Tec Family Of Nonreceptor Tyrosine Kinases Research Articles

Triple Degradation of BTK, IKZF1 and IKZF3 in B-Cell Malignancies

Bruton's Tyrosine Kinase (BTK), a TEC-family non-receptor tyrosine kinase, plays a critical role in B-cell development and function. Targeting of BTK with covalent inhibitors like ibrutinib has become a standard approach for many B-cell malignancies, including chronic lymphocytic leukemia (CLL), Waldenstrom macroglobulinemia, mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). Yet, many patients demonstrate intrinsic or acquired resistance to covalent BTK inhibition. The prognosis of patients who relapse after ibrutinib treatment for MCL or CLL is dismal, highlighting the urgent need for new approaches that overcome resistance to current BTK inhibitors. We hypothesized that degradation of BTK could be a better alternative to inhibition alone, as it would both: 1) maintain efficacy in cells harboring the ibrutinib-resistant BTK C418S mutation and 2) target non-catalytic functions of BTK. To address this, we turned to a small molecule-mediated protein degradation platform that utilizes an E3 ligase-targeting moiety linked to the ligand of a target of interest, so that the target can be marked for ubiquitination and subsequent proteasomal degradation. We previously showed that BTK is one of the most robustly degraded kinase targets using a nonspecific kinase inhibitor linked to a imide-based core followed by agnostic proteomics. We synthesized highly potent and selective degraders of BTK using imides as a base. To do so, the parent BTK inhibitor CGI1746 was linked to thalidomide using either polyethylene glycol (DD-03-007) linkers or saturated hydrocarbon chain (DD-03-171) linkers. After verifying that these degraders induce dimerization of BTK and CRBN and penetrate cells, we explored the pharmacological effects in vitro. Both DD-03-007 and DD-03-171 reduced BTK levels at concentrations as low as 40 nM within 4h of treatment. Furthermore, cellular BTK levels remained low for 24h after washout, showing that these degraders are capable of sustaining depletion of BTK for an extended period of time (Figure). DD-03-007 and DD-03-171 are both ligase and proteasome-dependent, as shown by co-treatment with bortezomib or MLN-4924 as well as competition experiments with lenalidomide or CGI-1746. Moreover, the degraders exhibited strong synergy with the HCK inhibitor A419259, suggesting that it would be possible to recapitulate ibrutinib's previously reported polypharmacology with an HCK inhibitor.We overexpressed wild-type or C481S BTK in TMD-8 cells and ran an antiproliferation assay, which showed that DD-03-007 overcame ibrutinib resistance associated with BTK C481S mutation.Next, we explored the effects of the degraders in MCL specifically. Proteomic analysis showed that DD-03-171 is a triple-degrader, as it degrades BTK but retains degradation activity on IKZF1 and IKZF3. Finally, we performed In vivo efficacy studies in cell line and patient-derived xenograft (PDX) models of MCL. The latter was obtained from a patient who had progressed on ibrutinib. In both models, DD-03-171 caused a significant reduction in tumor burden at an early timepoint. DD-03-171 also markedly extended survival compared to treatment with ibrutinib or lenalidomide alone (Figure). In conclusion, we developed highly potent and selective BTK degraders with activity in vivo against human MCL that induce the degradation of multiple factors essential for MCL survival. [Display omitted] DisclosuresTreon:Pharmacyclics: Consultancy, Other: Travel, Accommodations, Expenses, Research Funding; Janssen: Consultancy, Other: Travel, Accommodations, Expenses; Johnson & Johnson: Consultancy; BMS: Research Funding. Weinstock:Novartis, Dragonfly, Travera, DxTerity, Travera: Consultancy; Novartis: Consultancy, Research Funding; Novartis, Astra Zeneca, Abbvie, Aileron, Surface Oncology, Daiichi Sankyo: Research Funding; Genentech/Roche, Monsanto: Consultancy; Astra Zeneca, JAX, Samumed, Regeneron, Sun Pharma, Prescient: Patents & Royalties; Travera: Equity Ownership. Gray:Syros, Soltego, Petra, C4 Therapeutics: Equity Ownership.

