L. Yu, Department of Hematology, Huaian No. 1 Hospital, Nanjing Medical University, Huaian 223300, Jiangsu, China Fax: +86 517 84907078 Tel: +86 517 84952303 E-mail: firstname.lastname@example.org
The TEC family is ancient and constitutes the second largest family of cytoplasmic tyrosine kinases. In 1993, loss-of-function mutations in the BTK gene were reported as the cause of X-linked agammaglobulinemia. Of all the existing 90 tyrosine kinases in humans, Bruton’s tyrosine kinase (BTK) is the kinase for which most mutations have been identified. These experiments of nature collectively provide a form of mutation scanning with direct implications for the several hundred endogenous signaling proteins carrying domains also found in BTK. In 2009, an inactivating mutation in the ITK gene was shown to cause susceptibility to lethal Epstein–Barr virus infection. Both kinases represent interesting targets for inhibition: in the case of BTK, as an immunosuppressant, whereas there is evidence that the inhibition of inducible T-cell kinase (ITK) could influence the infectivity of HIV and also have anti-inflammatory activity. Since 2006, several patients carrying a fusion protein, originating from a translocation joining genes encoding the kinases ITK and spleen tyrosine kinase (SYK), have been shown to develop T-cell lymphoma. We review these disease processes and also describe the role of the N-terminal pleckstrin homology–Tec homology (PH–TH) domain doublet of BTK and ITK in the downstream intracellular signaling of such fusion proteins.
TEC family kinases (TFKs) evolved 600 million years ago prior to the existence of metazoans  and comprise five members in mammals: Bruton’s tyrosine kinase (BTK), inducible T-cell kinase (ITK), TEC, BMX [also known as epithelial and endothelial tyrosine kinase (ETK)] and TXK [also known as resting lymphocyte kinase (RLK)]. The phenotypes of loss-of-function mutations in mammals mainly affect the hematopoietic system, whereas, in fruit fly oogenesis, male genital development and life span are compromised, a phenotype partially reversed by the expression of human BTK . Many reviews, mainly concentrating on intracellular signaling, have been written on TFKs [3–7]. In this minireview, we focus on human disease, in which TFKs are showing increasing importance, both as an underlying cause, but recently also as potential targets for new drugs. The main emphasis is on BTK and ITK deficiency, as well as the translocation between ITK and spleen tyrosine kinase (SYK). Very recently, the TXK/TEC loci have also been associated with disease, namely the development of rheumatoid arthritis, in a genome-wide screen .
Mutations affecting BTK cause X-linked agammaglobulinemia (XLA) and provide insight into basic signaling mechanisms
In 1992, two TFKs were already known, namely TEC and ITK (reviewed in Ref. ). Even though information was available regarding their potential function, it was the identification of BTK, as the kinase affected in XLA [9,10], which immediately made TFKs known to the wider scientific community. In the same year, the xid (X-linked immunodeficiency) mouse was recognized as a spontaneously occurring animal disease model for inactivating mutations affecting this kinase [11,12]. However, the phenotype in the xid mouse is mild, whereas the identical mutation, causing the substitution of arginine 28 for cysteine (R28C), in humans  results in classical XLA, clearly demonstrating that there are species’ differences. Ellmeier et al.  reported that the combined inactivation of BTK and TEC in mice causes a phenotype resembling XLA, thus delineating species-specific redundancy. The R28C mutation, which abolishes binding to activation-induced phosphatidylinositol-3,4,5-trisphosphate in the cell membrane , was soon engineered and grafted onto other signaling molecules, such as v-akt murine thymoma viral oncogene (AKT) [also known as protein kinase B (PKB)]. In AKT, a cysteine substitution of the corresponding R25 in the pleckstrin homology (PH) domain also results in loss of function [16,17], thereby demonstrating related functions among selected PH domains. Thus, from the beginning, mutations in the BTK gene have contributed to our understanding of signaling mechanisms in general.
