Kaposi's Sarcoma-Associated Herpesvirus K-Rta Exhibits SUMO-Targeting Ubiquitin Ligase (STUbL) Like Activity and Is Essential for Viral Reactivation
Figure 4
K-Rta is a SUMO-targeting ubiquitin ligase.
In vitro ubiquitylation reactions were reconstituted with purified E1 (Ube1), E2 (UbcH5a), E3 (K-Rta) and HA-Ubiquitin. (A) SUMO-chains ubiquitylation. (a) Ubiquitylation was examined with anti-HA antibody. SUMO-chain and K-Rta were probed with anti-SUMO and anti-K-Rta antibodies, respectively (b, c). (B) In vitro ubiquitin reactions were performed using GST- SUMO-2 or GST-SUMO-2 chain as substrates. Purified GST-SUMO-2 and GST-SUMO-2 chain used in the reaction is shown in right panel. After ubiquitylation reactions, GST- SUMO beads were washed with high salt buffer (500 mM NaCl) containing 10% glycerol to eliminate any interacting protein. Covalently attached protein remained bound to GST-SUMO beads. The ubiquitylation reaction products were probed with an anti-HA antibody. Both SUMO and SUMO-chains were ubiquitylated only in the presence of K-Rta. (C) In vitro ubiquitylation reaction with K-Rta ΔSIM. (a) Ubiquitylation was examined with anti-HA antibody. The amount of K-Rta used in the reaction was shown with anti-K-Rta antibody. (b, c) The GST-SUMO-2 chain or His tagged artificial SUMO-2 chain was prepared and used as a substrate (left panels). Schematic diagram of SUMO-substrates is shown in bottom panels. Time course experiment was performed to examine efficacy of ubiquitinylation. The SUMO-substrates were isolated from the reaction at indicated time points by affinity resins, and ubiquitylation was examined by immunoblotting with anti-HA antibody. Small aliquots of reaction were taken from the reactions before isolation of SUMO-substrates and were used to confirm the amount of K-Rta in the reaction. (D) Inhibition of SUMO-conjugation in presence of K-Rta-Wt. In vitro SUMO-conjugation reactions were reconstituted and SUMO-2 was used as a substrate. SUMO mixture contains purified E1 (SAE1/2; 25 nM), E2 (Ubc9; 50 nM), 1 m ATP, and 2 µg of His-SUMO-2. Indicated antibodies were used to probe SUMO and K-Rta in the reaction (a). The intensity of the indicated bands was measured with image J program (b). (c) Purified PML protein was used as a substrate for SUMO-conjugation reaction. Ten microgram of purified PML protein was subjected to SDS-PAGE and stained with Coomassie (left panel). Indicated antibodies were used to probe PML and K-Rta in the reaction.