Spatial and temporal regulation of the Nedd4 family ubiquitin ligases through phospholipid binding

Abstract

The Nedd4 family of HECT ubiquitin ligases are essential regulators of cellular polarity, ion channel activity, motility, and inflammatory signaling. HECT family ubiquitin ligases employ a catalytic cysteine residue to target cellular substrates for mono or poly ubiquitination. The Nedd4 family has nine members each with an N‐terminal C2 domain. Little is known about how their cellular localization and catalytic activity are regulated by lipid binding. These domains bind phospholipids and are required for the localization of some family members to the plasma membrane. Using surface plasmon resonance we have investigated the specific lipid binding properties of the Nedd4 family C2 domains. We find that several of these domains bind to phosphoinositides with nanomolar affinity. We have used vesicular sedimentation assays and lipid blots to confirm these binding results. It is likely that phosphoinositide binding enables these proteins to be regulated spatially and temporally in cells. We are currently employing confocal microscopy and fluorescent C2 constructs to determine how cellular lipid binding regulates the ability of these ubiquitin ligases to target cellular substrates under normal and phospholipid‐modulated conditions. This research is supported by the American Heart Association (12PRE11960030) and the NIH (T32GM075762).