Mutation spectrum in XLA and genotype–phenotype correlations
Figure 1 depicts the linear organization of the domains in BTK and Fig. 2 shows missense mutations (amino acid substitutions) in a three-dimensional context in the various domains of BTK. Mutations affecting the R28 residue (marked in dark blue in Fig. 1) will result in the redistribution of electrostatic charges that are indispensible for ligand binding. Many of the mutations locate to highly structurally conserved regions, such as α-helices or β-sheets, whereas some are positioned in the connecting loops. Approximately one-third of all mutations in the BTK gene are missense and some of these reduce the stability of the protein. This is exemplified by mutations in the BTK motif of the Tec homology (TH) domain . This region is known to bind a Zn2+ ion, rendering stability to the adjacent PH domain. The substitution of conserved Zn2+-interacting amino acids results in the formation of a highly unstable protein, which is essentially undetectable in cell lysates. A more detailed description of missense mutations in the PH, TH, Src homology 2 (SH2) and kinase domains is given in Ref. .
Many other BTK missense mutant proteins are expressed at normal, or close to normal, levels and are instead functionally disabled. We will not survey the different BTK mutations, but instead refer to reports addressing this topic [19–22]. However, just to mention a few specifics, the online database for mutations in the BTK gene, designated BTKbase, http://bioinf.uta.fi/BTKbase/, contains more than 1100 entries [19–21]. This represents in excess of 970 unrelated families showing more than 600 unique molecular events. These numbers clearly demonstrate that, currently, most mutations are unique, i.e. only reported from a single family. This is especially true for frameshift mutations, even though recurrent mutations eventually will prevail here also as the overall number of mutations increases. Of the residues affected by missense mutations, proline residues are over-represented, presumably secondary to the strong influence of prolines on peptide folding . Thus, proline is a rigid amino acid creating a fixed kink in a protein chain.
Similar to the situation in many other genes, CpG dinucleotides in the BTK gene are more susceptible to mutation, approximately by an order of magnitude . Owing to the high frequency of CpG dinucleotides in arginine codons, the mutation spectrum provides a few highly significant genotype–phenotype correlations. Thus, certain codons, such as those encoding R13 and R288 in the PH and SH2 domains of BTK, respectively, are permissive for missense, but not for nonsense, changes, as there are no reported XLA patients with an R13 or R288 substitution, but plenty with stop codons . Conversely, for other arginine codons, corresponding to, for example, R520 and R525, located in the kinase domain, both nonsense and missense mutations cause XLA (P < 0.001). This provides immediate insight into potential conformational restrictions, as ‘tolerated’ BTK substitutions, exchanging R13 or R288 for other amino acids, presumably exist in the general population as rare, normal variants with maintained signaling function. To date, such rare variants have not been described, but, owing to their expected extremely low frequency, this outcome is anticipated. Recently, a rare variant, a nonpathogenic mutation predicted to affect the BTK SH3 domain by generating an A230V amino acid substitution, was reported . Structural analysis shows that this residue is located in the RT loop of the SH3 domain, which is involved in the recognition of interacting partners .
Although the genotype–phenotype correlation for highly selected residues is extremely strong, the overall correlation based on reported patients is weak, with only a modest over-representation of substitutions relative to frameshifts among patients with mild disease [20,21,25]. This is most probably a result of the fact that more subtle phenotypic changes only rarely lead to genetic analysis, and these mutations are therefore absent from the statistics. To this end, it seems likely that future genome sequencing efforts, where large populations are analyzed, will also identify individuals with mild disease, thereby providing the missing data.
