Nedd4 (Nedd4-1) is an E3 ubiquitin ligase involved in crucial biological processes such as growth factor receptor signaling. While canonical Nedd4-1 comprises a C2-WW(4)-HECT domain architecture, alternative splicing produces non-canonical isoforms that are poorly characterized. Here we characterized Nedd4-1(NE), a primate-specific isoform of Nedd4-1 that contains a large N-terminal Extension (NE) that replaces most of the C2 domain. We show that Nedd4-1(NE) mRNA is ubiquitously expressed in human tissues and cell lines. Moreover, we found that Nedd4-1(NE) is more active than the canonical Nedd4-1 isoform, likely due to the absence of a C2 domain-mediated autoinhibitory mechanism. Additionally, we identified two Thr/Ser phosphoresidues in the NE region that act as binding sites for 14-3-3 proteins, and show that phosphorylation on these sites reduces substrate binding. Finally, we show that the NE region can act as a binding site for the RPB2 subunit of RNA polymerase II, a unique substrate of Nedd4-1(NE) but not the canonical Nedd4-1. Taken together, our results demonstrate that alternative splicing of the ubiquitin ligase Nedd4-1 can produce isoforms that differ in their catalytic activity, binding partners and substrates, and mechanisms of regulation.
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