Abstract
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.
Highlights
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
Following up on this observation, we have previously found that the interaction of T cell-specific adapter protein (TSAD) with inducible T cell kinase (ITK) depends on the proline-rich region (PRR) of TSAD [10], indicating a role for the ITK Src homology 3 (SH3) domain in mediating this interaction
Because lymphocyte–specific kinase (LCK) SH3 and ITK SH3 bind to overlapping peptides in the TSAD PRR (Fig. 2A) and the ITK SH3 domain binds to the TSAD aa 262–269 peptide (Fig. 4), we directly assessed whether LCK SH3 and ITK SH3 may bind to adjacent sites on the same TSAD peptide
Summary
T-cell activation relies upon stimulation of specific intracellular signal-transduction pathways, where protein-tyrosine kinases (PTKs), phosphatases, and adapter molecules interact to convey the signal from the T-cell receptor (TCR) to the nucleus. The TEC family IL-2– inducible tyrosine kinase (ITK) is recruited to the membrane after TCR triggering and is activated by the SRC family PTK lymphocyte–specific kinase (LCK). The T cell–specific adapter protein (TSAD), encoded by the SH2D2A gene, is an adapter molecule containing a SRC homology 2 (SH2) domain and a proline-rich region (PRR) in addition to several tyrosine phosphorylation sites [4, 5]. Absence of TSAD results in deficient polarization of actin to the immunological synapse [8] as well as deficient activation-induced cell death [11] Both LCK and ITK consist of multiple domains, including one SH2 and one SRC homology 3 (SH3) domain [12]. Our findings suggest that, once active, ITK autophosphorylation of its own SH3 domain disrupts the ITK/ TSAD interaction, possibly creating an open binding site on TSAD for recruiting and activation of another ITK molecule by LCK
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