Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 6. Hic-5 increases the E3 activity of Cbl-c in vitro. A. In vitro E3 assays were performed as described in the methods with the recombinant WT or activated (Y341E) His-Cbl-c constructs as labeled above panel. Recombinant purified His-Hic-5 WT, C287A were added where indicated. The data for the E3 activity of the Y341E Cbl-c mutant were quantified for three experiments and are displayed on the graph. Values represent the mean ubiquitination +/– SE relative to Y341 Cbl-c alone. The dotted line represents the level of ubiquitination by Y341E Cbl-c in the absence of Hic-5. Two tailed p-values using a paired T-test comparing the results for Y341 Cbl-c + Hic-5 (WT or C287A) to Cbl-c are shown on the graph (NS = not significant). B. E3 assays were performed with either WT or Y341E recombinant Cbl-c as described above. Increasing amounts of recombinant His-Hic-5 was added as indicated above the panel. Purified active Src was added where indicated. C. In vitro E3 assays were performed with recombinant GST-Cbl-c RF (GST-RF) or GST-Cbl-c Y341E as described above. Recombinant His-Hic-5 or the His-LIM2 domain of Hic-5(LIM2) were added as indicated above the panel. In vitro E3 assays were performed at 30uC for 40 m in the presence and absence of E2 and then immunoblotted (IB) as indicated. MW markers in kDa are shown to the left of the panels. doi:10.1371/journal.pone.0049428.g006 activity [11,14]. In this study we demonstrate that purified recombinant Hic-5, but not the non-binding C287A mutant, increases the autoubiquitination of Cbl-c which is activated by a phosphomimetic Y to E mutation of the linker tyrosine or the deletion of the N-terminus in vitro while having no measureable effect on an unactivated Cbl-c (Fig. 6). For the most part, only E2 and RF proteins have been described to interact via the RF [3,4]. Regulation of E3 activity by the interaction between two different RF domains has been described in a number of cases. For example, BRCA has weak E3 activity that is markedly stimulated by heterodimerization with BARD1 [8]. MDM2 heterodimerizes with the closely related RF protein, MDMX, resulting in increased ubiquitination of p53 and decreased autoubiquitination [9,10]. In both cases, only one member of the dimer (BRCA1 for BRCA1/BARD and MDM2 for MDM2/MDMX) has demonstrated E3 activity while the second RF domain, without E3 activity of its own, serves to regulate the activity of the first RF domain [8,9,10]. RF mediated homodimerization of RF proteins has also been described [3]. For example, upon binding of SMAC or SMAC mimetics to cIAP1, the cIAP1 protein homodimerizes via the RF. This results in autoubiquitination and degradation of the cIAP1 protein [49]. The binding of Hic-5, a protein that is neither an E2 nor a RF protein, to the RF of an E3 appears to be a novel interaction. Interestingly, the interaction is mediated by the zinc coordinating LIM2 domain of Hic-5. Like the interactions described between the zinc coordinating RF of BARD1 and the RF of BRCA1, Hic-5 enhances E3 activity of the Cbl-c RF but Hic5 has no detectable E3 activity by itself. The isolated LIM2 of Hic-5 is sufficient for this increased activity, and further, the isolated LIM2 can increase the activity of the isolated RF of Cbl-c (Fig. 6). Thus, it appears that the effects on E3 activity are a direct consequence of the binding of the LIM domain of Hic-5 to the RF of Cbl-c. In cells, Hic-5 increases Cbl- c-mediated ubiquitination of a model substrate, the EGFR (Fig. 7). This suggests that the interaction of Hic-5 with Cbl-c does not just enhance autoubiquitination by Cbl-c but also increases the E3 activity of Cbl-c towards substrates. Our previous data suggest that the phosphoryation of the linker or deletion of the N-terminus of the Cbl proteins alters the interaction between the Cbl proteins and the E2. Structures of RF/E2 complexes indicate that regions surrounding the second zinc coordinating complex interact with PLOS ONE | www.plosone.org 10 November 2012 | Volume 7 | Issue 11 | e49428