The phenotype of XLA and the potential of BTK and ITK inhibitors
The outcome of defective BTK signaling in humans has been described previously in detail [26,27], and therefore we will only review this topic very briefly. Patients with XLA have a differentiation block resulting in an almost complete absence of B lymphocytes and plasma cells and very low levels of immunoglobulins of all classes. Humoral immune responses are essentially nonexistent. T cells are not affected, but myeloid cells show demonstrable abnormalities (see minireview by Ellmeier et al. ). Patients with XLA are very susceptible to pyogenic bacterial infections but, as these normally can be successfully treated with antibiotics, enteroviruses constitute a greater threat, owing to the fact that these infections are very difficult to treat . Prophylaxis in the form of γ-globulin replacement is standard for all patients [30,31].
Over the last few years, several companies have developed small-molecule inhibitors for BTK  and ITK [33,34]. ITK inhibitors may potentially be used for the treatment of inflammatory diseases  and, as discussed below, may also become part of the anti-HIV therapeutic arsenal. By blocking B-lymphocyte development, BTK inhibitors could potentially replace treatment with monoclonal antibodies directed against B-lymphocyte surface antigens, currently a multibillion dollar market. To this end, even after withdrawal, such monoclonals continue to suppress B-lymphocyte levels for long time periods, and it would be of great interest if the effect of BTK inhibitors could be more quickly reversed.
A mutation affecting ITK causes susceptibility to Epstein–Barr virus (EBV) infection
Although a multitude of disease-causing mutations in the BTK gene have been identified, it was only in 2009 that a spontaneous alteration in another human TFK gene was reported, namely in the ITK gene . ITK was discovered using a degenerate PCR screen for novel T-cell-expressed kinases [36,37]. This enzyme serves as an important player in inflammatory disorders, such as allergic asthma and atopic dermatitis [38,39]. In this minireview series, two articles describe the current understanding of ITK’s role in signaling and development [40,41].
Thus, in 2009, Huck et al.  identified two sisters from a consanguineous Turkish family who both died after developing severe immune dysregulation following infection with EBV. Detailed analysis revealed that they were homozygous for a missense mutation in the ITK gene, located on chromosome 5q31-5q32. This resulted in an amino acid substitution (R335W) in the SH2 domain of ITK, representing the first molecular cause of autosomal recessive lymphoproliferative disease. Arginine 335 is found in the ‘BG loop’ not involved in phosphotyrosine binding and mutation to tryptophan most probably causes instability of the SH2 domain. Thus, these patients had undetectable levels of ITK protein despite normal levels of mRNA. Consistent with this, in silico modeling predicted that the mutation would destabilize the SH2 domain and no R335W mutant protein was detected following overexpression in 293T cells . In 2011, Stepensky et al.  reported three cases from a single Arab family with a biallelic, nonsense mutation in the kinase domain. The nonsense mutation, C1764G, was predicted to cause a premature stop codon in the kinase domain, seemingly creating an unstable protein. All three presented with EBV-positive B-cell proliferation, which was diagnosed as Hodgkin’s lymphoma. Following chemotherapy, one patient went into stable remission and one developed severe hemophagocytic lymphohistiocytosis with multiorgan failure and died. The third patient underwent successful allogeneic bone marrow transplantation. The disease resembles ITK deficiency in mouse models with the absence of natural killer T cells (NKT cells).
Even though the patients with the R335W mutation completely lacked ITK protein, mutations in the ITK SH2 domain may have additional effects when the protein remains stable, by acting as a dominant negative form, or by interfering with other functional parts of the molecule. Thus, as a functional SH2 domain is necessary for enzymatic activity, it is likely that kinase activity is also compromised in certain mutants destabilizing the SH2 domain in TFKs [43,44]. So far, more than 30 missense mutations in the BTK SH2 domain have been described in patients with XLA, and the effects of these mutations have been analyzed in a large number of in vitro and in vivo studies . About 20 mutations affect residues directly involved in ligand binding, presumably abolishing the interaction with signaling partners. The remaining mutations alter amino acids located outside the ligand-binding pocket and reduce protein stability.
