title: Identification of NEK3 Kinase Threonine 165 as a Novel Regulatory Phosphorylation Site That Modulates Focal Adhesion Remodeling Necessary for Breast Cancer Cell Migration.
abstract: Accumulating evidence supports a role for prolactin (PRL) in the development and progression of human breast cancer. Although PRL is an established chemoattractant for breast cancer cells, the precise molecular mechanisms of how PRL regulates breast cancer cell motility and invasion are not fully understood. PRL activates the serine/threonine kinase NEK3, which was reported to enhance breast cancer cell migration, invasion, and the actin cytoskeletal reorganization necessary for these processes. However, the specific mechanisms of NEK3 activation in response to PRL signaling have not been defined. In this report, a novel PRL-inducible regulatory phosphorylation site within the activation segment of NEK3, threonine 165 (Thr-165), was identified. Phosphorylation at NEK3 Thr-165 was found to be dependent on activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway using both pharmacological inhibition and siRNA-mediated knockdown approaches. Strikingly, inhibition of phosphorylation at NEK3 Thr-165 by expression of a phospho-deficient mutant (NEK3-T165V) resulted in increased focal adhesion size, formation of zyxin-positive focal adhesions, and reorganization of the actin cytoskeleton into stress fibers. Concordantly, NEK3-T165V cells exhibited migratory defects. Together, these data support a modulatory role for phosphorylation at NEK3 Thr-165 in focal adhesion maturation and/or turnover to promote breast cancer cell migration.
authors.affiliation: From the Department of Pathology, Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611 and.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Harrington
authors.affiliation: the Department of Pathology, Virginia Commonwealth University, Richmond, Virginia 23298 charles.clevenger@vcuhealth.org.
authors.forename: Charles V
authors.initials: CV
authors.lastname: Clevenger
mesh_terms: D019943:Amino Acid Substitution; D001943:Breast Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D002465:Cell Movement; D004789:Enzyme Activation / Q000235:genetics; D005260:Female; D006801:Humans; D020935:MAP Kinase Signaling System; D061986:MCF-7 Cells; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D000072379:NIMA-Related Kinases / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000235:genetics; D011388:Prolactin / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics / Q000378:metabolism
chemical_list: D009363:Neoplasm Proteins; D013912:Threonine; D011388:Prolactin; C486224:NEK3 protein, human; D000072379:NIMA-Related Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3
keywords: NEK3; cell migration; extracellular-signal-regulated kinase (ERK); focal adhesion; prolactin; serine/threonine protein kinase; threonine phosphorylation