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Interactions between the Fyn SH3-domain and adaptor protein Cbp/PAG derived ligands, effects on kinase activity and

Silje A Solheim1, Evangelia Petsalaki, Anne J Stokka

  • 1The Biotechnology Centre of Oslo, University of Oslo, Norway.

The FEBS Journal
|August 30, 2008
PubMed

Insights

Phosphoprotein associated with glycosphingolipid-enriched domains (PAG) regulates T-cell activation by recruiting C-terminal Src kinase (Csk). This study reveals how Fyn kinase binding to PAG can be modulated, offering a new way to control Src family kinase activity.

Area of Science:

  • Immunology
  • Molecular Biology
  • Biochemistry

Background:

  • Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched domains (PAG) is a transmembrane adaptor protein crucial for T-cell activation regulation.
  • PAG recruits C-terminal Src kinase (Csk) to inhibit Src kinase activity via phosphorylation.
  • The Src family kinase Fyn binds to PAG through a dual-domain interaction, involving its SH3 and SH2 domains, which is critical for Csk recruitment.

Purpose of the Study:

  • To investigate the binding mechanism of the Fyn SH3 domain to peptide ligands derived from PAG microdomain sequences.
  • To characterize the kinetics and thermodynamics of Fyn SH3-domain/ligand interactions.
  • To explore the structural basis of Fyn SH3-domain binding to PAG and its functional implications.

Main Methods:

  • Surface Plasmon Resonance (SPR) for determining interaction kinetics and dissociation constants.
  • Circular Dichroism (CD) spectroscopy to assess the local structural changes upon ligand binding.
  • Molecular modeling to elucidate the detailed interaction mechanisms.
  • Kinase assays using full-length PAG proteins to evaluate functional effects.

Main Results:

  • Characterization of Fyn SH3-domain binding kinetics and affinity to various 14-mer peptide ligands.
  • Identification of structural changes induced by ligand association using CD spectroscopy.
  • Molecular modeling provided insights into the Fyn SH3-domain/peptide interactions.
  • Demonstrated correlation between ligand binding data and functional effects on PAG phosphorylation.

Conclusions:

  • The Fyn SH3-domain interaction with PAG-derived peptides is quantifiable and structurally influenced.
  • Minor modifications in the SH3-interacting motif of PAG can modulate Src family kinase tyrosine phosphorylation.
  • This study presents a potential strategy for controlling Src family kinase activity through substrate-based modulation.

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