Fig 1: BIRC6 mono- and di-ubiquitinates KRAS4A(A) In vitro ubiquitination of the indicated, previously validated recombinant substrates (caspase-7 or LC3B) or a negative control (PKM2) incubated with recombinant Strep-BIRC6, FLAG-UBA6 (an E1 ubiquitin-activating enzyme), and HA-ubiquitin, with or without MgATP. Unmodified and ubiquitinated proteins are revealed by immunoblot (IB).(B) In vitro ubiquitination reactions as in (A) utilizing recombinant KRAS4A or KRAS4B as substrates. Unmodified and ubiquitinated KRAS proteins are detected by anti-RAS and anti-Ub immunoblots. Mono- and di-ubiquitinated KRAS is revealed by the merged immunoblots. The in vitro reaction shows no isoform specificity.(C) In vitro ubiquitination reactions as in (A) utilizing the recombinant KRAS4B G domain (aa 1–165) as substrate, FLAG-UBA6, and buffer (Cont) or the indicated BIRC6 mutant (BIRmut, BIRC6C328S/C331S; ΔHelix, BIRC6Δ1,616–1,666; ΔUbl, BIRC6Δ3,819–4,068). Proteins are revealed by Coomassie stain (top) and anti-RAS immunoblot (bottom). Mono-ubiquitinated KRAS is the predominant species.(D) KRAS4A ubiquitination in intact cells. HA-KRAS4A-12V was expressed in BIRC6-deficient HEK293 cells with 8xHis-Ub with or without GFP-BIRC6. Cell lysates were analyzed by anti-HA immunoblot before (input) or after enrichment for ubiquitinated proteins with a cobalt affinity matrix. Mono-, di-, tri-, and polyubiquitinated KRAS4A were detected with anti-HA and anti-Ub immunoblots with mono- and di-ubiquitinated species predominant. Tubulin served as a loading control.(E) HA-KRAS4A-12V was expressed with 8×His-Ub in HEK293 cells expressing a CRISPR-Cas9 control single guide RNA (sgRNA) targeting Tomato (sgTM) or an sgRNA targeting BIRC6 (sgB6). BIRC6 was reintroduced in sgB6 cells by expressing GFP-BIRC6. Tubulin served as loading control.(F) Ubiquitination of KRAS4A and KRAS4B in intact, BIRC6-deficient cells transfected with empty vector (EV) or GFP-BIRC6 (B6), was assayed as in (D) except KRAS-12V was His-tagged and no exogenous Ub was expressed. Vinculin served as a loading control. Immunoreactive bands were quantified by Li-Cor Odyssey scan, and the percentage of ubiquitinated proteins was calculated as 100 × (mono + di + tri)/(unmodified + mono + di + tri) and plotted on the right (mean ± SD, n = 7 for KRAS4A and n = 4 for KRAS4B, **p < 0.01, Student’s t test).Experiments in (A)–(E) are representative of n ≥ 3. See also Figure S5.