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Functional Characterization of Nedd4.1 and Nedd4.2 HECT Domain Ligases
Author(s) -
AugustusWallace Allison Charlemagne,
Klein Jennifer M,
Haas Arthur L
Publication year - 2011
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.25.1_supplement.519.2
Subject(s) - nedd4 , domain (mathematical analysis) , ubiquitin , chemistry , ubiquitin ligase , biochemistry , mathematics , gene , mathematical analysis
Nedd4.1 and Nedd4.2 are distinct but evolutionarily‐related HECT domain ubiquitin ligases that function in fundamental cellular processes including mitosis, neuronal development, tumorigenesis and viral infection. Nedd4.1 targets substrates such as PTEN for 26S proteasome‐dependent degradation via formation of K48‐linked ubiquitin chains, while Nedd4.2 targets substrates for internalization and endosomal trafficking by formation of K63‐linked chains. The present study exploits the ability of HECT ligases to form free polyubiquitin chains in the absence of their cognate substrates as a reporter function in functional rate studies. In vitro screens demonstrate that both ligases show broad qualitative specificity for E2 paralogs. Quantitative rate studies demonstrate that Nedd4.1 is specific for HsUbc5B (K m = 13 ± 8 nM; k cat = 3.2 ± 2.0 × 10 −2 s −1 ) and its orthologs while Nedd4.2 is specific for HsUbcH7 (K m = 196 ± 71 nM, k cat = 8.9 ± 0.2 × 10 −2 s −1 ). Other studies indicate that C2‐HECT domain interactions that block substrate conjugation have no effect on the catalytic activity of Nedd4.2 in assembling polyubiquitin chains. These studies represent the first detailed mechanistic characterization of Nedd4.1 and Nedd4.2 that resolve a number of contradictory questions regarding substrate specificity. [Supported by GM034009 to A.L.H.]