The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell-specific adapter protein.

The Journal of biological chemistry 2019 Oct 18

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T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2-inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239-274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239-256 and aa 257-274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242-268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status. © 2019 Andersen et al.

Citation

Thorny Cesilie Bie Andersen, Per Eugen Kristiansen, Zsuzsa Huszenicza, Maria U Johansson, Ramakrishna Prabhu Gopalakrishnan, Hanna Kjelstrup, Scott Boyken, Vibeke Sundvold-Gjerstad, Stine Granum, Morten Sørli, Paul Hoff Backe, D Bruce Fulton, B Göran Karlsson, Amy H Andreotti, Anne Spurkland. The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell-specific adapter protein. The Journal of biological chemistry. 2019 Oct 18;294(42):15480-15494