The Interferon-inducible Ubiquitin-protein Isopeptide Ligase (E3) EFP Also Functions as an ISG15 E3 Ligase

Abstract

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3σ protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3σ in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3σ in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations. The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3σ protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3σ in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3σ in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations. ISG15 is one of several known Ubl ubiquitin-like modifiers (1.Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar, 2.Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). Upon type I interferon (IFN) 3The abbreviations used are: IFN, interferon; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; siRNA, small interfering RNA; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; shRNA, small hairpin RNA; ISRE, IFN-stimulated response element; Stat, signal transducer and activator of transcription. treatment, ISG15 forms covalent conjugates with cellular proteins, a process similar to ubiquitin modification (ubiquitination or ubiquitylation) (1.Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar). Ubiquitylation is now understood to be the dominant mechanism whereby cellular proteins are marked for degradation (3.Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar, 4.Hicke L. Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar). However, unlike ubiquitin modification, the role of ISG15 modification (ISGylation) has not been clearly defined, although ISG15 has been known since 1979 (5.Farrell P.J. Broeze R.J. Lengyel P. Nature. 1979; 279: 523-525Crossref PubMed Scopus (200) Google Scholar, 6.Ritchie K.J. Zhang D.-E. Semin. Cell Dev. Biol. 2004; 15: 237-246Crossref PubMed Scopus (119) Google Scholar). ISG15 is composed of two domains, each of which bears sequence identity to ubiquitin (33 and 32% for the N- and C-terminal domains, respectively) (7.Kim K.I. Zhang D.-E. Biochem. Biophys. Res. Commun. 2003; 307: 431-434Crossref PubMed Scopus (63) Google Scholar). The process of ISGylation is expected to be similar to that of protein ubiquitylation. Mass spectrometry analysis confirmed that an isopeptide bond can be formed between the C terminus of ISG15 and the lysine ϵ-amino group of the target protein (8.Zou W. Papov V. Malakhova O. Kim K.I. Dao C. Li J. Zhang D.-E. Biochem. Biophys. Res. Commun. 2005; 336: 61-68Crossref PubMed Scopus (66) Google Scholar). There are a series of distinct enzymes involved in the process of protein ubiquitylation and deubiquitylation, viz. the ubiquitin-activating enzyme (E1), ubiquitin carrier protein (E2), ubiquitin-protein isopeptide ligase (E3), and the Dub deubiquitining proteases (9.Weissman A.M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 169-178Crossref PubMed Scopus (1257) Google Scholar, 10.Wilkinson K.D. Cell. 2004; 119: 741-745Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). An E1 enzyme for ISGylation (UBE1L) has been cloned and shows high homology to the E1 enzyme UBE1 (11.Kok K. Hofstra R. Pilz A. van den Berg A. Terpstra P. Buys C.H. Carritt B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6071-6075Crossref PubMed Scopus (88) Google Scholar). UBE1L-deficient cells express only free ISG15 and do not show ISG15 conjugation, indicating that UBE1L is necessary for ISGylation (12.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (420) Google Scholar, 13.Malakhova O.A. Yan M. Malakhov M.P. Yuan Y. Ritchie K.J. Kim K.I. Peterson L.F. Shuai K. Zhang D.-E. Genes Dev. 2003; 17: 455-460Crossref PubMed Scopus (265) Google Scholar). UBP43 (USP18) has been identified as a de-ISGylating isopeptidase and is highly specific in removing ISG15, but not Nedd8 and SUMO (small ubiquitin-like modifier) from their conjugates (14.Malakhov 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). UBP43 is a member of the UBP (USP) family of ubiquitin-specific proteases and contains conserved Cys and His domains, which are common in all members of this family (15.Liu 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). Based on the information on UBE1L and UBP43, it is logical to hypothesize that ISG15 E2 and E3 are also members of the