Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 5. The second zinc coordinating complex of Hic-5 LIM2 is required for the interaction with Cbl-c. A. Schematic diagram of Hic-5 constructs used to map the interaction with Cbl-c. B. HEK293T cells were transfected with HA- Cbl-c and constructs for Hic-5 as indicated above the panel. HA-Cbl-c was immunoprecipitated with anti-HA beads and immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. * indicates immunoglobulin. C. HEK293T cells were transfected with HA-Cbl-c along with WT or mutants of Hic-5 which disrupt either the first or second zinc coordinating complexes of LIM2 (C287A and C313A respectively) as indicated above the figure. HA-Cbl-c was immunoprecipitated with anti-HA beads and immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. All transfections were balanced with empty vector controls; GFP was transfected as a control for transfection efficiency and is shown as a loading control. MWs in kDa are shown to the left of the panels. doi:10.1371/journal.pone.0049428.g005 [29,30]. Hic-5 has been implicated in cytoskeletal function, as a mediator of epithelial to mesenchymal transition, cellular senes- cence, and differentiation [29,31,33,34,35,36,37,38]. The nature of the interaction of Cbl-c and Hic-5 is unique. The interaction between the two proteins is mediated by the RF of Cbl-c and the LIM2 domain of Hic-5 (Figs. 2, 3, 4). Furthermore, we demonstrate that this interaction is mediated by specific zinc coordinating complexes within each of the specified domains (Figs. 3 and 4). The RF and LIM domains are each composed of two zinc coordinating complexes [30,44,45]. The disruption of the second zinc coordinating complex, but not the first, in the RF of Cbl-c resulted in the abrogation of the interaction with Hic-5. Disrupting the first zinc coordinating complex, but not the second, of the LIM2 domain of Hic-5 eliminates the interaction with Cbl- c. This suggests that the interaction between Hic-5 and Cbl-c is coordinated very specifically by the maintenance of these zinc finger structures. To our knowledge there have been no published reports of a RF-LIM domain interaction. Hic-5 interacts with Cbl-c but not with the closely related proteins Cbl or Cbl-b (Fig. 1C). While the RF domains of the three mammalian Cbl proteins are highly conserved, the RF of Cbl-c is the most divergent of all of the Cbl proteins. Cbl and Cbl-b are identical at 38 of the 40 amino acids in the core RF domain and the two non-identical amino acids are conservative changes (Fig. 4A). By contrast, Cbl-c differs at nine amino acids from either Cbl or Cbl-b. Swapping the RF of Cbl-c with the RF of Cbl- b confirmed that the Hic-5 interaction is mediated only by the RF of Cbl-c (Fig. 4C). It is interesting that disruption of the second zinc coordinating complex of Cbl-c, but not the first, abrogates the interaction between Cbl-c and Hic-5. Six of the nine amino acid differences between the RF of Cbl-c and the RFs of Cbl and Cbl-b are in the loops between the first and second coordination complexes. Together these data suggest that the binding of Hic-5 to Cbl-c is mediated by the region surrounding the second zinc coordination complex. Tyrosine phosphorylation of Cbl proteins by their substrates is necessary for E3 activity [7]. Previously we and others have shown that the N-terminus of Cbl-c inhibits E3 activity and that phosphorylation of tyrosine 341 in the linker region preceding the RF or deletion of the N-terminus leads to an increase in E3 PLOS ONE | www.plosone.org 9 November 2012 | Volume 7 | Issue 11 | e49428