The role of Phosphorylation in Claudin 1 mislocalization in human breast cancer cells
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Date
2015
Authors
Wang, Nan
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Abstract
Claudin 1, a major tight junction protein, is frequently deregulated and mislocalized to the cytoplasm in some human breast cancers. Mislocalization of claudin 1 has been recently shown to enhance the metastatic potential in other cancers, including melanoma, colon and liver cancers, and thus, may also promote metastasis in breast cancer. Moreover, the C-terminus of the claudin 1 protein has been shown to direct its membrane localization in normal epithelial cells. Furthermore, protein kinase activity (PKA/PKC) is important in regulating claudin 1 expression and localization in several cancers. Therefore, in breast cancer, it is possible that the phosphorylation of the PKA/PKC sites within the C-terminus may direct the localization of claudin 1. Thus, the hypothesis of the study is that mislocalization of claudin 1 in human breast cancer cells is regulated by phosphorylation.
First, to demonstrate whether the C-terminus of claudin 1 regulates its membrane localization in human breast cancer cells, GFP-tagged claudin 1 constructs lacking 24 amino acids of the C-terminus were generated. When these constructs were transfected into breast cancer cell lines (T47D and MCF-7), decreased membrane staining but increased cytoplasmic staining was observed compared to the full-length constructs that were observed primarily in the cell membrane. Next, in order to identify whether predicted phosphorylation sites within the C-terminal domain of claudin 1 protein were responsible for its mislocalization in human breast cancer cells, GFP-claudin 1 constructs were generated using site-directed mutagenesis that mimicked both constitutive phosphorylation and non-phosphorylation at PKC/PKA predicted target sites on the C-terminus. Following transfection in MCF-7 cells, constructs mimicking constitutive phosphorylation showed less membrane staining than their non-phosphorylatable counterparts.
Taken together, in human breast cancer cells, specific phosphorylation sites within the C-terminus of claudin 1 play a role in its subcellular localization. Thus, these results suggest that phosphorylation may be a mechanism regulating the mislocalization of claudin 1 to the cytoplasm in human breast cancer.
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Keywords
Breast Cancer, Claudin 1, Phosphorylation