Raji E. Joseph

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-gamma. This review highlights recent advances in our understanding of Itk kinase structure and enzymatic regulation, focusing on Itk protein domain interactions and mechanisms of substrate recognition. We also discuss the role of Itk in the development of conventional versus innate T-cell lineages, including both alphabeta and gammadelta T-cell subsets. Finally, we describe the complex role of Itk signaling in effector T-cell differentiation and the regulation of cytokine gene expression. Together, these data implicate Itk as an important modulator of T-cell signaling and function.

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 MgCl2/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.

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.

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-γ1 (PLC-γ1), leading to production of two second messengers, DAG and IP3. We have previously shown that phosphorylation of PLC-γ1 by Itk requires a direct, phosphotyrosine-independent interaction between the Src homology 2 (SH2) domain of PLC-γ1 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/PLCγ1 docking interaction attenuates T cell signaling. The binding surface on PLCγ1 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.

Conformational snapshots of Tec kinases during signaling

Summary:  The control of cellular signaling cascades is of utmost importance in regulating the immune response. Exquisitely precise protein–protein interactions and chemical modification of substrates by enzymatic catalysis are the fundamental components of the signals that alert immune cells to the presence of a foreign antigen. In particular, the phosphorylation events induced by protein kinase activity must be spatially and temporally regulated by specific interactions to maintain a normal and effective immune response. High resolution structures of many protein kinases along with supporting biochemical data are providing significant insight into the intricate regulatory mechanisms responsible for controlling cellular signaling. The Tec family kinases are immunologically important kinases for which regulatory details are beginning to emerge. This review focuses on bringing together structural insights gained over the years to develop an understanding of how domain interactions both within the Tec kinases and between the Tec kinases and other signaling molecules control immune cell function.

Activation loop dynamics determine the different catalytic efficiencies of B cell- and T cell-specific tec kinases.

Just six amino acid residues prevent the T cell kinase Itk from being as active as its B cell counterpart, Btk. Comparing Tec Kinase Dynamics T cell receptor (TCR) signals are mediated in part by the nonreceptor tyrosine kinase Itk, a member of the Tec family of kinases, whereas B cell receptor–dependent signaling involves another Tec kinase, Btk. Although both kinases have similar amino acid sequences, particularly in their kinase domains, Btk is more active than Itk. In a structure-function analysis, Joseph et al. showed that substitution of just six amino acid residues in Itk with the corresponding residues of Btk was sufficient to enhance the activity of Itk to a similar degree to that of Btk. Itk-deficient mouse T cells reconstituted with this mutant Itk exhibited excessive TCR-dependent signaling, which led the authors to suggest that the decreased kinase activity of Itk acts as a check to prevent inappropriate T cell activation. Itk (interleukin-2–inducible T cell kinase) and Btk (Bruton’s tyrosine kinase) are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of 6 of the 619 amino acid residues of Itk with the corresponding residues of Btk (and vice versa) was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties, depending on the cellular context. We suggest that the weaker catalytic activities of T cell–specific kinases serve to regulate cellular activation and prevent aberrant immune responses.

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 betaD strand. These results are extended into Btk (Brutons 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.

Disrupting the Intermolecular Self-Association of Itk Enhances T Cell Signaling

The Tec family tyrosine kinase (Itk), is a key component of the TCR signaling pathway. Biochemical studies have shown that Itk activation requires recruitment of Itk 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. However, the regulation of Itk enzymatic activity by Itk domain interactions is not yet well understood. In this study, we show that full-length Itk self-associates in an intermolecular fashion. Using this information, we have designed an Itk variant that exhibits reduced self-association but maintains normal binding to exogenous ligands via each of its regulatory domains. When expressed in insect cells, the Itk substrate phospholipase Cγ1 is phosphorylated more efficiently by the Itk variant than by wild-type Itk. Furthermore, expression of the Itk variant in primary murine T cells induced higher ERK activation and increased calcium flux following TCR stimulation compared with that of wild-type Itk. Our results indicate that the Tec kinase Itk is negatively regulated by intermolecular clustering and that disruption of this clustering leads to increased Itk kinase activity following TCR stimulation.

Substrate Specificity and Recognition Is Conferred by the Pleckstrin Homology Domain of the Dbl Family Guanine Nucleotide Exchange Factor P-Rex2

Dbl family guanine nucleotide exchange factors (GEFs) are characterized by the presence of a catalytic Dbl homology domain followed invariably by a lipid-binding pleckstrin homology (PH) domain. To date, substrate recognition and specificity of this family of GEFs has been reported to be mediated exclusively via the Dbl homology domain. Here we report the novel and unexpected finding that, in the Dbl family Rac-specific GEF P-Rex2, it is the PH domain that confers substrate specificity and recognition. Moreover, the β3β4 loop of the PH domain of P-Rex2 is the determinant for Rac1 recognition, as substitution of the β3β4 loop of the PH domain of Dbs (a RhoA- and Cdc42-specific GEF) with that of P-Rex2 confers Rac1-specific binding capability to the PH domain of Dbs. The contact interface between the PH domain of P-Rex2 and Rac1 involves the switch loop and helix 3 of Rac1. Moreover, substitution of helix 3 of Cdc42 with that of Rac1 now enables the PH domain of P-Rex2 to bind this Cdc42 chimera. Despite having the ability to recognize this chimeric Cdc42, P-Rex2 is unable to catalyze nucleotide exchange on Cdc42, suggesting that recognition of substrate and catalysis are two distinct events. Thus substrate recognition can now be added to the growing list of functions that are being attributed to the PH domain of Dbl family GEFs.

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.