Tec Homology Domain Research Articles

The zinc-binding region of IL-2 inducible T cell kinase (Itk) is required for interaction with Gα13 and activation of serum response factor

Tec family kinases play critical roles in the activation of immune cells. In particular, Itk is important for the activation of T cells via the T cell Receptor (TcR), however, molecules that cooperate with Itk to activate downstream targets remain little explored. Here we show that Itk interacts with the heterotrimeric G-protein α subunit Gα13 during TcR triggering. This interaction requires membrane localization of both partners, and is partially dependent on GDP- and GTP-bound states of Gα13. Furthermore, we find that Itk interacts with Gα13 via the zinc binding regions within its Tec homology domain. The interaction between Itk and Gα13 also results in tyrosine phosphorylation of Gα13, however this is not required for the interaction. Itk enhances Gα13 mediated activation of serum response factor (SRF) transcriptional activity dependent on its ability to interact with Gα13, but its kinase activity is not required to enhance SRF activity. These data reveal a new pathway regulated by Itk in cells, and suggest cross talk between Itk and G-protein signaling downstream of the TcR.

The Tec Family Kinase Itk Exists as a Folded Monomer in Vivo

Tec family tyrosine kinases transduce signals from antigen and other receptors. In particular, Itk plays an important role in T-cell development and activation. Itk has an N-terminal pleckstrin homology domain, a Tec Homology domain with a proline-rich region, SH3 and SH2 domains and a kinase domain, the structure each of which has been determined. However, the full structure of Itk and other Tec kinases remain elusive. Models of Itk suggest either a head to tail dimer, with the SH2 domain interacting with the SH3 domain, or a folded monomer with the SH3 domain interacting with the proline-rich region. We show here that in vivo Itk exists as a monomer, with the pleckstrin homology domain less than 80 A from the C terminus. Zn2+ coordinating residues in the Tec Homology domain, not the proline-rich region, are critical for this intramolecular interaction. These data have implications for our understanding of Tec family kinase structure.

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.

Etk/Bmx Transactivates Vascular Endothelial Growth Factor 2 and Recruits Phosphatidylinositol 3-Kinase to Mediate the Tumor Necrosis Factor-induced Angiogenic Pathway

Tumor necrosis factor (TNF), via its receptor 2 (TNFR2), induces Etk (or Bmx) activation and Etk-dependent endothelial cell (EC) migration and tube formation. Because TNF receptor 2 lacks an intrinsic kinase activity, we examined the kinase(s) mediating TNF-induced Etk activation. TNF induces a coordinated phosphorylation of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and Etk, which is blocked by VEGFR2-specific inhibitors. In response to TNF, Etk and VEGFR2 form a complex resulting in a reciprocal activation between the two kinases. Subsequently, the downstream phosphatidylinositol 3-kinase (PI3K)-Akt signaling (but not signaling through phospholipase C-gamma) was initiated and directly led to TNF-induced EC migration, which was significantly inhibited by VEGFR2-, PI3K-, or Akt-specific inhibitors. Phosphorylation of VEGFR2 at Tyr-801 and Tyr-1175, the critical sites for VEGF-induced PI3K-Akt signaling, was not involved in TNF-mediated Akt activation. However, TNF induces phosphorylation of Etk at Tyr-566, directly mediating the recruitment of the p85 subunit of PI3K. Furthermore, TNF- but not VEGF-induced activation of VEGFR2, Akt, and EC migration are blunted in EC genetically deficient with Etk. Taken together, our data demonstrated that TNF induces transactivation between Etk and VEGFR2, and Etk directly activates PI3K-Akt angiogenic signaling independent of VEGF-induced VEGFR2-PI3K-Akt signaling pathway.

Etk/Bmx as a tumor necrosis factor receptor type 2-specific kinase: role in endothelial cell migration and angiogenesis.

Tumor necrosis factor (TNF) is a cytokine that mediates many pathophysiologial processes, including angiogenesis. However, the molecular signaling involved in TNF-induced angiogenesis has not been determined. In this study, we examined the role of Etk/Bmx, an endothelial/epithelial tyrosine kinase involved in cell adhesion, migration, and survival in TNF-induced angiogenesis. We show that TNF activates Etk specifically through TNF receptor type 2 (TNFR2) as demonstrated by studies using a specific agonist to TNFR2 and TNFR2-deficient cells. Etk forms a preexisting complex with TNFR2 in a ligand-independent manner, and the association is through multiple domains (pleckstrin homology domain, TEC homology domain, and SH2 domain) of Etk and the C-terminal domain of TNFR2. The C-terminal 16-amino-acid residues of TNFR2 are critical for Etk association and activation, and this Etk-binding and activating motif in TNFR2 is not overlapped with the TNFR-associated factor type 2 (TRAF2)-binding sequence. Thus, TRAF2 is not involved in TNF-induced Etk activation, suggesting a novel mechanism for Etk activation by cytokine receptors. Moreover, a constitutively active form of Etk enhanced, whereas a dominant-negative Etk blocked, TNF-induced endothelial cell migration and tube formation. While most TNF actions have been attributed to TNFR1, our studies demonstrate that Etk is a TNFR2-specific kinase involved in TNF-induced angiogenic events.