The two patients with the R335W mutation had negligible levels of NKT cells. This suggests that NKT cells protect against increased susceptibility to EBV infection, EBV-positive B-cell proliferation and Hodgkin’s lymphoma. It has been postulated that NKT cells play a critical role in the immune response to EBV infection in humans [46,47]. Accordingly, the patient’s parents, who were heterozygous for this mutation, had low, but still detectable, numbers of NKT cells, and did not succumb to severe EBV infection. In mice, it has also been shown that NKT cells play important roles in protection against virus infections . The absence of ITK has been studied extensively in mouse models. ITK regulates a number of T-cell signaling pathways, including NKT cell development and functions; in ITK-deficient mice, the overall NKT percentage and numbers are decreased significantly [3,40,41,49–51]. Based on the data from the two patients and the results from animal research, human ITK mutation and ITK-deficient mice also share some other common features. Apart from the reduced number of NKT cells, naive T cells are also reduced in number, both CD4+ and CD8+. Moreover, especially within the CD8+ population, a subset with memory phenotype (CD44+, CD122+ in mice and CD45RO in humans) is increased [35,51,52]. This is also reflected in the transcriptome of both human  and mouse [51,53] CD8+ cells, which express very high levels of the transcription factor eomesodermin, whose own transcription is suppressed by ITK [35,51]. Another important transcriptional regulator is promyelocytic leukemia zinc finger protein, which is essential for NKT cell development and also plays a direct role in the generation of innate T cells with a memory phenotype [54,55]. Additional patients with other mutations were recently presented at the XIVth Meeting of the European Society for Immunodeficiencies, where Huck et al.  reported two new missense mutations and one family with a deletion in the ITK gene. EBV-associated lymphoproliferative disease was observed in patients with concomitant fever, lymphadenopathy, leukopenia and reduced numbers of NKT cells.
ITK – a potential target for HIV drug development
It is believed that 30 million people worldwide are currently infected with the virus that causes AIDS. Despite intensive scientific research over the past 27 years, HIV remains defiant and poses a serious challenge to public health . Although the introduction of powerful drugs has considerably improved the quality of life for patients with AIDS in industrialized countries, there is, at present, no definitive cure or vaccine. Therefore, the development of novel antiviral drugs should be a priority. Notably, the tools of modern molecular biology have enabled the design of nucleic acid analogs that could modulate gene expression in mammalian cells. Small interfering RNA is a case in point . To this end, we and other research groups have investigated RNA interference as a treatment regimen for HIV/AIDS [59,60]. By employing this approach, close to 70% inhibition of viral infection was achieved in cell lines stably transduced with an expression vector encoding short hairpin RNA against the CCR5 receptor. Similarly, viral replication was entirely compromised (> 90%) when cell lines expressing short hairpin RNA against the Rev protein were challenged with HIV .
More recently, we have demonstrated that proteasome inhibitors reduce the steady-state levels of TFKs in hematopoietic cell lines . As members of this family are known to be critical in inflammatory and infectious diseases, drugs that inhibit their activity or expression are of utmost importance. ITK has recently been shown to be crucial for HIV replication in susceptible cells at multiple levels . In resting human CD4+ T cells, the expression of ITK is extremely low and often undetectable in immunoblot analysis. The activation of CD4+ cells, however, dramatically induces transcription of the ITK gene and is key for the productive infection of HIV in these cells. Accordingly, the inhibition of ITK activity compromises HIV infection, gene expression and replication .
Our group has recently evaluated the effect of proteasome inhibitors on HIV infection and/or replication. To determine whether the depletion of ITK could affect HIV replication, we treated activated peripheral blood mononuclear cells with the clinically approved proteasome inhibitor bortezomib (Velcade) and challenged the cells with a strain of HIV. Surprisingly, HIV replication was dramatically blocked . Although other reasons could not be excluded, the overall reduction of ITK might be responsible for the potent viral inhibition. Moreover, novel proteasome inhibitors that are less toxic and more specific are currently in the pipeline for clinical approval , and several ITK-specific inhibitors have been developed [33,34].
