Marie Findsen Helstad,a, b, Johan Georg Visser,b, Bernd Thiede,c, Manuel Ramirez Garrastacho,c, Steven Ray
Haakon
Wilson,a, and Anne Spurkland,b
a Section of Chemical Life Science, Department of Chemistry, University of Oslo, Norway
b Department of Molecular Medicine, Institute of Basic Medical Sciences, University of Oslo, Norway
c Department
of Biosciences, Proteomics Core Facility, University of Oslo, Norway
Email: m.f.helstad@kjemi.uio.no
The function of immune cells are regulated by membrane receptors that trigger or inhibit phosphotyrosine (pTyr) dependent signalling pathways [1]. These pathways involve proteins with Src homology 2 (SH2) domains that bind phosphorylated tyrosines [1]. The T cell receptor (TCR) binds its extracellular ligand, resulting in exposure of tyrosine residues on the intracellular tails of the TCR [1]. This further recruits the lymphocyte specific tyrosine protein kinase (Lck), which activates T cells via pTyr cascades [1]. This activation induces expression of T cell specific adapter protein (TSAd), which is an adapter for Lck with unclear biological function. For the past decade, novel pTyr superbinders (SB) have been created as a tool to investigate the phosphoproteome of different cell types [2]. The proto-oncogene tyrosine protein kinase (Src) and its SH2 SB domain, designed with three amino acid mutation within the binding pocket, are reported to have greater affinity for pTyr proteins than SH2 wild type (WT) domains and pTyr specific antibodies (pY-Ab) [3]. In this study, we hypothesize that an Lck SH2 SB domain will allow for more efficient characterization of the phosphoproteome of activated T cells, and enable identification of potential differences when the Lck adapter TSAd is expressed or knocked out. Glutathione S-transferase (GST) tagged WT and SB Lck- and Src-SH2 domains were expressed in bacteria cells and isolated by glutathione sepharose beads, enabling pulldown (PD) experiments with pTyr proteins for western blot (WB) and nano liquid chromatography-mass spectrometry (nano LC-MS) experiments. Nano LC-MS analysis and WB experiments of PD with the four SH2 domains permit exploration of the binding capacity and identification of pTyr proteins. The pTyr proteins are assembled from pervanadate- or OKT3 stimulated Jurkat T cells. As supported by both nano LC-MS analysis and WB experiments, the SH2 SB domains of both Lck and Src was found to have greater affinity towards pTyr proteins compared to their SH2 WT domains. Initial analysis with nano LC-MS suggest that there may be a difference in the phosphoproteome of different T cell genotypes. Comparison of preliminary results using the two superbinders regarding enrichment of immune related proteins, indicate that the Lck SH2 SB domain perhaps is more specific, while the Src SH2 SB domain might be more comprehensive. This however requires further exploration.
References
[1] P. Borowicz et al., “Tyr192 Regulates Lymphocyte-Specific Tyrosine Kinase Activity in T Cells,” The Journal of Immunology, vol. 207, no. 4, pp. 1128–1137, Aug. 2021, doi: 10.4049/jimmunol.2001105.
[2] J. Li and X. Zhan, “Mass spectrometry analysis of phosphotyrosine-containing proteins,” Mass Spectrometry Reviews, vol. 43, no. 4, pp. 857–887, 2024, doi: 10.1002/mas.21836.
[3] Y. Yao et al., “One-Step SH2 Superbinder-Based Approach for Sensitive Analysis of Tyrosine Phosphoproteome,” J. Proteome Res., vol. 18, no. 4, pp. 1870–1879, Apr. 2019, doi: 10.1021/acs.jproteome.9b00045.