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Structural requirements for cub domain containing protein 1 (CDCP1) and Src dependent cell transformation.

ABSTRACT: 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.

SUBMITTER: Kollmorgen G

PROVIDER: S-EPMC3534080 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Structural requirements for cub domain containing protein 1 (CDCP1) and Src dependent cell transformation.

Kollmorgen Gwendlyn G Bossenmaier Birgit B Niederfellner Gerhard G Häring Hans-Ulrich HU Lammers Reiner R

PloS one 20121231 12

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 exo ...[more]

PMID: 23300860

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Project description:BackgroundDeregulated expression of the transmembrane glycoprotein CDCP1 (CUB domain-containing protein-1) has been detected in several cancers including colon, lung, gastric, breast, and pancreatic carcinomas. CDCP1 has been proposed to either positively or negatively regulate tumour metastasis. In this study we assessed the role of CDCP1 in properties of cells that are directly relevant to metastasis, namely adhesion and motility. In addition, association between CDCP1 and the tetraspanin protein CD9 was investigated.MethodsCDCP1 and CD9 protein expression was measured in a series of colon cancer cell lines by flow cytometry and Western blotting. Adhesion of Colo320 and SW480 cells was determined using a Matrigel adhesion assay. The chemotactic motility of SW480 cells in which CDCP1 expression had been reduced by RNA interference was analysed using the xCELLigence system Real-Time Cell Analyzer Dual Plates combined with 8 μm pore filters. Detergent-resistant membrane fractions were generated following density gradient centrifugation and the CDCP1 and CD9 protein composition of these fractions was determined by Western blotting. The potential association of the CDCP1 and CD9 proteins was assessed by co-immunoprecipitation.ResultsEngineered CDCP1 expression in Colo320 cells resulted in a reduction in cell adhesion to Matrigel. Treatment of SW480 cells with CDCP1 siRNA reduced serum-induced chemotaxis. CDCP1 and CD9 cell-surface protein and mRNA levels showed a positive correlation in colon cancer cell lines and the proteins formed a low-level, but detectable complex as judged by co-sedimentation of detergent lysates of HT-29 cells in sucrose gradients as well as by co-immunoprecipitation in SW480 cell lysates.ConclusionsA number of recent studies have assigned a potentially important role for the cell-surface protein CDCP1 in invasion and metastasis of a several types of human cancer cells. In this study, CDCP1 was shown to modulate cell-substratum adhesion and motility in colon cancer cell lines, with some variation depending on the colon cancer cell type. CDCP1 and CD9 were co-expressed at the mRNA and protein level and we obtained evidence for the presence of a molecular complex of these proteins in SW480 colon cancer cells.

Dataset Information

Structural requirements for cub domain containing protein 1 (CDCP1) and Src dependent cell transformation.

Publications

Structural requirements for cub domain containing protein 1 (CDCP1) and Src dependent cell transformation.

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