title: T cell specific adaptor protein (TSAd) promotes interaction of Nck with Lck and SLP-76 in T cells.
abstract: BACKGROUND
  The Lck and Src binding adaptor protein TSAd (T cell specific adaptor) regulates actin polymerization in T cells and endothelial cells. The molecular details as to how TSAd regulates this process remain to be elucidated.
  
  
  RESULTS
  To identify novel interaction partners for TSAd, we used a scoring matrix-assisted ligand algorithm (SMALI), and found that the Src homology 2 (SH2) domain of the actin regulator Non-catalytic region of tyrosine kinase adaptor protein (Nck) potentially binds to TSAd phosphorylated on Tyr(280) (pTyr(280)) and pTyr(305). These predictions were confirmed by peptide array analysis, showing direct binding of recombinant Nck SH2 to both pTyr(280) and pTyr(305) on TSAd. In addition, the SH3 domains of Nck interacted with the proline rich region (PRR) of TSAd. Pull-down and immunoprecipitation experiments further confirmed the Nck-TSAd interactions through Nck SH2 and SH3 domains. In line with this Nck and TSAd co-localized in Jurkat cells as assessed by confocal microscopy and imaging flow cytometry. Co-immunoprecipitation experiments in Jurkat TAg cells lacking TSAd revealed that TSAd promotes interaction of Nck with Lck and SLP-76, but not Vav1. TSAd expressing Jurkat cells contained more polymerized actin, an effect dependent on TSAd exon 7, which includes interactions sites for both Nck and Lck.
  
  
  CONCLUSIONS
  TSAd binds to and co-localizes with Nck. Expression of TSAd increases both Nck-Lck and Nck-SLP-76 interaction in T cells. Recruitment of Lck and SLP-76 to Nck by TSAd could be one mechanism by which TSAd promotes actin polymerization in activated T cells.
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway. c.d.hem@medisin.uio.no.
authors.forename: Cecilie Dahl
authors.initials: CD
authors.lastname: Hem
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway. v.s.gjerstad@medisin.uio.no.
authors.forename: Vibeke
authors.initials: V
authors.lastname: Sundvold-Gjerstad
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway. Stine.Granum@rr-research.no.
authors.forename: Stine
authors.initials: S
authors.lastname: Granum
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway. lise.koll@gmail.com.
authors.forename: Lise
authors.initials: L
authors.lastname: Koll
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway. greger.abrahamsen@medisin.uio.no.
authors.forename: Greger
authors.initials: G
authors.lastname: Abrahamsen
authors.affiliation: Institute of Enzymology, Research Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, 1117, Hungary. buday.laszlo@ttk.mta.hu.
authors.forename: Laszlo
authors.initials: L
authors.lastname: Buday
authors.affiliation: Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Oslo, 0317, Norway. anne.spurkland@medisin.uio.no.|Institute of Basal Medical Sciences, University of Oslo, PB 1105, Blindern, Oslo, 0317, Norway. anne.spurkland@medisin.uio.no.
authors.forename: Anne
authors.initials: A
authors.lastname: Spurkland
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D019169:Jurkat Cells; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000032:analysis / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015513:Oncogene Proteins / Q000032:analysis / Q000378:metabolism*; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism*; D060066:Protein Interaction Maps; D013601:T-Lymphocytes / Q000378:metabolism*; D018909:src Homology Domains
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C078332:Nck protein; D015513:Oncogene Proteins; D010750:Phosphoproteins; C405691:SH2D2A protein, human; C092947:SLP-76 signal Transducing adaptor proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)