biostudies-literatureMori YJapan Society for the Promotion of Science (JSPS)11513-11524https://www.ebi.ac.uk/biostudies/studies/S-EPMC6663864biostudies-literatureUnknown294(30)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 (<i>TACSTD</i>) 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 <i>claudin-7</i> 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.The Journal of biological chemistryTrophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility.PMC6663864KAKENHI (17K14998)Mori YYamashita TMorii EOjima KNakada HIwamoto SAkita KfalseTrophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility.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 (<i>TACSTD</i>) 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 <i>claudin-7</i> 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.2019-01-01T00:00:00Z2019 Jul2025-04-21T20:16:19.658Z2025-04-05T17:53:15.509ZS-EPMC66638643117709510.1074/jbc.RA119.00808410.1074/jbc.ra119.008084