Structure and Activation of Tec and Tec Family Kinases
The mechanisms which lead to the activation of Tec family kinases, in particular Btk and Itk, have been well studied in lymphocytes (Andreotti et al. 2010). The first regulatory step in the activation of Tec family kinases is their recruitment to the cytoplasmic membrane. This step is achieved through binding of the PH domain to the PI3K product phosphatidylinositol 3, 4, 5 trisphosphate (PIP3). Some notable differences however exist between the PH domains of Tec and Btk: whereas specific mutations in the Btk PH domain increased its binding to PIP3, these same mutations decreased the binding of the Tec PH domain to PIP3. In addition membrane recruitment or accumulation at the immunological synapse of Tec could also be mediated by its SH2 or SH3 domains, respectively. The further steps leading to the full activation of Tec kinases were extensively studied for Btk and Itk and to a lesser extent for Rlk and Tec. After membrane recruitment, Tec kinases are phosphorylated by Src and/or Syk kinases at a critical tyrosine residue within the conserved activation loop in the kinase domain which results in a conformational change allowing access of the substrate to the catalytic site of the kinase domain. In fibroblasts Tec is activated by the Src kinase Lyn and in T cells by Lck. Transphosphorylation is followed by an autophosphorylation of a tyrosine residue within the SH3 domain. Major phosphorylation sites were identified. These corresponded to conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. Tec however showed again some unique feature as the Tec-SH3 domain is phosphorylated at a nonhomologous site but conserved tyrosine residue, Y206. Another level of regulation of Tec kinase activity is provided by their intra- and intermolecular SH3/proline interactions. Whereas it was shown that for Itk this interaction occurs exclusively in an intramolecular fashion, for Tec, due to the presence of two consecutive proline-rich segments adjacent to its SH3 domain, these interactions occur both inter- and intramolecularly. These intra- and intermolecular SH3/proline interactions could certainly compete with exogenous ligands to regulate the activity and localization of Tec kinases.
Tec Expression Pattern and Its Participation in Signaling Pathways
The expression level of Tec is different among different cell types and tissues. In T and B cells, Tec is expressed approximately 15- to 17-fold lower than Itk or Btk, respectively. In B cells, Tec is expressed approximately 5-fold higher than in T cells. However it was shown that Tec is upregulated upon activation of T cells and is highly and differentially expressed in helper T-cell subsets. Tec expression is controlled by the transcription factors Sp1 and PU-1, and Tec was in addition shown to be under the transcriptional control of the NF-κB subunit p65⁄RelA in a dose-dependent manner, which was important for baseline expression levels but might also account for the observed cell type and cell activation state-specific expression levels (Yu et al. 2009).
Few studies address the role of Tec in signaling pathways of non-hematopoietic cells. Tec expression levels were shown to rise rapidly in the liver of rats after partial hepatectomy as well as in hepatocytes stimulated via hepatocyte growth factor (HGF) (Wang et al. 2006). In addition Tec was shown to promote hepatocyte proliferation and regeneration and has been implicated in the HGF-induced Erk signaling pathway (Li et al. 2011). Tec was also shown to play important functions in fibroblasts by regulating the actin polymerization and formation of stress fibers (Mao et al. 1998) and was also implicated in the unconventional secretion of fibroblast growth factor 2 (FGF2) (Mao et al. 1998). Tec was also connected to the stress signaling in the ischemic myocardium (Zhang et al. 2010).
