Bioscience (master's two years)

Interaction of Tec-SH3 domain with the adapter protein TSAd

TSAd is an adapter protein that is preferentially expressed in activated T and NK-cells. The role of TSAd in activated T cells is still unknown, but it seems to be required for proper activation of the T-cell, probably through interaction with the kinases Lck and Itk. TSAd is also expressed in endothelial cells, where it regulates vascular permeability upon VEGF stimulation.

As an adapter protein, TSAd have been found to interact with proteins through their SH2 and SH3 domain. Specifically it interacts with the Src kinase Lck through the Lck SH3 and SH3 domain, and with the Tec kinase Itk through the Itk-SH3 domain. It is highly likely that TSAd may also interact with other members of the Src and Tec kinase families. We are thus currently mapping the interaction of TSAd with the other Src and Tec family members. During the course of these studies, we have observed that the SH3 domain of the Tec kinase itself, binds strongly to TSAd. In the current project, we will examine this interaction further, using nuclear magnetic resonance spectroscopy (NMR).

Aims of study:

  1. Analyse the interaction of the isolated Tec SH3 domains with TSAd peptides known to bind to the Tec SH3 domain using NMR
  2. Compare the binding affinity of the Tec SH3 domain to TSAd with that of the Itk SH3 domain using NMR.
  3. Examine whether the Tec SH3 domain may compete or collaborate with the Lck SH3 domain in binding to TSAd peptides using NMR
  4. Examine whether Tec and TSAd interacts in vivo, by using transient transfection of Tec and TSAd cDNA into cells followed by immunoprecipitation or microscopy techniques.

Methods involved:

The master student will produce recombinant Tec-SH3 domain, labeled with N15, for NMR studies. Titration of peptides into the N15 labeled Tec-SH3 domain will reveal amino acid positions that are directly or indirectly affected by peptide binding. This will be done by HSQC-analysis. The solution structure of the Tec-SH3 domain is available in the protein database (pdb: 1GL5). It is thus relatively straight forward to map the affected amino acids onto the structure of the Tec-SH3 domain. The student will also learn to perform basic cell culture technology, including transient transfection of plasmids encoding the protein of interest, as well as perform immunoprecipitation and Western blot analysis.

Significance:

Both TSAd and Tec are genes that are induced upon activation of T cells. TSAd is expressed relatively early, within hours of the initial stimulation, while Tec is upregulated several days later. Analysis of how Tec interacts with TSAd and whether Tec interaction competes with Lck or Itk interaction (which we previously have studied by NMR), may contribute to the understanding of the role of TSAd and Tec in regulating T cell activation.

 

Supervisor Per Eugen Kristiansen, co-supervisor Anne Spurkland

 

Published Mar. 22, 2018 10:28 AM - Last modified Apr. 19, 2018 8:13 AM

Supervisor(s)

Scope (credits)

60