14-3-3-dependent inhibition of the deubiquitinating activity of UBPY and its cancellation in the M phase

The deubiquitinating enzyme UBPY, also known as USP8, regulates cargo sorting and membrane traffic at early endosomes. Here we demonstrate the regulatory mechanism of the UBPY catalytic activity. We identified 14-3-3 ε, γ, and ζ as UBPY-binding proteins using co-immunoprecipitation followed by mass spectrometric analysis. The 14-3-3 binding of UBPY was inhibited by mutating the consensus 14-3-3-binding motif RSYS680SP, by phosphatase treatment, and by competition with the Ser680-phosphorylated RSYS680SP peptide. Metabolic labeling with [32P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser680 is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser680 in a phosphorylation-dependent manner. The mutation at Ser680 led to enhanced ubiquitin isopeptidase activity of UBPY toward poly-ubiquitin chains and a cellular substrate, epidermal growth factor receptor, in vitro and in vivo. Moreover, addition of 14-3-3ε inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser680 and dissociated from 14-3-3s in the M phase, resulting in enhanced activity of UBPY during cell division. We conclude that UBPY is catalytically inhibited in a phosphorylation-dependent manner by 14-3-3s during the interphase, and this regulation is cancelled in the M phase.