Identification of a consensus site for TRAF6/p62 polyubiquitination

Abstract

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an ubiquitin ligase that regulates a diverse array of physiological processes via forming Lys-63 linked polyubiquitin chains. In this study, the lysine selection process for TRAF6/p62 ubiquitination was examined. The protein sequence of two characterized TRAF6/p62 substrates, NRIF and TrkA, revealed a conserved consensus pattern for the ubiquitination site of these two TRAF6 substrates. The consensus pattern established in the verified substrates was common to the other Trk receptor family members, TrkB and TrkC. Interestingly, Lysine 811 in TrkB was selected for ubiquination, and mutation of Lysine 811 diminished the formation of TRAF6/p62 complex that is necessary for effective ubiquination. Moreover, downstream signaling was affected upon binding of BDNF to the mutant TrkB receptor. These findings reveal a possible selection process for targeting a specific lysine residue by a single E3 ligase and underscore the role of the scaffold, p62, in this process. KeywordsUbiquitination; Ub ligase; TRAF6; p62; Trk Many adaptors have been identified in studies of other neuronal tyrosine kinases that may also prove to function in Trk receptor-mediated signaling [1]. Cytoplasmic protein p62 was identified as an interacting partner of atypical protein kinase C (PKC) [2] and has been shown to contain several protein-protein interacting modules that enable the protein to serve as a scaffold for activation of the transcription factor NF-κB [3]. The multidomain protein structure of p62 is suggestive of diverse protein-protein interactions and its link in cellular functions. The functional motifs in p62 include a Phox and Bem1p domain (PB1) domain that embeds an octicosapeptide Phox, Cdc and the atypical PKC-interaction domain (AID) (OPCA) motif, a ZZ zinc finger, a binding site for Tumor necrosis factor Receptor-Associated Factor 6 (TRAF6), two PEST sequences, and an Ubiquitin-associated (UBA) domain [4]. The Cterminal ubiquitin-associated domain (UBA) was discovered to bind non-covalently to ubiquitin [5]. In vitro binding studies have unveiled p62 as a unique ubiquitin-binding protein, which binds polyubiquitin non-covalently through its C-terminus [6].Ubiquitination of eukaryotic proteins regulates a broad range of cellular processes. E3 Ub ligases are known to interact with specific substrates either directly or through adaptor proteins. *Corresponding author. Fax: +1 (334) 844-5255, Email address: wootemw@auburn.edu (M. Wooten). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. . High substrate specificity of the ...

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