Interleukin-2 Tyrosine Kinase Research Articles

Itk tyrosine kinase substrate docking is mediated by a nonclassical SH2 domain surface of PLCγ1

Interleukin-2 tyrosine kinase (Itk) is a Tec family tyrosine kinase that mediates signaling processes after T cell receptor engagement. Activation of Itk requires recruitment to the membrane via its pleckstrin homology domain, phosphorylation of Itk by the Src kinase, Lck, and binding of Itk to the SLP-76/LAT adapter complex. After activation, Itk phosphorylates and activates phospholipase C-gamma1 (PLC-gamma1), leading to production of two second messengers, DAG and IP(3). We have previously shown that phosphorylation of PLC-gamma1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-gamma1 and the kinase domain of Itk. We now define this docking interface using a combination of mutagenesis and NMR spectroscopy and show that disruption of the Itk/PLCgamma1 docking interaction attenuates T cell signaling. The binding surface on PLCgamma1 that mediates recognition by Itk highlights a nonclassical binding activity of the well-studied SH2 domain providing further evidence that SH2 domains participate in important signaling interactions beyond recognition of phosphotyrosine.

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.

Solution Structure of the binary complex between the SH3 and SH2 domain of interleukin-2 tyrosine kinase

Solution Structure of the binary complex between the SH3 and SH2 domain of interleukin-2 tyrosine kinase

Proline Isomerization Preorganizes the Itk SH2 Domain for Binding to the Itk SH3 Domain

We report here the NMR-derived structure of the binary complex formed by the interleukin-2 tyrosine kinase (Itk) Src homology 3 (SH3) and Src homology 2 (SH2) domains. The interaction is independent of both a phosphotyrosine motif and a proline-rich sequence, the classical targets of the SH2 and SH3 domains, respectively. The Itk SH3/SH2 structure reveals the molecular details of this nonclassical interaction and provides a clear picture for how the previously described prolyl cis/ trans isomerization present in the Itk SH2 domain mediates SH3 binding. The higher-affinity cis SH2 conformer is preorganized to form a hydrophobic interface with the SH3 domain. The structure also provides insight into how autophosphorylation in the Itk SH3 domain might increase the affinity of the intermolecular SH3/SH2 interaction. Finally, we can compare this Itk complex with other examples of SH3 and SH2 domains engaging their ligands in a nonclassical manner. These small binding domains exhibit a surprising level of diversity in their binding repertoires.

Bacterial expression and purification of Interleukin-2 Tyrosine kinase: Single step separation of the chaperonin impurity

Biochemical and biophysical characterization of kinases requires large quantities of purified protein. Here, we report the bacterial expression and purification of active Itk kinase domain (a Tec family kinase) using ArcticExpress cells that co-express the chaperonin system Cpn60/10 from Oleispira antarctica. We describe a simple one step MgCl 2/ATP/KCl incubation procedure to remove the co-purifying chaperonin impurity. Chaperonin co-purification is a common problem encountered during protein purification and the simple incubation step described here completely overcomes this problem. The approach targets the chaperonin system rather than the protein of interest and is therefore widely applicable to other protein targets.

Murine Itk SH3 domain

Interleukin-2 tyrosine kinase (Itk) is a non-receptor tyrosine kinase of the Tec family that is activated upon antigen binding to the T cell receptor (Schwartzberg et al. 2005; Berg 2007). Itk is comprised of four regulatory domains: PH (Pleckstrin homology), TH (Tec homology), SH3, SH2 and the catalytic kinase domain. SH3 domains share a common fold consisting of five anti-parallel β strands that form a β barrel and bind canonical proline rich ligands as well as a variety of noncanonical ligands (Agrawal and Kishan 2002).

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.

Molecular Details of Itk Activation by Prolyl Isomerization and Phospholigand Binding: The NMR Structure of the Itk SH2 Domain Bound to a Phosphopeptide

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a critical component of the regulatory apparatus controlling the activity of this immunologically important enzyme. To gain insight into the structural features associated with the activated form of Itk, we have solved the NMR structure of the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY binding. Isomerization of a single prolyl imide bond in this domain is responsible for simultaneous existence of two distinct SH2 conformers. Prolyl isomerization directs ligand recognition: the trans conformer preferentially binds pY. The structure of the SH2/pY complex provides insight into the ligand specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC') couplings arising from hydrogen bonds has revealed propagation of structural changes from the pY binding pocket to the CD loop containing conformationally heterogeneous proline as well as to the alphaB helix, on the opposite site of the domain. These findings offer a structural framework for understanding the roles of prolyl isomerization and pY binding in Itk regulation.

Crystal Structures of Interleukin-2 Tyrosine Kinase and Their Implications for the Design of Selective Inhibitors

Interleukin-2 tyrosine kinase, Itk, is an important member of the Tec family of non-receptor tyrosine kinases that play a central role in signaling through antigen receptors such as the T-cell receptor, B-cell receptor, and Fcepsilon. Selective inhibition of Itk may be an important way of modulating many diseases involving heightened or inappropriate activation of the immune system. In addition to an unliganded nonphophorylated Itk catalytic kinase domain, we determined the crystal structures of the phosphorylated and nonphosphorylated kinase domain bound to staurosporine, a potent broad-spectrum kinase inhibitor. These structures are useful for the design of novel, highly potent and selective Itk inhibitors and provide insight into the influence of inhibitor binding and phosphorylation on the conformation of Itk.

Cyclophilin A is required for retinoic acid-induced neuronal differentiation in p19 cells.

