200 mM imidazole. Samples were evaluated by SDS-PAGE and staining with GelCode Blue Stain Reagent stained (Thermo Fischer Scientific, Rockford, IL). Proteins were dialyzed into 50 mM Bis-Tris pH 6.0, 20 mM NaCl, 10 uM ZnCl) using Fast Dialyzer chambers and RCIK membranes from Harvard Apar- atus (Holliston, MA). Production and Purification of GST Proteins Bacterial expression vectors were transformed in Rosetta bacteria. Selected colonies were grown in LB broth containing 100 mg/ml ampicillin. GST-Cbl-c RF constructs were grown in 10 ml overnight cultures and then diluted 1/10 in 90 ml of LB and grown at 37uC for 1 h. Protein production was induced with 2 mM isopropyl b-d-thiogalactoside at 37uC for 4 h. Cell pellets were resuspended in 5-ml of lysis buffer (50 mM Tris pH 8, 1 mM EDTA, 1% Triton-X 100, 5 mM DTT), sonicated and clarified by centrifugation. A 50% slurry of 300 ml of Glutathione Sepharose 4B (Amersham, Piscataway, NJ) was added to clarified lysate and incubated with rocking overnight at 4uC. Beads were pelleted by centrifugation and washed 5 times in lysis buffer and then 5 times in cold PBS. Proteins bound to GSH-beads were resuspended to a 50% slurry in PBS for storage. Results Yeast Two-hybrid Identifies Hic-5 as a Cbl-c Interacting Protein Using partial and full length constructs of Cbl-c as bait in a yeast two-hybrid screen of breast cancer/prostate cancer, spleen, and brain cDNA libraries, the LIM domain containing proteins Hic-5 and Trip-6 were identified as potential Cbl-c interacting proteins. Both Hic-5 and Trip-6 were identified when full length Cbl-c (amino acids 1–474) and a construct containing the last 114 amino acids (360–474) were used as bait. To confirm the interactions of Cbl-c with Hic-5 and Trip-6, full-length cDNAs of Hic-5 and Trip-6 were co-expressed with HA-epitope tagged Cbl-c in HEK293T cells. Immunoprecipitation of HA-Cbl-c showed co- immunoprecipitation of Hic-5 but not of Trip-6 (Fig. 1A and B). These data confirmed the interaction of Cbl-c with Hic-5. The interaction between the endogenous proteins was established by the demonstration of specific co-immunoprecipitation of Hic-5 from lysates from cells that express both proteins but not from cells that express high levels of endogenous Hic-5 but lack detectable Cbl-c using an anti-Cbl-c antibody (Fig. 1C). When immunopre- cipitating Cbl-c from the CFPAC-1 cell line, we sometimes see higher molecular weight bands (see above asterisk in Fig. 1C). The identity of these bands is not known but could represent post- translationally modified forms of Cbl-c. Cbl-c is the most divergent of the three mammalian Cbl proteins [17,19]. To test the interaction of Hic-5 with each of the Cbl proteins, Hic-5 was co-expressed with HA-epitope tagged Cbl, Cbl-b, and Cbl-c in HEK293T cells, followed by immunoprecip- itation of Cbl proteins with an anti-HA affinity matrix. Hic-5 co- precipitated with Cbl-c, but not with Cbl or Cbl-b, suggesting a selective interaction between Cbl-c and Hic-5 (Fig. 1D). Hic-5 has been described to localize to focal adhesions, the cytosol, and to the nucleus [42,43]. To investigate the site of interaction between Hic-5 and Cbl-c, CFP-Hic-5 and YFP-Cbl-c were transfected into HeLa cells and visualized using confocal microscopy (Fig. 2). Cbl-c and Hic-5 overlapped in a diffuse pattern (Fig. 2, panels 1–3; Pearson’s Correlation Coefficient = 0.70 +/–0.15). Hic-5 was also found in a punctate distribution on the basal surface of the cells consistent with its known localization to focal adhesions but Cbl-c was not localized to these structures Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 (Fig. 2, panels 4–6; Pearson’s Correlation Coefficient = 20.06 +/ 20.07). To confirm that the punctuate