Function of Lipid Binding Ability of Nedd4 on Its Regulation of PTEN and Other Substrates

Nedd4 (neural precursor cell expressed, developmentally down-regulated 4) proteins are a family of E3 ubiquitin ligases. These enzymes play important roles in various cellular activities by transferring ubiquitin to various substrates. Several key regulators of cellular homeostasis, such as the tumor suppresser PTEN, are Nedd4 substrates. The ubiquitination of PTEN by Nedd4 proteins is involved in cellular proliferation and neuron survival. Early studies showed that both Nedd4-1 and PTEN can bind phospholipids and localize to cellular membranes. Here we further explored the lipid-binding properties of Nedd4-1 and found that calcium promotes Nedd4-1 lipid binding affinity to phosphatidylserine. Ubiquitination assays showed that the increasing lipid binding affinity stimulated by calcium can enhance the ubiquitination of PTEN by Nedd4-1. Intracellular calcium fluctuations also alter the cellular localization of PTEN. To determine if calcium induced binding to phosphatidylserine regulated other Nedd4-1 substrates, we investigated the previously identified Nedd4-1 substrate Ebola virus matrix protein VP40. We confirmed that ubiquitination promotes VP40 induced virus-like particle binding (VLP) and for the first time demonstrate that Nedd4-1 ubiquitinates VP40 in vitro. As intracellular levels of calcium rise, virus like particle (VLP) budding of VP40 is stimulated. This finding implies that cytosolic calcium levels may stimulate VLP budding by localizing Nedd4 proteins to the cytosolic plasma membrane interface, which is enriched with phosphatidylserine.