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.     

Emt/Itk associates with activated TCR complexes: role of the pleckstrin homology domain

Expressed in mast and T-cells/inducible T cell tyrosine kinase (Emt/Itk) is a protein tyrosine kinase required for T cell Ag receptor (TCR)-induced activation and development. A physical interaction between Emt/Itk and TCR has not been described previously. Here, we have utilized laser scanning confocal microscopy to demonstrate that Ab-mediated engagement of the CD3epsilon chain induces the membrane colocalization of Emt/Itk with TCR/CD3. Removal of the Emt/Itk pleckstrin homology domain (DeltaPH-Emt/Itk) abrogates the association of the kinase with the cell membrane, as well as its activation-induced colocalization with the TCR complex and subsequent tyrosine phosphorylation. The addition of a membrane localization sequence to DeltaPH-Emt/Itk from Lck restores all of these deficiencies except the activation-induced tyrosine phosphorylation. Our data suggest that the PH domain of Emt/Itk can be replaced with another membrane localization signal without affecting the membrane targeting and activation-induced colocalization of the kinase with the TCR. However, the PH domain is indispensable for the activation-induced tyrosine phosphorylation of the kinase.     

Structural characterization of a proline-driven conformational switch within the Itk SH2 domain

Interleukin-2 tyrosine kinase (Itk) is a T cell-specific kinase required for a proper immune response following T cell receptor engagement. In addition to the kinase domain, Itk is composed of several noncatalytic regulatory domains, including a Src homology 2 (SH2) domain that contains a conformationally heterogeneous Pro residue. Cis-trans isomerization of a single prolyl imide bond within the SH2 domain mediates conformer-specific ligand recognition that may have functional implications in T cell signaling. To better understand the mechanism by which a proline switch regulates ligand binding, we have used NMR spectroscopy to determine two structures of Itk SH2 corresponding to the cis and trans imide bond-containing conformers. The structures indicate that the heterogeneous Pro residue acts as a hinge that modulates ligand recognition by controlling the relative orientation of protein-binding surfaces.     

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.     

SH2-Dependent Autophosphorylation within the Tec Family Kinase Itk

The Tec family kinase, Itk (interleukin-2 tyrosine kinase), undergoes an in cis autophosphorylation on Y180 within its Src homology 3 (SH3) domain. Autophosphorylation of the Itk SH3 domain by the Itk kinase domain is strictly dependent on the presence of the intervening Src homology 2 (SH2) domain. A direct docking interaction between the Itk kinase and SH2 domains brings the Itk SH3 domain into the active site where Y180 is then phosphorylated. We now identify the residues on the surface of the Itk SH2 domain responsible for substrate docking and show that this SH2 surface mediates autophosphorylation in the full-length Itk molecule. The canonical phospholigand binding site on the SH2 domain is not involved in substrate docking, instead the docking site consists of side chains from three loop regions (AB, EF and BG) and part of the βD strand. These results are extended into Btk (Bruton's tyrosine kinase), a Tec family kinase linked to the B-cell deficiency X-linked agammaglobulinemia (XLA). Our results suggest that some XLA-causing mutations might impair Btk phosphorylation.     

Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade

Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain directs the constitutive association of Itk with buoyant membranes that are the primary site of TCR activation and are enriched in both Lck and LAT. This association is required for the transphosphorylation of Itk. Second, the Itk proline-rich region binds to Grb2 and LAT. Third, the Itk Src homology (SH3) 3 and SH2 domains interact cooperatively with Syk-phosphorylated SLP-76. Notably, SLP-76 contains a predicted binding motif for the Itk SH2 domain and binds to full-length Itk in vitro. Finally, we show that kinase-inactive Itk can antagonize the SLP-76-dependent activation of NF-AT. The inhibition of NF-AT activation depends on the Itk pleckstrin homology domain, proline-rich region, and SH2 domain. Together, these observations suggest that multivalent interactions recruit Itk to LAT-nucleated signaling complexes and facilitate the activation of LAT-associated phospholipase Cgamma1 by Itk.     

