Enigma blocks Cbl-c-mediated RET9MEN2A ubiquitination.

<p>(<b>A</b>) HEK293T cells were transfected with RET9MEN2A along with GST-Cbl-c, FLAG-Enigma or FLAG-EnigmaΔLIM2-3 alone and in combination as indicated above the blots. All transfections included an equal amount of HA-ubiquitin. At 40h post-transfection, all cells were treated with 20nM MG-132 for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for RET and HA-ubiquitin. Co-immunoprecipitation of Cbl-c and Enigma were evaluated as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control. (<b>B</b>) RET truncation constructs used for mapping Cbl-c and Enigma interaction sites. Structural domains include the extracellular cadherin-like repeats (C1-4), a cysteine rich region (CR), a hydrophobic transmembrane region (TM), a split cytoplasmic tyrosine kinase domain (TK1 and TK2), and intracellular tyrosines subsequently mutated to an F or stop (*) to create each construct used in this study. (<b>C</b>) HEK293T cells were transfected with GFP-tagged Enigma both alone and in combination with RET9MEN2A or one of a series of RET9MEN2A mutant constructs as indicated above each blot. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for GFP-Enigma. Molecular weight standards are indicated on the left of each panel and Hsc70 serves as loading control. (<b>D</b>) HEK293T cells were transfected with each of the RET9MEN2A constructs both alone and in combination with GST-Cbl-c as indicated above each blot. All transfections were balanced with empty vector controls, and cells were collected 48 h post-transfection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. GST pull-downs were performed on 300 µg of each of the whole cell lysates and immunoblotted for Cbl-c, phosphotyrosine (pY), and RET as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control.</p

Cbl binds RET and MEN2A kinases.

<p>(<b>A</b>) HEK293T cells were transfected separately with pJ7Ω plasmids encoding each of the following four RET kinase isoforms: RET 9, RET 51, and their MEN2A (C634R) mutant isoforms. Each was transfected both with and without GST-Cbl-c. Whole cell lysates were collected at 48h post-transfection and GST pull-downs were performed on 300 µg of whole cell lysates and immunoblotted (IB) as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel β-actin serves as a loading control. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c both with and without RET9MEN2A. Replicate plates were treated with either 500 µM Sorafenib (+) or DMSO as vehicle control (-) for 90m prior to collection. Whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysates using anti-RET antibody with Protein A/G Sepharose and immunoblotted for RET and phosphotyrosine (pY) as indicated. GST pull-downs were performed, as described above, and immunoblotted (IB) for RET, phosphotyrosine (pY), and GST-Cbl-c as indicated. Molecular weight standards are indicated to the left of each panel and Hsc70 serve as loading controls.</p

Enigma abrogates RETMEN2A ubiquitination.

<p>(<b>A</b>) RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates described above and immunoblotted for HA-ubiquitin and phosphotyrosine (pY) as indicated on the right. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c with and without FLAG-Enigma along with either RET9MEN2A or RET51MEN2A. Cells were collected 48h post-transfection, and whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for RET and HA-ubiquitin as indicated on the right. Molecular weight standards are indicated on the left and Ponceau S staining serves as a measure of protein loading. (<b>C</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above the blots. All transfections were performed in triplicate. At 24h post-transfection, triplicate plates were pooled and replated. At 40h post-transfection, replicate plates were treated with 100ng/mL cycloheximide (CHX) for either 3 or 5h as indicated. Control plates received an equivalent volume of DMSO vehicle control for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates was immunoblotted as indicated. RET steady state levels were then assessed using β-actin as a loading control. (<b>D</b>) Densitometric analysis of RET steady state levels in the presence of GST-Cbl-c and Enigma, either alone or in combination. Levels were all compared to Vector transfected cells in the absence of cycloheximide. Error bars denote mean ± SE (n  = 3). (<b>E</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above each blot. At 24h post-transfection, all cells were starved of FBS for 24h prior to cell harvesting. A total of 20 µgs of each of the whole cell lysates were immunoblotted as indicated to the right of each panel. Phospho-MAPK steady state levels were assessed using MAPK and Hsc70 as loading controls. Molecular weight standards are indicated on the left of each panel. (<b>F</b>) HEK293T cells were transfected with wild-type RET and the RET co-receptor, GFRα1 along with GST-Cbl-c and Enigma both alone and in combination. Vector controls included empty GST vector and all transfections were performed in duplicate and included HA-tagged ubiquitin. At 24h post-transfection, duplicate plates were pooled and re-plated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 24h, then stimulated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087116#pone.0087116-Boulay1" target="_blank">[43]</a>, using 30ng/mL GDNF and 100ng/mL exogenous GFRα1 (+) or water control (-) for 15m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess RET ubiquitination, RET immunoprecipitations were performed on 300ugs of whole cell lysate and immunoblotted for HA-ubiquitin as indicated on the right. (<b>G</b>) HEK293T cells were transfected with EGFR along with GST-Cbl-c and Enigma both alone and in combination. All transfections were performed in duplicate. At 24h post-transfection, duplicate plates were pooled and replated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 8h, then stimulated with 100ng/mL EGF (+) or water control (-) for 10m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess EGFR ubiquitination, EGFR immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for EGFR and HA-ubiquitin as indicated on the right. Molecular weight standards are shown to the left of each panel and tubulin serves as loading control.</p

