title: Interaction of SPIN90 with syndapin is implicated in clathrin-mediated endocytic pathway in fibroblasts.
abstract: SPIN90, a 90-kDa Nck-interacting protein with a SH3 domain, plays a role in sarcomere formation and myofibril assembly, and its phosphorylation is modulated by cell adhesion and Erk activation. Here we demonstrate that SPIN90 participates in receptor-mediated endocytic pathway in fibroblasts. We identified syndapin (synaptic dynamin-binding protein) as a SPIN90 interacting protein using yeast two-hybrid screening. SPIN90 directly binds the SH3 domain of syndapin via its proline rich domain in vitro and in vivo and also associates with clathrin. Over-expression of SPIN90-full length in COS-7 cells inhibited transferrin uptake, a marker of endocytosis. Interestingly, SPIN90-PRD, a syndapin-binding domain, significantly inhibited endocytosis, and the inhibition was reversed by co-expression of syndapin. Depleting SPIN90 through antibody microinjection or Knocking it down using siRNAs also significantly inhibited transferrin internalization. Moreover, early endosomal marker proteins (EEA1 and Rab5) appeared to closely associate or partially co-localize with SPIN90 in endosomes and an internalized FITC-dextran and Texas Red-EGF were found on the endosomes in association with SPIN90. Time-lapse video showed that GFP-SPIN90 travels with moving vesicles within living cells. Taken together, these findings suggest that SPIN90 is implicated in receptor-mediated endocytic pathway in fibroblasts.
authors.affiliation: Department of Life Science and Molecular Disease Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
authors.forename: Sung Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Choi
authors.forename: Kyoung Woo
authors.initials: KW
authors.lastname: Lee
authors.forename: Nan Hyung
authors.initials: NH
authors.lastname: Hong
authors.forename: Bong Hwan
authors.initials: BH
authors.lastname: Sung
authors.forename: Kyu Yeong
authors.initials: KY
authors.lastname: Choi
authors.forename: Seon-Myung
authors.initials: SM
authors.lastname: Kim
authors.forename: Sunghoe
authors.initials: S
authors.lastname: Chang
authors.forename: Soo Hyun
authors.initials: SH
authors.lastname: Eom
authors.forename: Woo Keun
authors.initials: WK
authors.lastname: Song
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary / Q000235:genetics; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D066298:In Vitro Techniques; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D020798:Two-Hybrid System Techniques; D018909:src Homology Domains
chemical_list: D002352:Carrier Proteins; D002966:Clathrin; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; C117738:Pacsin1 protein, rat; C492181:Pacsin2 protein, rat; D034741:RNA, Small Interfering; C491670:Wipf1 protein, rat