TCR/CD3-Induced activation and binding of Emt/Itk to linker of activated T cell complexes: requirement for the Src homology 2 domain

Expressed in mast and T cells/inducible T cell tyrosine kinase (Emt/Itk), a Tec family protein tyrosine kinase, is critical for the development and activation of T lymphocytes. The mechanism through which Emt/Itk mediates its effector functions is poorly understood. In this study, we show that the Emt/Itk Src homology 2 (SH2) domain is critical for the transphosphorylation and activation of Emt/Itk catalytic activity that is mediated by TCR/CD3 engagement. Furthermore, we find that the Emt/Itk SH2 domain is essential for the formation of TCR/CD3-inducible Emt/Itk-LAT complexes, whereas the SH3 domain and catalytic activity are not required. The Emt/Itk-linker of activated T cells (LAT) complexes are biologically important because Jurkat T cells with deficient LAT expression (JCaM2) fail to increase Emt/Itk tyrosine phosphorylation upon TCR/CD3 stimulation. Confocal microscopy reveals that in activated cells, LAT complexes colocalize with TCR/CD3. The present data suggest that upon TCR/CD3 engagement, the Emt/Itk SH2 domain mediates the formation of a molecular complex containing Emt/Itk, LAT, and TCR/CD3; this complex is essential for Emt/Itk activation and function.     

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.     

A novel function for the Tec family tyrosine kinase Itk in activation of beta 1 integrins by the T-cell receptor

Stimulation of T cells via the CD3--T-cell receptor (TCR) complex results in rapid increases in beta 1 integrin-mediated adhesion via poorly defined intracellular signaling events. We demonstrate that TCR-mediated activation of beta 1 integrins requires activation of the Tec family tyrosine kinase Itk and phosphatidylinositol 3-kinase (PI 3-K)-dependent recruitment of Itk to detergent-insoluble glycosphingolipid-enriched microdomains (DIGs) via binding of the pleckstrin homology domain of Itk to the PI 3-K product PI(3,4,5)-P(3). Activation of PI 3-K and the src family kinase Lck, via stimulation of the CD4 co-receptor, can initiate beta 1 integrin activation that is dependent on Itk function. Targeting of Itk specifically to DIGs, coupled with CD4 stimulation, can also activate beta 1 integrin function independently of TCR stimulation. Changes in beta 1 integrin function mediated by TCR activation of Itk are also accompanied by Itk-dependent modulation of the actin cytoskeleton. Thus, TCR-mediated activation of beta 1 integrins involves membrane relocalization and activation of Itk via coordinate action of PI 3-K and a src family tyrosine kinase.     

Tec kinase Itk forms membrane clusters specifically in the vicinity of recruiting receptors

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 A 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.     

Structural analysis of the lymphocyte-specific kinase Lck in complex with non-selective and Src family selective kinase inhibitors.

BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.     

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.     

Characterization of Itk tyrosine kinase: contribution of noncatalytic domains to enzymatic activity.

Itk is a Tec family tyrosine kinase found in T cells that is activated upon ligation of the T cell receptor (TCR/CD3), CD2, or CD28. Itk contains five domains in addition to the catalytic domain: pleckstrin homology, Tec homology which contains a proline-rich region, Src homology 3, and Src homology 2. To provide a basis for understanding the contribution of these various domains to catalysis, recombinant Itk was purified and its substrate specificity determined by steady-state kinetic methods. Measurements of the rates of phosphorylation of various protein substrates, including Src associated in mitosis 68K protein (SAM68), CD28, linker for activation of T cells, and CD3 zeta, at a fixed concentration indicated that SAM68 was phosphorylated most rapidly. Wild-type Itk and three Itk mutants were characterized by comparing their activity (k(cat)) using the SAM68 substrate. A deletion mutant removing the pleckstrin homology domain and part of the Tec homology domain (Itk(Delta152)) had approximately 10-fold less activity than wild type, a mutant with an altered proline-rich domain (P158A,P159A) had a more dramatic 100-fold loss of activity, and the catalytic domain alone was essentially inactive. Itk(Delta152) had K(m) values for ATP and SAM68 nearly identical to those of the wild-type enzyme, while Itk(P158A,P159A) had approximately 3-fold higher K(m) values for each substrate. SAM68 phosphorylation by the wild-type and mutant enzymes in the presence of several tyrosine kinase inhibitors were compared using a homogeneous time-resolved fluorescence assay. Both the Itk(Delta152) deletion mutant and the Itk(P158A,P159A) mutant had IC(50) values similar to those of the wild-type enzyme for staurosporine, PP1, and damnacanthal. These comparisons, taken together with the similar K(m) values for ATP and SAM68 substrate between the wild-type and the mutant enzymes, indicate that the amino acids in the N-terminal 152 residues and proline-rich domains enhance catalysis by affecting turnover rate rather than substrate binding.     

