Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog, Hic 5 Philip E. Ryan1,2, Stephen C. Kales1, Rajgopal Yadavalli1, Marion M. Nau1, Han Zhang 1 , Stanley Lipkowitz1* 1 Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America, 2 The George Washington University, Institute for Biomedical Sciences, Washington, District of Columbia, United States of America Abstract Cbl proteins (Cbl, Cbl-b and Cbl-c) are ubiquitin ligases that are critical regulators of tyrosine kinase signaling. In this study we identify a new Cbl-c interacting protein, Hydrogen peroxide Induced Construct 5 (Hic-5). The two proteins interact through a novel interaction mediated by the RING finger of Cbl-c and the LIM2 domain of Hic-5. Further, this interaction is mediated and dependent on specific zinc coordinating complexes within the RING finger and LIM domain. Binding of Hic-5 to Cbl-c leads to an increase in the ubiquitin ligase activity of Cbl-c once Cbl-c has been activated by Src phosphorylation or through an activating phosphomimetic mutation. In addition, co-transfection of Hic-5 with Cbl-c leads to an increase in Cbl- c mediated ubiquitination of the EGFR. These data suggest that Hic-5 enhances Cbl-c ubiquitin ligase activity once Cbl-c has been phosphorylated and activated. Interactions between heterologous RING fingers have been shown to activate E3s. This is the first demonstration of enhancement of ubiquitin ligase activity of a RING finger ubiquitin ligase by the direct interaction of a LIM zinc coordinating domain. Citation: Ryan PE, Kales SC, Yadavalli R, Nau MM, Zhang H, et al. (2012) Cbl-c Ubiquitin Ligase Activity Is Increased via the Interaction of Its RING Finger Domain with a LIM Domain of the Paxillin Homolog, Hic 5. PLoS ONE 7(11): e49428. doi:10.1371/journal.pone.0049428 Editor: Michael Massiah, George Washington University, United States of America Received September 5, 2012; Accepted October 8, 2012; Published November 7, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lipkowis@mail.nih.gov Introduction The covalent attachment of ubiquitin to proteins (known as ubiquitination or ubiquitylation) plays a fundamental role in regulating diverse cell processes including protein degradation, membrane protein trafficking, protein localization, enzyme activation, and DNA repair (reviewed in [1,2]). Covalent attachment of ubiquitin to a substrate occurs through a sequential activation and conjugation of ubiquitin to a target protein by a series of three reactions [1,2]. This is initiated by the ATP- dependent covalent attachment of the ubiquitin molecule to the active site cysteine of the ubiquitin activating enzyme (E1). The ubiquitin molecule is then transferred to via a transesterificaiton reaction to the active site cysteine on an ubiquitin-conjugating enzyme (E2). Subsequently, the E2 interacts directly with an ubiquitin ligase (E3) which facilitates the transfer of the ubiquitin molecule to the substrate. RING finger (RF) proteins constitute the majority of E3s, and accordingly they are fundamental regulators of many key cellular processes [3,4]. The RF is comprised of ,40– 60 amino acids that form a coordination complex with two zinc ions [3,4,5]. The RF interacts with the E2 to mediate transfer of ubiquitin from the active site of the E2 in most cases to an acceptor lysine on target proteins or the growing end of ubiquitin chains. The E3 activity of RF proteins is regulated by covalent modifications of the substrate (e.g., phosphorylation) [6,7], homo- and heterodimerization mediated by the RF domains (e.g., Mdm2 homodimers, Mdm2/MdmX heterodimers, and BARD1/BRCA1 heterodimers) [8,9,10], covalent modification of the RF protein (e.g., phosphorylation and sumoylation) [7,11,12,13,14], and interaction with non-RF proteins (e.g., MAGE proteins) [15]. Cbl proteins are RF E3s that negatively regulate signaling by many tyrosine kinases (e.g., EGFR, Met, and Src) and tyrosine kinase-dependent pathways (e.g., T-cell receptor). There are three mammalian Cbl proteins: Cbl (a.k.a., c-Cbl, Cbl2, and RNF55), Cbl-b (a.k.a., RNF56), and Cbl-c (a.k.a., Cbl-3, Cbl-SL, and RNF57) [16,17] (We have used the HUGO nomenclature for the Cbl proteins. The nomenclature is as follows: Cbl refers to the first mammalian family member identified (a.k.a. c-Cbl, Cbl2, and RNF55); Cbl-b refers to the second mammalian Cbl protein identified (a.k.a. RNF56); Cbl-c refers to the third Cbl protein identified (a.k.a. Cbl-3, Cbl-SL, RNF57)). Cbl proteins have a highly conserved N-terminus consisting of a tyrosine kinase binding (TKB) domain that binds to specific phosphorylated tyrosines on substrates, a catalytic RF domain, and an alpha helical linker region separating the TKB and RF domains [16,17]. The C-termini of the proteins are less highly conserved, although all three mammalian Cbl proteins have a proline rich (PR) region that mediates interaction with SH3-domain containing proteins. Cbl and Cbl-b have ubiquitin associated (UBA) domains at their C-termini that mediate homodimerization or ubiquitin binding, respectively. The E3 activity of the Cbl proteins is negatively regulated by the N-terminus of the proteins and activity is PLOS ONE | www.plosone.org 1 November 2012 | Volume 7 | Issue 11 | e49428