BMX-Mediated Regulation of Multiple Tyrosine Kinases Contributes to Castration Resistance in Prostate Cancer.

Prostate cancer responds to therapies that suppress androgen receptor (AR) activity (androgen deprivation therapy, ADT) but invariably progresses to castration-resistant prostate cancer (CRPC). The Tec family nonreceptor tyrosine kinase BMX is activated downstream of PI3K and has been implicated in regulation of multiple pathways and in the development of cancers including prostate cancer. However, its precise mechanisms of action, and particularly its endogenous substrates, remain to be established. Here, we demonstrate that BMX expression in prostate cancer is suppressed directly by AR via binding to the BMX gene and that BMX expression is subsequently rapidly increased in response to ADT. BMX contributed to CRPC development in cell line and xenograft models by positively regulating the activities of multiple receptor tyrosine kinases through phosphorylation of a phosphotyrosine-tyrosine (pYY) motif in their activation loop, generating pYpY that is required for full kinase activity. To assess BMX activity in vivo, we generated a BMX substrate-specific antibody (anti-pYpY) and found that its reactivity correlated with BMX expression in clinical samples, supporting pYY as an in vivo substrate. Inhibition of BMX with ibrutinib (developed as an inhibitor of the related Tec kinase BTK) or another BMX inhibitor BMX-IN-1 markedly enhanced the response to castration in a prostate cancer xenograft model. These data indicate that increased BMX in response to ADT contributes to enhanced tyrosine kinase signaling and the subsequent emergence of CRPC, and that combination therapies targeting AR and BMX may be effective in a subset of patients.Significance: The tyrosine kinase BMX is negatively regulated by androgen and contributes to castration-resistant prostate cancer by enhancing the phosphorylation and activation of multiple receptor tyrosine kinases following ADT. Cancer Res; 78(18); 5203-15. ©2018 AACR.

Ovarian polarity and cell shape determination by Btk29A in Drosophila.

Drosophila Btk29A is a Tec family nonreceptor tyrosine kinase, the ortholog of which causes X-linked agammaglobulinemia in humans when mutant. In Btk29AficP mutant ovaries, multiple defects are observed: extrapolar cells form ectopically; osk mRNA fails to accumulate posteriorly in mature oocytes; the shape and alignment of follicle cells are grossly distorted. All these phenotypes are rescued by selectively overexpressing the type 2 isoform of wild-type Btk29A in follicle cells. Expression of certain proteins enriched in adherens junctions is markedly affected in Btk29AficP mutants; the anterior-posterior gradient normally observed in the expression of DE-Cadherin and Armadillo are lost and Canoe is sequestered from adherens junctions. Intriguingly, tyrosine phosphorylation of Canoe is reduced in Btk29AficP mutants. It is proposed that Btk29A is required for the establishment of egg chamber polarity presumably through the regulation of subcellular localization of its downstream proteins, including Cno.

Discovery of 2-((3-Acrylamido-4-methylphenyl)amino)-N-(2-methyl-5-(3,4,5-trimethoxybenzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamide (CHMFL-BMX-078) as a Highly Potent and Selective Type II Irreversible Bone Marrow Kinase in the X Chromosome (BMX) Kinase Inhibitor.

BMX is a member of TEC family nonreceptor tyrosine kinase and is involved in a variety of critical physiological and pathological processes. Through combination of irreversible inhibitor design and type II inhibitor design approaches, we have discovered a highly selective and potent type II irreversible BMX kinase inhibitor compound 41 (CHMFL-BMX-078), which exhibited an IC50 of 11 nM by formation of a covalent bond with cysteine 496 residue in the DFG-out inactive conformation of BMX. It displayed a high selectivity profile (S score(1) = 0.01) against the 468 kinases/mutants in the KINOMEscan evaluation and achieved at least 40-fold selectivity over BTK kinase. Given the fact that BMX mediated signaling pathway is still not fully understood, compound 41 would serve as a useful pharmacological tool to elucidate the detailed mechanism of BMX mediated signaling pathways.

