Interaction of SPIN90 with syndapin is implicated in clathrin-mediated endocytic pathway in fibroblasts.
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.
Kim
Sung Hyun
SH
Department of Life Science and Molecular Disease Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea.
Choi
Hyun Jin
HJ
Lee
Kyoung Woo
KW
Hong
Nan Hyung
NH
Sung
Bong Hwan
BH
Choi
Kyu Yeong
KY
Kim
Seon-Myung
SM
Chang
Sunghoe
S
Eom
Soo Hyun
SH
Song
Woo Keun
WK
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
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