TEC-family kinases: regulators of T-helper-cell differentiation.

The TEC-family protein tyrosine kinases ITK, RLK and TEC have been identified as key components of T-cell-receptor signalling that contribute to the regulation of phospholipase C-gamma, the mobilization of Ca(2+) and the activation of mitogen-activated protein kinases. Recent data also show that TEC kinases contribute to T-cell-receptor-driven actin reorganization and cell polarization, which are required for productive T-cell activation. Functional studies have implicated TEC kinases as important mediators of pathways that control the differentiation of CD4(+) T helper cells. Here, we review studies of signalling pathways that involve TEC kinases and how these pathways might contribute to the regulation of T-helper-cell differentiation and function.

Tec kinases: shaping T-cell activation through actin.

Following stimulation, T cells undergo marked actin-dependent changes in shape that are required for productive cellular interactions and movement during immune responses. Reorganization of the actin cytoskeletal is also necessary for the formation of an immunological synapse - the convergence of several signaling molecules at the plasma membrane that occurs after effective T-cell receptor (TCR) signaling. Much emerging evidence indicates that the Tec family of tyrosine kinases has a role in actin cytoskeleton reorganization. Specifically, T cells that lack or express mutant versions of the Tec kinase Itk show impaired TCR-induced actin polymerization, cell polarization and regulation of the signaling events involved in cytoskeletal reorganization. These data, as well as other findings, support roles for Tec kinases in actin cytoskeleton regulation.

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP.

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.

Sf-Stk kinase activity and the Grb2 binding site are required for Epo-independent growth of primary erythroblasts infected with Friend virus.

During the initial stage of Friend virus-induced erythroleukemia in mice, interaction of the viral protein gp55 with the erythropoietin receptor, and other host factors, drives the expansion of erythroid precursor cells. Recently, we demonstrated that the Friend virus susceptibility locus, Fv2, which is required for the expansion of infected cells, encodes a naturally occurring, N-terminally truncated form of the Stk receptor tyrosine kinase (Sf-Stk). Here we show that in vitro expression of Sf-Stk confers Friend virus sensitivity to erythroid progenitor cells from Fv2(rr) mice. Furthermore, our data reveal that Sf-Stk kinase activity and Y436, but not Y429, are required for Epo-independent colony formation following Friend virus infection. Introduction of a mutation that results in failure to bind Grb2 abrogates the ability of Sf-Stk to induce colonies in response to Friend virus, while the Grb2 binding site from EGFR restores this response. Consistent with the ability of Grb2 to recruit SOS and Gab1, the Ras/MAPK and PI3K pathways are activated by Sf-Stk, and both of these pathways are required for gp55-mediated erythroblast proliferation. These data clearly demonstrate a requirement for signaling through Sf-Stk in the Epo-independent expansion of Friend virus-infected cells, and suggest a pivotal role for Grb2 in this response.

Functional hierarchy of the N-terminal tyrosines of SLP-76.

The adaptor protein Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) plays a central role in T cell activation and T cell development. SLP-76 has three functional modules: an acidic domain with three key tyrosines, a central proline-rich domain, and a C-terminal Src homology 2 domain. Of these, mutation of the three N-terminal tyrosines (Y112, Y128, and Y145) results in the most profound effects on T cell development and function. Y112 and Y128 associate with Vav and Nck, two proteins shown to be important for TCR-induced phosphorylation of proximal signaling substrates, Ca(2+) flux, and actin reorganization. Y145 has been shown to be important for optimal association of SLP-76 with inducible tyrosine kinase, a key regulator of T cell function. To investigate further the role of the phosphorylatable tyrosines of SLP-76 in TCR signaling, cell lines and primary T cells expressing SLP-76 with mutations in individual or paired tyrosine residues were analyzed. These studies show that Tyr(145) of SLP-76 is the most critical tyrosine for both T cell function in vitro and T cell development in vivo.

Tec kinases regulate TCR-mediated recruitment of signaling molecules and integrin-dependent cell adhesion.

T cells deficient in the Tec kinases Itk or Itk and Rlk exhibit defective TCR-stimulated proliferation, IL-2 production, and activation of phospholipase C-gamma. Evidence also implicates Tec kinases in actin cytoskeleton regulation, which is necessary for cell adhesion and formation of the immune synapse in T lymphocytes. In this study we show that Tec kinases are required for TCR-mediated up-regulation of adhesion via the LFA-1 integrin. We also demonstrate that the defect in adhesion is associated with defective clustering of LFA-1 and talin at the site of interaction of Rlk-/-Itk-/- and Itk-/- T cells with anti-TCR-coated beads. Defective recruitment of Vav1, protein kinase Ctheta, and Pyk2 was also observed in Rlk-/-Itk-/- and Itk-/- T cells. Stimulation with ICAM-2 in conjunction with anti-TCR-coated beads enhanced polarization of Vav1, protein kinase Ctheta, and Pyk2 in wild-type cells, demonstrating a role for integrins in potentiating the recruitment of signaling molecules in T cells. Increased recruitment of signaling molecules was most pronounced under conditions of low TCR stimulation. Under these suboptimal TCR stimulation conditions, ICAM-2 could also enhance the recruitment of signaling molecules in Itk-/-, but not Rlk-/-Itk-/- T cells. Thus, Tec kinases play key roles in regulating TCR-mediated polarization of integrins and signaling molecules to the site of TCR stimulation as well as the up-regulation of integrin adhesion.

Tec family kinases in T lymphocyte development and function.

The Tec family tyrosine kinases are now recognized as important mediators of antigen receptor signaling in lymphocytes. Three members of this family, Itk, Rlk, and Tec, are expressed in T cells and activated in response to T cell receptor (TCR) engagement. Although initial studies demonstrated a role for these proteins in TCR-mediated activation of phospholipase C-gamma, recent data indicate that Tec family kinases also regulate actin cytoskeletal reorganization and cellular adhesion following TCR stimulation. In addition, Tec family kinases are activated downstream of G protein-coupled chemokine receptors, where they play parallel roles in the regulation of Rho GTPases, cell polarization, adhesion, and migration. In all these systems, however, Tec family kinases are not essential signaling components, but instead function to modulate or amplify signaling pathways. Although they quantitatively reduce proximal signaling, mutations that eliminate Tec family kinases in T cells nonetheless qualitatively alter T cell development and differentiation.

Beyond calcium: new signaling pathways for Tec family kinases.

The Tec kinases represent the second largest family of mammalian non-receptor tyrosine kinases and are distinguished by the presence of distinct proline-rich regions and pleckstrin homology domains that are required for proper regulation and activation. Best studied in lymphocyte and mast cells, these kinases are critical for the full activation of phospholipase-C gamma (PLC-gamma) and Ca(2+) mobilization downstream of antigen receptors. However, it has become increasingly clear that these kinases are activated downstream of many cell-surface receptors, including receptor tyrosine kinases, cytokine receptors, integrins and G-protein-coupled receptors. Evidence suggests that the Tec kinases influence a wide range of signaling pathways controlling activation of MAP kinases, actin reorganization, transcriptional regulation, cell survival and cellular transformation. Their impact on cellular physiology suggests that the Tec kinases help regulate multiple cellular processes beyond Ca(2+) mobilization.