title: Tyr724 phosphorylation of ELMO1 by Src is involved in cell spreading and migration via Rac1 activation.
abstract: BACKGROUND
  The complex of Dock180/ELMO1 that functions as a bipartite guanine nucleotide exchange factor for Rac is essential for diverse physiological and pathological processes of cells such as cell migration, phagocytosis, and invasion of cancer cells. Among the Src-family tyrosine kinases (SFKs), it has been reported that Hck directly phosphorylates ELMO1, regulating phagocytosis by promoting activation of Rac1; however, the involvement of other SFKs in ELMO1 phosphorylation has remained unknown. Here, we identified novel tyrosine (Y) residues of ELMO1 phosphorylated by SFKs, and examined the effects on Rac1 activity, cell adhesion, spreading, and cell motility on extracellular matrix (ECM).
  
  
  RESULTS
  In this study, we unveiled that Src and Fyn can induce tyrosine phosphorylation of ELMO1 in in vivo and in vitro phosphorylation assays. Mutational analyses identified both Y720 and Y724 residues of ELMO1 as Src-mediated phosphorylation sites, preferentially on Y724. Single substitution of Y724 to Phe abrogated Rac1 activation triggered by Src. To elucidate the biological function of pY724, we established NIH3T3 cells stably expressing wild-type ELMO1 or its Y724F mutant together with Dock180. Among them, Y724-deficient cells exhibited a depletion of Rac1 activity with diminished phosphorylation of ELMO1 even upon the ECM-stimulation. It is noteworthy that NIH3T3 cells with ELMO1 Y724F were strikingly defective to promote cell spreading on fibronectin-coated dish, concomitantly exhibiting immature assemblies of actin stress fibers and focal adhesions. Eventually, ELMO1 Y724F significantly impaired cell migration.
  
  
  CONCLUSIONS
  These results define that Src-mediated Y724 phosphorylation in ELMO1 plays a critical role for cell spreading via activation of Rac1, leading to promotion of cell migration. As the overexpression and/or hyperactivation of Src have been shown in a wide variety of human cancers, Src-mediated phosphorylation of Y724 in ELMO1 may regulate cancer cell adhesion to the ECM, invasion into surrounding tissues, and subsequent distant metastasis.
authors.affiliation: Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. y-makino@iam.u-tokyo.ac.jp.|Laboratory of Pathology and Development, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, 113-0032, Japan. y-makino@iam.u-tokyo.ac.jp.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Makino
authors.affiliation: Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. tsudam@med.hokudai.ac.jp.
authors.forename: Masumi
authors.initials: M
authors.lastname: Tsuda
authors.affiliation: Department of Cell Physiology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. yohba@med.hokudai.ac.jp.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ohba
authors.affiliation: Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. hnishihara@med.hokudai.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nishihara
authors.affiliation: Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. h-sawa@czc.hokudai.ac.jp.|Hokkaido University Research Center for Zoonosis Control, Sapporo, 001-0020, Japan. h-sawa@czc.hokudai.ac.jp.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Sawa
authors.affiliation: Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. path@higashi-tokushukai.or.jp.|Sapporo Higashi Tokushukai Hospital, Sapporo, 065-0033, Japan. path@higashi-tokushukai.or.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Nagashima
authors.affiliation: Department of Cancer Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. tanaka@med.hokudai.ac.jp.|Department of Translational Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo, 060-8638, Japan. tanaka@med.hokudai.ac.jp.
authors.forename: Shinya
authors.initials: S
authors.lastname: Tanaka
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002465:Cell Movement; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D017354:Point Mutation; D014443:Tyrosine / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C465214:ELMO1 protein, human; C510359:ELMO1 protein, mouse; D014443:Tyrosine; D019061:src-Family Kinases; D020830:rac1 GTP-Binding Protein