Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 4. Mutation of the first zinc coordinating complex of the RF of Cbl-c disrupts Hic-5 binding. A. Sequence alignment of the RFs of Cbl, Cbl-b and Cbl-c. Amino acids in red show the nine amino acids that are divergent between the RF of Cbl-c and the RFs of Cbl and Cbl-b. The two positions highlighted in blue mark the amino acids shared between Cbl-c and Cbl-b that differ from Cbl. The two cysteines mutated to alanine in Figure 3D and E are indicated below the sequence. B. Schematic diagram of Cbl-c, Cbl-b and chimeric constructs with the swapped RF domains. Shaded boxes represent Cbl-c sequence. White boxes represent Cbl-b sequence. All constructs were HA-epitope tagged on the N-terminus. C. HEK293T cells were transfected with HA-Cbl-c, Cbl-b, or the chimeric proteins with swapped RF domains and Hic-5 as indicated above the panels. The Cbl proteins were immunoprecipiated with anti-HA beads and the immunoprecipitates (IP) or cell lysates (lysates) 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 control for loading. D. HEK293T cells were transfected with HA-Cbl-c, RF mutants of HA-Cbl-c, HA-Cbl-c DRF, and Hic-5 as indicated above the panels. The Cbl-c proteins were immunoprecipitated with anti-HA beads and the immunoprecipitates (IP) or cell lysates (lysates) 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 control for loading. E. Purified recombinant GST-tagged Cbl-c RF proteins (WT, C351A or C366A mutants) bound to GSH-sepharose beads were added to a bacterial lysate expressing recombinant His-tagged Hic-5 or empty His vector as indicated above the panel. Proteins were precipitated with GSH-sepharose beads and immunoblotted (IB) as indicated to the right of the panels. Expression of His-Hic-5 in the bacterial lysates is shown in the lower panel. MWs in kDa are shown to the left of the panels. doi:10.1371/journal.pone.0049428.g004 Overall, Hic-5 enhanced both autoubiquitination of activated Cbl-c in vitro and substrate ubiquitination by activated Cbl-c in a cellular model. Discussion In this study we used a yeast two-hybrid screen to identify a new Cbl-c interacting protein, Hic-5 (Fig. 1). Hic-5 is a LIM containing protein in the paxillin family that consists of eight protein-protein interacting domains: four LD domains and four LIM domains PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49428