Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 7. Hic-5 enhances Cbl-c mediated ubiquitination of EGFR in cells. A. HEK293T cells were transfected with EGFR, HA-epitope tagged ubiquitin, with and without Cbl-c in the presence and absence of Hic-5 as indicated above the panels. B. HEK293T cells were transfected with EGFR, HA-epitope tagged ubiquitin and Cbl-c alone and with either Hic-5 WT or Hic-5 C287A. C. HEK293T cells were transfected with EGFR, HA-epitope tagged ubiquitin, GST-Cbl-c, GST-Cbl-c Y341F, and Hic-5 WT as indicated. All transfections were performed in duplicate. Cells were serum starved for 18 h, and one plate of each pair was stimulated with 10 ng/ml EGF for 15 min. EGFR was immunoprecipitated and 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 shown as a loading control. MWs in kDa are shown to the left of the panels. doi:10.1371/journal.pone.0049428.g007 the loops on the E2 protein [3,4]. Thus, Hic-5 binding is likely to occur in a region that may alter E2 binding. The proximity of the binding site of Hic-5 to the binding site for the E2 suggests that it may affect the interaction between the Cbl-c RF and the E2. Alternatively, the binding of Hic-5 to the Cbl-c RF could allow Hic-5 to interact with the E2 and induce allosteric changes that enhance ubiquitin transfer. Further studies are needed to determine the structural nature of the interaction between the LIM2 of Hic-5 and the RF of Cbl-c and to determine the mechanism underlying the enhancement of E3 activity. To our knowledge this is the first report showing a LIM domain having a direct interaction with a RF domain. We demonstrate clearly here that this interaction contributes to the regulation of E3 activity for Cbl-c. 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