ubiquitin E2 and E3 families. Recently, Kim et al. (16.Kim K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (185) Google Scholar) and Zhao et al. (17.Zhao C. Beaudenon S.L. Kelley M.L. Waddell M.B. Yuan W. Schulman B.A. Huibregtse J.M. Krug R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7578-7582Crossref PubMed Scopus (248) Google Scholar) reported the identification of the ubiquitin E2 enzyme UBC8 (UBCH8/UbcM8) as an ISG15-conjugating enzyme. Using the small interfering RNA (siRNA) approach, both groups indicated that UBC8 is a predominant ISG15 E2 enzyme for IFN-induced protein ISGylation in HeLa cells. Interestingly, UBC8 also functions as a ubiquitin E2 enzyme (18.Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), indicating the possible cross-talk of protein ISGylation and ubiquitylation. In the ubiquitin system, the transfer of ubiquitin from E2 enzymes to target proteins is mediated by E3 enzymes, which interact with both E2 and its target proteins and function as a bridge between E2 and the target protein (19.Hochstrasser M. Nat. Cell Biol. 2000; 2: E153-E157Crossref PubMed Scopus (368) Google Scholar). UBC8 has been shown to interact with a number of ubiquitin E3 ligases to mediate protein ubiquitylation, such as E6AP, HHARI, Parkin, cIAP, Dorfin, EFP, RLIM, and Staring (18.Kumar S. Kao W.H. Howley P.M. J. Biol. Chem. 1997; 272: 13548-13554Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 20.Moynihan T.P. Ardley H.C. Nuber U. Rose S.A. Jones P.F. Markham A.F. Scheffner M. Robinson P.A. J. Biol. Chem. 1999; 274: 30963-30968Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21.Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 22.Hu S. Yang X. J. Biol. Chem. 2003; 278: 10055-10060Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 23.Niwa J. Ishigaki S. Doyu M. Suzuki T. Tanaka K. Sobue G. Biochem. Biophys. Res. Commun. 2001; 281: 706-713Crossref PubMed Scopus (71) Google Scholar, 24.Urano T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Nature. 2002; 417: 871-875Crossref PubMed Scopus (308) Google Scholar, 25.Kramer O.H. Zhu P. Ostendorff H.P. Golebiewski M. Tiefenbach J. Peters M.A. Brill B. Groner B. Bach I. Heinzel T. Gottlicher M. EMBO J. 2003; 22: 3411-3420Crossref PubMed Scopus (441) Google Scholar, 26.Chin L.S. Vavalle J.P. Li L. J. Biol. Chem. 2002; 277: 35071-35079Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). The fact that UBC8 functions as a dual E2 enzyme for both ISG15 and ubiquitin raises the possibility that some UBC8-interacting ubiquitin E3 ligases can function as the ISG15 E3 ligase, mediating the interaction between UBC8 and the ISG15 target proteins and helping in the transfer of ISG15 from UBC8 to target proteins. Here, we report that a UBC8-interacting protein, EFP (estrogen-responsive finger protein), can help in the ISG15 modification of 14-3-3σ protein both in transfection experiments and in vivo. EFP depends on its RING finger domain to support the ISGylation of 14-3-3σ. These data indicate that EFP is an ISG15 E3 ligase for 14-3-3σ. This is the first study reporting the identification of ISG15 E3 in vivo, and we provide evidence that EFP protein is a common component in the ubiquitin and ISG15 conjugation pathways. Plasmid Construction—Plasmids pCAGGS-mISG15, pCAGGS-His6-mISG15, pFLAG-CMV2-UBC8, and pCAGGS-HA-UBE1L have been described previously (16.Kim K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (185) Google Scholar). pcDNA3-UBC8 and pcDNA3-UBE1L by the of UBC8 and UBE1L the EFP was the and the an 14-3-3σ was the was the was a from of from of Cell and 293T cells in with and cells in and cells in and by as described previously D.-E. Mol. Cell. Biol. PubMed Scopus Google Scholar). The MCF-7 cell was in with and cells by The in the which contains and with and M. B. K. Y. A. T. J. Res. 2003; PubMed Scopus Google Scholar). was from RNA from and MCF-7 cells was to the of RNA from each was on a and with cells with phosphate-buffered and in and was to cell and for with and and in and and cells in and with a of protein with the and in EFP and 14-3-3σ from the indicated ISG15 was by ISG15 has been described previously O.A. Yan M. Malakhov M.P. Yuan Y. Ritchie K.J. Kim K.I. Peterson L.F. Shuai K. Zhang D.-E. Genes Dev. 2003; 17: 455-460Crossref PubMed Scopus (265) Google Scholar). was from was as described previously M.P. Kim K.I. Malakhova O.A. Zhang D.