Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 Figure 3. Hic-5 interacts with the RF of Cbl-c. A. Schematic diagram of Cbl-c constructs used to map the interaction with Hic-5. All constructs were HA-epitope tagged on the N-terminus. B. HEK293T cells were transfected with various HA- Cbl-c constructs and Hic-5 as indicated above panels. 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 control for loading. MWs in kDa are shown to the left of the panels. doi:10.1371/journal.pone.0049428.g003 tination of Cbl-c Y341E while addition of Hic-5 containing the C287A mutation did not (Fig. 6A, compare lane 7 to lanes 6 and 8). Quantification of the data by densitometry for three experiments demonstrated that WT Hic-5 resulted in an approximately 1.5 fold increase in Cbl-c autoubiquitination while Hic-5 C287A did not significantly enhance Cbl-c autoubiquitina- tion (Fig. 6A, graph). To further study the increase in Cbl-c E3 activity, we incubated Cbl-c Y341E with increasing concentrations of Hic-5 which resulted in a dose dependent increase in E3 activity (Fig. 6B, lanes 8–11). As previously shown [14], incubation of WT Cbl-c with Src results in a stimulation of E3 activity (Fig. 6B, compare lanes 2 and 3). The addition of Hic-5 resulted in a dose dependent stimulation of the E3 activity (Fig. 6B, compare lanes 4–6 to lane 3). These data suggest that Hic-5 can increase the activity of Cbl-c only after the E3 is activated by phosphorylation. Previous work has demonstrated that deletion of the N-terminus of Cbl-c also enhances the E3 activity of Cbl-c and the isolated RF of Cbl-c has robust E3 activity [14]. Thus, we tested whether Hic- 5 could increase the E3 activity of the isolated Cbl-c RF. Similar to the phosphomimetic Cbl-c Y341 E mutant, Hic-5 increased the autoubiquitination of the isolated RF of Cbl-c (Fig. 6C, compare lanes 3 and 7 to lanes 2 and 6, respectively). Also, we found that a purified recombinant protein containing only the LIM2 domain of Hic-5 enhanced the activity of either the Cbl-c RF or Cbl-c Y341E (Fig. 6C, lanes 4 and 8). Hic-5 Enhances Cbl-c Mediated Ubiquitination of EGFR Ubiquitination of the activated EGFR is a well studied function of Cbl proteins [6,7,46,47,48]. To investigate whether the interaction between Cbl-c and Hic-5 results in increased E3 activity of Cbl-c in cells, we tested whether Hic-5 could affect the ability of Cbl-c to ubiquitinate the EGFR. HEK293T cells were transfected with the EGFR alone, with WT Hic-5 with and without WT Cbl-c. All transfections included an HA-epitope tagged ubiquitin. Ubiquitination of the EGFR was assessed by immunoprecipitation of the EGFR and immunoblotting with anti- HA. Cbl-c increased the ubiquitination of the EGFR upon EGF activation (Fig. 7A, compare lanes 2 and 4), and this was further increased by the co-expression of Hic-5 (Fig. 7A, lane 8). Subsequent co-expression studies using Hic-5 C287A in addition to WT Hic-5 showed that the non-binding Hic-5 C287A mutant did not increase the ubiquitination of EGFR by Cbl-c (Fig. 7B). Hic-5 enhanced autoubiquitination of Cbl-c in vitro only when Cbl-c was activated by phosphorylation or by a phosphomimetic mutation of the linker tyrosine Y341 (Fig. 6). Previous work has shown that mutation of Y341 to phenylalanine (F) abrogates Cbl-c E3 activity [14]. To test whether activation of Cbl-c by phosphorylation on the linker tyrosine was required for Hic-5 to enhance the ubiquitination cells were transfected with EGFR +/– WT or Y341F GST-Cbl-c in the absence or presence of WT Hic-5 (Fig. 7C). Ubiquitination of EGFR by GST-Cbl-c was enhanced by Hic-5 (Fig. 7C, top panel, compare lanes 3 and 4 to lanes 7 and 8). EGF did not induce ubiquitination of the EGFR by GST-Cbl-c Y341F and Hic-5 did not enhance ubiquitination of EGFR by the mutant Cbl protein (Fig. 7C, top panel, lanes 9–12). GST pull down of the Cbl protein demonstrated that EGF induced significant tyrosine phosphorylaton of GST-Cbl-c, but this was decreased in GST-Cbl-c Y341F (Fig. 7C, third panel, compare lanes 4 and 8 to lanes 10 and 12). Interestingly, the phosphor- ylation of the WT Cbl-c protein was accompanied by a slower migrating band on the gel that was absent in the Y341F mutant Cbl-c (Fig. 7C, fourth panel, compare lanes 4 and 8 to lanes 10 and 12). We often observe ubiquitination of unstimulated EGFR when the EGFR is overexpressed with a Cbl protein in 293T cells (e.g. see Fig. 7B, lane 1 or Fig. 7C, lane 3). Interestingly, the presence of Hic-5 increases the level of ubiquitination of the unstimulated EGFR in the presence of Cbl-c (Compare Fig. 7A or Fig 7C, lanes 3 and 7). We believe this is due to the presence of activated EGFR when the EGFR is overexpressed due either to endogenous ligand or forced proximity due to overexpression. PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49428