Tec Kinase Itk Forms Membrane Clusters Specifically in the Vicinity of Recruiting Receptors

Abstract

The Tec family of tyrosine kinases transduces signals from antigen and other receptors in cells of the hematopoietic system. In particular, interleukin-2 inducible T cell kinase (Itk) plays an important role in modulating T cell development and activation. Itk is activated by receptors via a phosphatidylinositol 3-kinase-mediated pathway, which results in recruitment of Itk to the plasma membrane via its pleckstrin homology domain. We show here that membrane localization of Itk results in the formation of clusters of at least two molecules within 80 Å of each other, which is dependent on the integrity of its pleckstrin homology domain. By contrast, the proline-rich region within the Tec homology domain, SH3 or SH2 domains, or kinase activity were not required for this event. More importantly, these clusters of Itk molecules form in distinct regions of the plasma membrane as only receptors that recruit phosphatidylinositol 3-kinase reside in the same membrane vicinity as the recruited Itk. Our results indicate that Itk forms dimers in the membrane and that receptors that recruit Itk do so to specific membrane regions.The Tec family of non-receptor tyrosine kinases is the second largest family of non-receptor tyrosine kinases (1). This family, which includes Itk, 2 is involved in transducing signals from a number of receptors, including the T cell receptor, B cell receptor, Fc⑀R, c-Kit, CD28, CD2, CD32, and the erythropoietin receptor (1-6). These kinases play important roles in regulating cytokine and immune receptor signals that regulate the immune response (7,8). Itk in particular is involved in early T cell receptor signaling and regulates increases in intracellular calcium and activation of the nuclear factor of activated T cells family of transcription factors (9, 10). In addition, Itk regulates the development of Th2 cells and their subsequent cytokine secretion, thereby modulating the immune response (10 -13). A common feature of these kinases is that they require the activation of PI3 kinase, as well as Src kinase activation for their activity (14 -17). These kinases, including Itk, have a PH domain that allows them to be recruited to the plasma membrane by an activated PI3 kinase (1). Thus, membrane recruitment is a critical component of their activation, and Itk can be found at the plasma membrane of cells, although other events are required for its full activity (17)(18)(19). It is, however, not clear whether this is general membrane localization of the protein in anticipation of activation or a specific localization in certain regions of the plasma membrane.The structure of Itk in cells is not known, although it has been suggested to form dimers in its inactive state, with a resultant monomerization upon activation (20,21). We have examined these issues using a split YFP system (22) and find that Itk forms dimers or higher order clusters; however, it does so only at the plasma membrane and only in the vicinity of a receptor that can recruit PI3 kinase. Our data shed new light on the regulation, struct...

Loss-of-function mutations within the IL-2 inducible kinase ITK in patients with EBV-associated lymphoproliferative diseases

“…R29C also impedes membrane recruitment of ITK as demonstrated by exclusive staining of the cytoplasm excluding the cellular membranes. 32 These results collectively suggest that ITK R29H lost its ability to recruit to the plasma membrane both in vitro and in vivo.…”

“…Under inactive conditions, ITK remains as a monomer within the cytoplasm of a T cell. 32,46 Following TCR activation, ITK becomes activated and incorporated into a multi-protein signalosome by membrane binding via its PH domain. 11 It seems likely but remains to be seen whether R29H or other mutations also differently affect ITK kinase activity.…”

Interleukin-2-inducible T Cell Kinase (Itk) Network Edge Dependence for the Maturation of iNKT Cell

“…We compared these two mutants as it is possible that the folding of the kinase-deleted mutant may be different from the WT kinase. The structure of full-length Itk is not known, but based on a number of experiments using isolated domains, and other approaches in cells, we and others have proposed one of two models for folding of this protein, either an intramolecular folded monomer, or a intermolecular folded dimer (36,37,41). Deletion of the kinase domain in both models could potentially result in enhanced interactions with the SH2 and SH3 domains.…”

The Tec Family Kinase Itk Exists as a Folded Monomer in Vivo

“…During stimulation of the TcR, phosphatidylinositol 3-kinase is activated, resulting in the formation of cell membrane phosphoinositides, to which the PH domain of Itk binds. Itk also forms dimers specifically at the plasma membrane in the vicinity of receptors that activate phosphatidylinositol 3-kinase (17). Once Itk is recruited to the membrane, it is phosphorylated by Src family kinases (18,19).…”