-E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). RNA for expression was used for small hairpin RNA for The for are EFP and The for are and the for are and The with and and the the of EFP in the 293T cells with and expression and The expression of EFP and the ISGylation of 14-3-3σ the of EFP on IFN-induced 14-3-3σ ISGylation in vivo, we by of with the 293T cells. MCF-7 cells in with or cells with cells with PBS, and cells in cell MCF-7 cells and cell with the expression of EFP and the ISGylation of 14-3-3σ EFP upon the and of ISG15 are similar to of ubiquitin J. M. J.M. A.L. Kim J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), the mechanism of ISGylation is expected to be similar to that of protein ubiquitylation and is to the activity of the E1, E2, and E3 both ISG15 expression and protein ISGylation are highly upon Furthermore, all known components of the protein ISGylation system ISG15, UBE1L, UBP43, and are proteins K.J. Zhang D.-E. Semin. Cell Dev. Biol. 2004; 15: 237-246Crossref PubMed Scopus (119) Google Scholar, K.I. Malakhova O.A. K. Yan M. B. Zhang D.-E. J. 2005; PubMed Scopus Google Scholar). that ISG15 E3 ligases are UBC8-interacting ubiquitin E3 ligases that also be by Based on T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Nature. 2002; 417: 871-875Crossref PubMed Scopus (308) Google Scholar, M. B. K. Y. A. T. J. Res. 2003; PubMed Scopus Google Scholar) and information from we identified one protein that the In expression et al. M. B. K. Y. A. T. J. Res. 2003; PubMed Scopus Google Scholar) that EFP is one of the IFN-stimulated in cell and EFP is by treatment, analysis was with RNA from cells and MCF-7 cells. EFP has been reported to be an in MCF-7 cells K. Inoue S. A. M. T. K. Muramatsu M. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar). that EFP be by a EFP and the ISG15 also by as a the of EFP is the of protein analysis was with from cells and MCF-7 cells. that the protein of EFP, a a ISG15 protein was also by treatment. The of to ISG15 modification, ISG15, UBE1L, UBP43, and the IFN-stimulated response K.J. Zhang D.-E. Semin. Cell Dev. Biol. 2004; 15: 237-246Crossref PubMed Scopus (119) Google Scholar, K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (185) Google Scholar), which is for by type I its interaction with the IFN-stimulated and Jr., PubMed Scopus Google Scholar, Y. T. M. Y. T. T. EMBO J. 1999; PubMed Scopus Google Scholar). shown in the of the EFP K. Inoue S. A. M. T. K. Muramatsu M. Biochem. Biophys. Res. Commun. 1997; PubMed Scopus Google Scholar) also contains an The that the EFP contains the sequence and that EFP protein is by support the of EFP protein in IFN-stimulated 14-3-3σ by ISG15 in the 293T protein is a RING ubiquitin E3 ligase that 14-3-3σ and 14-3-3σ in a T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Nature. 2002; 417: 871-875Crossref PubMed Scopus (308) Google Scholar). EFP can interact with UBC8 its RING finger domain and the transfer of ubiquitin from UBC8 to 14-3-3σ T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Nature. 2002; 417: 871-875Crossref PubMed Scopus (308) Google Scholar). Recently, UBC8 has been identified as an ISG15 enzyme (16.Kim K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (185) Google Scholar, C. Beaudenon S.L. Kelley M.L. Waddell M.B. Yuan W. Schulman B.A. Huibregtse J.M. Krug R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7578-7582Crossref PubMed Scopus (248) Google Scholar). These support the possible role of EFP in the ISG15 modification of 14-3-3σ protein. 14-3-3σ be by ISG15, we UBE1L, and 293T cells. and proteins by and the was used in analysis to 14-3-3σ. shown in 14-3-3σ with of ISG15 the a that the of a 14-3-3σ protein was This was in cell with 14-3-3σ and ISG15 Expression of UBE1L and UBC8 the of this and with of UBE1L and UBC8 expression strongly the of 14-3-3σ The of 14-3-3σ protein by in the set of the of 14-3-3σ protein of 14-3-3σ by by of 14-3-3σ for The was is known that proteins can and that is B. Jones Y. A. Nature. PubMed Scopus Google Scholar). is that 14-3-3σ can with which was also by the interaction with the ISGylation of 14-3-3σ. 293T cells with UBE1L and and expression as to and with expression and protein ISGylation as described in the to EFP not and can support 14-3-3σ 293T cells with