenovirus construct was made using the pADeasy system (Qbiochem). Amplified clones were verified by sequencing. Antibodies—The Skp2 antibody used for immunoprecipitation was purchased from Zymed Laboratories Inc.. Skp2 (His-435) and Cdk2 (Glu-119) antibodies used in immunoblotting analysis were purchased from Santa Cruz. His, HA, and V5 antibodies were purchased from Amersham Biosciences, Babco, and Invitrogen, respectively. The affinity-purified polyclonal ISG15 antibody was kindly supplied by Dr. Arthur Haas and does not cross-react with ubiquitin (19Loeb K.R. Haas A.L. J. Biol. Chem. 1992; 267: 7806-7813Abstract Full Text PDF PubMed Google Scholar). Cell Extracts—Unless stated otherwise, cells were harvested in mammalian lysis buffer (150 mm NaCl, 0.1% Nonidet P-40, and 50 mm Tris-HCl, pH 7.5) by scraping, incubating on ice for 10 min, and then centrifuging at 14,000g (40° C) for 15 min; the supernatant was retained. Yeast Two-hybrid Screen—The MatchMaker (BD Biosciences Clontech) yeast two-hybrid system was used to screen for Skp2-interacting proteins. Yeast strain AH109 was transformed with plasmid pGBKT-Skp2 containing full-length Skp2 fused to the GAL4 DNA-binding domain. Transformed cells were streaked on plates lacking tryptophan to select for single clones. This strain was then transformed with a human lymphoma cDNA library (HL4006AE; Invitrogen) cloned into the vector pACT1 that fuses cDNAs to the GAL4 activating domain. 2 × 106 clones were screened on plates lacking histidine (+10 mm 3AT) or adenine and histidine (+10 mm 3AT). Interaction of positive clones was verified by retransforming clones into the original Skp2 bait strain and assaying for interaction. Skp2 and UBP43 constructs were then cloned into yeast expression vectors compatible with the ProQuest two-hybrid system from Invitrogen. Constructs were transformed into AH109 and screened on plates lacking leucine, tryptophan, or histidine (+30 mm 3AT). Production of Recombinant Proteins in Bacteria and GST Purification—Escherichia coli BL21 (Invitrogen) cells were used to express the vector, pDEST15 (Invitrogen) containing GST or a GST-Skp2 fusion protein. One liter of each strain was induced with 100 mm isopropyl β-d-thiogalactoside for 2 h. Protein was isolated using a 3× freeze-thaw protocol with a dry ice and ethanol bath. Extracts were purified over glutathione-Sepharose 4B matrix (Amersham Biosciences) according to manufacturer's instructions. Extracts from HEK 293 cells transfected with indicated plasmids were added to the matrix. Bound fractions were by using the indicated cells on a were with expressing Skp2 with an and UBP43 with a V5 was with cells were harvested in mammalian lysis The was to a and bound proteins were with and to was into (Invitrogen) containing an UBP43 was into (Invitrogen) containing V5 and and constructs were a from Dr. and cells, transfected with indicated were treated for with 10 and cells were with adenovirus for h. Skp2 and Skp2 knockout were in the of were then for with of with containing mm and mm and at and Cell were and then for V5 immunoprecipitation with antibody from Invitrogen. Proteins were by and by were added the and the and IFN-β and were treated with mouse and were harvested cell growth × cells were in treated with of mouse and harvested in each cell number was by with a was used to cells. A of cells was UBP43 with Skp2-interacting proteins, we a yeast two-hybrid screen and clones for Skp2 proteins and of the cDNA two identified and novel Skp2 In of screen, we isolated Skp1 and Skp1 is the protein that Skp2 to the SCF complex via the Skp2 F-box (18Malakhov M.P. Kim K.I. Malakhova O.A. Jacobs B.S. Borden E.C. Zhang D.E. J. Biol. Chem. 2003; 278: 16608-16613Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). is a protein identified to with Skp2 and substrate recognition of A.C. P.D. Z. Pagano M. 2000; PubMed Scopus Google Scholar, D. G. B. M. Pagano M. Hershko A. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). the other clones we on a for a of the UBP43 C that was isolated in the original screen and full-length UBP43, not the were to with Skp2, as by of and growth on plates lacking histidine isolate the Skp2-interacting of UBP43, we made of the We a between and of UBP43 that seems to be involved in with Skp2. The construct containing the as well as a construct lacking the demonstrated interaction. a construct containing interaction. was with Skp2 was We therefore that the between and of UBP43 is required for the between UBP43 and Skp2. Skp2 Interaction with UBP43 the Skp2 we the of UBP43 and Skp2 in that full-length Skp2 can UBP43 expressed in the human cell A construct lacking the and C to constructs lacking the or both the and F-box the and C with The of Skp2 has been implicated in substrate Our data suggest that UBP43 is a substrate of Skp2 and UBP43 in the of UBP43 with Skp2, we in coli that expressed GST-Skp2 or GST were bound to by with from human embryonic 293 cells that expressed that not to UBP43 in of UBP43 by Skp2 and by that UBP43 with the of Skp2 that UBP43 is a substrate of UBP43 protein levels be controlled by protein this we expressed Skp2 and UBP43 in cells. UBP43 levels were strongly Skp2 was expressed with UBP43 was expressed Skp2 functions in an E3 ligase complex that substrates for degradation by the 26 S we UBP43 accumulates in cells that have been treated with the proteasome Indeed, these UBP43 can be Together, these results suggest that UBP43 is degraded in the proteasome most by SCFSkp2-mediated of UBP43 and ISG15 in levels of UBP43 are in cells that are of Skp2, we immunoblotting of from or in UBP43 levels are in with type that UBP43 levels are in cells and are with the We also a in UBP43 levels in with not In to the in UBP43 levels in an in ISG15 levels was This that in the of Skp2, UBP43 levels are up-regulated, resulting in cleavage of ISG15 from substrates and a in Skp2 UBP43 in degradation of proteins is by multi-ubiquitination of targeted UBP43 is ubiquitinated in we transfected cells with plasmids Skp2, V5-tagged UBP43, and UBP43, in the of Skp2, in higher with ubiquitination These were of the proteasome we immunoprecipitation with antibodies to UBP43 protein. were that were that these ubiquitinated UBP43 2 and was by Skp2 and was or was 1 and these to ubiquitinated UBP43 then of the proteasome in an and a to higher This is the as in In this UBP43 was on by immunoblotting the on in these the of transfected UBP43 was with Skp2, we not a in UBP43 levels Skp2 We that UBP43 is ubiquitinated in Skp2 can the of ubiquitinated UBP43, we normal cells with V5-tagged UBP43 expressed from an adenovirus with levels of that proteasome and UBP43 ubiquitination is by levels of Skp2. We the level of Skp2 at or levels not whereas UBP43 levels were higher to of ubiquitinated In these demonstrate that UBP43 is ubiquitinated in vivo by Skp2 and is then degraded in a Skp2 of UBP43 in results that UBP43 levels were controlled by SCFSkp2-mediated degradation via the pathway. UBP43 degradation in we in cells. UBP43 has a of UBP43 is to these the the of the UBP43 cells were treated with a proteasome UBP43 was We results in human cells not In treatment of by analysis of UBP43 protein levels the results not Skp2 expression in degradation of UBP43 not We the analysis in and The of UBP43 was in Skp2 cells with in knockout cells and Skp2 was in the levels of UBP43 at the and then at that level for the of the We that Skp2 can rapid degradation of UBP43 via the pathway. Skp2 the ISG15 Conjugation ISG15 conjugation to substrates is induced IFN-β Skp2 has an on ISG15 conjugation by of regulation of UBP43, we Skp2 in the human cell treatment with LPS, cells Skp2 a in ISG15 conjugation with cells transfected with vector the other induced with IFN-β a in ISG15 conjugation and an in ISG15 with We that Skp2 can the level of ISG15 most via degradation of from levels of ISG15 these are to of the type I IFN (17Ritchie K.J. Malakhov M.P. Hetherington C.J. Zhou L. Little M.T. Malakhova O.A. Sipe J.C. Orkin S.H. Zhang D.E. Genes Dev. 2002; 16: 2207-2212Crossref PubMed Scopus (134) Google Scholar). we that which have levels of ISG15 be to the of IFN-β we the of toward IFN-β. Skp2 cells a in cell growth IFN-β In the growth of was by IFN-β at h. These data suggest that of Skp2 or the response to most of the higher levels of UBP43 The function of ISG15 modification is not well It is that carefully controlled ISG15 conjugation and deconjugation to substrates is for the of a cell and of an This that the level and activity of enzymes that ISG15 including and UBP43, need