Schematic representation of major CDCP1 features important for focus formation.

<p>Only upon overexpression of Src and CDCP1, cells become transformed, and all changes in CDCP1 except for the processing mutant reduce transformation efficiency. (A–K) Left line, CDCP1 with five intracellular tyrosine residues (circles below horizontal line that represents the plasma membrane), right line with circle, Src kinase; fat print, overexpression; filled circles, tyrosine residues phosphorylated upon transient overexpression. X - no focus formation; arrows indicate focus formation. (D) mutant Y734F, (E) addback mutant Y734, (F) mutant C689, 690G, (G) mutant ΔNT217, (H) mutant ΔNT370, (I) mutant K365A, R368A, (K) myristoylation defective Src G2A mutant.</p

Role of CDCP1-domains for cellular transformation.

<p>(A) Schematic presentation of the mutants used. (B–F) NIH3T3 cells were infected with Src and CDCP1 or the indicated deletion mutants, grown and stained as above. In separate experiments, the mutants ΔNT370, K365A,R368A, C689,690G and Δ5K were compared in their transformation efficiency to wild type CDCP1. Please note, that the number of CDCP1 virus in (D) was reduced in comparison to the other experiments.</p

Structural Requirements for Cub Domain Containing Protein 1 (CDCP1) and Src Dependent Cell Transformation

<div><p>Cub domain containing protein 1 (CDCP1) is strongly expressed in tumors derived from lung, colon, ovary, or kidney. It is a membrane protein that is phosphorylated and then bound by Src family kinases. Although expression and phosphorylation of CDCP1 have been investigated in many tumor cell lines, the CDCP1 features responsible for transformation have not been fully evaluated. This is in part due to the lack of an experimental system in which cellular transformation depends on expression of exogenous CDCP1 and Src. Here we use retrovirus mediated co-overexpression of c-Src and CDCP1 to induce focus formation of NIH3T3 cells. Employing different mutants of CDCP1 we show that for a full transformation capacity, the intact amino- and carboxy-termini of CDCP1 are essential. Mutation of any of the core intracellular tyrosine residues (Y734, Y743, or Y762) abolished transformation, and mutation of a palmitoylation motif (C689,690G) strongly reduced it. Src kinase binding to CDCP1 was not required since Src with a defective SH2 domain generated even more CDCP1 dependent foci whereas Src myristoylation was necessary. Taken together, the focus formation assay allowed us to define structural requirements of CDCP1/Src dependent transformation and to characterize the interaction of CDCP1 and Src.</p> </div

Co-overexpression of Src and CDCP1 leads to focus formation.

<p>(A) Fifty thousand NIH3T3 cells were infected with 500.000 retroviruses encoding CDCP1 or Src individually or together. After 48 h the cells were reseeded in a 10 cm-dish and grown for 3 weeks, with 3 media changes per week. In parallel, a 2% aliquot was taken from the CDCP1/Src infected cells and reseeded together with 150.000 parental NIH3T3 cells (1∶50 dilution). Finally, the cells were stained with crystal violett. (B) Parental NIH3T3 cells, cells infected with the CDCP1 encoding retrovirus or individual foci derived from infections at lower m.o.i. than in <i>A</i> that had been isolated and expanded, were lysed, aliquots with similar amounts of protein run on an SDS-PAGE, proteins transferred to nitrocellulose and the filter blotted with the indicated antibodies. αPY, anti-phosphotyrosine. Size markers (kDa) are indicated.</p

Src and CDCP1 dependent focus formation require tyrosine phosphorylation of CDCP1.

<p>(A) NIH3T3 cells were infected as above with Src alone or with Src and the indicated CDCP1 forms. After three weeks, the cells were stained with crystal violett. Y734 - all codons for tyrosine residues but Y734 were mutated to encode Phe; Y734/743/762F - the codons for these tyrosines were mutated to Phe. (B) CDCP1 and the indicated tyrosine mutants were transiently overexpressed in 293 cells. After treatment with peroxovanadate the cells were lysed and CDCP1 immunoprecipitated with the Cub1 antibody. Immunoprecipitates were analyzed by SDS-PAGE and Western blotting with a phosphotyrosine-antibody (αPY; upper panel). In parallel, an aliquot from the cell lysate was analyzed for CDCP1 expression with an antibody against CDCP1 (lower panel). (C) Similar to (B), 293 cells were transfected with plasmids encoding CDCP1 and its mutants, and in addition c-Src. Cells were analyzed as above and the Cub1 immunoprecipitates detected with a phosphotyrosine specific antibody, whereas in an aliquot of the cell lysate CDCP1 and c-Src expression were tested (lower panel). αPY, anti-phosphotyrosine.</p

Effect of Src-mutations on CDCP1 dependent transformation.

