ISG15 modification of the eIF4E cognate 4EHP enhances cap structure-binding activity of 4EHP

Abstract

The expression of the ubiquitin-like molecule ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. 4EHP is an mRNA 5 cap structure-binding protein and acts as a translation suppressor by competing with eIF4E for binding to the cap structure. Here, we report that 4EHP is modified by ISG15 and ISGylated 4EHP has a much higher cap structure-binding activity. These data suggest that ISGylation of 4EHP may play an important role in cap structuredependent translation control in immune responses.Supplemental material is available at http://www.genesdev.org.Received July 19, 2006; revised version accepted December 19, 2006. ISG15 was the first reported ubiquitin-like modifier (ubl) (Haas et al. 1987). It forms covalent conjugates with cellular proteins similar to protein ubiquitylation (Loeb and Haas 1992). ISG15 expression and protein modification by ISG15 (ISGylation) are strongly activated by Type I interferon (IFN) (Farrell et al. 1979;Loeb and Haas 1992). IFNs are critical cytokines involved in innate immune responses (Garcia-Sastre and Biron 2006). These facts suggest that ISG15 modification may modulate certain immune responses related to pathogen infections and various stresses. Similar to protein ubiquitylation, ISGylation is also regulated by a set of enzymes. UBE1L is an ISG15 E1 enzyme, which shows high homology with the ubiquitin E1 (Yuan and Krug 2001). USP18 (UBP43) has been identified as a deISGylating isopeptidase (Malakhov et al. 2002). Recently, ubiquitin E2 enzymes UbcH6 and UbcH8 have been determined to function also as ISG15 conjugating enzymes (Kim et al. 2004;Zhao et al. 2004;. UbcH8 is reported to interact with several ubiquitin E3 ligases to mediate protein ubiquitylation . The fact that UbcH8 functions as a dual E2 enzyme for both ubiquitin and ISG15 raises the possibility that some UbcH8-interacting ubiquitin E3 ligases can function as an ISG15 E3 ligase. In fact, we have recently reported that the UbcH8 partner Efp ubiquitin ligase functions as an E3 for 14-3-3 ISGylation (Zou and Zhang 2006).The binding of eukaryotic translation initiation factor 4F (eIF4F) to the mRNA 5Ј cap structure is the rate-limiting step of cap structure-dependent translation initiation in eukaryotes (Gingras et al. 1999). eIF4F contains cap-binding protein eIF4E, scaffold protein eIF4G, and RNA helicase eIF4A. The interaction between eIF4E and eIF4G results in a conformational change of both proteins and enhances the association between eIF4E and the RNA cap structure (Gross et al. 2003). There are three eIF4E-family members in mammals termed eIF4E-1 (eIF4E), eIF4E-2 (4EHP and 4E-LP), and eIF4E-3 (Rom et al. 1998;Joshi et al. 2004). Like prototypical eIF4E, 4EHP is expressed ubiquitously; however, expression of eIF4E-3 is detected only in heart, skeletal muscle, lung, and spleen (Joshi et al. 2004). Similar to eIF4E, both 4EHP and eIF4E-3 bind to the RNA 5Ј cap...

Activation of Double-stranded RNA-activated Protein Kinase (PKR) by Interferon-stimulated Gene 15 (ISG15) Modification Down-regulates Protein Translation

“…Plasmid Construction-Construction of pcDNA3.1-hUBE1L, pcDNA3.1-mUbcH8, and pCAGGS-His 6 -mISG15 was described previously (10). FLAG-Herc5 was described previously (13).…”

“…Human SUMO1 (NM_003352.4) cDNA was amplified by RT-PCR using the mRNA of HeLa cells. FLAG-SUMO1-PKR, FLAG-ISG15-PKR, and FLAG-PKR-ISG15 fusion proteins were constructed as described previously (10). Site-directed point mutations were generated by the QuikChange XL Site-directed Mutagenesis kit (Stratagene).…”

Expression, regulation and function of the ISGylation system in prostate cancer

“…So far, the biological function of ISGylation has only been elucidated for some cellular target proteins. These include the functional inhibition of enzymes Takeuchi et al, 2006;Zou et al, 2007;Malakhova and Zhang, 2008) and the enhancement of the cap structure-binding activity of the translational suppressor, 4EHP (Okumura et al, 2007). Recent studies have shown that ISGylation negatively regulates the ubiquitin-proteasome pathway by direct interference with polyubiquitination (Desai et al, 2006).…”

Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation

“…As a ubiquitin-like modifier, ISG15 can form covalent conjugates to many cellular proteins, a process termed "protein ISGylation" (12,13). A series of distinct enzymes are involved in the process of protein ISGylation, including ubiquitin-activating enzyme E1 homolog (UBE1L) (14,15), a conjugating enzyme, ubiquitin carrier protein L6 (UBCH8) (E2) (16,17), protein ligases (E3) (18)(19)(20)(21), and an ISG15-specific protease USP18 (22,23), as well as some viral proteins (24). Interestingly, ISG15 and the majority of its modification enzymes are encoded by ISGs (25), indicating that protein ISGylation is a tightly regulated process and plays important roles in immune responses (26).…”