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. 2014 Dec 20;21(18):2498–2514. doi: 10.1089/ars.2014.5843

FIG. 5.

FIG. 5.

PTEN targets the Neh6 degradation domain of NRF2. (A) GSK-3β phosphorylates NRF2 at Ser335 and Ser338. HEK293T were transfected with fusion protein EYFP-Neh6EK/FK-V5 under conditions of GSK-3 inhibition (co-transfection of hypomorphic GSK-3βY216 mutant plus incubation with 10 μM SB216763) and activation (co-transfection of active GSK-3βΔ9). MG132 (30 μM) was added to prevent protein degradation. Mass spectrometry analysis identified five phosphorylated Ser residues at positions 335, 338, 342, 347, and 365 (indicated by *) but phosphorylation of Ser335 and Ser338 was found only under conditions of GSK-3 stimulation. Annotated mass spectra of the identified phosphorylated peptides are provided in Supplementary Tables S3–S12. (B) PTEN induces the degradation of NRF2 at the level of its Neh6 phosphodegron. HEK293T cells were co-transfected with expression vectors for CFP-Neh2, EGFP-Neh6, EGFP, and either empty vector or myr-PTEN. Then, cells were maintained in low-serum for 16 h and pulse-chased with 100 μg/ml CHX. Whole-protein lysates were immunoblotted with anti-GFP antibody (upper blot) or anti-PTEN antibody (lower blot) showing over-expression of myr-PTEN. (C, D) Determination of half lives. Graphs depict the natural logarithm of the relative protein levels of the indicated proteins as a function of CHX chase time. Protein half life was determined using the linear part of the degradation curves. (E) CFP-Neh2 but not EGFP-Neh6 is modulated by KEAP1. HEK293T cells were co-transfected with expression vectors for CFP-Neh2, EGFP-Neh6, and EGFP and either vector or HA-tagged KEAP-1. Then, the cells were maintained in low-serum medium for 16 h and pulse-chased with 100 μg/ml CHX at the indicated time points. (F, G) Determination of half lives. (H) EGFP-Neh6 but not CFP-Neh2 is modulated by GSK-3β/β-TrCP. HEK293T cells were co-transfected with expression vectors for CFP-Neh2, EGFP-Neh6, EGFP, Flag-β-TrCP, and either HA-tagged GSK-3βY216F (hypomorphic version of GSK-3β that retains only residual activity, plus incubation in 10 μM SB216763) or GSK-3βΔ9 (constitutively active version) and pulse-chased with CHX. (I, J) Determination of half lives. β-TrCP, beta-transducin repeat containing protein; CFP, cyan fluorescent protein; CHX, cycloheximide; EGFP, enhanced green fluorescent protein; EYFP, enhanced yellow fluorescent protein.