to be Indeed, mice lacking UBP43 are neuronal hypersensitivity to IFN, and demonstrate in hematopoietic (17Ritchie K.J. Malakhov M.P. Hetherington C.J. Zhou L. Little M.T. Malakhova O.A. Sipe J.C. Orkin S.H. Zhang D.E. Genes Dev. 2002; 16: 2207-2212Crossref PubMed Scopus (134) Google Scholar). the other expression of UBP43 differentiation in cell (13Liu L.Q. Ilaria Jr., R. Kingsley P.D. Iwama A. van Etten R.A. Palis J. Zhang D.E. Mol. Cell. Biol. 1999; 19: 3029-3038Crossref PubMed Scopus (132) Google Scholar). In the E1 enzyme for ISG15, UBE1L, is in for suggesting that the of ISG15 conjugation to levels of UBP43 are controlled at the level of transcription by and IFN type 1 (14Kang D. Jiang H. Wu Q. Pestka S. Fisher P.B. Gene. 2001; 267: 233-242Crossref PubMed Scopus (59) Google Scholar, O. Malakhov M. Hetherington C. Zhang D.E. J. Biol. Chem. 2002; 277: 14703-14711Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We demonstrate that the SCFSkp2 ubiquitin ligase the UBP43 protein level by degradation via the pathway. Our data demonstrate that Skp2 to UBP43 and resulting in UBP43 degradation via the proteasome. In lacking Skp2, levels of ISG15 are and ISG15 conjugates are with UBP43 the other levels of Skp2 in an in proteins. It was that UBP43 protein was an that was by Skp2 levels not The of both ISG15 and deconjugating pathways that ISG15 modification is a process that to be carefully controlled for normal cellular function and Indeed, expression of ISG15 in cell In this degradation play a role to the levels of UBP43 according to the growth and stress of a Skp2 is regulated at the level of transcription and protein degradation C. H. M. A. Reed S.I. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. E. A. C. M. Nat. Cell Biol. 1999; 1: PubMed Scopus Google Scholar). Skp2 protein is in and cells, as cells S and in We have an between Skp2 and UBP43 levels in cells not with the role of Skp2 in We have not been to UBP43 levels the cell cycle, UBP43 levels are in cells not treated with IFN or LPS, and the antibody does not Our data not the that one F-box protein in the degradation of in UBP43 is with a of 2 UBP43 levels are in and Skp2 the levels to that Skp2 does have a major on UBP43 protein The recognition of substrates by SCF complexes is by substrate at residues C. H. M. M. M. J. 2000; 19: PubMed Scopus Google Scholar, Genes Dev. 1999; PubMed Scopus Google Scholar, S. Q. T. M. 2001; PubMed Scopus Google Scholar, S. C. Science. PubMed Scopus Google Scholar, S. R. C. M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, R. S. C. Mol. Cell. Biol. 2001; PubMed Scopus (63) Google Scholar, C. R. J. C. S. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar, Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). This is also for SCFSkp2 (9Carrano A.C. Eytan E. Hershko A. Pagano M. Nat. Cell Biol. 1999; 1: 193-199Crossref PubMed Scopus (1335) Google Scholar, D. Lukas J. Reed S.I. Genes. Dev. 2002; 16: 2946-2957Crossref PubMed Scopus (238) Google Scholar, C. H. M. A. Reed S.I. Mol. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. Science. 278: PubMed Scopus Google Scholar, T. J. J. Zhang H. Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). In the protein was to be required for of and p130 A.C. P.D. Z. Pagano M. 2000; PubMed Scopus Google Scholar, D. G. B. M. Pagano M. Hershko A. Nat. Cell Biol. 2001; PubMed Scopus Google Scholar). We have that UBP43 is is important for and is for with Skp2 are ISG15 conjugation plays an important role in stress response, and is also implicated in controlling cell proliferation and It is that proteins ISG15 expression and suggesting that ISG15 activity H. Zhang H. Biol. 1999; 9: Full Text Full Text PDF PubMed Scopus Google Scholar, J. 2001; PubMed Scopus Google Scholar). It is that Skp2 the levels of cell regulators as we show here, a in stress A between cell and stress response at the level of degradation has been the yeast complex the and as well as the transcription involved in the response to and has an important role in the cellular response to It will be to the mechanism that Skp2 to cellular stress and the role of ISG15 conjugation in this Skp2 cell and cellular stress signaling will as a for pathways that cell and We for the Arthur Haas for ISG15 M. Liu and B. for and Y. and of the for and