Tec in the Myeloid System
Mast cells are important in the immunological response to pathogenic parasites like helminths and protozoa and are key players in type I hypersensitivity reactions. They express the high-affinity Fc receptor type I for IgE (FcεRI) which binds IgE. The cross-linking of the IgE/FcεRI complex with antigen triggers the activation of the mast cells and leads to their effector functions like the release of histamine, leukotrienes (LT), and cytokines. Tec was shown to be activated following FcεRI stimulation in mast cells and to participate in the signaling pathway downstream of the FcεRI (Schmidt et al. 2009). In vitro, bone marrow derived mast cells (BMDC) generated by addition of IL-3 or IL-3/SCF to Tec-/- BM were indistinguishable from wild-type (WT) BMMC with respect to histological staining characteristics and expression levels of FcεRI and ckit. However higher mast cell numbers were recovered after Tec −/− BMMC cultures in comparison to WT BMMC cultures. Functionally, it was shown that Tec played an important role in the generation of LTC4, one of the most potent bronchoconstrictive LT. In addition, Tec was shown to differentially regulate the cytokine release in mast cells. Biochemically, Tec −/− BMMC showed a reduction in intracellular calcium levels as well as an increase in Erk1/2 phosphorylation.
Macrophages are important as a first line of defense against many extracellular bacteria and fungi and developed distinct mechanisms to specifically recognize particular microorganisms. Expression of the C-type lectin receptor (CLR) dectin-1 on macrophages is important to mediate protection against fungal pathogens. Recognition of fungal pathogens over dectin-1 leads to Syk-dependent activation of a noncanonical caspase-8-dependent inflammasome. Based on studies involving Tec −/− mice and bone marrow-derived macrophages (BMMs), Tec was shown to be required for the assembly of the caspase-8 inflammasome and to be part of a sensing machinery emanating from dectin-1 (Zwolanek et al. 2014). Mice deficient for Tec were resistant to fungal sepsis in two different systems of murine infection models. Toll-like receptors (TLR) are another type of pathogen-sensing receptors in macrophages. A phosphoproteome study based on Btk −/− and Tec −/− /Btk −/− BMMs revealed that Tec can compensate for Btk in TLR signaling (Tampella et al. 2015). In addition by comparing signaling over TLRs in different macrophage subsets from WT and Tec −/− /Btk −/− mice, it could be shown that Tec and Btk act either as positive or negative regulators of TLR signaling, dependent on the presence of inhibitory immunoreceptor complexes like Trem2 in particular macrophage subsets.
In addition to compensatory mechanisms in TLR signaling, Tec was also shown to be involved in the regulation of M-CSF receptor signaling in BMMs. M-CSF is a crucial cytokine which strongly influences the differentiation, proliferation, and survival of macrophages. Retrieval of M-CSF from BMM cultures results in apoptosis of the cells, pinpointing out the importance of this cytokine for macrophage survival. BMMs were generated from WT and Tec −/− /Btk −/− BMM with low and high M-CSF concentrations. In contrast to WT BMMs, Tec −/− /Btk −/− BMMs did not survive with low M-CSF concentrations. The Tec −/− /Btk −/− BMM cultures could however be rescued with high M-CSF concentrations, showing that Tec and Btk are important for a proper sensing of M-CSF levels by the developing macrophages. GM-CSF is also used in cell culture systems to generate BMMs from BM, but besides macrophages also BM-derived dendritic cells (BMDCs) develop. It was observed that Tec and Btk were important for the expression of the GM-CSF receptor α (GM-CSFRα) in BMMs but not in BMDCs. Phagocytosis is an important function of macrophages, as it represents the endocytosis of microbial pathogens leading to their clearance. It was also shown in the murine macrophage cell line RAW 264.7 by siRNA-mediated inhibition of Tec and/or Btk expression that FCγR-mediated phagocytosis was strongly impaired, showing also a contribution of Tec and Btk to phagocytic processes. Macrophages besides their phagocytic function are also important mediators for other cells of the immune system by releasing cytokines. One important cytokine produced by macrophages is IL-8, which attracts mainly neutrophils to the site of infection. It was shown that siRNA-mediated knockdown of Tec in the human monocytic cell line THP-1 lead to a reduction in IL-8 production of these cells after LPS stimulation. Overexpression of Tec in RAW264.7 cells leads to an increase in IL-8 promoter activity.