Stable transfectants with expression of small interfering RNA for targeting cyclophilin A (CypA) in p19 cells lose their potential for retinoic acid (RA)-induced neuronal differentiation but not Me(2)SO-induced mesodermal differentiation. This difference suggests that CypA is specifically required for the RA-induced neuronal pathway. In addition to the loss of RA-induced RA receptor beta expression and retinoic acid response element (RARE)-binding activity, a dramatic reduction in RA-induced RARE-mediated luciferase activity in the CypA knockdown cell line suggests that CypA affects RARE-mediated regulation of gene expression. Silent mutation of target sequences confirms the specificity of RNA interference in p19 embryonal carcinoma cells. Collectively, our data reveal that a novel function of CypA is required in the processing of RA-induced neuronal differentiation in p19 embryonal carcinoma cells.

Ligand specificity modulated by prolyl imide bond Cis/Trans isomerization in the Itk SH2 domain: a quantitative NMR study.

The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) binds two separate ligands: a phosphotyrosine-containing peptide and the Itk Src homology 3 (SH3) domain. Binding specificity for these ligands is regulated via cis/trans isomerization of the Asn 286-Pro 287 imide bond in the Itk SH2 domain. In this study, we develop a novel method of analyzing chemical shift perturbation and cross-peak volumes to measure the affinities of both ligands for each SH2 conformer. We find that the cis imide bond containing SH2 conformer exhibits a 3.5-fold higher affinity for the Itk SH3 domain compared with binding of the trans conformer to the same ligand, while the trans conformer binds phosphopeptide with a 4-fold greater affinity than the cis-containing SH2 conformer. In addition to furthering the understanding of this system, the method presented here will be of general application in quantitatively determining the specificities of conformationally heterogeneous systems that use a molecular switch to regulate binding between multiple distinct ligands.

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.

Proline Isomerization Regulates ITK

Interleukin-2 tyrosine kinase (ITK) is a T cell-specific member of the Tec family of nonreceptor tyrosine kinases. Brazin et al . determined by nuclear magnetic resonance (NMR) studies that the ITK SH2 domain existed in two conformations reflecting the protein with Pro 287 bond in the cis or trans configuration. The SH2 domain of ITK can bind in a phosphotyrosine-dependent manner (substrate binding) or can interact in a phosphotyrosine-independent manner with the SH3 domain of another ITK molecule. Chemical shift perturbations in the presence of the ligands showed that the cis conformation was required for the phosphotyrosine-independent binding to ITK SH3 domains, and the trans conformation was required for phosphopeptide binding. The ITK SH2 domain interacted specifically with the peptidyl-prolyl cis/trans isomerase cyclophilin A (CypA), as measured by NMR. In vitro and in vivo CypA interacted with and inhibited ITK kinase activity. The authors propose that CypA is an endogenous regulator of ITK activity, and the ITK ligand recognition is governed by the conformation of the Asn 286 -Pro 287 bond. K. N. Brazin, R. J. Mallis, D. B. Fulton, A. H. Andreotti, Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A. Proc. Natl. Acad. Sci. U.S.A. 99 , 1899-1904 (2002). [Abstract] [Full Text]

Proline Isomerization Regulates ITK

Interleukin-2 tyrosine kinase (ITK) is a T cell-specific member of the Tec family of nonreceptor tyrosine kinases. Brazin et al. determined by nuclear magnetic resonance (NMR) studies that the ITK SH2 domain existed in two conformations reflecting the protein with Pro287 bond in the cis or trans configuration. The SH2 domain of ITK can bind in a phosphotyrosine-dependent manner (substrate binding) or can interact in a phosphotyrosine-independent manner with the SH3 domain of another ITK molecule. Chemical shift perturbations in the presence of the ligands showed that the cis conformation was required for the phosphotyrosine-independent binding to ITK SH3 domains, and the trans conformation was required for phosphopeptide binding. The ITK SH2 domain interacted specifically with the peptidyl-prolyl cis/trans isomerase cyclophilin A (CypA), as measured by NMR. In vitro and in vivo CypA interacted with and inhibited ITK kinase activity. The authors propose that CypA is an endogenous regulator of ITK activity, and the ITK ligand recognition is governed by the conformation of the Asn286-Pro287 bond.K. N. Brazin, R. J. Mallis, D. B. Fulton, A. H. Andreotti, Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A. Proc. Natl. Acad. Sci. U.S.A. 99, 1899-1904 (2002).[Abstract][Full Text]

Regulation of the tyrosine kinase Itk by the peptidyl-prolyl isomerase cyclophilin A.

Interleukin-2 tyrosine kinase (Itk) is a nonreceptor protein tyrosine kinase of the Tec family that participates in the intracellular signaling events leading to T cell activation. Tec family members contain the conserved SH3, SH2, and catalytic domains common to many kinase families, but they are distinguished by unique sequences outside of this region. The mechanism by which Itk and related Tec kinases are regulated is not well understood. Our studies indicate that Itk catalytic activity is inhibited by the peptidyl prolyl isomerase activity of cyclophilin A (CypA). NMR structural studies combined with mutational analysis show that a proline-dependent conformational switch within the Itk SH2 domain regulates substrate recognition and mediates regulatory interactions with the active site of CypA. CypA and Itk form a stable complex in Jurkat T cells that is disrupted by treatment with cyclosporin A. Moreover, the phosphorylation levels of Itk and a downstream substrate of Itk, PLCgamma1, are increased in Jurkat T cells that have been treated with cyclosporin A. These findings support a novel mode of tyrosine kinase regulation for a Tec family member and provide a molecular basis for understanding a cellular function of the ubiquitous peptidyl prolyl isomerase, CypA.

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.