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

• Published in: The Journal of biological chemistry 2019-10, Vol.294 (42), p.15480-15494
• Main Authors: Andersen, Thorny Cesilie Bie, Kristiansen, Per Eugen, Huszenicza, Zsuzsa, Johansson, Maria U., Gopalakrishnan, Ramakrishna Prabhu, Kjelstrup, Hanna, Boyken, Scott, Sundvold-Gjerstad, Vibeke, Granum, Stine, Sørli, Morten, Backe, Paul Hoff, Fulton, D. Bruce, Karlsson, B. Göran, Andreotti, Amy H., Spurkland, Anne
• Format: Article
• Language: English
• Subjects: adaptor protein; Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Amino Acid Motifs; basis; binding; Biochemistry & Molecular Biology; cell signaling; cell-activation; chemical-shift; HEK293 Cells; high-affinity; Humans; IL-2-inducible T cell kinase (ITK); immunity; Immunologi inom det medicinska området; Immunology in the medical area; kinase; LCK proto-oncogene SRC family tyrosine kinase; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - chemistry; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - genetics; Lymphocyte Specific Protein Tyrosine Kinase p56(lck) - metabolism; negative regulation; NMR; nuclear magnetic resonance; peptide; Phosphorylation; protein; Protein Binding; protein kinase; protein phosphorylation; protein structure; protein-protein interaction; Protein-Tyrosine Kinases - chemistry; Protein-Tyrosine Kinases - genetics; Protein-Tyrosine Kinases - metabolism; recognition domains; SH2D2A; Signal Transduction; specificity; Src homology 3 domain (SH3 domain); src Homology Domains; structural; T cell-specific adapter protein; T cell-specific adapter protein (TSAD); T-cell; TSAD; tyrosine kinase; tyrosine-protein kinase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.008318

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.