Neutrophils are important myeloid cells which are attracted by cytokines like IL-8 to sites of infection where they help as a first line of defense to fight pathogenic microbes. It could be shown that Tec is phosphorylated and activated in human neutrophils in a Src-dependent manner following exposure to monosodium urate (MSU) crystals (Popa-Nita et al. 2008). By using siRNA silencing of Tec in human primary neutrophils, it could be also shown that Tec was important for the production of IL-1β and IL-8 following stimulation with MSU crystals. Tec was therefore implicated in the initiation and perpetuation of gout. In addition it was shown that Tec-deficient neutrophils had a defect in the caspase-8-driven signalosome following infection of Tec −/− mice with Candida albicans (Zwolanek et al. 2014).
Dendritic cells are crucial sensors of microbial pathogens, and they are the most potent antigen-presenting cells (APCs). Tec was shown to modulate the cytokine secretion of murine BMDC. Tec-deficient BMDC secreted more IL-1β, IL-23, IL-6, TGFβ1, and IL-12 following stimulation with heat killed Streptococcus pneumoniae (S. pneumoniae) (Boucheron et al. 2010). These cytokines are T helper (Th) 17 driving factors, a T-cell subset which is important for a proper immune response to extracellular bacteria like S. pneumoniae and also to fungal pathogens.
Important functions of Tec and Btk have also been described in platelet activation over G protein-coupled receptors as well as in osteoclastogenesis over signaling by the receptor activator of nuclear factor-κB ligand (RANKL), involving in both cases activation of phospholipase Cγ2 (PLCγ2) (Koprulu and Ellmeier 2009).
Tec in the Lymphoid System
B cells are important cells of the adaptive immune system and are able to produce immunoglobulins (Ig) specific to pathogens. Tec was first implicated in the IL-3 signaling pathway in pro-B-cell lines. In a yeast two-hybrid system, the docking protein BRDG1 was identified as an interaction partner of Tec. Tec was also found to be expressed in human and murine B cells and to be activated downstream of the BCR. Tec was also shown to interact with Dok-1 via TH, SH2, and kinase domain in a pro-B-cell line. B-cell development was however not affected in Tec −/− mice, in contrast to Btk −/− mice, where a severe block in B-cell development was observed. This block was however even stronger in Tec −/− /Btk −/− mice, resulting in the absence of mature B cells. Tec can therefore compensate for Btk function in B cells. Studies with DT40 chicken B-cell lines showed that overexpression of Tec was able to induce stable NFAT activation, and this activation was independent of the tyrosine kinases Lyn, Syk, or Btk or the adapter Grb2, Grap, or BLNK. Tec-dependent NFAT activation required however PLCγ2 overexpression.
T cells are the second crucial cell type belonging to the adaptive immune system. They can be divided in CD8+ and CD4+ T cells. CD8+ T cells are important to fight viral infections and tumors. CD4+ T cells can differentiate toward virus-specific Th1 cells, Th2 cells which support B-cell responses, or Th17 cells which control extracellular bacterial and fungal pathogens. All these subsets secrete IL-2, and in addition and in addition Th-specific cytokines like IFNg for Th1 cells, IL-4 for Th2 cells and IL-17A for Th17 cells.
