Figure 3: Usp9x deubiquitinates Ets-1 and regulates its degradation. | Nature Communications

Figure 3: Usp9x deubiquitinates Ets-1 and regulates its degradation.

From: Usp9x regulates Ets-1 ubiquitination and stability to control NRAS expression and tumorigenicity in melanoma

Figure 3

(a) Heat maps of differentially ubiquitinated proteins. NRAS mutant SK-Mel147 cells were exposed to control and Usp9x KD or G9 treatment as noted. The number of unique peptides and proteins reproducibly detected is shown. (b) Schematic diagram of the human Ets-1 protein showing the PNT (pointed domain, aa 53–136), TAD (transactivation domain, aa 137–242) and ETS domains. The putative site of ubiquitination (MNYEK*LSR) in human Ets-1 is shown and is conserved in mammalian species (right). (c) Immunoblot of ETS family proteins and NRAS in NRAS mutant melanoma cells with and without Usp9x KD. Actin served as a loading control. (d) Reciprocal immunoprecipitation of Usp9x and Ets-1 with endogenous Ets-1 and Usp9x in NRAS mutant SK-Mel2 cells. Immunoblotting was performed to detect Ets-1 or Usp9x in pulldowns and a portion of the input sample. (e) Top—Ectopically expressed FLAG-Usp9x (full-length) or FLAG-Usp9x-CDM (catalytic domain mutant, C1566A) was co-expressed with HA-Ets-1 in HEK293T cells. HA (Ets-1) immunoprecipitation was followed by immunoblotting of FLAG (Usp9x—top) or HA (Ets-1—bottom). Input lysate was also immunoblotted. Center—Ectopically expressed FLAG-Usp9x deletion constructs (FLAG-Usp9x E1, FLAG-Usp9x E1/CDM (catalytic domain mutant—C1566A), FLAG-Usp9x E5 (C-terminal deletion)) (illustrated in the bottom panel) were co-expressed with HA-Ets-1 in HEK293T cells. HA (Ets-1) immunoprecipitation was followed by FLAG (Usp9x) or HA (Ets-1) immunoblotting. Input lysate was also immunoblotted. Bottom—Map and summary of the Usp9x deletion constructs and their Ets-1 binding activity. The position of the ubiquitin C-terminal hydrolase (UCH) in the catalytic domain is shown by bold letters. Numbers and letters designate highlighted amino acids. (f) Immunoblot for Usp9x, Ets-1 and actin in control and Usp9x KD WM1366 NRAS mutant cells treated±MG132 for 8 h (10 μM). (g) HEK293T cells ectopically expressing FLAG-Ets-1 and HA-Ubiquitin were subjected to control or Usp9x KD (left) or treated with vehicle or G9 (2.5 μM, 6 h—right). FLAG immunoprecipitation was followed by HA blotting to detect Ub-Ets-1 levels. Immunoblot for FLAG (Ets-1) in the pulldowns (top) and input lysate (Usp9x and actin—bottom) is shown.

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