Ibrutinib repurposing: from B-cell malignancies to solid tumors.

Ibrutinib (Imbruvica®, also known as PCI-32765) is a first-in-class, irreversible small-molecule inhibitor of Bruton's Tyrosine Kinase (BTK) that binds covalently to cysteine C481 within the ATP-binding pocket. Since BTK is a Tec family non-receptor tyrosine kinase that is specifically required for B-cell antigen receptor (BCR) signaling, ibrutinib was initially developed for the treatment of B-cell malignancies. Currently, it is approved for first-line treatment of chronic lymphocytic leukemia, and for the treatment of mantle-cell lymphoma and Waldenstrom's macroglobulinemia patients that have received at least one previous therapy. However, BTK is also involved in signaling pathways downstream of many other receptors and its expression is not restricted to B cells. In fact, it is expressed in all hematopoietic lineages with the exception of T lymphocytes. Taking advantage of this aspect of BTK biology, some groups, including ours, have exploited the utility of inhibiting BTK in different cell types other than B cells. In particular, we validated the critical role of BTK for mast cell degranulation in mouse models of pancreatic cancer, namely insulinoma [1] and pancreatic ductal adenocarcinoma (PDAC) [2]. Ibrutinib was effective in both scenarios, leading to vasculature collapse and tumor regression in insulinoma and to a potent and unexpected anti- fibrotic effect in PDAC, where it synergized with standard of care chemotherapy to extend mice survival. This latter observation suggests that the anti- fibrotic activity of ibrutinib could be beneficial also for the treatment of other fibrotic diseases, an aspect that deserves further studies. More recently, Gunderson and colleagues contributed to the understanding of ibrutinib therapeutic impact in PDAC by describing how BTK regulates B-cell and macrophage-mediated T-cell suppression and demonstrating that ibrutinib restores T-cell dependent anti-tumor responses and triggers growth inhibition [3]. These studies combined led to the initiation of a phase 2/3 clinical trial for the use of ibrutinib in combination with nab- paclitaxel and gemcitabine in metastatic PDAC. Importantly, emerging data obtained from various mouse models of cancer suggests that beneficial use of ibrutinib could extend beyond pancreatic cancer to other solid tumors. For example, myeloid-derived suppressor cells (MDSCs) express BTK and are present in the stroma of many different tumors, causing immunosuppression and evasion of anti-tumor immune responses. Consistently, treatment of breast cancer and melanoma mouse models with ibrutinib reduced the number of MDSCs in the spleen and the tumor, and combination therapy with anti-PD-L1 resulted in reduced mammary tumor growth [4]. Moreover, synergy of ibrutinib with immune checkpoint blockade has also been reported in mouse models of lymphoma, breast and colon cancer, but in a BTK-independent manner [5]. In this case, suppression of tumor growth was due to inhibition of the interleukin-2–inducible T-cell kinase (ITK), an enzyme required by Th2 T cells, allowing a shift of T-cell immune responses to a Th1 bias. However, off-target (BTK-independent) effects of ibrutinib are not limited to ITK, since its inhibitory activity affects several other kinases including Tec, EGFR, HER2, HER3, HER4, JAK3, Blk, Fgr, Hck, Lck, Yes/YES1, Bmx/Etk and Txk. While this lack of selectivity could explain some of its side effects, it could be exploited for the treatment of tumors without BTK dependency. Indeed, two recent reports exemplify this approach by showing promising in vitro data against EGFR-mutant or overexpressed non-small cell lung cancer (NSCLC) [6] and HER2-positive breast cancer. Finally, expression of BTK variants was found in cancer cells of breast, prostate [7] and colon tumors [8]. In all cases, its downregulation by RNAi or its inhibition by pharmacological means, including ibrutinib, leads to impaired proliferation and/or cell death. All these findings broaden the spectrum of tumor types potentially susceptible to treatment with ibrutinib. Its use against solid tumors, alone or in combination with standard therapies or novel immune-based approaches, is increasingly becoming a clinically viable option. Indeed, a number of clinical trials are already underway for the treatment of different solid tumor types, such as EGFR mutant NSCLC (http://ClinicalTrials.gov Identifier: NCT02321540), refractory stage IV cutaneous melanoma (NCT02581930), metastatic pancreatic adenocarcinoma (NCT02562898 and NCT02436668), localized prostate cancer (NCT02643667), advanced gastrointestinal and genitourinary tumors (NCT02599324), advanced carcinoid and pancreatic neuroendocrine tumors (NCT02575300) and other relapsed or refractory solid tumors including lung and breast (NCT02403271). Results of these trials will shed light on the applicability of ibrutinib in human patients beyond hematological malignancies and quickly open the way to new immediate clinical applications. Nevertheless, further work will be needed in order to establish, for every tumor type, the contribution of BTK-related responses versus inhibition of other kinases, either in tumor cells themselves or in the tumor stroma. Ibrutinib constitutes a prime example of therapeutic switching, but there are likely many other drugs already in clinical use that are candidates for repositioning, and could offer new, safe and easily-implementable opportunities for cancer patients.

Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site.

Btk29A-mediated tyrosine phosphorylation of armadillo/β-catenin promotes ring canal growth in Drosophila oogenesis.

Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29AficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29AficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29AficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.

Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor.

Btk and Etk/BMX are Tec-family non-receptor tyrosine kinases. Btk has previously been reported to be expressed primarily in B cells and has an important role in immune responses and B-cell malignancies. Etk has been shown previously to provide a strong survival and metastasis signal in human prostate cancer cells, and to confer androgen independence and drug resistance. While the role of Etk in prostate carcinogenesis is well established, the functions of Btk in prostate cancer have never been investigated, likely due to the perception that Btk is a hematopoietic, but not epithelial, kinase. Herein, we found that Btk is overexpressed in prostate cancer tissues and prostate cancer cells. The level of Btk in prostate cancer tissues correlates with cancer grades. Knockdown of Btk expression selectively inhibits the growth of prostate cancer cells, but not that of the normal prostate epithelial cells, which express very little Btk. Dual inhibition of Btk and Etk has an additive inhibitory effect on prostate cancer cell growth. To explore Btk and Etk as targets for prostate cancer, we developed a small molecule dual inhibitor of Btk and Etk, CTN06. Treatment of PC3 and other prostate cancer cells, but not immortalized prostate epithelial cells with CTN06 resulted in effective cell killing, accompanied by the attenuation of Btk/Etk signals. The killing effect of CTN06 is more potent than that of commonly used inhibitors against Src, Raf/VEGFR and EGFR. CTN06 induces apoptosis as well as autophagy in human prostate cancer cells, and is a chemo-sensitizer for docetaxel (DTX), a standard of care for metastatic prostate cancer patients. CTN06 also impeded the migration of human prostate cancer cells based on a ‘wound healing' assay. The anti-cancer effect of CTN06 was further validated in vivo in a PC3 xenograft mouse model.

