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

Journal Article · Mon Oct 01 04:00:00 EDT 2007 · Experimental Cell Research

DOI:https://doi.org/10.1016/j.yexcr.2007.07.028· OSTI ID:21045895

Mizuno, Emi; Kitamura, Naomi ^[1]; Komada, Masayuki ^[1]

---

1. Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-16 Nagatsuta, Midori-ku, Yokohama 226-8501 (Japan)

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 {epsilon}, {gamma}, and {zeta} 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 RSYS{sup 680}SP, by phosphatase treatment, and by competition with the Ser{sup 680}-phosphorylated RSYS{sup 680}SP peptide. Metabolic labeling with [{sup 32}P]orthophosphate and immunoblotting using antibody against the phosphorylated 14-3-3-binding motif showed that Ser{sup 680} is a major phosphorylation site in UBPY. These results indicated that 14-3-3s bind to the region surrounding Ser{sup 680} in a phosphorylation-dependent manner. The mutation at Ser{sup 680} 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 14-3-3{epsilon} inhibited the UBPY activity in vitro. Finally, UBPY was dephosphorylated at Ser{sup 680} 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.

• OSTI ID: 21045895
• Journal Information: Experimental Cell Research, Journal Name: Experimental Cell Research Journal Issue: 16 Vol. 313; ISSN 0014-4827; ISSN ECREAL
• Country of Publication: United States
• Language: English

Related Subjects

60 APPLIED LIFE SCIENCES
ANTIBODIES
CELL DIVISION
ENZYMES
GROWTH FACTORS
IN VITRO
IN VIVO
MASS SPECTROSCOPY
MUTATIONS
PEPTIDES
PHOSPHORUS 32
PHOSPHORYLATION
RECEPTORS