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J Biol Chem. 2016 Jan 22;291(4):2033-42. doi: 10.1074/jbc.M115.689869. Epub 2015 Nov 24.

The Vps27/Hrs/STAM (VHS) Domain of the Signal-transducing Adaptor Molecule (STAM) Directs Associated Molecule with the SH3 Domain of STAM (AMSH) Specificity to Longer Ubiquitin Chains and Dictates the Position of Cleavage.

Author information

1
From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and.
2
the Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807.
3
From the Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel and reuvenw@ekmd.huji.ac.il.

Abstract

The deubiquitinating enzyme associated molecule with the SH3 domain of STAM (AMSH) is crucial for the removal of ubiquitin molecules during receptor-mediated endocytosis and lysosomal receptor sorting. AMSH interacts with signal transducing adapter molecule (STAM) 1 or 2, which enhances the activity of AMSH through an unknown mechanism. This stimulation is dependent on the ubiquitin-interacting motif of STAM. Here we investigate the specific mechanism of AMSH stimulation by STAM proteins and the role of the STAM Vps27/Hrs/STAM domain. We show that, in the presence of STAM, the length of the ubiquitin chains affects the apparent cleavage rate. Through measurement of the chain cleavage kinetics, we found that, although the kcat of Lys(63)-linked ubiquitin chain cleavage was comparable for di- and tri-ubiquitin, the Km value was lower for tri-ubiquitin. This increased affinity for longer chains was dependent on the Vps27/Hrs/STAM domain of STAM and required that the substrate ubiquitin chain contain homogenous Lys(63)-linkages. In addition, STAM directed AMSH cleavage toward the distal isopeptide bond in tri-ubiquitin chains. Finally, we generated a structural model of AMSH-STAM to show how the complex binds Lys(63)-linked ubiquitin chains and cleaves at the distal end. These data show how a deubiquitinating enzyme-interacting protein dictates the efficiency and specificity of substrate cleavage.

KEYWORDS:

AMSH; STAM; VHS; deubiquitylation (deubiquitination); endosomal sorting complex required for transport (ESCRT); metalloprotease; polyubiquitin chain; ubiquitin; ubiquitin-interacting motif

PMID:
26601948
PMCID:
PMC4722476
DOI:
10.1074/jbc.M115.689869
[Indexed for MEDLINE]
Free PMC Article

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