ITK signals tune CD8+ T cell homeostatic proliferation and anti-tumor immunity (LYM4P.747)

Abstract IL-2 inducible T cell kinase (ITK) is a Tec family non-receptor tyrosine kinase. Loss of ITK perturbs CD8+ T cell homeostasis and leads to spontaneous acquisition of innate memory/effector phenotype. However, Itk-/- naïve CD8+ T cells develop on an OTI Rag-/- background, and we use these cells to show here that ITK tunes lymphopenia-induced proliferation (LIP) and antigen sensitivity during CD8+ T cell homeostatic expansion. Experimental analysis and computational simulations revealed that in a lymphopenic environment, naïve Itk-/- CD8+ T cells exhibit massive and immediate homeostatic expansion, accompanied by significant death and followed by population collapse. The enhanced immediate expansion of Itk-/- cells is CD8+ T cell-intrinsic and is independent of recipient MHCI, but dependent on donor CD8+ T cell-T cell interaction. The lack of ITK resulted in enhanced antigen sensitivity and effector program in homeostatically expanded (HP) CD8+ T cells. These Itk-/- HP CD8+ T cells exhibit robust anti-tumor immunity in an antigen specific manner, independent of CD4+ T cell help. These data suggest that ITK intrinsically regulates TcR tuning of homeostatic cytokines during homeostasis of CD8+ T cells and that targeting ITK may have clinical benefit in cancer therapy by generating superior anti-tumor responses.

Bruton's tyrosine kinase: potential target in human multiple myeloma

Bruton's tyrosine kinase (BTK), a Tec family non-receptor tyrosine kinase that is required for B cell development, is critical for the initiation and maintenance of human B-cell malignancies. However, the expression of BTK and the role that BTK plays in the pathogenesis of multiple myeloma (MM) remain seldom reported. In this study we examined the expression and screened for gene mutations of BTK in MM cells. We showed that BTK was elevated and activated in a dexamethasone-resistant cell line and in two out of nine (22.2%) patients’ cells. Interestingly, patients with higher BTK expression had a poorer prognosis. In addition, a single nucleotide polymorphism (SNP) at cDNA position 2062 (T2062C) in the BTK gene was recorded in six out of eight (75%) patients and in U266 cells. This SNP in MM cells was not detected in other malignant hematopoietic cells of different lineages. These results suggest that the function of BTK warrants further investigation, and BTK expression might be used as a prognostic indicator for patients with MM.

Rescue of the aggregation prone Itk Pleckstrin Homology domain by two mutations derived from the related kinases, Btk and Tec

IL-2 inducible T-cell kinase (Itk) is a Tec family non-receptor tyrosine kinase involved in signaling downstream of the T-cell receptor. Itk contains an amino-terminal Pleckstrin Homology (PH) domain that binds phosphatidylinositol (3,4,5)-trisphosphate, recruiting Itk to the plasma membrane upon T-cell receptor activation. In addition to phosphoinositide binding, accumulating data suggest that the Itk PH domain likely mediates additional interactions outside of the phosphoinositide ligand binding pocket. The structural basis for additional PH domain functions remains elusive because of the poor recombinant expression and in vitro solution behavior of the Itk PH domain. Here, we determine that the lone α-helix in the Itk PH domain is responsible for the poor solution properties and that mutation of just two residues in the Itk α-helix to the corresponding amino acids in Btk or Tec dramatically improves the soluble recombinant expression and solution behavior of the Itk PH domain. We present this double mutant as a valuable tool to characterize the structure and function of the Itk PH domain. It is also interesting to note that the precise sites of mutation identified in this study appear as somatic mutations associated with cancerous tissue. Collectively, the findings suggest that the two helical residues in the Itk PH domain may serve an important and unique structural role in wild-type Itk that differentiates this tyrosine kinase from its related family members.

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

Invariant natural killer T (iNKT) cells are a unique subset of innate T lymphocytes that are selected by CD1d. They have diverse immune regulatory functions via the rapid production of interferon-γ (IFN-γ) and interleukin-4 (IL-4). In the absence of signaling nodes Itk and Txk, Tec family non-receptor tyrosine kinases, mice exhibit a significant block in iNKT cell development. We now show here that although the Itk node is required for iNKT cell maturation, the kinase domain edge of Itk is not required for continued maturation iNKT cells in the thymus compared with Itk-null mice. This rescue is dependent on the expression of the Txk node. Furthermore, this kinase domain independent edge rescue correlates with the increased expression of the transcription factors T-bet, the IL-2/IL-15 receptor β chain CD122, and suppression of eomesodermin expression. By contrast, α-galactosyl ceramide induced cytokine secretion is dependent on the kinase domain edge of Itk. These findings indicate that the Itk node uses a kinase domain independent edge, a scaffolding function, in the signaling pathway leading to the maturation of iNKT cells. Furthermore, the findings indicate that phosphorylation of substrates by the Itk node is only partially required for maturation of iNKT cells, while functional activation of iNKT cells is dependent on the kinase domain/activity edge of Itk.

