title: The focal adhesion targeting domain of p130Cas confers a mechanosensing function.
abstract: Members of the Cas family of focal adhesion proteins contain a highly conserved C-terminal focal adhesion targeting (FAT) domain. To determine the role of the FAT domain in these proteins, we compared wild-type exogenous NEDD9 with a hybrid construct in which the NEDD9 FAT domain had been exchanged for the p130Cas (also known as BCAR1) FAT domain. Fluorescence recovery after photobleaching (FRAP) revealed significantly slowed exchange of the fusion protein at focal adhesions and significantly slower two-dimensional migration. No differences were detected in cell stiffness as measured using atomic force microscopy (AFM) and in cell adhesion forces measured with a magnetic tweezer device. Thus, the slowed migration was not due to changes in cell stiffness or adhesion strength. Analysis of cell migration on surfaces of increasing rigidity revealed a striking reduction of cell motility in cells expressing the p130Cas FAT domain. The p130Cas FAT domain induced rigidity-dependent phosphorylation of tyrosine residues within NEDD9. This in turn reduced post-translational cleavage of NEDD9, which we show inhibits NEDD9-induced migration. Collectively, our data therefore suggest that the p130Cas FAT domain uniquely confers a mechanosensing function.
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.|Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales 2000, Australia.
authors.forename: Peta M
authors.initials: PM
authors.lastname: Bradbury
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Kylie
authors.initials: K
authors.lastname: Turner
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Camilla
authors.initials: C
authors.lastname: Mitchell
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Kaitlyn R
authors.initials: KR
authors.lastname: Griffin
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Shiloh
authors.initials: S
authors.lastname: Middlemiss
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Loretta
authors.initials: L
authors.lastname: Lau
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Dagg
authors.affiliation: Australian National Fabrication Facility-Queensland node, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, Queensland 4067, Australia.
authors.forename: Elena
authors.initials: E
authors.lastname: Taran
authors.affiliation: Tissue Engineering and Microfluidics Laboratory, Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, Queensland 4067, Australia.
authors.forename: Justin
authors.initials: J
authors.lastname: Cooper-White
authors.affiliation: Department of Physics, Friedrich-Alexander University of Erlangen-Nuremberg, 91054 Erlangen, Germany.
authors.forename: Ben
authors.initials: B
authors.lastname: Fabry
authors.affiliation: Children's Cancer Research Unit, Kids Research Institute, The Children's Hospital at Westmead, Westmead, New South Wales 2145, Australia geraldine.oneill@health.nsw.gov.au.|Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales 2000, Australia.
authors.forename: Geraldine M
authors.initials: GM
authors.lastname: O'Neill
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002465:Cell Movement; D050719:Crk-Associated Substrate Protein / Q000737:chemistry* / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000187:drug effects; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D000072417:Protein Domains; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D050719:Crk-Associated Substrate Protein; C099942:NEDD9 protein, human; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D013752:Tetracycline
keywords: Cell migration; FAT; Focal adhesion; Mechanosensing; NEDD9; P130Cas