Figure 5.

Phosphorylation of USP48 by GSK3β increases USP48 activity. A) Inhibition of GSK3β has no effect on association between USP48 and TRAF2. Beas2B cells were treated with or without TWS119 (10 μM) for 1 h. Cell lysates were then immunoprecipitated with TRAF2 antibody, followed by immunoblotting with USP48 antibody. Input cell lysates were immunoblotted with USP48, TRAF2, and β-actin antibodies. B) Deletion of phosphorylation motif of USP48 has no effect on USP48 interaction with TRAF2. Beas2B cells were transfected with empty vector, Usp48-v5, or Usp48Δ886–890-v5 as indicated and incubated for 48 h. Cell lysates were then immunoprecipitated with TRAF2 antibody, followed by immunoblotting with V5 antibody. Input cell lysates were immunoblotted with V5, TRAF2, and β-actin antibodies. C) Inhibition of GSK3β attenuates USP48 effect on deubiquitination of TRAF2. Beas2B cells were transfected with empty vector or Usp48-v5 and incubated for 48 h. Cells were then treated with or without TWS119 (10 μM) for 1 h. Denatured cell lysates were subjected to immunoprecipitation with TRAF2 antibody, followed by immunoblotting with ubiquitin antibody. Input lysates were immunoblotted with V5, TRAF2, and β-actin antibodies. D) Phosphorylation motif deletion mutant of USP48 loses DUB activity. Recombinant USP48 wild-type protein, USP48C98S mutant protein, and USP48Δ886–890 mutant protein cleavage of Lys48 Ub chain linkages were analyzed by immunoblotting with ubiquitin antibody. V5 antibody was used for input lysate immunoblotting. E) GSK3βS9A increases USP48 DUB activity. Dimeric Ub cleavage analysis was performed of immunoprecipitates derived from recombinant USP48 incubated with or without GSK3βS9A. Reaction mixtures were analyzed with ubiquitin antibody. V5 and HA antibodies were used for input lysate immunoblotting (n = 3). *P < 0.01 compared to Usp48-v5. Shown are representative blots from at least 3 independent experiments. F) Scheme showing GSK3β-mediated USP48 phosphorylation increases USP48 DUB activity.