NEDD4-2 is an E3 ubiquitin ligase that plays an essential role in regulating kidney homeostasis through many substrates ranging from ion channels and transporters to signalling proteins. Nedd4-2 knock-out mice display many features of chronic kidney disease. Accumulating evidence both from our laboratory and others suggests a protective role for NEDD4-2 against kindy damage through regulation of its substrates. While numerous NEDD4-2 substrates have been characterised, many remain undiscovered. Therefore, the aim of this project is to identify novel NEDD4-2 targets in the kidney to better understand the contribution of NEDD4-2 to kidney health and disease.
In this study, we employed proteomic analyses to reveal novel potential substrates of NEDD4-2 in kidney cells. From this screen, phospholipid scramblase 1 (PLSCR1) was identified as a potential candidate. PLSCR1 is a scramblase important for externalising the apoptotic marker phosphatidyl serine (PS), which acts as an ‘eat me’ signal for macrophage clearance during apoptosis. Notably, Nedd4-2 KO kidney cells were found to have a higher level of PLSCR1. Biochemistry assays confirmed the interaction and ubiquitination of PLSCR1 by NEDD4-2. Annexin V staining of externalised PS was used as a measure of PLSCR1 scrambling activity. Our results revealed increased PS flipping in response to apoptotic stimuli in the absence of NEDD4-2 and this phenotype was reversed when NEDD4-2 expression was restored. These results indicate the regulation of PLSCR1 scrambling activity by NEDD4-2. In this study, we report for the first time, PLSCR1 as a novel target of NEDD4-2 in the kidney highlighting a potential role for NEDD4-2 in the apoptotic events associated with kidney damage. These results provide further insight into the protective role of NEDD4-2 in kidney health.