Substrate clustering potently regulates the activity of WW-HECT domain–containing ubiquitin ligases

The Nedd4 family of HECT domain–containing E3 ligases ubiquitinate many transcription factors and signaling proteins, and their activity is tightly regulated. Normally, intramolecular interactions curb the catalytic activity of the HECT domain, but these can be broken by the binding of PY motifs, found on substrate molecules and adaptors, to the WW domains characteristic of this E3 ligase family. This raises the prospect of substrates automatically activating the ligases, frustrating the purpose of ligase regulation. Here we show that soluble protein substrates and adaptors such as α arrestins, even with multiple PY elements, cannot activate ligase activity efficiently. However, we found that polymerization or membrane tethering of these substrates dramatically increases the ligase activity both in vivo and in vitro. Aggregation of luciferase-containing substrates upon heat shock had a similar effect and could also expose cryptic PY elements in the substrates. We inferred that ligase activation critically requires a substantial array of clustered PY motifs and that the formation of such arrays on membranes or in polymeric aggregates may be an essential step in this mode of ligase regulation. We conclude that recruitment of α arrestins to membrane receptors and aggregation of unstable proteins after heat shock may be physiologically relevant mechanisms for triggering ubiquitination by Nedd4 family HECT domain–containing E3 ligases.