title: Girdin is phosphorylated on tyrosine 1798 when associated with structures required for migration.
abstract: The mammalian protein Girdin interacts with several key molecules such as actin, and it functions as a regulator of the cytoskeleton. Silencing of Girdin mRNA results in defective migration in a variety of cultured cells. Moreover, knockout of Girdin causes phenotypes related to defective migration, including hypoplasia of olfactory bulbs and a widened rostral migratory stream (RMS) in mice. To elucidate the molecular basis underlying cellular migration, we generated site- and phosphorylation state-specific antibodies against human Girdin peptides carrying four putative phosphorylation sites (serine1386 [S1386], S1416, tyrosine1764 [Y1764] and Y1798) that had been identified by mutagenesis analyses or mass spectrometric studies. We found that these residues were phosphorylated in an epidermal growth factor (EGF)-dependent manner. Among the four antibodies we developed, the antibody that targeted Girdin when phosphorylated at Y1798 (pY1798) worked well for immunohistochemistry of paraffin-embedded tissues as well as for cultured cells. Immunocytochemistry of HEK293FT cells transfected with an EGF receptor expression plasmid exhibited punctate signals with pY1798. These signals colocalized with those of endocytosed EGF receptors after EGF stimulation. Signals from pY1798 were also observed on lamellipodia, filopodia, focal adhesion and stress fibers in NIH3T3 cells under conventional culture conditions. Immunohistochemistry of paraffin-embedded mouse brain at P14 using anti-pY1798 antibody displayed signals at the hilum-side (internal side) of the dentate gyrus of the hippocampus, the RMS, the accessory olfactory bulb and the olfactory bulb in which Girdin expression was detected. Primary culture of RMS neurons showed punctate signals of pY1798 at the tips of leading processes as well as in the cytoplasm, whereas no signals were observed when neurons were treated with Src inhibitor, PP2. Our data revealed the changes in the phosphorylation status of Y1798 in Girdin when it associated with migration-related structures in vitro and in vivo.
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Omori
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Department of Endocrinology and Diabetes, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Electronic address: Masato-a@med.nagoya-u.ac.jp.
authors.forename: Masato
authors.initials: M
authors.lastname: Asai
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kuga
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Ushida
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Tetsushi
authors.initials: T
authors.lastname: Izuchi
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Mii
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Enomoto
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Naoya
authors.initials: N
authors.lastname: Asai
authors.affiliation: Department of Surgery, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan.
authors.forename: Masato
authors.initials: M
authors.lastname: Nagino
authors.affiliation: Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan; Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Electronic address: mtakaha@med.nagoya-u.ac.jp.
authors.forename: Masahide
authors.initials: M
authors.lastname: Takahashi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D002465:Cell Movement; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D010766:Phosphorylation; D014443:Tyrosine / Q000032:analysis / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D008840:Microfilament Proteins; D014443:Tyrosine; D033921:Vesicular Transport Proteins; C502451:CCDC88A protein, human; C527550:Ccdc88a protein, mouse
keywords: Cell migration; Focal adhesion; Girdin; Tyrosine phosphorylation