A specific intermolecular association between the regulatory domains of a Tec family kinase

Interleukin-2 tyrosine kinase (Itk), is a T-cell specific tyrosine kinase of the Tec family. We have examined a novel intermolecular interaction between the SH3 and SH2 domains of Itk. In addition to the interaction between the isolated domains, we have found that the dual SH3/SH2 domain-containing fragment of Itk self-associates in a specific manner in solution. Tec family members contain the SH3, SH2 and catalytic domains common to many kinase families but are distinguished by a unique amino-terminal sequence, which contains a proline-rich stretch. Previous work has identified an intramolecular regulatory association between the proline-rich region and the adjacent SH3 domain of Itk. The intermolecular interaction between the SH3 and SH2 domains of Itk that we describe provides a possible mechanism for displacement of this intramolecular regulatory sequence, a step that may be required for full Tec kinase activation. Additionally, localization of the interacting surfaces on both the SH3 and SH2 domains by chemical shift mapping has provided information about the molecular details of this recognition event. The interaction involves the conserved aromatic binding pocket of the SH3 domain and a newly defined binding surface on the SH2 domain. The interacting residues on the SH2 domain do not conform to the consensus motif for an SH3 proline-rich ligand. Interestingly, we note a striking correlation between the SH2 residues that mediate this interaction and those residues that, when mutated in the Tec family member Btk, cause the hereditary immune disorder, X-linked agamaglobulinemia.     

A remote substrate docking mechanism for the tec family tyrosine kinases

During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain.     

Mechanism-based design of a protein kinase inhibitor

Protein kinase inhibitors have applications as anticancer therapeutic agents and biological tools in cell signaling. Based on a phosphoryl transfer mechanism involving a dissociative transition state, a potent and selective bisubstrate inhibitor for the insulin receptor tyrosine kinase was synthesized by linking ATPgammaS to a peptide substrate analog via a two-carbon spacer. The compound was a high affinity competitive inhibitor against both nucleotide and peptide substrates and showed a slow off-rate. A crystal structure of this inhibitor bound to the tyrosine kinase domain of the insulin receptor confirmed the key design features inspired by a dissociative transition state, and revealed that the linker takes part in the octahedral coordination of an active site Mg2+. These studies suggest a general strategy for the development of selective protein kinase inhibitors.     

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both T_H1 and T_H2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.     

Differential regulation of human NK cell-mediated cytotoxicity by the tyrosine kinase Itk

NK cells are effector lymphocytes that can recognize and eliminate virally infected and transformed cells. NK cells express distinct activating receptors, including an ITAM-containing FcR complex that recognizes Ab-coated targets, and the DNAX-activating protein of 10 kDa-containing NKG2D receptor complex that recognizes stress-induced ligands. The regulatory role of specific tyrosine kinases in these pathways is incompletely understood. In this study, we show that, in activated human NK cells, the tyrosine kinase IL-2-inducible T cell kinase (Itk), differentially regulates distinct NK-activating receptors. Enhanced expression of Itk leads to increases in calcium mobilization, granule release, and cytotoxicity upon stimulation of the ITAM-containing FcR, suggesting that Itk positively regulates FcR-initiated cytotoxicity. In contrast, enhanced Itk expression decreases cytotoxicity and granule release downstream of the DNAX-activating protein of 10 kDa-containing NKG2D receptor, suggesting that Itk is involved in a pathway of negative regulation of NKG2D-initiated granule-mediated killing. Using a kinase mutant, we show that the catalytic activity of Itk is required for both the positive and negative regulation of these pathways. Complementary experiments where Itk expression was suppressed also showed differential regulation of the two pathways. These findings suggest that Itk plays a complex role in regulating the functions initiated by distinct NK cell-activating receptors. Moreover, understanding how these pathways may be differentially regulated has relevance in the setting of autoimmune diseases and antitumor immune responses where NK cells play key regulatory roles.     

Inducible T cell tyrosine kinase regulates actin-dependent cytoskeletal events induced by the T cell antigen receptor

The tec family kinase, inducible T cell tyrosine kinase (Itk), is critical for both development and activation of T lymphocytes. We have found that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events. Expression of Src homology (SH) 2 domain mutant Itk transgenes into Jurkat T cells inhibits these events. Furthermore, Itk(-/-) murine T cells display significant defects in TCR/CD3-induced actin polymerization. In addition, Jurkat cells deficient in linker for activation of T cells expression, an adaptor critical for Itk activation, display impaired cytoskeletal events and expression of SH3 mutant Itk transgenes reconstitutes this impairment. Interestingly, expression of an Itk kinase-dead mutant transgene into Jurkat cells has no effect on cytoskeletal events. Collectively, these data suggest that Itk regulates TCR/CD3-induced actin-dependent cytoskeletal events, possibly in a kinase-independent fashion.