Negative regulation of ISG15 E3 ligase EFP through its autoISGylation

Abstract

The function of ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) has been an enigma for many years. Recently, the research of ISGylation has been accelerated by the identification of the enzymes involved in the ISG15 conjugation process. Our previous study identified the interferon inducible protein EFP as an ISG15 isopeptide ligase (E3) for 14-3-3sigma. In this study, we show that ISG15 E3 ligase EFP can be modified by ISG15. Two ubiquitin E2 conjugating enzymes, UbcH6 and UbcH8, can support ISGylation of EFP. The Ring-finger domain of EFP is important for its ISGylation. Full-length EFP can enhance the ISGylation of Ring domain deleted EFP, indicating EFP can function as an ISG15 E3 ligase for itself. We also determined the ISGylation site of EFP and created its ISGylation resistant mutant EFP-K117R. Compared to the wild-type EFP, this mutant further increases the ISGylation of 14-3-3sigma. Thus we propose that autoISGylation of EFP negatively regulates its ISG15 E3 ligase activity for 14-3-3sigma.

Figure 1

EFP can be ISGylated in a 293T cell conjugation system. (A) 293T cells were transfected with Flag-EFP, HA-UBE1L and His-ISG15 expression constructs as indicated. Cells were harvested 48 hours after transfection. Protein of cell extracts (500 μg) was subjected to Ni-NTA pull down and Western blotted (WB) with Flag antibody. (B) Protein of cell extracts (500 μg) was subjected to immunoprecipitation with Flag antibody and Western blotted with ISG15 antibody. The positions of unmodified EFP and ISGylated EFP are indicated. (C) Twenty μg of protein from transfected cells was separated by SDS-PAGE and analyzed by Western blotting with antibodies specific for Flag (EFP), HA (UBE1L) and ISG15. (D) MCF-7 cells were treated with 0 (-), 500 (+), and 2,000 (++) U/ml of human IFN-α for 48 hours. Cell extracts (1,000 μg) were immunoprecipitated (IP) with EFP antibody and Western blotted with antibodies specific for ISG15. The proteins from 2,000 U/ml IFN-α treated cells were also immunoprecipitated with Flag antibody as a negative control. The positions of ISGylated EFP, and the immunoglobulin heavy chain (HC) are shown. (E) The expression of EFP and protein ISGylation were analyzed by Western blotting with antibodies specific for EFP and ISG15. The positions of molecular weight markers (kDa) are shown on the left side of each panel.

Figure 2

The effects of UbcH6, UbcH8 and RING-finger domain on EFP autoISGylation. 293T cells were transfected with expression plasmids encoding His-ISG15, HA-EFP, UBE1L, UbcH6 (A), and UbcH8 (B). Cells were harvested 48 hours after transfection. Proteins (500 μg) were subjected to Ni-NTA pull down and western blotted with HA. (C) Schematic representation of wild-type EFP and EFP mutants. The reported domains interacting with UbcH8 and 14-3-3σ are shown. (D) 293T cells were transfected with plasmids encoding His-ISG15, HA-EFP and HA-EFP mutants as indicated. Protein of cell extracts (500 μg) was subjected to immunoprecipitation with HA antibody and Western blotted with ISG15 antibody.

Figure 3

EFP can enhances the ISGylation of Ring domain deleted EFP. (A) 293T cells were transfected with plasmids encoding Flag-EFP, Flag-EFP(1-403) and HA-EFP constructs as indicated. Protein of cell extracts (500 μg) was subjected to immunoprecipitation with Flag antibody and Western blotted with HA antibody. (B) Schematic representation of different EFP constructs. The reported domains interacting with UbcH8 and 14-3-3σ are shown. (C) 293T cells were transfected with plasmids encoding His-ISG15, Flag-EFP, Flag-EFP (1-403) and HA-EFP-ΔRing constructs as indicated. 48 hours later, protein of cell extracts (500 μg) was subjected to Ni-NTA pull down and western-blotted with anti-HA antibody. Protein expression was analyzed by direct western blot with HA and Flag antibody. The amount of Ni-NTA enriched free ISG15 was shown by Ponceau staining.

Figure 4

ISGylation of EFP inhibits its ISG15 E3 ligase activity for 14-3-3σ. (A) 293T cells were transfected with HA tagged wt EFP (lanes 1 and 2) and mutant EFP-K117R (lanes 3 and 4) in the absence or presence of His-ISG15 as indicated. 48 hours after transfection, 500 μg of protein in cell lysates were subjected to Ni-NTA pull down and Western blotted with HA antibody. (B) 293T cells were transfected with plasmids encoding His-ISG15, HA-EFP, HA-EFP-K117R, and Flag-14-3-3σ as indicated. 48 hours later, protein of cell extracts (500 μg) was subjected to Ni-NTA pull down and western-blotted with anti-Flag and anti-HA antibody. Protein expression was analyzed by direct western blotting with HA and Flag antibody. The amount of Ni-NTA enriched free ISG15 was shown by Ponceau staining.