The proline rich region of the Tec homology domain of ITK regulates its activity

Inducible T-cell kinase (ITK) is a member of the Tec family of tyrosine kinases that are involved in signals emanating from cytokine receptors, antigen receptors and other lymphoid cell surface receptors. Stimulation of tyrosine phosphorylation and activation of ITK by the T-cell antigen receptor, CD28 and CD2 requires the presence of the Src family kinase Lck in T-cells. We have previously demonstrated that the activation of ITK by Src family kinases uses a phosphatidylinositol 3-kinase pathway, which recruits ITK to the membrane via its pleckstrin homology (PH) domain where it is acted upon by Src. We have further explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that deletion of the proline rich sequence found in the Tec homology domain of ITK results in reduced basal activity of ITK approximately 50%. These differences in the basal activity of ITK were observed when the PH domain was deleted or when the kinase was membrane targeted. Furthermore, this deletion reduces the ability of the Src family kinase Lck to activate ITK, as well as to induce the ITK mediated tyrosine phosphorylation of its substrate PLCγ1. By contrast, deletion of the SH3 domain of ITK resulted in a two-fold increase in the basal activity of ITK, and allowed this mutant to have an enhanced response to Lck. These results suggest that the proline rich region within the Tec homology domain of ITK regulates its basal activity and its response to Src family kinase signals.

Interaction between Btk TH and SH3 domain

Several mechanisms are involved in the regulation of cellular signaling. Bruton tyrosine kinase (Btk) of the Tec family contains in the Tec homology (TH) domain a proline-rich region (PRR) capable of interacting with several SH3 domains. The Btk has the SH3 domain adjacent to the TH domain. CD and fluorescence spectroscopy were used to study the binding of two peptides corresponding to segments in the PRR to the Btk SH3 domain. The peptide for the N-terminal half of the PRR binds specifically, whereas the other peptide had hardly any affinity. The TH domain has about four times lower affinity to the SH3 domain than the peptide, 17.0 vs 3.9 microM. The interaction was further tested with an SH3 domain construct that contained the PRR. The two peptides cannot compete for the binding to the extended protein and the TH domain has two times lower affinity to the extended SH3 domain. The intra- or intermolecular interaction between the TH and SH3 domain might have regulatory function also in the other Tec family members.

PKCbeta modulates antigen receptor signaling via regulation of Btk membrane localization.

Mutations in Bruton's tyrosine kinase (Btk) result in X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. While targeted disruption of the protein kinase C-beta (PKCbeta) gene in mice results in an immunodeficiency similar to xid, the overall tyrosine phosphorylation of Btk is significantly enhanced in PKCbeta-deficient B cells. We provide direct evidence that PKCbeta acts as a feedback loop inhibitor of Btk activation. Inhibition of PKCbeta results in a dramatic increase in B-cell receptor (BCR)-mediated Ca2+ signaling. We identified a highly conserved PKCbeta serine phosphorylation site in a short linker within the Tec homology domain of Btk. Mutation of this phosphorylation site led to enhanced tyrosine phosphorylation and membrane association of Btk, and augmented BCR and FcepsilonRI-mediated signaling in B and mast cells, respectively. These findings provide a novel mechanism whereby reversible translocation of Btk/Tec kinases regulates the threshold for immunoreceptor signaling and thereby modulates lymphocyte activation.

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.

Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase

Bruton's tyrosine kinase (Btk), a nonreceptor cytoplasmic tyrosine kinase belonging to the Tec family of kinases, has been shown to be critical for B cell proliferation, differentiation, and signaling. Loss-of-function mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA), a primary immunodeficiency in humans, and the less severe condition xid in mice. Although Btk is mainly localized in the cytoplasm under steady state conditions, it translocates to the plasma membrane upon growth factor stimulation and cross-linking of the B cell receptor. Nevertheless, in ectopically as well as endogenously Btk-expressing cells, it can also translocate to the nucleus. Deletion of the pleckstrin homology (PH) domain (DeltaPH1) leads, however, to an even redistribution of Btk within the nucleus and cytoplasm in the majority of transfected cells. In contrast, an SH3-deleted (DeltaSH3) mutant of Btk has been found to be predominantly nuclear. We also demonstrate that the nuclear accumulation of DeltaPH1 is dependent on Src expression. This nucleocytoplasmic shuttling is sensitive to the exportin 1/CRM1-inactivating drug, leptomycin B, indicating that Btk utilizes functional nuclear export signals. In addition, while the DeltaPH1 mutant of Btk was found to be active and tyrosine-phosphorylated in vivo, DeltaSH3 displayed decreased autokinase activity and was not phosphorylated. Our findings indicate that the nucleocytoplasmic shuttling of Btk has implications regarding potential targets inside the nucleus, which may be critical in gene regulation during B cell development and differentiation.

