title: Cortactin phosphorylated by ERK1/2 localizes to sites of dynamic actin regulation and is required for carcinoma lamellipodia persistence.
abstract: BACKGROUND
  Tumor cell motility and invasion is governed by dynamic regulation of the cortical actin cytoskeleton. The actin-binding protein cortactin is commonly upregulated in multiple cancer types and is associated with increased cell migration. Cortactin regulates actin nucleation through the actin related protein (Arp)2/3 complex and stabilizes the cortical actin cytoskeleton. Cortactin is regulated by multiple phosphorylation events, including phosphorylation of S405 and S418 by extracellular regulated kinases (ERK)1/2. ERK1/2 phosphorylation of cortactin has emerged as an important positive regulatory modification, enabling cortactin to bind and activate the Arp2/3 regulator neuronal Wiskott-Aldrich syndrome protein (N-WASp), promoting actin polymerization and enhancing tumor cell movement.
  
  
  RESULTS
  In this report we have developed phosphorylation-specific antibodies against phosphorylated cortactin S405 and S418 to analyze the subcellular localization of this cortactin form in tumor cells and patient samples by microscopy. We evaluated the interplay between cortactin S405 and S418 phosphorylation with cortactin tyrosine phosphorylation in regulating cortactin conformational forms by Western blotting. Cortactin is simultaneously phosphorylated at S405/418 and Y421 in tumor cells, and through the use of point mutant constructs we determined that serine and tyrosine phosphorylation events lack any co-dependency. Expression of S405/418 phosphorylation-null constructs impaired carcinoma motility and adhesion, and also inhibited lamellipodia persistence monitored by live cell imaging.
  
  
  CONCLUSIONS
  Cortactin phosphorylated at S405/418 is localized to sites of dynamic actin assembly in tumor cells. Concurrent phosphorylation of cortactin by ERK1/2 and tyrosine kinases enables cells with the ability to regulate actin dynamics through N-WASp and other effector proteins by synchronizing upstream regulatory pathways, confirming cortactin as an important integration point in actin-based signal transduction. Reduced lamellipodia persistence in cells with S405/418A expression identifies an essential motility-based process reliant on ERK1/2 signaling, providing additional understanding as to how this pathway impacts tumor cell migration.
authors.affiliation: Department of Neurobiology and Anatomy, Program in Cancer Cell Biology, Mary Babb Randolph Cancer Center, West Virginia University, Morgantown, West Virginia, United States of America.
authors.forename: Laura C
authors.initials: LC
authors.lastname: Kelley
authors.forename: Karen E
authors.initials: KE
authors.lastname: Hayes
authors.forename: Amanda Gatesman
authors.initials: AG
authors.lastname: Ammer
authors.forename: Karen H
authors.initials: KH
authors.lastname: Martin
authors.forename: Scott A
authors.initials: SA
authors.lastname: Weed
mesh_terms: D000199:Actins / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002465:Cell Movement; D051356:Cortactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation; D011487:Protein Conformation / Q000187:drug effects; D011554:Pseudopodia / Q000378:metabolism; D034622:RNA Interference; D012694:Serine / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism
chemical_list: D000199:Actins; C495868:CTTN protein, human; D051356:Cortactin; D051316:Wiskott-Aldrich Syndrome Protein; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D012694:Serine; D004815:Epidermal Growth Factor; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3