T-cell signaling regulated by the Tec family kinase, Itk

Abstract

The Tec family tyrosine kinases regulate lymphocyte development, activation, and differentiation. In T cells, the predominant Tec kinase is Itk, which functions downstream of the T-cell receptor to regulate phospholipase C-gamma. This review highlights recent advances in our understanding of Itk kinase structure and enzymatic regulation, focusing on Itk protein domain interactions and mechanisms of substrate recognition. We also discuss the role of Itk in the development of conventional versus innate T-cell lineages, including both alphabeta and gammadelta T-cell subsets. Finally, we describe the complex role of Itk signaling in effector T-cell differentiation and the regulation of cytokine gene expression. Together, these data implicate Itk as an important modulator of T-cell signaling and function.

Figure 1.

Itk domain organization and it's role in T cell signaling. (A) The domain structure of Itk. (B) Schematic of T-cell signaling emphasizing the proximal signaling complex downstream of the TCR as described in the text. The trio of tyrosine kinases, Zap-70, Lck, and Itk that initiate signaling following TCR engagement are shown as gray, green, and brown circles, respectively. Arrows indicate early phosphorylation events on TCR/pMHC recognition. The amino-terminal region of Lck binds to the cytoplasmic tail of CD4 via coordination of Zn⁺⁺ (Huse et al. 1998). Active Lck phosphorylates the ITAMs, Zap-70, and Itk, which in turn phosphorylate their targets Slp-76, LAT (by Zap-70) and PLCγ1 (by Itk). Activated PLCγ1 then hydrolyzes PIP₂ (dashed arrow) to produce DAG and IP₃ leading to increased calcium flux. Multiple copies of these signaling molecules likely cooperate in the phosphorylation cascade and so stoichiometry as shown is overly simplified. Moreover, negative regulation of the signaling cascade in the form of phosphatases, kinases and other types of molecules also plays a significant role in T-cell function but are not represented here.

Figure 2.

Activation/inactivation of Itk. (A) Comparison of the domain structures of Itk, Csk and Src kinases. The domains are colored as follows: PH-BH is brown, PRR is yellow, SH3 is green, SH2 is orange, SH2-Kinase linker and Kinase domain are blue. All three kinases share the SH3-SH2-kinase cassette but differ with respect to resulting catalytic activity of the isolated kinase domain. Itk and Csk kinase domains alone show very poor catalytic activity whereas the Src kinase domain readily phosphorylates substrates. (B) Model of active Itk based on biochemical data and the high-resolution structure of Csk (PDB code: 1K9A). The SH2-Kinase linker and SH3-SH2 linker are both arranged along the back of the small kinase lobe. Activating interactions between the kinase domain and the SH2-kinase linker in particular are also supported by a recent structure of Btk (Marcotte et al. 2010). SLP-76 is depicted by the black line and is shown binding to both Itk SH3 and SH2. The PH-BH domain is placed so that its carboxy-terminus is close in space to the amino-terminus of the SH3 domain. The intervening sequence is the PRR, which would be available in this model for binding an SH3 domain from another protein. PH domain is shown bound to its PIP₃ ligand. (C) Model of Itk adopting the extended conformation seen for Btk in SAXS studies (Marquez et al. 2003). Of note is the fact that the intramolecular PRR/SH3 interaction is sterically allowed and that the SH2-Kinase linker region is likely in an extended conformation and not the active conformation shown in (B). (D) Native gel electrophoresis for the SH3-SH2-Kinase fragment of Itk and for full length Itk showing ladders indicative of mulitmerization. Sample concentration for both purified proteins is approximately 5 micromolar. (E) Model of Itk multimerization based on the structure of the binary Itk SH3/SH2 complex (PDB code: 2K79). Doubled headed arrows indicate PH domain self-association. Whether Itk multimerization occurs exclusively at the membrane remains to be determined. It is of note that the SH3/SH2 interface defined in PDB 2K79 is accessible in the models shown in both (B) and (C) suggesting that either configuration could multimerize. In (B), (C) and (E) the amino and carboxy-termimi of full length Itk are indicated by N and C, respectively.

Figure 3.

Itk function in T helper cell differentiation. At the left, the three major lineages of effector T cells are shown, along with the cytokine signals and transcription factors that regulate their differentiation from naïve CD4⁺ T cells. Unlike T_H1 cells, which coexpress Itk and Rlk, T_H2 cells express only Itk. The status of Rlk expression in T_H17 cells is currently unknown. In the absence of Itk (right panel), NFAT_c1 activity is greatly reduced, leading to impaired production of IL-4 by T_H2 cells and IL-17A by T_H17 cells. Itk^-/- T_H1 cells, which continue to express Rlk, have a more modest defect in effector cytokine production.

Figure 4.

Regulation of T-cell lineages by Itk. In the thymus, progenitor T cells (CD4⁻CD8⁻CD3⁻; DN) develop into γδ T cells and αβ T cells. Cells developing in the αβ T-cell lineage progress through an intermediate CD4⁺CD8⁺ (DP) stage prior to their maturation into mature conventional αβ T cells, CD1d-restricted αβ NKT cells and other subsets of innate-like αβ T cells. Conventional αβ T cells, which are selected on classical MHC molecules expressed on thymic stromal cells, and αβ NKT cells, which are selected on the MHC class IB molecule, CD1d, expressed on hematopoietic cells (HC) and dependent on SLAM receptor signaling, are greatly reduced in the absence of Itk. In contrast, innate-like αβ T cells, which are also selected by recognition of MHC molecules on HC and require SLAM receptor signaling, and “innate-like” γδ T cells (γδ NKT) are substantially increased in number in Itk^-/- mice. A third subset of cells, illustrated by conventional γδ T cells, appear unaffected by the presence versus the absence of Itk. Currently, the cell–cell interactions required for γδ T-cell development are unknown.