The Vps27/Hrs/STAM (VHS) domain of the signaltransducing adaptor molecule (STAM) directs associated molecule with the SH3 domain of STAM (AMSH) specificity to longer ubiquitin chains and dictates the position of cleavage

Nardeen Baiady, Prasanth Padala, Bayan Mashahreh, Einav Cohen-Kfir, Emily A. Todd, Kelly E. Du Pont, Christopher E. Berndsen, Reuven Wiener*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

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 bySTAMproteins and the role of theSTAMVps27/ Hrs/STAM domain.Weshow that, in the presence ofSTAM,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 Lys63-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 Lys63-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 Lys63-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.

Original languageAmerican English
Pages (from-to)2033-2042
Number of pages10
JournalJournal of Biological Chemistry
Volume291
Issue number4
DOIs
StatePublished - 22 Jan 2016

Bibliographical note

Funding Information:
This work was supported by United States-Israel Binational Science Foundation Grant 2013261, National Science Foundation Research Experience for Undergraduates Grant CHE-1461175, and Marie Curie Career Integration Grant PCIG13-GA-2013-630755. The authors declare that they have no conflicts of interest with the contents of this article.

Publisher Copyright:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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