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Neddylation‐Deficient K712R CUL3 Δ403‐459 Double Mutant Attenuates Proteasome‐Dependent KLHL3 Degradation
Author(s) -
Cornelius Ryan J.,
Zhang Chong,
Erspamer Kayla J.,
Terker Andrew S.,
Singer Jeffrey D.,
Yang ChaoLing,
Ellison David H.
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.965.6
Subject(s) - neddylation , mg132 , cullin , ubiquitin , biology , microbiology and biotechnology , ubiquitin ligase , proteasome , biochemistry , proteasome inhibitor , gene
Cullin 3 (CUL3) is a member of the cullin RING E3 ubiquitin ligase family that mediates ubiquitylation and degradation of multiple cellular proteins via the proteasome. CUL3 facilitates ubiquitylation‐dependent WNK kinase degradation through the substrate adaptor Kelch‐like 3 (KLHL3). Activation of CUL3 requires a covalent modification called neddylation, in which a NEDD8 molecule is attached to a specific lysine residue. A form of Familial hyperkalemic hypertension (FHHt, or pseudohypoaldosteronism type II) has been shown to result from deletion of CUL3 exon 9 (consisting of 57 amino acid residues; CUL3 Δ403‐459). We showed previously that CUL3 Δ403‐459 is more heavily neddylated; this led to enhanced KLHL3 degradation and increased abundance of WNKs. Here, we sought to determine the molecular mechanism for this effect. We transfected HEK293 cells with a FLAG‐tagged neddylation‐deficient CUL3 plasmid construct in which a point‐mutation (K712R) was introduced at the neddylation site. Immunoprecipitation demonstrated that neddylation was nearly absent in both WT K712R CUL3 and K712R CUL3 Δ403‐459. When compared to CUL3 Δ403‐459, K712R CUL3 Δ403‐459 exhibited less capacity to degrade KLHL3. As a result, WNK4 abundance was lower when KLHL3 was co‐expressed with the K712R CUL3 Δ403‐459, compared with CUL3 Δ403‐459. To examine the mechanism of KLHL3 degradation, we incubated cells with the proteasome inhibitor MG132 for 18 h. This almost completely inhibited KLHL3 degradation; in contrast, inhibition of autophagy with two separate drugs, chloroquine and 3‐methyladenine, had only a modest effect. The results indicate that, 1) neddylation at the 712 lysine residue is important for CUL3 Δ403‐459 function, and 2) KLHL3 degradation, mediated by CUL3 Δ403‐459, occurs predominately via the proteasomal pathway. In summary, the FHHt‐causing CUL3 Δ403‐459 mutation exhibits increased neddylation at lysine 712, which causes proteasomal‐dependent degradation of KLHL3. This likely leads to increased WNK kinase abundance and human disease. Support or Funding Information Work was supported by National Institutes of Health Grants 2R01DK051496‐15A1 and 5T32DK067864‐10.