Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION The binding of eukaryotic translation initiation factor ISG15 modification of the 4F (eIF4F) to the mRNA 5Ј cap structure is the rate-lim- eIF4E cognate 4EHP enhances iting step of cap structure-dependent translation initia- tion in eukaryotes (Gingras et al. 1999). eIF4F contains cap structure-binding activity cap-binding protein eIF4E, scaffold protein eIF4G, and of 4EHP RNA helicase eIF4A. The interaction between eIF4E and eIF4G results in a conformational change of both pro- Fumihiko Okumura, Weiguo Zou, teins and enhances the association between eIF4E and 1 the RNA cap structure (Gross et al. 2003). There are and Dong-Er Zhang three eIF4E-family members in mammals termed Department of Molecular and Experimental Medicine, The eIF4E-1 (eIF4E), eIF4E-2 (4EHP and 4E-LP), and eIF4E-3 Scripps Research Institute, La Jolla, California 92037, USA (Rom et al. 1998; Joshi et al. 2004). Like prototypical eIF4E, 4EHP is expressed ubiquitously; however, expres- The expression of the ubiquitin-like molecule ISG15 and sion of eIF4E-3 is detected only in heart, skeletal muscle, protein modification by ISG15 (ISGylation) are strongly lung, and spleen (Joshi et al. 2004). Similar to eIF4E, both Ј activated by interferon, genotoxic stress, and pathogen 4EHP and eIF4E-3 bind to the RNA 5 cap structure (Rom et al. 1998; Joshi et al. 2004). Furthermore, both eIF4E infection, suggesting that ISG15 plays an important role -؅ and eIF4E-3 are able to bind to eIF4G to facilitate trans in innate immune responses. 4EHP is an mRNA 5 cap lation initiation (Rom et al. 1998; Joshi et al. 2004). How- structure-binding protein and acts as a translation sup- ever, 4EHP does not interact with eIF4G (Rom et al. pressor by competing with eIF4E for binding to the cap 1998; Joshi et al. 2004) and thus cannot function in ribo- structure. Here, we report that 4EHP is modified by some recruitment. This suggests that 4EHP competes ISG15 and ISGylated 4EHP has a much higher cap struc- with eIF4E or eIF4E-3 for binding to the RNA 5Ј cap ture-binding activity. These data suggest that ISGylation structure and prevents translation. In fact, the Dro- of 4EHP may play an important role in cap structure- sophila 4EHP homolog (d4EHP) has recently been re- dependent translation control in immune responses. ported to be an mRNA-specific translation inhibitor (Cho et al. 2005). Supplemental material is available at http://www.genesdev.org. Here, we report that 4EHP is a target of ISG15 modi- fication. The UbcH8-interacting E3 ligase HHARI (hu- Received July 19, 2006; revised version accepted December man homolog of Drosophila ariadne) (Moynihan et al. 19, 2006. 1999; Tan et al. 2003) promotes ISGylation of 4EHP. More importantly, ISG15 modification of 4EHP substan- ISG15 was the first reported ubiquitin-like modifier (ubl) tially enhances its cap structure-binding activity. This is (Haas et al. 1987). It forms covalent conjugates with cel- the first report that shows “gain of function” due to lular proteins similar to protein ubiquitylation (Loeb and ISG15 modification and suggests a role for protein Haas 1992). ISG15 expression and protein modification ISGylation in cap structure-dependent translation con- by ISG15 (ISGylation) are strongly activated by Type I trol in innate immune responses. interferon (IFN) (Farrell et al. 1979; Loeb and Haas 1992). IFNs are critical cytokines involved in innate immune Results and Discussion responses (Garcia-Sastre and Biron 2006). These facts suggest that ISG15 modification may modulate certain 4EHP is an ISG15 target protein immune responses related to pathogen infections and Given that UbcH8 is able to act as a conjugating enzyme various stresses. Similar to protein ubiquitylation, in both the ubiquitylation and ISGylation pathways, we ISGylation is also regulated by a set of enzymes. UBE1L studied whether the UbcH8-interacting RING–IBR– is an ISG15 E1 enzyme, which shows high homology RING-type ubiquitin ligase HHARI can function as an with the ubiquitin E1 (Yuan and Krug 2001). USP18 ISG15 ligase to modify its target protein 4EHP (Moyni- (UBP43) has been identified as a deISGylating isopepti- han et al. 1999; Tan et al. 2003). 