Development of a proper immune system requires the selection of lymphocytes expressing a useful repertoire of antigen receptors that can respond to foreign or dangerous antigens but not to self. For T-Cells developing in the thymus, these selection processes include both positive and negative selection of immature CD4 and CD8 cells, helping shape the mature T-Cell repertoire. These processes are regulated in large part through the interactions between the TCR (T-Cell Antigen Receptor) expressed on a given thymocyte and peptides presented in the context of either Class-II or Class-I MHC molecules. In T-Cells, at least three Tec Kinases are expressed: Tec, RLK (Resting Lymphocyte Kinase)/TXK and ITK (Inducible T-Cell Kinase), which have expression primarily restricted to the T-Cell lineage and mast cells (Ref.1, 2 & 3). The Tec Kinases represent the second largest family of mammalian non-RTKs (Receptor Tyrosine Kinases) which are distinguished by the presence of distinct PRR (Proline-Rich Regions) and PH (Pleckstrin Homology) domains. These kinases influence a wide range of signaling pathways controlling activation of MAPKs (Mitogen Activated Protein Kinases), Actin reorganization, transcriptional regulation, cell survival and cellular transformation (Ref.7).
The T-Helper cells recognize antigens when processed by APCs (Antigen Presenting Cells) containing MHC Class-II and CD4 (CD4 Antigen). CD4 is an accessory protein essential for MHC Class-II/TCR/CD3 interaction and plays a more general role in mediating cell recognition events than merely those of cellular immune response. Tec PTKs (Protein-Tyrosine Kinases) is activated following TCR/CD3 or CD28 ligation (Ref.4). The TEC-family kinases are characterized by a common domain organization: they have an amino-terminal PIP3 (Phosphatidylinositol-3, 4, 5-Trisphosphate)-binding pleckstrin-homology domain, w
hich is followed by a Tec-homology domain that contains one or two proline-rich regions, then SH3 (SRC Homology-3) and SH2 protein-interaction domains, and a carboxy terminal kinase domain (Ref.5). The Tec kinases are the only tyrosine kinases that have pleckstrin-homology domains, which inducibly recruit TEC-family members to the plasma membrane by binding the PI3K(Phosphatidylinositol 3-Kinase) product PIP3, thereby promoting their activation. Activation of TEC-family kinases requires several interrelated steps: first, recruitment to the plasma membrane through interactions between their pleckstrin homology domains and the products of PI3K and/or other proteins; second, phosphorylation by SRC-Family Kinases; and third, interactions with other proteins that bring the TEC-Family Kinases into antigen-receptor signaling complexes. In addition, TEC-Family Kinases are also regulated by conformational changes directed by intra-and intermolecular interactions involving their SH2 domains, SH3 domains and PRRs.
Engagement of the TCR by peptide-MHC complexes, in conjunction with co-stimulatory molecules at the cell surface of APCs (Antigen Presenting Cells), leads to rapid activation of the SRC-Family Kinase Lck, phosphorylation of the Zeta-chain of the TCR proximal signaling molecule CD3, and recruitment and activation of the protein kinase ZAP70 (Zeta-chain associated protein kinase of 70 kDa) and of PI3K. Lck along with Fyn remains attached to the cytoplasmic domain of CD4. Fyn phosphorylates WASP by binding to it (Ref.11). ZAP70 then phosphorylates the adaptors LAT (Linker for Activation of T-Cells) and SLP76 (SH2-Domain Containing Leukocyte Protein of 76 kDa), which together function as a platform for the recruitment of several key signaling molecules, including the enzyme PLC-Gamma (Phospholipase-C-Gamma), the adaptors GRB2 (Growth Factor Receptor Bound Protein-2), SOS, a related molecule, GADS (GRB2-Related Adaptor Protein-2), which binds to SLP76, Nck (Non-Catalytic Region of Tyrosine Kinase) and ADAP (Adhesion and Degranulation Promoting Adaptor Protein), and the guanine nucleotide-exchange factor Vav1 (Ref.9 & 10). This TCR-signaling complex is crucial for downstream effector functions, including mobilization of Ca2+, activation of MAPKs (Mitogen-Activated Protein Kinases) and downstream transcription factors, and regulation of the actin cytoskeleton. PLC-Gamma is an enzyme that catalyzes PIP2 (Phosphatidylinositol-4, 5-Bisphosphate) catabolism to generate both IP3 (Inositol-1, 4, 5-Trisphosphate), which is required for Ca2+ mobilization, and DAG (Diacylglycerol). IP3 binds to IP3R (IP3 Receptors) on intracellular organelles to release intracellular Ca2+, which leads to an influx of Ca2+ from extracellular sources via CaCn (Ca2+ Channels). DAG activates DAG-binding proteins such as PKCs (Protein Kinase-C) and RasGRP (Ras Guanyl Releasing Protein), which are important for downstream activation of MAPKs such as JNK (JUN amino-terminal kinase), ERK1 (Extracellular-Signal-Regulated Kinase-1) and ERK2 and other downstream transcription factors AP-1 (Activator Protein-1), NF-KappaB (Nuclear Factor-Kappa B) and NFAT (Nuclear Factor of Activated T-cells). Kinase-inactive ITK antagonizes the SLP76-dependent activation of NFAT (Ref.8 & 9). In addition to activation by DAG, PKC-Theta is activated through a Vav1 and Rac-mediated pathway. The LAT-SLP76 complex also functions as a platform for the accumulation of molecules including Vav1, Rac, CDC42 (Cell Division Cycle-42), WASP (Wiskott-Aldrich Syndrome Protein), ARP2 (Actin-Related Protein-2) and ARP3 that regulate the polymerization of F-Actin. PKC-Theta then regulates the IKK (Inhibitor of Kappa Light Polypeptide Gene Enhancer in B-Cells Kinase) complex and ultimately results in translocation of NF-KappaB to the nucleus, followed by degradation of I-KappaBs (Inhibitor of Kappa Light Chain Gene Enhancer in B-Cells). Calcium ions also promote NF-KappaB translocation indirectly through activation of Calm (Calmodulin), which in turn facilitates CaMKII activation. Once active, Calm indirectly enhances NFAT (Nuclear Factor of Activated T-Cells) translocation to the nucleus via Caln (Calcineurin) (Ref.12 & 13). These molecules, together with other downstream effectors, control TCR-mediated T-Cell polarization, adhesion and migration. Together, these signaling intermediates are crucial for the production of cytokines and the expression of activation markers by T-Cells.
ITK, therefore, seems to be a crucial component of pathways that are required for the reorganization of actin, the induction of cell polarization and the recruitment of molecules to the immunological synapse (including PKC-Theta, a key adhesion molecule that helps to stabilize contacts with the APC, leading to productive T-Cell activation. Modification of T-Cell signaling through mutation of Tec Kinases leads to altered thymic development. It decreases both Ca2+ mobilization and MAPK activation in thymocytes and leads to switches from positive selection to death by neglect and from negative selection to positive selection (Ref.1 & 2). T-Cells lacking ITK or ITK and RLK, all show varying defects in PLC-Gamma phosphorylation associated with decreased IP3 production and Ca2+ mobilization. In particular, the late phase of Ca2+ influx from extracellular stores is most affected in these cells (Ref.6 & 12).