Abstract
Filopodia are dynamic membrane extensions generated by F-actin bundling and are involved in cancer cell migration, invasion and metastasis. Fascin is the crucial actin-bundling protein in filopodia, with phosphorylation at fascin serine 39 being well characterized to regulate fascin-mediated actin bundling in filopodia. However, increasing evidence indicates that fascin is phosphorylated at a number of sites. Whether phosphorylation at other sites also regulates fascin function is unknown. In this study, we show that four potential phosphorylation sites in fascin, specifically tyrosine 23, serine 38, serine 39 and serine 274, regulate cell behavior and filopodia formation in esophageal squamous cancer cells. Expression of non-phosphorylatable mutations at each of the four sites promoted anchorage-independent growth, cell motility and filopodia formation, whereas phosphomimetic mutations at each of these sites inhibited these cell behaviors, implying that fascin function in esophageal squamous cancer is regulated by fascin phosphorylation at multiple sites. Furthermore, phosphorylation at S38 and S39 cooperatively regulated cell behavior and filopodia formation, with dual dephosphorylation at both S38 and S39 residues maximally enhancing cell proliferation, migration and filopodia formation, and phosphorylation at any of the two phosphorylatable sites resulting in reduced enhancement. Taken together, our results reveal that phosphorylation at fascin amino acids Y23, S38, S39 and S274, in combination, downregulates the extent of anchorage-independent growth, cell migration and filopodia formation in esophageal squamous cancer cells.
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Acknowledgements
We thank Dr. Stanley Li Lin from the Department of Cell Biology and Genetics of Shantou University Medical College for assistance in revising the manuscript. This work was supported by Grants from the National Natural Science Foundation of China (Nos. 81360331, 81472613 and 81602630), the Natural Science Foundation of China-Guangdong Joint Fund (Nos. U1301227 and U0932001), the Science and Technology Planning Project of Guang Dong Province (Nos. 2014A030304060 and 2014A020212286) and the Department of Education, Guangdong Government under the Top-tier University Development Scheme for Research and Control of Infectious Diseases.
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EML and LYX involved in conception and design and study supervision. JJX, EML, LYX and FMZ participated in development of methodology. FMZ, XNW and HSS acquired data. FMZ, XNW and HSS contributed to analysis and interpretation of data. FMZ, EML, LYX and PJN involved in writing, review and/or revision of the manuscript. LDL, FMZ, XNW, HSS and ZPD contributed to administrative, technical or material support.
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Zeng, FM., Wang, XN., Shi, HS. et al. Fascin phosphorylation sites combine to regulate esophageal squamous cancer cell behavior. Amino Acids 49, 943–955 (2017). https://doi.org/10.1007/s00726-017-2398-1
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DOI: https://doi.org/10.1007/s00726-017-2398-1