title: EBP50 phosphorylation by Cdc2/Cyclin B kinase affects actin cytoskeleton reorganization and regulates functions of human breast cancer cell line MDA-MB-231.
abstract: The actin cytoskeleton plays an important role in cell shape determination, adhesion and cell cycle progression. Ezrinradixin-moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na(+)-H(+) exchanger regulatory factor 1 (NHERF1), associates with actin cytoskeleton and is related to cell cycle progression. Its Ser279 and Ser301 residues are phosphorylated by cyclin-dependent kinase 2 (cdc2)/cyclin B during the mitosis phase. However, the biological significance of EBP50 phosphorylation mediated by cdc2/cyclin B is not clear. In the present study, MDA-MB-231 cells with low levels of endogenous EBP50 protein were stably transfected with constructs of EBP50 wild type (WT), phosphodeficient (serine 279 and serine 301 mutated to alanine-S279A/S301A) or phospho-mimetic (serine 279 and serine 301 mutated to aspartic acid-S279D/S301D) mutants. Subsequently, multiple phenotypes of these cells were characterized. Failure of cdc2/cyclin B-mediated EBP50 phosphorylation in cells expressing S279A/S301A (AA cells) significantly increased F-actin content, enhanced the adherence of cells to the extracellular matrix, altered cell morphology and caused defects in cytokinesis, as reflected in the formation of giant cells with heteroploid DNA and multinucleation or giant nuclei. Furthermore, knockdown of EBP50 expression in AA cells rescued cell defects such as the cytokinesis failure and abnormal cell morphology. EBP50 S279A/ S301A had a weaker binding affinity with actin than EBP50 S279D/S301D, which might explain the increase of F-actin content in the AA cells. The present results suggest that cdc2/cyclin B-mediated EBP50 phosphorylation may play a role in the regulation of various cell functions by affecting actin cytoskeleton reorganization.
authors.affiliation: Department of Biochemistry and Molecular Biology, Capital Medical University, Beijing, 100069, China.
authors.forename: Chaoyuan
authors.initials: C
authors.lastname: Sun
authors.forename: Junfang
authors.initials: J
authors.lastname: Zheng
authors.forename: Shan
authors.initials: S
authors.lastname: Cheng
authors.forename: Duiping
authors.initials: D
authors.lastname: Feng
authors.forename: Junqi
authors.initials: J
authors.lastname: He
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D016203:CDC2 Protein Kinase; D019556:COS Cells; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D019926:Cyclin B / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases; D048749:Cytokinesis; D005109:Extracellular Matrix / Q000378:metabolism; D005260:Female; D006801:Humans; D050505:Mutant Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D058105:Polymerization; D011485:Protein Binding; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*
chemical_list: D000199:Actins; D019926:Cyclin B; D050505:Mutant Proteins; D010750:Phosphoproteins; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; D018844:Cyclin-Dependent Kinases