Substrate Specificity and Regulation of Nedd4 proteins

Nedd4-1 and Nedd4-2 are closely related E3 ubiquitin protein ligases that contain a C2 domain, 3-4 WW domains, and a catalytic ubiquitin ligase HECT domain. The WW domains of Nedd4 proteins recognize substrates for ubiquitination by binding the sequence L/PPxY (the PY-motif) found in target proteins. Nedd4-2 functions as a suppressor of the epithelial Na+ channel (ENaC), which interacts with Nedd4-2 WW domains via PY-motifs located at its C-terminus. The importance of Nedd4-2 mediated ENaC regulation is highlighted by the fact that mutations affecting the ENaC PY-motifs cause Liddle syndrome, a hereditary hypertension. Since all Nedd4 family members recognize the same core sequence in their target proteins, the question was raised of how substrate specificity for Nedd4 family members is achieved. Using intrinsic tryptophan florescence to measure the binding affinity of Nedd4-1/-2 WW domains for their substrate PY-motifs, we demonstrate the importance of both PY-motif and WW domain residues, outside the core binding residues, in determining the specificity of WW domain-ligand interactions. Little was known about regulation of catalytic activity for this family of E3 ligases, and hence was the second focus of my work. Notably, Nedd4-2 contains a PY-motif within its HECT domain, raising the possibility that its catalytic activity is regulated by an interaction between its WW domains and HECT domain. Here I present evidence supporting a model in which a low-affinity interaction between the Nedd4-2 WW domains and its HECT domain regulate Nedd4-2 stability by preventing self-ubiquitination and subsequent degradation. Furthermore, evidence is presented suggesting that interaction between Nedd4-2 and the RING-E3 ligase Rnf11, a Nedd4-2 substrate, may also serve to regulate Nedd4-2 stability, as this interaction leads to decreased Nedd4-2 self-ubiquitination. Collectively, the studies presented here further our understanding of the substrate specificity and regulation of Nedd4-1 and Nedd4-2.Ph