Hic-5 interacts with the RF of Cbl-c.

<p><b>A.</b> 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>B.</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.</p

Hic-5 colocalizes with Cbl-c.

<p>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 NA 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).</p

Mutation of the first zinc coordinating complex of the RF of Cbl-c disrupts Hic-5 binding.

<p><b>A.</b> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049428#pone-0049428-g003" target="_blank">Figure 3D and E</a> are indicated below the sequence. <b>B.</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. <b>C.</b> 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. <b>D.</b> HEK293T cells were transfected with HA-Cbl-c, RF mutants of HA-Cbl-c, HA-Cbl-c ΔRF, 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. <b>E.</b> 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.</p

Hic-5 interacts with Cbl-c.

<p><b>A.</b> 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>B.</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. <b>C.</b> 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. <b>D.</b> 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.</p

Hic-5 enhances Cbl-c mediated ubiquitination of EGFR in cells.

<p><b>A.</b> 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>B.</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. <b>C.</b> 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.</p

The second zinc coordinating complex of Hic-5 LIM2 is required for the interaction with Cbl-c.

<p><b>A.</b> Schematic diagram of Hic-5 constructs used to map the interaction with Cbl-c. <b>B.</b> HEK293T cells were transfected with HA- Cbl-c and constructs for Hic-5 as indicated above the panel. HA-Cbl-c was immunoprecipitated with anti-HA beads and immunoprecipitates (IP) or cell lysates (lysate) were immunoblotted (IB) as indicated to the right of the panels. * indicates immunoglobulin. <b>C.</b> HEK293T cells were transfected with HA-Cbl-c along with WT or mutants of Hic-5 which disrupt either the first or second zinc coordinating complexes of LIM2 (C287A and C313A respectively) as indicated above the figure. HA-Cbl-c was immunoprecipitated with anti-HA beads and 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; GFP was transfected as a control for transfection efficiency and is shown as a loading control. MWs in kDa are shown to the left of the panels.</p

Hic-5 increases the E3 activity of Cbl-c <i>in vitro</i>.

<p><b>A.. </b><i>In vitro</i> E3 assays were performed as described in the methods with the recombinant WT or activated (Y341E) His-Cbl-c constructs as labeled above panel. Recombinant purified His-Hic-5 WT, C287A were added where indicated. The data for the E3 activity of the Y341E Cbl-c mutant were quantified for three experiments and are displayed on the graph. Values represent the mean ubiquitination +/– SE relative to Y341 Cbl-c alone. The dotted line represents the level of ubiquitination by Y341E Cbl-c in the absence of Hic-5. Two tailed p-values using a paired T-test comparing the results for Y341 Cbl-c + Hic-5 (WT or C287A) to Cbl-c are shown on the graph (NS  =  not significant). <b>B.</b> E3 assays were performed with either WT or Y341E recombinant Cbl-c as described above. Increasing amounts of recombinant His-Hic-5 was added as indicated above the panel. Purified active Src was added where indicated. <b>C.. </b><i>In vitro</i> E3 assays were performed with recombinant GST-Cbl-c RF (GST-RF) or GST-Cbl-c Y341E as described above. Recombinant His-Hic-5 or the His-LIM2 domain of Hic-5(LIM2) were added as indicated above the panel. <i>In vitro</i> E3 assays were performed at 30°C for 40 m in the presence and absence of E2 and then immunoblotted (IB) as indicated. MW markers in kDa are shown to the left of the panels.</p