Itk Controls the Spatiotemporal Organization of T Cell Activation

Abstract

During T cell activation by antigen-presenting cells (APCs), the diverse spatiotemporal organization of components of T cell signaling pathways modulates the efficiency of activation. Here, we found that loss of the tyrosine kinase interleukin-2 (IL-2)–inducible T cell kinase (Itk) in mice altered the spatiotemporal distributions of 14 of 16 sensors of T cell signaling molecules in the region of the interface between the T cell and the APC, which reduced the segregation of signaling intermediates into distinct spatiotemporal patterns. Activation of the Rho family guanosine triphosphatase Cdc42 at the center of the cell-cell interface was impaired, although the total cellular amount of active Cdc42 remained intact. The defect in Cdc42 localization resulted in impaired actin accumulation at the T cell–APC interface in Itk-deficient T cells. Reconstitution of cells with active Cdc42 that was specifically directed to the center of the interface restored actin accumulation in Itk-deficient T cells. Itk also controlled the central localization of the guanine nucleotide exchange factor SLAT [Switch-associated protein 70 (SWAP-70)–like adaptor of T cells], which may contribute to the activation of Cdc42 at the center of the interface. Together, these data illustrate how control of the spatiotemporal organization of T cell signaling controls critical aspects of T cell function.

DEF6, a Novel Substrate for the Tec Kinase ITK, Contains a Glutamine-rich Aggregation-prone Region and Forms Cytoplasmic Granules that Co-localize with P-bodies

“…Together these results suggest that phosphorylation of DEF6 by ITK causes cellular redistribution and aggregate formation and they are consistent with the recent observation that DEF6 localization in T cells is dependent upon ITK function (18).…”

“…However, some aspects of DEF6-deficiency, such as effects on Ca 2ϩ signaling, activation of the transcription factor NFAT and IL-17A expression have been described in ITK-deficient mice (15,42,43) which prompted us to test whether DEF6 was a substrate for ITK, a member of the Tec-family of tyrosine kinases. Most recently an important functional link between ITK and DEF6 has been established at the immune synapse where ITK has been identified as necessary for the localization of DEF6 to this region and for actin remodeling in this signal-transducing structure (18). Here we show that DEF6 is a direct substrate for ITK and is phosphorylated at tyrosines 210 and 222.…”

“…Our data provide a biochemical link to the recently described phenomenon that the spatiotemporal organization of cdc42 and DEF6 is ITK-dependent. ITK selectively recruits DEF6 to the center of the interface between the T cell and antigen presenting cell (18). In addition, we demonstrate that a mutant of DEF6 (Y210E-Y222E) that mimics its phosphorylation by ITK causes it to assemble into cytoplasmic granules that are similar in appearance to stress granules and/or mRNA processing bodies (P-bodies); structures involved in the regulation of mRNA translation (19,20).…”

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

“…However, a question of major importance is how well do developmental defects in transgenic mice predict the behavior of a small molecule Itk inhibitor, and consequently, what is the real therapeutic potential of inhibiting Itk? There are two issues to consider, the first being how to compare T cell development in a knock-out mouse with inhibition of peripheral T cell activation; the second is how to compare a knockout with a small molecule inhibitor because Itk plays a key role in the spatiotemporal assembly and organization of the receptor TCR signaling complex, and knockout of Itk causes disruption to multiple components of the TCR complex (41). A small molecule binding to the catalytic site of the kinase is unlikely to result in the same degree of disruption of the whole TCR complex.…”

Systems Imaging of the Immune Synapse

“…Thus at the system scale uneven signaling distributions govern the information flow through signaling networks [3–6]. Supporting the importance of subcellular location for function in T cell activation, loss-of-function mutations of various signaling intermediates consistently yield diminished localization [7–11]. In addition, through the imaging of large numbers of signaling intermediates, subcellular structures mediating signal integration have emerged [12, 13].…”