RN181, a novel ubiquitin E3 ligase that interacts with the KVGFFKR motif of platelet integrin a IIb b 3 Teresa M. Brophy a , Markus Raab a , Heide Daxecker a , Kevin G. Culligan a , Ingo Lehmann b , Anthony J. Chubb c , Achim Treumann a,b , Niamh Moran a, * a Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, 123 St. Stephens Green, Dublin 2, Ireland b Proteomic and Mass-Spectrometry Facility, Royal College of Surgeons in Ireland, Ireland c Department of Pharmaceutical and Medicinal Chemistry, Royal College of Surgeons in Ireland, Ireland article info Article history: Received 11 February 2008 Available online 10 March 2008 Keywords: Integrin Platelet RN181 Ubiquitin E3 ligase abstract We previously identified proteins that bind with high affinity to a peptide corresponding to the cytoplas- mic regulatory domain (KVGFFKR) of the platelet-specific integrin subunit a IIb . These included a hypo- thetical protein termed HSPC238, recently renamed as RING finger protein, RN181. Here, we establish the presence of RN181 in human platelets by RT-PCR, Western blotting and mass spectrometry and con- firm its affinity for the platelet integrin. We demonstrate that RN181 has ubiquitin E3 ligase activity and that all other components of the ubiquitination pathway are abundant in platelets, suggesting a novel link of integrin signal transduction pathways with ubiquitin-conjugation events. Ó 2008 Elsevier Inc. All rights reserved. Integrins comprise a large family of cell-surface adhesion mol- ecules that mediate cell–cell or cell–matrix interactions [1]. Fol- lowing cellular activation, signals are conveyed from the short cytoplasmic tails of integrin subunits to their large extracellular domains, causing alteration of the integrin conformation and affin- ity state for ligand(s) [2–4]. This inside-out activation of integrins permits the regulated binding of ligands. The transduction of sig- nals from the extracellular domain back into the cell is termed out- side-in signaling. Integrins therefore co-ordinate the responsiveness of cells to the extracellular environment. The acti- vation state of an integrin is critical for its function and is therefore highly regulated. However, despite this, little is known of the molecular mechanisms involved in transducing integrin-depen- dent signaling events. The short cytoplasmic domains of integrins contain motifs that are highly conserved and have been implicated in the regulation of integrin activation. One of the most extensively studied motifs is the membrane-proximal KxGFFKR motif, contained within the a- helical structure of a-subunit tails, responsible for the regulation of integrin activation [5–7]. Mutation or deletion of the GFFKR re- gion generates constitutively activated integrins, capable of bind- ing ligand without the need for prior activation [8]. The search for integrin-regulating cytoplasmic proteins has therefore focused on the binding capacity to this motif [9]. In the human platelet, this has identified a number of integrin binding proteins including the protein-phosphatase PP1c [10] and regulatory proteins such as cal- cium- and integrin-binding protein (CIB1) [9], ancient ubiquitous protein-1 (AUP-1) [11], Chloride channel regulatory protein (ICln) [12] and triose phosphate isomerase [13]. This wide assortment of proteins may bind transiently to integrin cytoplasmic tails dur- ing the vastly dynamic process of platelet activation and aggrega- tion. However, the mechanism by which the KVGFFKR motif of the platelet integrin a IIb -subunit regulates integrin activation is still not fully elucidated. In a bid to identify new KVGFFKR-binding proteins, we previ- ously screened a high-density protein expression array of 37,200 recombinant human proteins for proteins capable of specifically interacting with a biotinylated KVGFFKR peptide [12]. In this pres- ent study, we further investigate one such a IIb b 3 -interacting pro- tein termed HSPC238 and recently renamed as RN181 in Swissprot. We now demonstrate the presence of RN181 in human platelets and confirm its ability to interact with the platelet-spe- cific integrin a IIb b 3 . RN181 contains a RING (Really Interesting New Gene)-type zinc-finger domain suggestive of a possible role in protein ubiqui- tination pathways. Ubiquitination is a post-translational modifica- tion that regulates cellular degradation of proteins, protein trafficking and signaling networks [14,15]. In a cell-free assay, we demonstrate that RN181 is a novel platelet RING-finger-containing ubiquitin E3 ligase suggesting a role for RN181-mediated ubiquiti- nation in integrin and platelet signaling. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.02.142 * Corresponding author. Fax: +353 14022453. E-mail address: nmoran@rcsi.ie (N. Moran). Biochemical and Biophysical Research Communications 369 (2008) 1088–1093 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc