Tec family kinases Itk and Rlk / Txk in T lymphocytes: cross-regulation of cytokine production and T-cell fates

Developing thymocytes and T cells express the Tec kinases Itk, Rlk/Txk and Tec, which are critical modulators of T-cell receptor signaling, required for full activation of phospholipase Cγ, and downstream Ca(2+) and ERK-mediated signaling pathways. Over the last 10 years, data have implicated the Tec family kinases Itk and Rlk/Txk as important regulators of cytokine production by CD4(+) effector T-cell populations. Emerging data now suggest that the Tec family kinases not only influence cytokine-producing T-cell populations in the periphery, but also regulate the development of distinct innate-type cytokine-producing T-cell populations in the thymus. Together, these results suggest that the Tec family kinases play critical roles in helping shape immune responses via their effects on the differentiation and function of distinct cytokine-producing, effector T-cell populations.     

Tec kinases regulate TCR-mediated recruitment of signaling molecules and integrin-dependent cell adhesion

T cells deficient in the Tec kinases Itk or Itk and Rlk exhibit defective TCR-stimulated proliferation, IL-2 production, and activation of phospholipase C-gamma. Evidence also implicates Tec kinases in actin cytoskeleton regulation, which is necessary for cell adhesion and formation of the immune synapse in T lymphocytes. In this study we show that Tec kinases are required for TCR-mediated up-regulation of adhesion via the LFA-1 integrin. We also demonstrate that the defect in adhesion is associated with defective clustering of LFA-1 and talin at the site of interaction of Rlk-/-Itk-/- and Itk-/- T cells with anti-TCR-coated beads. Defective recruitment of Vav1, protein kinase Ctheta, and Pyk2 was also observed in Rlk-/-Itk-/- and Itk-/- T cells. Stimulation with ICAM-2 in conjunction with anti-TCR-coated beads enhanced polarization of Vav1, protein kinase Ctheta, and Pyk2 in wild-type cells, demonstrating a role for integrins in potentiating the recruitment of signaling molecules in T cells. Increased recruitment of signaling molecules was most pronounced under conditions of low TCR stimulation. Under these suboptimal TCR stimulation conditions, ICAM-2 could also enhance the recruitment of signaling molecules in Itk-/-, but not Rlk-/-Itk-/- T cells. Thus, Tec kinases play key roles in regulating TCR-mediated polarization of integrins and signaling molecules to the site of TCR stimulation as well as the up-regulation of integrin adhesion.     

Tec kinases Itk and Rlk are required for CD8+ T cell responses to virus infection independent of their role in CD4+ T cell help

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4(+) T cells, particularly in the phosphorylation of phospholipase C-gamma1 after TCR activation; furthermore, both Itk and Rlk are important in CD4(+) T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8(+) T cell signaling and function. Using Itk(-/-) and Itk(-/-)Rlk(-/-) mice, we examined the roles of these Tec family kinases in CD8(+) T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-gamma1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8(+) T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4(+) T cell help, thereby substantiating the important role of Tec kinases in CD8(+) T cell signaling.     

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.     

Subtle defects in pre-TCR signaling in the absence of the Tec kinase Itk

alphabeta T cell development in the thymus is dependent on signaling through the TCR. The first of these signals is mediated by the pre-TCR, which is responsible for promoting pre-T cell proliferation and the differentiation of CD4(-)8(-)3(-) (DN) thymocytes into CD4(+)8(+)3(+) (DP) cells. In many cases, T cell signaling proteins known to be essential for TCR signaling in mature T cells are also required for pre-TCR signaling in DN thymocytes. Therefore, it came as a surprise to discover that mice lacking the Tec kinases Itk and Rlk, enzymes required for efficient activation of phospholipase C-gamma1 in mature T cells, showed no obvious defects in pre-TCR-dependent selection events in the thymus. In this report, we demonstrate that DN thymocytes lacking Itk, or Itk and Rlk, are impaired in their ability to generate normal numbers of DP thymocytes, especially when placed in direct competition with WT DN thymocytes. We also show that Itk is required for maximal pre-TCR signaling in DN thymocytes. These data demonstrate that the Tec kinases Itk and Rlk are involved in, but are not essential for, pre-TCR signaling in the thymus, suggesting that there is an alternative mechanism for activating phospholipase C-gamma1 in DN thymocytes that is not operating in DP thymocytes and mature T cells.     

Tec kinases: shaping T-cell activation through actin

Following stimulation, T cells undergo marked actin-dependent changes in shape that are required for productive cellular interactions and movement during immune responses. Reorganization of the actin cytoskeletal is also necessary for the formation of an immunological synapse - the convergence of several signaling molecules at the plasma membrane that occurs after effective T-cell receptor (TCR) signaling. Much emerging evidence indicates that the Tec family of tyrosine kinases has a role in actin cytoskeleton reorganization. Specifically, T cells that lack or express mutant versions of the Tec kinase Itk show impaired TCR-induced actin polymerization, cell polarization and regulation of the signaling events involved in cytoskeletal reorganization. These data, as well as other findings, support roles for Tec kinases in actin cytoskeleton regulation.     

T-Cell Signaling Regulated by the Tec Family Kinase, Itk

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-γ. 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 αβ and γδ 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.The Tec family nonreceptor tyrosine kinases, Tec, Btk, Itk/Emt/Tsk, Rlk/Txk, and Bmx/Etk, are expressed primarily in hematopoietic cells and serve as important mediators of antigen receptor signaling in lymphocytes (Berg et al. 2005; Felices et al. 2007; Readinger et al. 2009). The demonstration that the human B-cell immunodeficiency, X-linked agammaglobulinemia (XLA), is caused by mutations in Btk first underscored the importance of this tyrosine kinase family in lymphocyte development and antigen receptor signaling (Rawlings et al. 1993; Thomas et al. 1993; Tsukada et al. 1993; Vetrie et al. 1993). T lymphocytes express three Tec kinases: Itk, Rlk and Tec. To date, only Itk has been found to have a clearly defined function in T cells, leading to the conclusion that Itk is the predominant Tec kinase in T cells. In this review, we will cover recent findings that highlight the critical role of Itk in T-cell signaling and function.     

Itk/Emt/Tsk activation in response to CD3 cross-linking in Jurkat T cells requires ZAP-70 and Lat and is independent of membrane recruitment.

The Tec family tyrosine kinase, Itk has been implicated in T cell antigen receptor (TCR) signaling, yet little is known about Itk regulation. Here, we investigate the role of the tyrosine kinase ZAP-70 in regulating Itk. Whereas Itk was activated in Jurkat T cells in response to CD3 cross-linking, Itk activation was defective in the ZAP-70-deficient P116 Jurkat T cell line. Itk responsiveness to TCR engagement was restored in P116 cells stably transfected with ZAP-70 cDNA. ZAP-70 itself could not directly phosphorylate the Itk kinase domain, indicating an indirect regulation of Itk activity. No role was found for ZAP-70 in regulating Itk recruitment to the plasma membrane, an event that has been suggested to be rate-limiting for the activation of Tec family kinases. Indeed, Itk was found to be constitutively targeted to the membrane fraction in both Jurkat and P116 cells. Lat, a prominent in vivo substrate of ZAP-70 that mediates assembly of multimolecular signaling complexes at the plasma membrane of T cells was also found to be required for TCR-stimulated Itk activation. Itk could not be activated by CD3 cross-linking in a Lat-negative cell line, unless Lat expression was restored. Lat and Itk were observed to co-associate in response to CD3 cross-linking in Jurkat T cells, but not in P116 T cells. The Lat-Itk association correlated with Lat tyrosine phosphorylation, which was deficient in the P116 T cells. These data suggest that ZAP-70 and Lat play important, probably sequential, roles in regulating the activation of Itk following TCR engagement.     

Mechanism and functional significance of Itk autophosphorylation

Tec family non-receptor tyrosine kinases (Itk, Btk, Tec, Rlk and Bmx) are characterized by the presence of an autophosphorylation site within the non-catalytic Src homology 3 (SH3) domain. The full-length Itk mutant containing phenylalanine in place of the autophosphorylated tyrosine has been studied in Itk-deficient primary T cells. These studies revealed that the non-phosphorylated enzyme restores Itk mediated signaling only partially. In spite of these insights, the precise role of the Tec kinase autophosphorylation site is unclear and the mechanism of the autophosphorylation reaction within the Tec kinases is not known. Here, we show both in vitro and in vivo that Itk autophosphorylation on Y180 within the SH3 domain occurs exclusively via an intramolecular, in cis mechanism. Using an in vitro kinase assay, we show that mutation of the Itk autophosphorylation site Y180 to Phe decreases kinase activity of the full-length enzyme by increasing Km for a peptide substrate. Moreover, mutation of Y180 to Glu, a residue chosen to mimic the phosphorylated tyrosine, alters the ligand-binding capability of the Itk SH3 domain in a ligand-dependent fashion. NMR chemical shift mapping gives residue-specific structural insight into the effect of the Y180E mutation on ligand binding. These data provide a molecular level context with which to interpret in vivo functional data and allow development of a structural model for Itk autophosphorylation.     

The linker between SH2 and kinase domains positively regulates catalysis of the Tec family kinases

Tec family nonreceptor tyrosine kinases are key immunological enzymes that control processes that range from T and B cell development to reorganization of the actin cytoskeleton. The full-length Tec kinases have been resistant to crystallization. This lack of structural data and the paucity of in vitro biochemical data for this kinase family leave a void in our understanding of Tec kinase regulation. In this report we have used interleukin-2 tyrosine kinase (Itk) as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay has uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity. The precise residues that allosterically regulate Itk are conserved among Tec kinases, pointing to the conserved nature of this regulatory mechanism within the family. These findings indicate that Tec kinases are not regulated in the same manner as the Src kinases but rather share some of the regulatory features of Csk instead.     

Reconstitution of Btk signaling by the atypical tec family tyrosine kinases Bmx and Txk

Bruton's tyrosine kinase (Btk) is mutated in X-linked agammaglobulinemia patients and plays an essential role in B cell receptor signal transduction. Btk is a member of the Tec family of nonreceptor protein-tyrosine kinases that includes Bmx, Itk, Tec, and Txk. Cell lines deficient for Btk are impaired in phospholipase C-gamma2 (PLCgamma2)-dependent signaling. Itk and Tec have recently been shown to reconstitute PLCgamma2-dependent signaling in Btk-deficient human cells, but it is not known whether the atypical Tec family members, Bmx and Txk, can reconstitute function. Here we reconstitute Btk-deficient DT40 B cells with Bmx and Txk to compare their function with other Tec kinases. We show that in common with Itk and Tec, Bmx reconstituted PLCgamma2-dependent responses including calcium mobilization, extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) activation, and apoptosis. Txk also restored PLCgamma2/calcium signaling but, unlike other Tec kinases, functioned in a phosphatidylinositol 3-kinase-independent manner and failed to reconstitute apoptosis. These results are consistent with a common role for Tec kinases as amplifiers of PLCgamma2-dependent signal transduction, but suggest that the pleckstrin homology domain of Tec kinases, absent in Txk, is essential for apoptosis.     

Tec family kinases: regulation of FcεRI-mediated mast-cell activation

Mast cells express the high-affinity receptor for IgE (FcεRI) and are key players in type I hypersensitivity reactions. They are critically involved in the development of allergic rhinitis, allergic asthma and systemic anaphylaxis, however, they also regulate normal physiological processes that link innate and adaptive immune responses. Thus, their activation has to be tightly controlled. One group of signaling molecules that are activated upon FcεRI stimulation is formed by Tec family kinases, and three members of this kinase family (Btk, Itk and Tec) are expressed in mast cells. Many studies have revealed important functions of Tec kinases in signaling pathways downstream of the antigen receptors in lymphocytes. This review summarizes the current knowledge about the function of Tec family kinases in FcεRI-mediated signaling pathways in mast cell.     

Itk is not essential for CD28 signaling in naive T cells

Itk, a member of the Tec family of tyrosine kinases, is critical for TCR signaling, leading to the activation of phospholipase C gamma1. Early biochemical studies performed in tumor cell lines also implicated Itk in CD28 signaling. These data were complemented by functional studies on primary Itk-/- T cells that suggested a negative role for Itk in CD28 signaling. In this report, we describe a thorough analysis of CD28-mediated responses in T cells lacking Itk. Using purified naive CD4+ T cells from Itk-/- mice, we examine a range of responses dependent on CD28 costimulation. We also analyze Akt and glycogen synthase kinase-3beta phosphorylation in response to stimulation of CD28 alone. Overall, these experiments demonstrate that CD28 signaling, as well as CD28-mediated costimulation of TCR signaling, function efficiently in the absence of Itk. These findings indicate that Itk is not essential for CD28 signaling in primary naive CD4+ T cells.     

The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization

Cell polarization is required for virtually all functions of T cells, including transendothelial migration in response to chemokines. However, the molecular pathways that establish T cell polarity are poorly understood. We show that the activation of the partitioning defective (Par) polarity complex is a key event during Rap1- and chemokine-induced T cell polarization. Intracellular localization and activation of the Par complex are initiated by Rap1 and require Cdc42 activity. The Rac activator Tiam1 associates with both Rap1 and components of the Par complex, and thereby may function to connect the Par polarity complex to Rap1 and to regulate the Rac-mediated actin remodelling required for T cell polarization. Consistent with these findings, Tiam1-deficient T cells are impaired in Rap1- and chemokine-induced polarization and chemotaxis. Our studies implicate Tiam1 and the Par polarity complex in polarization of T cells, and provide a mechanism by which chemokines and Rap1 regulate T cell polarization and chemotaxis.     

The Tec family of tyrosine kinases in T cells,amplifiers of T cell receptor signals

ITK and Rlk/Txk are the predominant Tec family of tyrosine kinases expressed in T cells, and are involved in T cell antigen receptor mediated activation of T cells. These kinases require prior activation of Lck, Zap-70 and PI3-kinase for efficient activation. They share major substrates with both Lck and Zap-70, however the pathways they regulate are unclear. Recent evidence suggests that these kinases may not activate unique pathways, but instead serve as amplifiers for the upstream kinases Lck and Zap-70. This review will discuss the evidence for this view.     

Rac1 mediates collapse of microvilli on chemokine-activated T lymphocytes

Lymphocytes circulate in the blood and upon chemokine activation rapidly bind, where needed, to microvasculature to mediate immune surveillance. Resorption of microvilli is an early morphological alteration induced by chemokines that facilitates lymphocyte emigration. However, the antecedent molecular mechanisms remain largely undefined. We demonstrate that Rac1 plays a fundamental role in chemokine-induced microvillar breakdown in human T lymphocytes. The supporting evidence includes: first, chemokine induces Rac1 activation within 5 s via a signaling pathway that involves Galphai. Second, constitutively active Rac1 mediates microvilli disintegration. Third, blocking Rac1 function by cell permeant C-terminal "Trojan" peptides corresponding to Rac1 (but not Rac2, Rho, or Cdc42) blocks microvillar loss induced by the chemokine stromal cell-derived factor 1alpha (SDF-1alpha). Furthermore, we demonstrate that the molecular mechanism of Rac1 action involves dephosphorylation-induced inactivation of the ezrin/radixin/moesin (ERM) family of actin regulators; such inactivation is known to detach the membrane from the underlying actin cytoskeleton, thereby facilitating disassembly of actin-based peripheral processes. Specifically, ERM dephosphorylation is induced by constitutively active Rac1 and stromal cell-derived factor 1alpha-induced ERM dephosphorylation is blocked by either the dominant negative Rac1 construct or by Rac1 C-terminal peptides. Importantly, the basic residues at the C terminus of Rac1 are critical to Rac1's participation in ERM dephosphorylation and in microvillar retraction. Together, these data elucidate new roles for Rac1 in early signal transduction and cytoskeletal rearrangement of T lymphocytes responding to chemokine.     

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.