In Vivo Significance of ITK-SLP-76 Interaction in Cytokine Production

In vitro data have suggested that activation of the inducible T-cell kinase (ITK) requires an interaction with the adaptor protein SLP-76. One means for this interaction involves binding of the ITK SH3 domain to the polyproline-rich (PR) region of SLP-76. However, the biological significance of this association in live cells and the consequences of its disruption have not been demonstrated. Here, we utilized a polyarginine-rich, cell-permeable peptide that represents the portion of the SLP-76 PR region that interacts with the ITK SH3 domain as a competitive inhibitor to disrupt the association between ITK and SLP-76 in live cells. We demonstrate that treatment of cells with this peptide, by either in vitro incubation or intraperitoneal injection of the peptide in mice, inhibits the T-cell receptor (TCR)-induced association between ITK and SLP-76, recruitment and transphosphorylation of ITK, actin polarization at the T-cell contact site, and expression of Th2 cytokines. The inhibition is specific, as indicated by lack of effects by the polyarginine vehicle alone or a scrambled sequence of the cargo peptide. In view of the role of ITK as a regulator of Th2 cytokine expression, the data underscore the significance of ITK as a target for pharmacological intervention.The Tec family of tyrosine kinases plays a critical role in lymphocyte development and activation through antigen receptors (4, 40). The inducible T-cell kinase (ITK), a member of the Tec family, regulates selection during thymocyte development and controls the generation of effective Th2 responses (15, 40). Phosphorylation of ITK on Tyr 511 by the Src family kinase LCK occurs early upon the engagement of the T-cell antigen receptor (TCR) and is critical for the enzymatic activation of ITK (18, 44). Upon its activation, ITK phosphorylates phospholipase C-γ1 (PLC-γ1) on tyrosines 775 and 783, an event critical for phospholipase activity (3, 5) and ensuing intracellular and capacitative Ca²⁺ mobilization (26). In this fashion, ITK regulates downstream signaling events that regulate biological responses, such as cytokine production (4, 40).ITK is organized in modular domains that play critical roles in its activation (47). Upon T-cell engagement, ITK colocalizes with the TCR, a process dependent on the pleckstrin homology (PH) domain of ITK and its interaction with PIP3 at the plasma membrane (11, 19). Activation of ITK also requires interaction with adaptor proteins, such as SLP-76 and LAT (8, 10). The SH2 domain of ITK appears to be critical for its interaction with LAT, whereas both the SH2 and SH3 domains are required for interaction with SLP-76 (8, 10). In vitro studies have demonstrated that the SH3 domain of ITK interacts with the proline-rich (PR) region of SLP-76, and it has been speculated that this interaction is critical for the activation of ITK (6, 8). However, the biological significance of the interaction has not been demonstrated in live cells. In the present investigation, we used a cell-permeable peptide as a competitive inhibitor of the interaction between ITK and SLP-76. To this end, we synthesized a 12-amino-acid peptide, which represents the PR region of SLP-76 that binds to the ITK-SH3 domain, and rendered it cell permeable by the addition of nine arginines at its N-proximal end. Here, we show that this cell-permeable peptide, henceforth called R9-QQP, is readily taken up by both Jurkat T cells and murine splenocytes and disrupts events that are mediated by the engagement of the TCR. Thus, association of ITK and SLP-76, recruitment of ITK and actin polarization at the T-cell contact site, LCK-mediated transphosphorylation of ITK on tyrosine 511, and production of Th2 cytokines are inhibited by R9-QQP in a dose-dependent and peptide-specific manner. The data presented here are novel and significant because they provide the first demonstration of the biological relevance of the specific interaction between the ITK-SH3 domain and the SLP-76 PR region in live cells. Furthermore, the data underscore the potential of cell-permeable peptides as useful probes for dissecting signal transduction pathways in live cells, and in view of the regulatory role that ITK plays in Th2 cytokine production, they have implications for the pharmacological manipulation of ITK in disease situations.