<p>(A) NIH3T3 cells were infected with CDCP1 and the indicated Src mutants, grown and stained as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053050#pone-0053050-g001" target="_blank">Figure 1</a>. (B,C) HEK293 cells overexpressing the indicated proteins were lysed, an aliquot of the lysate was used for expression analysis and the rest immunoprecipitated with the antibody Cub1. Proteins were size separated by SDS-PAGE, transferred to nitrocellulose and blotted with antibodies against CDCP1, Src or phosphotyrosine (αPY). Src-KA - kinase inactive Src; Src-WA - defective SH3 domain; Src-RA - defective SH2 domain; Src-G2A - no myristoylation.</p

Deciphering the Stepwise Binding Mode of HRG1β to HER3 by Surface Plasmon Resonance and Interaction Map

<div><p>For the development of efficient anti-cancer therapeutics against the HER receptor family it is indispensable to understand the mechanistic model of the HER receptor activation upon ligand binding. Due to its high complexity the binding mode of Heregulin 1 beta (HRG1β) with its receptor HER3 is so far not understood. Analysis of the interaction of HRG1β with surface immobilized HER3 extracellular domain by time-resolved Surface Plasmon Resonance (SPR) was so far not interpretable using any regular analysis method as the interaction was highly complex. Here, we show that Interaction Map (IM) made it possible to shed light on this interaction. IM allowed deciphering the rate limiting kinetic contributions from complex SPR sensorgrams and thereby enabling the extraction of discrete kinetic rate components from the apparently heterogeneous interactions. We could resolve details from the complex avidity-driven binding mode of HRG1β with HER3 by using a combination of SPR and IM data. Our findings contribute to the general understanding that a major conformational change of HER3 during its activation is induced by a complex sequential HRG1β docking mode.</p></div

Kinetic interaction parameters calculated by Interaction Map.

<p>Kinetic values of the four SPR assays shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116870#pone.0116870.t001" target="_blank">table 1</a>, calculated by Interaction Map (TraceDrawer Software 1.6, Ridgeview Instruments AB). Listed are mean values of three replicates (±standard deviation).</p><p>Kinetic interaction parameters calculated by Interaction Map.</p

Deciphering the binding behavior of HRG1β to HER3, investigated by different SPR assay setups.

<p>To determine the interaction of HRG1β with HER3 in presence and absence of anti-HER3 antibody mAb208, four different SPR assay setups were designed. The descriptive symbols illustrate the corresponding assay setups. Arrows indicate injection of analytes (left column). Measured biomolecular interactions were evaluated using a regular Langmuir model (middle column) and where applicable using a two-state reaction model (right column) by Biacore Evaluation Software 2.0. The curve fittings are highlighted in red. The curve corresponding to the highest concentration is indicated in each sensorgram. Report points were used to additionally characterize the shape of the sensorgrams. They are indicated by asterisk: BL_early (*) is the binding signal shortly before the end of the analyte injection. BL_late (**) is the binding signal 100 seconds after the end of the injection. SL (***) is the stability late signal at the end of the dissociation phase. Three replicates of each concentration are shown in black in each sensorgram (n = 3). The third highest concentration of each assay was injected twice (n = 6). (A—C) Murine antibody mAb208 was captured by immobilized rabbit anti-mouse antibody on CM5 sensor chip surface. (A) Injection of HER3 (ECD3). (B) Injection of pre-incubated HER3/HRG1β. (C) Injection of HRG1β. (D) Biotinylated HER3-Avi (bi-ECD3) was captured on a streptavidin-coated CAP sensor chip. Subsequently, HRG1β was injected.</p

Direct estrogen receptor (ER) / HER family crosstalk mediating sensitivity to lumretuzumab and pertuzumab in ER+ breast cancer

<div><p>Bidirectional cross talk between members of the human epidermal growth factor family of receptors (HER) and the estrogen receptor (ER) is believed to underlie resistance mechanisms that develop in response to treatment with anti-HER agents and endocrine therapy. We investigated the interaction between HER2, HER3 and the ER <i>in vitro</i> using human embryonic kidney cells transfected with human HER2, HER3, and ERα. We also investigated the additive efficacy of combination regimens consisting of anti-HER3 (lumretuzumab), anti-HER2 (pertuzumab), and endocrine (fulvestrant) therapy <i>in vivo</i>. Our data show that both HER2 and HER3 can directly complex with the ER and can mediate phosphorylation of the ER. Phosphorylation of the ER was only observed in cells that expressed both HER2 and ERα or in heregulin-stimulated cells that expressed both HER3 and ERα. Using a mouse xenograft model of ER+/HER2-low (HER2 immunohistochemistry 1+ or 2+ without gene amplification) human breast cancer we show that the combination of lumretuzumab and pertuzumab is highly efficacious and induces long-lasting tumor regression <i>in vivo</i> and adding endocrine therapy (fulvestrant) to this combination further improved efficacy. In addition, a prolonged clinical response was observed with the combination of lumretuzumab and pertuzumab in a patient with ER+/HER2-low breast cancer who had failed endocrine therapy. These preclinical data confirm that direct cross talk exists between HER2/HER3 and ER which may explain the resistance mechanisms to endocrine therapy and monoclonal antibodies that target HER2 and HER3. Our data also indicate that the triplet of anti-HER2, anti-HER3, and endocrine therapy might be an efficacious combination for treating patients with ER+/HER2-low breast cancer, which is an area of significant unmet medical need.</p></div