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United States Department of Agriculture

Agricultural Research Service

Title: Proteomic analysis reveals Hrs ubiquitin-interacting motif-mediated ubiquitin signaling in multiple cellular processes

Authors
item Wei Pridgeon, Yuping
item Olzmann, James - EMORY UNIVERSITY
item Kirk, Elizabeth - EMORY UNIVERSITY
item Chin, Lih-Shenemory U - EMORY UNIVERSITY
item Li, Lian - EMORY UNIVERSITY

Submitted to: FEBS Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 24, 2008
Publication Date: January 1, 2009
Citation: Prdigeon, J.W., Webber, E.A., Sha, D., Li, L., Chin, L.S. 2009. Proteomic analysis reveals Hrs ubiquitin-interacting motif-mediated ubiquitin signaling in multiple cellular processes. The FEBS Journal. 276(1):118-131.

Interpretive Summary: Ubiquitination is a post-translational modification of proteins. Increasing evidence points to critical importance of protein ubiquitination in the control of diverse cellular processes, from DNA repair and transcription regulation to vesicular trafficking and virus budding. Moreover, dysregulated ubiquitination has been implicated in the pathogenesis of many human diseases, including cancer and neurodegenerative disorders. Elucidation of the molecular mechanisms by which cells recognize and sort ubiquitinated proteins is thus essential for understanding ubiquitin signaling in normal physiology and diseases. The ubiquitin-interacting motif (UIM) is a conserved ubiquitin recognition module. UIMs are found in many proteins implicated in a variety of cellular processes, including endocytosis, endosome-to-lysosome trafficking, DNA repair, mRNA splicing, and neurodegeneration. The UIM domain of Hrs has been shown to bind ubiquitin in vitro and facilitate the sorting of a transferrin receptor-ubiquitin fusion protein to lysosomes. However, the identities of ubiquitinated cargo and other cellular proteins that are recognized by the Hrs UIM domain remain unknown. In order to gain insight into the role of Hrs UIM-mediated ubiquitin signaling in cells, we performed a genome-scale screen for Hrs UIM-interacting proteins in human brain by using a combined in vitro expression cloning (IVEC) and GST-pull down approach. Here, we report the identification of a set of proteins that are specifically recognized by the UIM domain of Hrs. Our results reveal the involvement of Hrs UIM-mediated protein interactions in the coordination of multiple steps in endosomal trafficking as well as in the regulation of cell signaling, cytoskeleton and membrane dynamics, and other cellular processes.

Technical Abstract: The ubiquitin-interacting motif (UIM) is a conserved ubiquitin binding module found in many proteins, including hepatocyte growth factor regulated tyrosine kinase substrate (Hrs), a key component of the endosomal sorting machinery. Here we show that the Hrs UIM domain preferentially binds K48- and K63-linked polyubiquitin chains over monoubiquitin. By using an in vitro expression cloning (IVEC) screening approach, we have identified 47 proteins that are specifically recognized by the UIM domain of Hrs. The validity and specificity of the IVEC screen have been confirmed by subsequent characterization of two identified proteins, Munc18-1 and Hsc70. Surprisingly, only a small fraction of the identified Hrs UIM-interacting proteins are membrane cargo proteins. Major classes of the identified proteins include components of the vesicular trafficking machinery, cell signaling molecules, membrane protein-associated adaptor proteins, components of cytoskeleton and cytoskeleton-dependent transport, and enzymes involved in ubiquitination, lipid metabolism, and other metabolic processes. Furthermore, we have identified 6 novel proteins. Our findings provide new insights into the UIM domain interacting proteins and suggest the involvement of Hrs UIM domain in multiple cellular processes in addition to endosomal sorting.

Last Modified: 11/21/2014
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