structures were focal adhesions, cells transfected with CFP-Hic-5 or YFP-Cbl-c were stained with an antibody for vinculin, a protein specific for focal adhesions. Hic-5 and vinculin colocalized to the punctuate structures confirming that these are focal adhesions (Fig. 2, panels 7–9; Pearson’s Correlation Coefficient = 0.70 +/20.23). No colocalization of Cbl-c and vinculin was found (Fig. 2, panels 10– 12; Pearson’s Correlation Coefficient = 20.11 +/2 0.10). The overlap of Cbl-c and Hic-5 in a diffuse compartment and the lack of colocalization of Cbl-c and Hic-5 in a clearly identifiable compartment (e.g., focal adhesions) makes the deter- mination of the site of their interaction inconclusive. Hic-5 Interacts with the RING Finger of Cbl-c To map the site of the interaction of Cbl-c with Hic-5, a series of deletion mutations of Cbl-c were coexpressed with Hic-5 in HEK293T cells and assessed for co-immunoprecipitation with Hic-5 (Fig. 3). The deletion of the last 124 amino acids of Cbl-c including the RF abrogated binding to Hic-5 (Fig. 3B, lane 8). This is consistent with the identification of Hic-5 in the yeast two-hybrid screen by a Cbl-c bait containing the last 114 amino acids. In contrast, successive deletions of the C-terminus up to the RF did not affect binding of Cbl-c to Hic-5A (Fig. 3B, lanes 5–7). Also, a naturally occurring splice variant (Cbl-c Short) that deletes the second half of the SH2 domain of Cbl-c (and abrogates binding to tyrosine phosphorylated proteins) did not affect binding to Hic-5 (Fig. 3B, lane 4). These results identified the RF as a critical domain for the interaction between Cbl-c and Hic-5. The RF of Cbl-c differs from the RF of Cbl and Cbl-b by nine of 40 amino acids (Fig. 4A). To further test the specificity of the interaction between Hic-5 and the RF of Cbl-c, chimeric proteins of Cbl-b containing the RF of Cbl-c and Cbl-c containing the RF of Cbl-b were expressed along with Hic-5 in HEK293T cells and their interactions assayed by co-precipitation (Fig. 4B and C). As controls, the non-chimeric versions of these constructs for Cbl-b and Cbl-c were tested (Fig. 4B and C). Hic-5 co-precipitated with Cbl-c and with the chimeric Cbl-b containing the Cbl-c RF but not with either Cbl-b or the chimeric protein of Cbl-c containing the Cbl-b RF. Together, these data indicated that the RF of Cbl-c is critical for the interaction with Hic-5. The Cbl RF is a cross braced C3HC4 zinc coordinating motif [4,44]. Using site-directed mutagenesis, we tested the effect of mutating the first cysteine in the first zinc coordinating complex (C351A) of the RF of Cbl-c, the first cysteine in the second zinc coordinating complex (C366A) in the RF of Cbl-c, or both on the ability of Cbl-c to interact with Hic-5. The Cbl-c and Hic-5 interaction was abrogated in the C366A mutant and in the double mutant but not with the C351A mutant (Fig. 4D, compare lanes 5 and 6 to lanes 3 and 4). This suggests that the second zinc coordination complex of the Cbl-c RF is necessary for the interaction. To further test the specificity of the interaction between the RF of Cbl-c and Hic-5, we performed pull down assays using a recombinant purified GST-tagged protein contain- ing the isolated RF of Cbl-c (WT, C351A and C366A) and a bacterial lysate expressing recombinant His-tagged Hic-5. Hic-5 precipitated with GST-RF WT and GST-RF C351A mutant (Fig. 4E, lanes 4 and 6) but not with GST or GST-RF 366A (Fig. 4E, Lanes 2 and 8). Cbl-c Interacts with the LIM2 of Hic-5 Hic-5 is a member of the paxillin family of LIM proteins that are characterized by N-terminal LD protein-interacting domains and four C-terminal LIM domains [29,30]. The minimal PLOS ONE | www.plosone.org 4 November 2012 | Volume 7 | Issue 11 | e49428