Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility.

Yugo Mori, Kaoru Akita, Kazuki Ojima, Shungo Iwamoto, Tomoko Yamashita, Eiichi Morii, Hiroshi Nakada

The Journal of biological chemistry 2019 Jul 26

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Dysfunction of tight junctions is a critical step during the initial stage of tumor progression. Trophoblast cell surface antigen 2 (Trop-2) belongs to the family of tumor-associated calcium signal transducer (TACSTD) and is required for the stability of claudin-7 and claudin-1, which are often dysregulated or lost in carcinogenesis. Here, we investigated the effects of Trop-2 phosphorylation on cell motility. Analyses using HCT116 cells expressing WT Trop-2 (HCT116/WT) or Trop-2 alanine-substituted at Ser-303 (HCT116/S303A) or Ser-322 (HCT116/S322A) revealed that Trop-2 is phosphorylated at Ser-322. Furthermore, coimmunoprecipitation and Transwell assays indicated that Trop-2 S322A interacted with claudin-7 the strongest, and a phosphomimetic variant, Trop-2 S322E, the weakest and that HCT116/S322E cells have the highest motility and HCT116/S322A cells the lowest. All cell lines had similar levels of claudin-7 mRNA, but levels of claudin-7 protein were markedly decreased in the HCT116/S322E cells, suggesting posttranscriptional control of claudin-7. Moreover, claudin-7 was clearly localized to cell-cell borders in HCT116/S322A cells but was diffusely distributed on the membrane and partially localized in the cytoplasm of HCT116/S322E and HCT116/WT cells. These observations suggested that Trop-2 phosphorylation plays a role in the decrease or mislocalization of claudin-7. Using protein kinase C (PKC) inhibitors and PKC-specific siRNAs, we found that PKCα and PKCδ are responsible for Trop-2 phosphorylation. Of note, chemical PKC inhibition and PKCα- and PKCδ-specific siRNAs reduced motility. In summary, our findings provide evidence that Trop-2 is phosphorylated at Ser-322 by PKCα/δ and that this phosphorylation enhances cell motility and decreases claudin-7 localization to cellular borders. © 2019 Mori et al.

Citation

Yugo Mori, Kaoru Akita, Kazuki Ojima, Shungo Iwamoto, Tomoko Yamashita, Eiichi Morii, Hiroshi Nakada. Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility. The Journal of biological chemistry. 2019 Jul 26;294(30):11513-11524