and expression as to and with The expression of proteins transfection was by 14-3-3σ by ISG15 upon in MCF-7 14-3-3σ is to ISG15 modification, we with and MCF-7 cells with or by The by ISG15 or 14-3-3σ. shown in a with the of 14-3-3σ with a ISG15 was in the but not in the conjugation was not in the of the The also by shown in an was in the but not in the The of this to the ISG15 in The of the in to that of the predominant 14-3-3σ in the 293T ISG15-conjugating system described in also the ISGylation of 14-3-3σ in HeLa cells and cells not EFP ISGylation of that 14-3-3σ is a target of ISG15 modification to EFP can function as an ISG15 E3 ligase for 14-3-3σ. first used the 293T system to the of EFP on 14-3-3σ was in 293T cells with the UBE1L, and UBC8 expression in the or of The proteins with and by described 14-3-3σ was it was with UBE1L, and UBC8 the of the of 14-3-3σ was by expression with the 14-3-3σ signal was also was with and EFP expression this signal and not in of 14-3-3σ by by of 14-3-3σ. The cell by for of and ISG15 conjugation EFP the of it not the of protein the of EFP in the ISGylation of the roles of two other UBC8-interacting ubiquitin E3 and HHARI, T.P. Ardley H.C. Nuber U. Rose S.A. Jones P.F. Markham A.F. Scheffner M. Robinson P.A. J. Biol. Chem. 1999; 274: 30963-30968Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 21.Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). shown in the expression of only EFP but not or and in of the of EFP in for EFP the IFN-induced ISGylation of that EFP 14-3-3σ ISGylation in 293T cell transfection we EFP in a in 14-3-3σ ISGylation in cells. cellular EFP expression the a expression or R. R. 2002; PubMed Scopus Google Scholar). we the expression of EFP and the ISGylation of 14-3-3σ in 293T cells. for and 293T cells with of a of and of EFP the of EFP protein and 14-3-3σ ISGylation in a the of EFP on IFN-induced ISGylation of MCF-7 cells with and that also express the the cells with PBS, and cells in cell The of EFP and proteins shown in the EFP protein was strongly by expression and by expression in cells. interferon treatment, the protein of EFP was and the EFP expression was clearly by EFP The expression of both on the 14-3-3σ protein However, the of ISGylation was decreased by EFP expression confirmed that the of 14-3-3σ was in the and These that EFP is necessary in for 14-3-3σ The RING for EFP to as an ISG15 E3 E3 been two the and RING finger which the conserved and RING finger domains, (3.Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar). RING are formed by conserved and that two J. Mol. Biol. 2000; PubMed Scopus Google Scholar). study has shown that the RING finger domain is important for EFP in the interaction with UBC8 and in the of 14-3-3σ T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Nature. 2002; 417: 871-875Crossref PubMed Scopus (308) Google Scholar). the ISG15 E3 ligase activity of EFP is RING finger we a number of EFP and their E3 ligase for 14-3-3σ 293T cells with and with EFP expression RING EFP not support the ISGylation of 14-3-3σ of conserved in the EFP RING finger domain with also the function of EFP and the the RING finger domain not support the ISGylation of 14-3-3σ. These that the RING finger domain is for EFP to function as an ISG15 E3 ligase for 14-3-3σ. The protein ISG15 is highly upon (1.Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar, P.J. Broeze R.J. Lengyel P. Nature. 1979; 279: 523-525Crossref PubMed Scopus (200) Google Scholar, D.C. Jr., J. Biol. Chem. Full Text PDF PubMed Google Scholar) and is as a free protein and a target in cells A.L. J. Biol. Chem. Full Text PDF PubMed Google Scholar). ISG15 has been known for (5.Farrell P.J. Broeze R.J. Lengyel P. Nature. 1979; 279: 523-525Crossref PubMed Scopus (200) Google Scholar), the process of ISGylation and the of ISGylation have not been well Recently, on ISGylation has been by the identification of the enzymes involved in the ISG15 conjugation the ISG15 E1 enzyme UBE1L, the E2 enzyme and the de-ISGylating enzyme UBP43 (12.Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (420) Google Scholar, 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, K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (185) Google Scholar, C. Beaudenon S.L. Kelley M.L. Waddell M.B. Yuan W. Schulman B.A. Huibregtse J.M. Krug R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7578-7582Crossref PubMed Scopus (248) Google Scholar). number of ISG15 target proteins to with A.L. L. P. R. A. J. 2002; PubMed Scopus Google Scholar) and and M.P. Kim K.I. Malakhova O.A. Zhang D.