Tec family non-receptor tyrosine kinase ITK is important for the development of skin-specific intraepithelial T cells through regulation of their skin-homing property (96.17)

Abstract Proper localization of immune cells during the development is critical for their optimal functions. Murine skin-specific intraepithelial T cells (sIELs) are a unique tissue-specific innate T cell population that function in the first line of defense to maintain the local tissue integrity. The sIELs originate from their fetal thymic progenitors that specifically express canonical Vγ3+ TCR. Although it has been shown that the expression of skin-homing molecules, such as CCR10, is associated with the TCR-mediated cellular selection of the progenitors for their development into sIELs, molecules that mediate the selection-associated homing molecule expression is not fully understood. Here we report that ITK-null mice have a defective sIEL development due to impaired expression of the homing molecules in the fetal thymic sIEL progenitors. In the absence of ITK, the fetal thymic Vγ3+ cells failed to upregulate the expression of the chemokine receptors important for their thymus-exiting and skin-homing, including S1P1 and CCR10, which was associated with a gradient accumulation of the Vγ3 cells in the fetal thymus and a severe defect in their seeding of the skin. On the other hand, the ITK deficiency did not affect the TCR- or IL-15 receptor-mediated proliferation of the progenitor cells. These results indicate that ITK-mediated signaling is critical for the acquirement of proper homing property by the sIEL progenitors for their peripheral localization.

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.

Tec kinases, actin, and cell adhesion

The Tec family non-receptor tyrosine kinases have been recognized for their roles in the regulation of phospholipase C-gamma and Ca(2+) mobilization downstream from antigen receptors on lymphocytes. Recent data, however, show that the Tec family kinase interleukin-2-inducible T-cell kinase (Itk) also participates in pathways regulating the actin cytoskeleton and 'inside-out' signaling to integrins downstream from the T-cell antigen receptor. Data suggest that Itk may function in a kinase-independent fashion to regulate proper recruitment of the Vav1 guanine nucleotide exchange factor. By enhancing actin cytoskeleton reorganization, recruitment of signaling molecules to the immune synapse, and integrin clustering in response to both antigen and chemokine receptors, the Tec kinases serve as modulators or amplifiers that can increase the duration of T-cell signaling and regulate T-cell functional responses.

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.

Modulation of tracheal smooth muscle responses in inducible T-cell kinase knockout mice

It has been shown that the Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) plays a role in the activation of naïve T cells and in the differentiation of T helper (T H2)-type cells producing cytokines in a model of allergic inflammation, thereby possibly indirectly mediating hyperresponsivenes of airway smooth muscle tone. Using excised tracheae from wild type (WT) mice and those lacking ITK, we conducted a series of in vitro experiments in which isometric smooth muscle tones were assessed in response to several agonists to determine whether the absence of ITK would affect the responsiveness of tracheal smooth muscle cells. The resulting change in contractile responses was evaluated by measuring agonist cumulative concentration-response curves (CCRC). Our results indicate that the cholinergic agonist acetylcholine (ACh) and its analog carbachol (CCh) exhibited comparable CCRC profiles in contracting isolated tracheae from both WT and ITK −/− mice, with no alteration in their efficacies. However, the EC 50 values for the two agonists were found to be significantly higher in ITK −/− tracheae than in those from WT mice, suggesting an alteration of the potencies of these cholinergic agonists in the trachea of ITK −/− mice. Moreover, we found that the depolarizing agent potassium chloride (KCl) had a significantly lower efficacy in contracting ITK −/− tracheae compared to those from WT mice. This difference in KCl efficacy was abolished in the presence of a calcium (Ca 2+) voltage-dependent channel (VDC) agonist, Bay K8644, suggesting a modulation of the KCl induced permeability of VDC Ca 2+ channels in the trachea of ITK −/− mice. Taken together, these results suggest that the presence of ITK may play a modulating role in the pharmacomechanical as well as in the electromechanical coupling of airway smooth muscle contraction.

