RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.
Carbonic anhydrase 12 (CA12) is a membrane-associated enzyme that is highly expressed on many human cancers. It is a poor prognostic marker and hence an attractive target for cancer therapy. This study aimed to develop a humanized CA12-antibody with anti-cancer activity.Antibody libraries were constructed and screened by the Retrocyte display®. Antibody binding and blocking properties were determined by ELISA, flow cytometry and enzymatic activity assays. Spheroid viability was determined by Cell-Titer-Fluor assay.We developed a novel humanized CA12-specific antibody, 4AG4, which recognized CA12 as an antigen and blocked CA12 enzymatic activity. Our humanized CA12-antibody significantly inhibited spheroid growth of lung adenocarcinoma A549-cells in vitro by blocking CA12 enzymatic activity. Similar anti-tumor effects were recapitulated with CA12-gene knockout of A549-cells.Our newly identified humanized CA12-antibody with anti-cancer activity, represents a new tool for the treatment of CA12-positive tumors.
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