4EHP could be modified dase (Malakhov et al. 2002). Recently, ubiquitin E2 en- by ISG15 with or without exogenous expression of zymes UbcH6 and UbcH8 have been determined to func- HHARI when 4EHP was overexpressed in the presence of tion also as ISG15 conjugating enzymes (Kim et al. 2004; ISG15 E1 and E2. We observed two ISGylated Flag-4EHP Zhao et al. 2004; Takeuchi et al. 2005). UbcH8 is re- bands (Fig. 1A). ISGylation of 4EHP was slightly en- ported to interact with several ubiquitin E3 ligases to hanced by HHARI expression (Fig. 1A,B). However, mediate protein ubiquitylation (Dao and Zhang 2005). HHARI(⌬C) that lost the UbcH8-interacting RING–IBR– The fact that UbcH8 functions as a dual E2 enzyme for RING domain (Moynihan et al. 1999; Tan et al. 2003) did both ubiquitin and ISG15 raises the possibility that some not increase the ISGylation of 4EHP (Fig. 1B). We further UbcH8-interacting ubiquitin E3 ligases can function as examined the ISGylation of endogenous 4EHP (Fig. 1C). an ISG15 E3 ligase. In fact, we have recently reported ISGylation of endogenous 4EHP was clearly detected that the UbcH8 partner Efp ubiquitin ligase functions as with the expression of HA-HHARI. These data showed ␴ an E3 for 14–3–3 ISGylation (Zou and Zhang 2006). that HHARI is an ISG15 ligase for 4EHP. Endogenous [Keywords: ISG15 (UCRP); 4EHP; ubiquitin; ISGylation; RNA cap struc- 4EHP also gave two bands of ISGylation signal, even ture; mRNA translation; interferon] though the top band overlapped with a nonspecific band 1Corresponding author. E-MAIL [email protected]; FAX (858) 784-9593. (Fig. 1C). We also studied the ISGylation of endogenous Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1521607. 4EHP in response to IFN stimulation using UBP43 GENES & DEVELOPMENT 21:255–260 © 2007 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/07; www.genesdev.org 255 Downloaded from genesdev.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Okumura et al. tion using Ni-NTA agarose and anti-Flag M2 agarose (Fig. 2A). Bands A and B were subjected to mass spec- trometry analysis. As shown in Figure 2B, the peptides from band A are underlined (#A1–A6), and those from band B are boxed (#B1–B4). If any lysine residue of 4EHP was modified by ISG15, the modified lysine residue could not be recognized by trypsin and the unique pep- tide that contains an additional Gly–Gly from ISG15 might be detected by nano LC-MS/MS analysis (Zou et al. 2005). The mass of one peptide (#B3) from band B matched the Gly–Gly modification (Fig. 2B). However, we could not detect the peptide that was modified by ISG15 in band A. To confirm that the candidate Lys 222 is one of the ISGylation sites, we mutated this Lys to Arg, and then examined whether this mutant 4EHP(K222R) was modified by ISG15 (Fig. 2C). Mutant 4EHP(K222R) lost the bottom ISGylated band and still kept the top ISGylated band. This result demonstrates Figure 1. 4EHP is an ISGylation target. (A) ISGylation of 4EHP. 293T cells were transfected with HA-HHARI, Flag-4EHP, UBE1L, UbcH8, and His6-ISG15 expression plasmids as indicated. Thirty-six hours after transfection, cell lysates were subjected to Ni-NTA pull- down and immunoblot (IB) analysis with anti-Flag antibody. The positions of unmodified and ISGylated 4EHP are shown on the right side as well as the molecular weight markers (in kilodaltons). (B) HHARI enhances ISGylation of 4EHP. Either Flag-HHARI (WT) or C-terminal-deleted Flag-HHARI (⌬C) was transfected to 293T cells with HA-4EHP and the His6-ISGylation system (UBE1L, UbcH8, and His6-ISG15). Cell lysates were subjected to Ni-NTA pull-down and immunoblot (IB) analysis. (C) ISGylation of endogenous 4EHP. 293T cells were transfected with UBE1L, UbcH8, HA-HHARI, and His6-ISG15 expression plasmids as indicated. Cell lysates were sub- jected to Ni-NTA pull-down and immunoblot (IB) analysis. (D) ISGylation of endogenous 4EHP dependent on IFN␣ treatment. KT-1dUBP43 cells were stimulated by 1000 U/mL of IFN␣-2a for 48 h. The cell lysates were subjected to immunoprecipitation (IP) with anti-ISG15 antibody and immunoblot (IB) analysis. (E) eIF4E is not ISGylated. 293T cells were transfected with HA-eIF4E or HA-4EHP with or without the His6-ISGylation system as indicated. Cell ly- sates were subjected to Ni-NTA pull-down and immunoblot (IB) analysis. The positions of unmodified eIF4E and 4EHP are shown as well as ISGylated 4EHP on the right side. knockdown KT-1/A3 cells, which give more ISGylated pro- Figure 2. The ISGylation sites of 4EHP. (A) Coomassie staining of tein than parental cells (Fig. 1D). Two bands of ISGylated purified Flag-4EHP and ISGylated Flag-4EHP. Both bands A and B, 4EHP protein were also detected on a Western blot. To- which are ISGylated Flag-4EHP, were subjected to nano LC-MS/MS gether, these results indicate that 4EHP is an ISG15 tar- analysis. (B) Identified peptides by nano LC-MS/MS analysis. Pep- tides that came from band A are underlined (#A1–A6), and those that get and HHARI functions as an ISG15 E3 ligase for 4EHP came from band B are boxed (#B1–B4). The lysine residue that was modification in the presence of the ISGylation system. identified to be modified by Gly–Gly is indicated by the arrow.