increased upon phosphorylation of a tyrosine in the linker region [11,14]. While Cbl and Cbl-b have been well-studied and characterized, less is known about Cbl-c. Cbl-c is expressed exclusively in epithelial cells while Cbl and Cbl-b are widely expressed in mammalian tissues [18,19,20,21]. Like Cbl and Cbl-b, the N- terminus of Cbl-c is composed of the highly conserved TKB, RF, and linker region [17,19]. The C-terminus of the Cbl-c protein diverges from the other two Cbl proteins by having a shorter PR domain and lacking a UBA domain. Cbl-c, like Cbl and Cbl-b, is a functional E3 that can ubiquitinate and downregulate the EGFR, v-Src, and RET in cells [7,19,22,23]. Mice for Cbl and Cbl-b have clear immunological and hematological defects that help to define their physiological roles, but mice for Cbl-c are viable and fertile with no clear abnormality [18,24,25,26,27]. Thus the physiological role of Cbl-c is not clear. To elucidate the function of Cbl-c, we sought to identify Cbl-c interacting proteins utilizing a yeast two-hybrid screen and identified Hic-5 (a.k.a., TGFb1I1, ARA55) [28,29] as a binding partner. Hic-5 is a member of the LIM domain containing paxillin family and the mRNA is widely expressed in normal tissues including epithelial tissues such as the lung, liver, and kidney and the mRNA is expressed in normal epithelial cells in culture [29,30]. Interestingly, the mRNA is low or absent in transformed cells from hematopoietic and epithelial malignancies [29]. Hic-5 has been shown to be involved in many events involving cytoskeletal rearrangements, including focal adhesion formation, nuclear/cytoplasmic transport, cell migration, myotube formation, and epithelial to mesenchymal transition [29,31,32,33,34,35,36,37,38]. The Cbl-c/Hic-5 interaction is mediated by the RF of Cbl-c and the second LIM domain of Hic-5. Importantly, we established that Hic-5 enhances the E3 activity of Cbl-c both in vitro and in cells. Thus we have identified a novel interaction between two distinct zinc coordinating structures leading to enhancement of the Cbl-c RF E3 activity. Materials and Methods Materials Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin sulfate were obtained from Invitrogen (Carlsbad, CA). Dulbecco’s phosphate buffered saline (DPBS) was purchased from Mediatech Inc. (Herndon, VA). Recombinant human EGF was purchased from BD Biosciences, Inc. (San Jose, CA). Tissue culture plasticware and other laboratory consumables were purchased from commercial sources. Yeast Two-Hybrid Screening Yeast two-hybrid screening was carried out at Myriad Genetics (Salt Lake City, UT) using a partial (amino acids 360–474) or a full length Cbl-c as the bait with a mating-based method. The Cbl-c cDNAs were cloned into pGBT.superB creating an open reading frame for Cbl-c fused to the GAL4 DNA-binding domain. The bait plasmid was introduced into Myriad’s ProNet yeast strain PNY200 (MATa ura3-52 ade2-101 trp1-901 his3-D200 leu2-3112 gal4D gal80D). The bait yeast cells were allowed to mate with Myriad’s ProNet MATa yeast cells, BK100 (MATa ura3-52 trp1- 901 his3-D200 leu2-3112 gal4D gal80D GAL2-ADE2 LYS2::GAL1- HIS3 met2::GAL7-lacZ) containing three independent cDNA libraries from human breast/prostate cancer, spleen and brain. After mating, at least 5 million diploid yeast cells were obtained from each library and selected on His- and Ade-lacking medium. The auxotrophy is suppressed if the bait and prey proteins interact. The prey plasmids were isolated from the positive Modulation of Cbl-c E3 Activity by LIM2 of Hic-5 colonies, and the interaction was confirmed by expression of third reporter gene (lacZ). cDNAs in the positive prey plasmids were sequenced. Expression Constructs The expression plasmids for full length HA-epitope tagged Cbl, Cbl-b, and Cbl-c, as well as Cbl-c Short and the control vector (pCEFL) have been described previously [19,39]. HA-epitope deletion constructs for Cbl-c were created using PCR (DPro, DCT and DRF) or site-directed mutatgenesis (DCT2) to introduce a stop codon using Quick Change Kit (Stratagene, La Jolla, CA). HA- epitope Cbl-c mutants (C351A and C366A) were generated by site-directed mutagenesis using Quick Change Kit (Stratagene, La Jolla, CA). N-terminus tagged YFP-Cbl-c was constructed by first cloning Cbl-c ORF into the Hind III and Kpn I sites of pEGFP C3 (Clonetech, Mountain View, CA). The GFP protein was replaced with the YFP protein by cloning the YFP protein into the Age I and BsrG I sites of GFP. The pCMV-Hic-5 expression vector was obtained from Thermo Fisher Scientific/Open Biosystems (Huntsville, AL). Site directed mutagenesis of pCMV-Hic-5 to generate the C287A and C313A mutants was performed using the Quick Change Kit (Stratagene, La Jolla, CA). N-terminus tagged Hic5-eCFP in pReceiver-M3 was purchased from GeneCopoeia (EX-U1273-M32, Rockville, MD). The GFP expression plasmid used as a loading control for transfections (pCDNA3.1/zeo) was obtained from Invitrogen (Carlsbad, CA). The GST-tagged Cbl-c RF was created by PCR amplification of the Cbl-c RF and subsequent cloning of the PCR product into the 59 BamHI and 39 EcoRI restriction sites of pGEX-5X-1. His- tagged constructs for Cbl-c, Cbl-c Y341E, Hic-5, Hic-5 C287A, Hic-5 LIM2, and Hic-5 LIM2 C287A were created by amplifying the appropriate sequences using PCR and cloning the PCR products into the pTrc-HisA vector at the XhoI and HindIII restriction sites. pTrc-HisA was purchased from Invitrogen (Carlsbad, CA). pET15b-UbcH5b was a provided by Dr. Allan Weissman. All of the constructs were confirmed by DNA sequencing. Cell Culture and Transfections Human embryonic kidney HEK293T, CFPAC-1, and HeLa cells were obtained from ATCC and maintained in culture in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin sulfate. HEK293T cells were transfected using calcium phosphate (Profection; Promega Corp., Madison, WI). Following transfection cells were grown 48 h prior to the preparation of cell lysates. CFPAC-1 and HeLa cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Transfections were allowed to incubate 18 h prior to rescue, and cells were grown an additional 48 h before being harvested. Immunoblotting and Immunoprecipitation To harvest proteins, cells were washed twice in ice-cold DPBS containing 200 mM sodium orthovanadate (Fisher Chemicals, Fairlawn, NJ) and then lysed in ice-cold lysis buffer (10 mM Tris- HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 100 mM iodoacetamide (Sigma-Aldrich Corp., St. Louis, MO), 2 mM sodium orthovanadate, and protease inhibitors (Complete tabsH, Roche Diagnostics Corp., Indianap- olis, IN). The lysates were cleared of debris by centrifugation at 16,000 6g for 15 min at 4uC. Supernatant protein concentrations were determined using a BioRad protein assay (BioRad, Hercules, CA). For immunoblotting, lysates (2 mg protein/ml) were boiled in loading buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 2% SDS, 1 mg/ml bromphenol blue, 0.3573 M b- PLOS ONE | www.plosone.org 2 November 2012 | Volume 7 | Issue 11 | e49428