Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme.

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.