Extensive studies on the function of Tec in T cells have been performed (Yang et al. 2000; Boucheron and Ellmeier 2012). Tec was shown to be expressed at low levels in primary human and murine resting T cells. Tec was shown to bind CD28 via its SH3 domain and to be activated following TCR and CD28 cross-linking in murine hybridoma cells, leading to an enhanced transcription of the IL-2 and IL-4 promoters. In murine splenocytes, Tec depletion by antisense strategy resulted in a reduction in IL-2 gene induction. Tec was also found to regulate specifically NFAT activity in Jurkat T cells in absence of T-cell stimulation, which was a unique feature of Tec, and could not be observed by overexpression of Btk or Itk. In contrast the transcription factors AP-1 or NF-κB were unaffected, although one study reported constitutive association of Tec with PKCθ in Jurkat T-cell lines and AP-1 activation following Tec overexpression. The regulation of NFAT activity was at least partially dependent on kinase activity of Tec and completely dependent on intact PH and SH2 domains. Some kinase-independent functions were therefore proposed for Tec in the activation of NFAT, and it was suggested that Tec had also important adaptor functions. Activation of Tec via Lck was shown to be important for Tec to exert its functions. In addition, in contrast to PLCγ1 activation via Itk which required the adaptor molecules SLP76 and LAT, PLCγ1 activation via Tec was independent of these adaptor molecules. This unique property of Tec in T-cell signaling was also observed by fluorescence microscopy of transfected Jurkat cells or a murine T-cell clone, as Tec was observed in punctated structures at the cell membrane, suggesting unique subcellular compartmentalization.
The inositol phosphatases SHIP1 and 2 were found to bind Tec, and SHIP phosphatases negatively regulated Tec by preventing its membrane localization. This finding suggests the existence of a Dok/SHIP/Tec negative regulatory signalosome, as SHIP phosphatases and Tec were shown to interact with Dok family adaptor proteins.
However Tec −/− mice did not display major immunological alterations. Indeed IL-2 production and proliferation were not altered in absence of Tec. A possible explanation could be the low expression levels of Tec in naïve primary T cells. However no alterations were also observed when CD4+ T cells were polarized toward Th1 and Th2 cells, despite a predicted function of Tec in IL-2 and IL-4 secretion. In particular a function for Tec in Th2 cells was expected, as Tec expression was higher in these cells compared to Th1 cells. Compensatory mechanisms by other Tec kinases expressed in Tec −/− T cells might overcome the need for Tec regulation of proliferation and IL-2 secretion of T cells and of the differentiation and IL-4 secretion of Th2 cells. However Itk −/− T cells have a profound defect in these processes which point at a dominant function of Itk in overall T-cell activation, proliferation, and differentiation. In contrast a specific role for Tec in Th17 cell differentiation could be observed (Boucheron and Ellmeier 2012). Tec −/− mice had increased frequencies of IL-17A secreting peripheral CD4+ T cells, and it could be shown that this increase in Th17 cells could also mediate protection in a model of S. pneumonia infection. A T-cell intrinsic function for Tec in the generation of Th17 cells could be shown. Therefore Tec was described as a negative regulator of Th17 differentiation. Later on, the Th17-driving transcription factor cMaf was shown to be specifically tyrosine phosphorylated in Hek293 cells by Tec, but not other Tec kinases, and phosphorylated cMaf could bind more efficiently to the IL-4 and IL-21 promoters. Phosphorylation of cMaf was important for optimal secretion of IL-21, a cytokine known to support Th17 differentiation. Therefore Tec was also associated with a positive regulation of Th17 cells.
Tec has been implicated in many cell types and regulatory processes during cell differentiation, proliferation, and survival. Redundant functions with other Tec kinases made it difficult to dissect the specific role of Tec in particular cellular processes. However biochemical and genetic studies allowed understanding the most important functions of Tec. So far Tec has been associated with the proper sensing of cytokines, of specific pathogens, and was shown to have important modulatory functions in a broad spectrum of cellular subsets and functions. In contrast to other Tec kinases, Tec expression levels are regulated either by cellular stress as it was observed following partial hepatectomy in liver tissues, or following HGF administration in hepatocytes, or after TCR activation in primary T cells. Tec expression levels are therefore tightly controlled. Being involved in many receptor-driven pathways, further investigations might still uncover novel aspects of cellular processes regulated by Tec. Concerning its implications in human disease, rheumatoid arthritis has been linked with single-nucleotide polymorphisms (SNP) at the Tec and Tec/Rlk loci. In addition to a possible implication in the generation and perpetuation of gout, its function in macrophages, neutrophils, and Th17 cells makes Tec an interesting drug target to modulate immune functions during disease.