Premium
c‐Src Directly Interacts with the C‐terminal Domain of Occludin by involving SH2 and SH3 domains
Author(s) -
Seth Ankur,
Samak Geetha,
Zola Courtney E,
Rao R.K.
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.1004.10
Subject(s) - occludin , proto oncogene tyrosine protein kinase src , sh3 domain , immunoprecipitation , chemistry , microbiology and biotechnology , tight junction , sh2 domain , biology , biochemistry , kinase , gene
The effect of H 2 O 2 on c‐Src translocation to tight junction (TJ) and the direct binding to occludin were investigated. Caco‐2 cell monolayers were exposed to H 2 O 2 . Src activation was measured by FRET‐based Src kinase assay and immunoblot analysis for c‐Src(pY418). Association of c‐Src with TJ proteins was determined by co‐immunoprecipitation and confocal microscopy. Direct binding of c‐Src with C‐terminal domain of occludin determined by pair‐wise binding using c‐Src and GST‐Occludin‐C (C‐terminal domain). GST‐Occludin‐C(Ocldn) Δ 378–385 and GST‐Ocldn Y379/383D were used to determine whether interaction is an Enzyme‐Substrate binding. Recombinant SH2 and SH3 domains of c‐Src were used to determine their role in occludin binding. H 2 O 2 activated c‐Src and increased co‐immunoprecipitation of occludin with c‐Src(pY418). H 2 O 2 increased localization of c‐Src(pY418) at the TJ. c‐Src binds to GST‐Ocldn‐C as well as GST‐Ocldn Δ378–385 and GST‐Ocldn Y379/383D indicating that the interaction is not just an enzyme‐substrate binding. SH2 and SH3 domains dose‐dependently attenuated c‐Src binding to GST‐Ocldn WT and GST‐Ocldn Δ378–385 indicating that these domains are involved in c‐Src binding to occludin. Incubation of GST‐SH2 and GST‐SH3 with Caco‐2 cell extract pulled down occludin. Results indicate that H 2 O 2 induces translocation of c‐Src to TJ by direct binding to occludin, which involves SH2 and SH3 domains.