Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 1. Hic-5 interacts with Cbl-c. A. HEK293T cells were transfected with HA-Cbl-c alone, Hic-5, or the combination of HA-Cbl-c and Hic-5 as indicated above panels. B. HEK293T cells were transfected with HA-Cbl-c alone, Trip-6, or the combination of HA-Cbl-c and Trip-6 as indicated above panels. In A and B, HA-Cbl-c was immunoprecipitated from the cell lysates. 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. Green fluorescent protein (GFP) was transfected as a control for transfection efficiency and is shown as a loading control. C. Endogenous Cbl-c was immunoprecipitated from whole cell lysates from MB231 (which expresses Hic-5 but no Cbl-c) and CFPAC-1 (which expresses both Hic-5 and Cbl-c) cell lines and immunoblotted for Cbl-c and Hic-5. Immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. The arrow indicates immunoprecipitated Cbl-c and the asterisk indicates the immunoglobulin heavy chain. D. HEK293T cells were transfected with HA-Cbl, HA-Cbl-b, HA- Cbl-c alone, Hic-5 alone, or each HA epitope tagged Cbl protein with Hic-5 as indicated above the panels. Cbl proteins were immunoprecipitated and immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. The arrow indicates immunoprecipitated Hic-5 and the asterisk indicates an artifact band that includes the immunoglobulin heavy chain. MWs in kDa are shown to the left of the panels. doi:10.1371/journal.pone.0049428.g001 interacting region of Hic-5 identified by the Cbl-c baits (amino acids 125–316) included the second two LD domains and the first two LIM domains. Full length Hic-5 and a deletion mutant removing LIM domains 3 and 4 co-precipitated with Cbl-c (Fig. 5B, lanes 4 and 6) but deletion of LIM domains 2–4 abrogated the interaction with Cbl-c (Fig. 5B, lane 8). These data suggest that LIM domain 2 of Hic-5 is necessary for interaction with Cbl-c. The Hic-5 LIM2 domain is a C2HC3D zinc finger [29]. While the RF and LIM domains are both zinc fingers that have two zinc coordinating complexes, they are structurally different and the LIM domains have no reported E3 activity [45]. For either structure, mutation of any of the zinc coordinating amino acids disrupts the coordination of the zinc atom [29,44,45]. Again, we used site-directed mutagenesis to mutate the first cysteine of the first zinc coordinating complex of LIM2 (C287A) or the first cysteine of the second zinc coordinating complex of LIM2 (C313A) in the full length Hic-5 protein. The interaction between Cbl-c and Hic-5 was disrupted by mutation of the first zinc coordinating complex of the Hic-5 LIM2 domain but not by PLOS ONE | www.plosone.org 5 November 2012 | Volume 7 | Issue 11 | e49428