Figure 5.

MKRN1 functions as an E3 ligase by inducing ubiquitination and subsequent proteasomal degradation of p14ARF. A) MKRN1 induces proteasomal degradation of p14ARF. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, or GFP followed by 3-h treatment with 20 µM MG132 or LLnL. The lysates were immunoblotted with anti-FLAG, HA, and GFP antibodies. B) E3 ligase activity of MKRN1 is required for p14ARF degradation. H1299 cells were transfected with mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, HA/H307E (E3 ligase activity–deficient mutant), or GFP. The cell lysates were immunoblotted using anti-FLAG, HA, and GFP antibodies. C) Both MKRN1 WT and H307E interact with p14ARF. Mixtures of plasmids expressing p14ARF/FLAG, HA/MKRN1, or HA/H307E were transfected into 293T cells followed by immunoprecipitation using anti-HA antibodies and detection of the immunoprecipitated proteins with anti-FLAG and HA antibodies. D) MKRN1 ubiquitinates p14ARF. H1299 cells were transfected with mixtures of plasmids expressing His/Ub, p14ARF/FLAG, HA/MKRN1, or mock as indicated, with or without MG132. The cell lysates were pulled down with Ni²⁺-NTA resin and detected using anti-HA and FLAG antibodies. E) MKRN1 H307E is defective in inducing p14ARF ubiquitination. H1299 cells were transfected with mixtures of plasmids expressing His/Ub, p14ARF/FLAG, HA/MKRN1, or mock as indicated. The cell lysates were pulled down with Ni²⁺-NTA resin and detected with anti-FLAG and HA antibodies. F) MKRN1 depletion suppresses ubiquitination of endogenous p14ARF. HeLa cells were transfected with control or MKRN1 siRNAs followed by MG132 treatment. The cell lysates were immunoprecipitated with anti-p14ARF antibodies, and the immunoprecipitates were detected with anti-p14ARF and HA ubiquitin antibodies. All of the ubiquitination analyses were performed under conditions of denaturation. MKRN1 = Makorin ring finger protein 1; p14ARF = p14 alternative reading frame; Ni²⁺-NTA = Ni²⁺-nitrilotriacetic acid.