Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 2. Hic-5 colocalizes with Cbl-c. HeLa cells were transfected with CFP-Hic-5 and/or YFP-Cbl-c, fixed, and imaged using a Zeiss LSM 510 META confocal microscope with a 63X /1.3 immersion-oil Plan-Neofluar objective as described in the methods section. Images for each protein are shown in grey scale and for merged images in color. Panels 1–3 demonstrate the diffuse localization of YFP-Cbl-c and CFP-Hic-5. Panels 4–6 demonstrate the punctate localization of CFP-Hic-5, but not YFP-Cbl-c. In the merged images (panels 3 and 6) YFP-Cbl-c is shown in green, CFP-Hic-5 is shown in red and the overlapping signal is shown in yellow. To demonstrate that the punctate distribution of CFP-Hic-5 represents localization to focal adhesions, cells were stained using an anti-vinculin antibody as a marker of focal adhesions. Panels 7–9 demonstrate the colocalization of CFP- Hic-5 and vinculin in the punctate structures, consistent with colocalization in focal adhesions. Panels 10–12 show no colocalization of YFP-Cbl-c with vinculin. In the merged images for the vinculin staining (panels 9 and 12) YFP-Cbl-c and CFP-Hic-5 are shown in green, vinculin is shown in red, and the overlapping signal is shown in yellow. The IMARIS software was used for colocalization analysis, and Pearson’s correlation coefficient (PCC) was calculated for each image analysis on 5–10 cells for each combination (see text for results). doi:10.1371/journal.pone.0049428.g002 mutation of the second zinc coordinating complex of the Hic-5 LIM2 domain (Fig. 5C, compare lane 6 to lane 4 and 8). These data coupled with those of the RF mutant data suggest a specific interaction between the region around the second zinc coordi- nating complex of the Cbl-c RF and the region around the first zinc coordinating complex of the second LIM domain of Hic-5. Hic-5 Enhances Cbl-c E3 Activity in vitro Cbl proteins are E3s whose activity depends on the RF [7]. Considering the unique nature of the interaction between Hic-5 and the RF domain of Cbl-c we tested whether this interaction would affect the E3 activity of Cbl-c. Using an in vitro E3 assay [14] His-tagged purified recombinant Cbl-c WT was incubated with recombinant E1, E2 and Ub in the presence or absence of purified Hic-5 or the non-binding Hic-5 C287A mutant. Under these conditions, there was no detectable E3 activity and Hic-5 did not lead to increased activity of WT Cbl-c (Fig. 6A, lanes 2–4). Previous work has demonstrated that phosphorylation or a phosphomimetic Y to E mutation of a tyrosine located on the alpha-helical linker that precedes the RF dramatically increases the activity of Cbl proteins when measured by in vitro E3 autoubiquitination assays [11,14]. We next tested whether Hic-5 would affect the activity of a phosphomimetic mutant (Cbl-c Y341E). An E3 assay with Cbl-c Y341E either alone, with Hic-5 or with Hic-5 C287A showed that Hic-5 increased the autoubiqui- PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49428