-E. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), by proteins that function in diverse cellular RNA stress and N.V. Papov V. W. Li J. Virgin H.W. Zhang D.-E. Biochem. Biophys. Res. Commun. 2005; 336: PubMed Scopus Google Scholar, C. C. Huibregtse J.M. S. Krug R.M. Proc. Natl. Acad. Sci. U. S. A. 2005; PubMed Scopus Google Scholar). Based on the between the ubiquitin and ISG15 we that ISG15 E3 ligases are members of the ubiquitin E3 interact with the ISG15 E2 enzyme and be by Recently, E3, Zhao et al. (17.Zhao C. Beaudenon S.L. Kelley M.L. Waddell M.B. Yuan W. Schulman B.A. Huibregtse J.M. Krug R.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 7578-7582Crossref PubMed Scopus (248) Google Scholar) that ISG15 conjugation can be by a ubiquitin E3 ligase in However, are in data this In this we have that the RING finger ubiquitin E3 ligase EFP can ISG15 conjugation to 14-3-3σ upon treatment. have also that the RING which is for the ubiquitin E3 ligase activity of EFP T. Saito T. Tsukui T. Fujita M. Hosoi T. Muramatsu M. Ouchi Y. Inoue S. Nature. 2002; 417: 871-875Crossref PubMed Scopus (308) Google Scholar), is for 14-3-3σ These that EFP as an ISG15 E3 ligase for 14-3-3σ. this is the first report on the identification of an ISG15 E3 ligase in vivo. These indicate that the pathways of ubiquitin and ISG15 in vivo. UBC8 and its E3 ligase EFP have the to both ubiquitin and ISG15 are the of enzymes for their in ubiquitin or ISG15 is a highly conserved is and and roles in cellular (3.Pickart C.M. Annu. Rev. Biochem. 2001; 70: 503-533Crossref PubMed Scopus (2922) Google Scholar, L. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In ISG15 has been identified only in and its sequence is that of ubiquitin Zhang D.-E. 2005; PubMed Scopus Google Scholar), suggesting that ISG15 not have ISG15 expression is and is strongly during stress type I in the of ISG15, UBC8 and EFP can function as ubiquitylation Upon the of ISG15 also as protein ISGylation members of the ubiquitin E2 family are in a These E2 enzymes can activated ubiquitin from However, only a of members is the only identified can activated ISG15 from ISG15 with ubiquitin for its E2 it is highly Furthermore, the of ubiquitin or ISG15 in the specific functions of EFP and other UBC8-interacting E3 The fact that both UBC8 and EFP are the common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental in distinct ISG15 expression and ISGylation are by type I Expression of all known components of the ISGylation system, ISG15, UBE1L, UBP43, and is by K.J. Zhang D.-E. Semin. Cell Dev. Biol. 2004; 15: 237-246Crossref PubMed Scopus (119) Google Scholar, K.I. Giannakopoulos N.V. Virgin H.W. Zhang D.-E. Mol. Cell. Biol. 2004; 24: 9592-9600Crossref PubMed Scopus (185) Google Scholar, K.I. Malakhova O.A. K. Yan M. B. Zhang D.-E. J. 2005; PubMed Scopus Google Scholar). The of the ISRE, which is for by type I In this we have also shown that EFP expression is upon and that the of EFP contains an EFP is also an S. A. Hosoi T. S. H. T. A. Ouchi Y. H. Muramatsu M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar). EFP protein is its of expression is not high in Suzuki T. Inoue S. K. A. H. S. T. H. Mol. Cell. 2004; PubMed Scopus Google Scholar). is possible that its expression in of cells is on type I and that the function of EFP in cells is to ISGylation of its The function of ISG15 modification has been an for ISG15 has been in a of on its to ubiquitin and other ubiquitin-like proteins, the and analysis of A. O. S. I. Mol. Cell. Biol. 2005; PubMed Scopus Google Scholar) and ISG15 K.I. Yan M. Malakhova O. W. Zhang D.-E. Cell. Biol. PubMed Scopus Google Scholar) not suggesting that ISG15 is a process that is by be to the of ISGylation for some target proteins. In this we have identified 14-3-3σ as a in target of ISG15 we have that EFP is an ISG15 E3 ligase for 14-3-3σ. the number and of and the of proteins in cellular PubMed Scopus (185) Google Scholar, and G. B. Scholar), is an target for study of the of ISG15 These findings analysis and of the of protein for and for the for and members of the D.-E. for and the