Mutation Screening of the BTK Gene in 56 Families With X-Linked Agammaglobulinemia (XLA): 47 Unique Mutations Without Correlation to Clinical Course

To determine the utility of single-stranded conformation polymorphism (SSCP) analysis for mutation screening in the BTK (Bruton's tyrosine kinase) gene, we investigated 56 X-linked agammaglobulinemia (XLA) families. To obtain genotype/ phenotype correlations, predicted protein aberrations were correlated with the clinical course of the disease. This study included 56 patients with XLA, with or without a positive family history, who were diagnosed on the basis of their clinical features, low peripheral B-cell count, and low immunoglobulin levels. Ten patients with isolated hypogammaglobulinemia and 50 healthy males served as controls. SSCP analysis was performed for the entire BTK gene, including the exon-intron boundaries and the promoter region. Structural implications of the missense mutations were investigated by molecular modeling, and the functional consequences of some mutations also were evaluated by in vitro kinase assays and Western blot analysis. We report the largest series of patients with XLA to date. All but 5 of the 56 index patients with XLA screened with SSCP analysis showed BTK gene abnormalities, and in 2 of the 5 SSCP-negative patients, no BTK protein was found by Western blot analysis. There were 51 mutations, including 37 novel ones, distributed across the entire gene. This report contains the first promoter mutation as well as 14 novel missense mutations with the first ones described for the Tec homology domain and the glycine-rich motif in the SH1 domain. Each index patient had a different mutation, except for four mutations, each in two unrelated individuals. This result supports the strong tendency for private mutations in this disease. No mutations were found in the controls. Our results demonstrate that molecular genetic testing by SSCP analysis provides an accurate tool for the definitive diagnosis of XLA and the discrimination of borderline cases, such as certain hypogammaglobulinemia or common variable immunodeficiency patients with overlapping clinical features. Genotype/ phenotype correlations are not currently possible, making prediction of the clinical course based on molecular genetic data infeasible.

Missense mutations affecting a conserved cysteine pair in the TH domain of Btk

Tec family protein tyrosine kinases have in their N-terminus two domains. The PH domain is followed by Tec homology (TH) domain, which consists of two motifs. The first pattern, Btk motif, is also present in some Ras GAP molecules. C-terminal half of the TH domain, a proline-rich region, has been shown to bind to SH3 domains. Mutations in Bruton's tyrosine kinase (Btk) belonging to the Tec family cause X-linked agammaglobulinemia (XLA) due to developmental arrest of B cells. Here we present the first missense mutations in the TH domain. The substitutions affect a conserved pair of cysteines, residues 154 and 155, involved in Zn 2+ binding and thereby the mutations alter protein folding and stability.

Tec protein-tyrosine kinase is an effector molecule of Lyn protein-tyrosine kinase.

The Tec family is a recently emerging subfamily among nonreceptor type protein-tyrosine kinases (PTKs) consisting of Tec, Txk, Btk, Bmx, and Itk/Tsk/Emt. They have a long amino-terminal unique region containing a pleckstrin homology domain and a Tec-homology domain. We could previously show that, through the Tec-homology domain, Tec is bound to Lyn kinase both in vitro and in vivo. Because Tec is coexpressed with Lyn in many hematopoietic cell types, it has been intriguing to investigate the biological role of the Tec-Lyn association. Here we demonstrate that Lyn can phosphorylate tyrosine residues of the Tec protein, and thereby activate Tec in 3T3 fibroblasts. However, coexpression of Tec has little effect on the phospho-tyrosine-contents of Lyn. By using the in vitro kinase assay and the yeast system, we could prove that the Tec protein is a direct substrate of the Lyn kinase both in vitro and in vivo. From this evidence we conclude that Tec acts downstream of Lyn in intracellular signaling pathways. This is a novel case where one PTK is phosphorylated and regulated by another.

An SH3-binding Site Conserved in Bruton's Tyrosine Kinase and Related Tyrosine Kinases Mediates Specific Protein Interactions in Vitro and in Vivo

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

Tec homology (TH) adjacent to the PH domain

The pleckstrin homology (PH) domain is extended in the Btk kinase family by a region designated the TH (Tec homology) domain, which consists of about 80 residues preceding the SH3 domain. The TH domain contains a conserved 27 amino acid stretch designated the Btk motif and a proline-rich region. Sequence similarity was found to a putative Ras GTPase activating protein and a human interferon-γ binding protein both in the PH domain and the Btk motif region. SLK1/SSP31 protein kinase and a non-catalytic p85 subunit of PI-3 kinase had similarity only with the proline rich region. The identification of a PH domain extension in some signal transduction proteins in different species suggests that this region is involved in protein—protein interactions.