Attenuation of immunological symptoms of allergic asthma in mice lacking the tyrosine kinase ITK.

Allergic asthma patients manifest airway inflammation and some show increases in eosinophils, T(H)2 cells, and cytokines, increased mucous production in the lung, and elevated serum IgE. This T(H)2-type response suggests a prominent role for T(H)2 cells and their cytokines in the pathology of this disease. The Tec family nonreceptor tyrosine kinase inducible T cell kinase (ITK) has been shown to play a role in the differentiation and/or function of T(H)2-type cells, suggesting that ITK may represent a good target for the control of asthma. Using a murine model of allergic asthma, we show here that ITK is involved in the development of immunological symptoms seen in this model. We show that mice lacking ITK have drastically reduced lung inflammation, eosinophil infiltration, and mucous production following induction of allergic asthma. Notably, T cell influx into the lung was reduced in mice lacking ITK. T cells from ITK(-/-) mice also exhibited reduced proliferation and cytokine secretion, in particular IL-5 and IL-13, in response to challenge with the allergen OVA, despite elevated levels of total IgE and increased OVA-specific IgE responses. Our results suggest that the tyrosine kinase ITK preferentially regulates the secretion of the T(H)2 cytokines IL-5 and IL-13 and may be an attractive target for antiasthmatic drugs.

Altering T-cell activation by targeting the multidomain tyrosine kinase Itk

The Tec family non-receptor tyrosine kinase Itk is expressed in T cells, natural killer (NK) cells and mast cells. The role of this multidomain kinase in T cells has been linked to T-cell receptor–CD3 (TCR–CD3) signaling and Itk regulates and amplifies signals through the costimulatory receptors CD28 and CD2. Itk binds a specific subset of membrane inositol phospholipids through its pleckstrin homology (PH) domain; it forms functional molecular complexes with a variety of signaling proteins through its Tec homology (TH), Src homology 3 (SH3) and SH2 domains; and it phosphorylates several protein substrates on tyrosine residues in cells. Among >500 protein kinases expressed in the human proteome, we propose that Itk is a validated T-cell target suitable for pharmaceutical intervention. Targeted disruption of protein–protein interactions between Itk and some of its binding partners, and inhibition of the intrinsic kinase activity of Itk, could provide platforms through which to alter T-cell activation in immunological and inflammatory disorders.

Ｔｈ１細胞分化におけるＴｘｋの機能と役割

Imbalance of Th1/Th2 responses is associated with the development of human autoimmune diseases and allergic diseases. We have studied a role of Txk, a member of Tec family non-receptor tyrosine kinase for the Th1/Th2 cell development.We found that Txk is expressed Th1/Th0 cells with IFN-γ producing potential. Txk transfection leads to severalfold increase in IFN-γ production; neither IL-2 nor IL-4 production were affected. Thus, Txk acts as a Th1 cell specific transcription factor.Furthermore, we found that Txk expression is enhanced by Th1 cytokines but is inhibited by Th2 cytokines. Txk expression of the peripheral blood T cells is decreased in patients with allergic diseases. Collectively, Txk expression is modulated by with Th1/Th2 cytokines and is importantly involved in immune aberration in patients with immunological disorders. These results suggest that Txk is a possible therapeutic target of the diseases with Th1/Th2 imbalance.