Transforming activity of the ITK–SYK fusion protein
Under physiological growth conditions, SYK seems to be autoinhibited and is believed to exist in a closed conformation [65–67]. Following cellular stimulation, SYK becomes phosphorylated by an SRC family kinase and binds to the immunoreceptor tyrosine-based activation motifs at the inner surface of the plasma membrane. Binding to immunoreceptor tyrosine-based activation motifs fixes the molecule in an extended configuration, thereby stabilizing the noninhibited state. Additional phosphorylation events involving multiple tyrosines, in particular those at the carboxyl terminal tail, facilitate the interaction of SYK with the adapter proteins BLNK (also known as SLP-65) and SLP-76, making it fully active.
SYK has been linked to the development and maintenance of hematological malignancies . Moreover, as a result of chromosomal translocation, a chimera, consisting of the dimerizing TEL protein and SYK, was formed and has been shown to cause a rare form of myelodysplastic syndrome .
Recently, ITK was the first and only known Tec family member reported to undergo a chromosomal translocation event leading to a chimeric kinase with transforming capacity, the hallmark of which is unspecified peripheral T-cell lymphoma . Consequently, the PH–TH domain doublet of ITK fuses directly with the linker B kinase region of SYK. The PH domain of TFKs usually binds to phosphatidylinositol-3,4,5-trisphosphate, thereby bringing them in close proximity to other membrane-tethered signaling proteins. In SYK, the linker B region contains key tyrosines that are subject to auto- and/or transphosphorylation, and that mediate interaction with Vav, c-Cbl and the p85α subunit of phosphatidylinositol 3-kinase, whereas the kinase domain harbors two unique tyrosines (the paired activation loop tyrosines) critical for activation and signaling [65–67]. The fusion event creates a novel kinase with a unique composition that probably favors an open conformation structure, with the potential for constitutive activation. Thus, ITK–SYK, but not ITK or SYK themselves, is capable of transforming NIH-3T3 cells . In addition, we and others have demonstrated that the activation and plasma membrane localization of the fusion construct are dependent on phosphatidylinositol 3-kinase signaling, and that ITK–SYK phosphorylates the adapter proteins SLP-76 and BLNK in the absence of external stimuli [70–72].
More recently, a transgenic mouse expressing the ITK–SYK fusion under the control of a T-cell-specific promoter , as well as another mouse model in which bone marrow cells were transduced with a vector expressing ITK–SYK , have been described. Expression of the chimera resulted in the formation of highly malignant peripheral T-cell lymphomas in mice, with a phenotype resembling that described in human patients. In T cells from transgenic mice, the ITK–SYK fusion was found to translocate to lipid rafts and was able to constitutively phosphorylate T-cell receptor-associated signaling proteins. It is noteworthy that, when the same fusion construct was specifically expressed in the B-cell lineage of these animals, it did not induce the formation of B-cell lymphomas. Thus, transgenic mice with a CD19 promoter-mediated expression of ITK–SYK failed to develop B-cell lymphoma but, instead, yielded T-cell tumors, albeit with considerable delay, probably caused by promoter leakiness . Unexpectedly, in the transduced model, the R29C mutant (corresponding to BTK R28C), which lacks the membrane-targeting ability, showed enhanced tumorigenicity. These findings underline the surprisingly stark differences between B and T lymphocytes with regard to their response to different TFK fusions, and also raises the important question of the outcome of the corresponding translocation involving BTK in B lymphocytes generating BTK–SYK. Will such a fusion behave differently from ITK–SYK in terms of transformation capacity, membrane localization and phosphorylation of key residues?
Comparison between the activation of ITK–SYK and BTK–SYK
To determine its activation capacity, we constructed the corresponding fusion kinase BTK–SYK, harboring the PH–TH domain doublet (amino acids 1–196) of BTK fused with the linker B kinase region of SYK (306–635 amino acids) (Fig. 2). We used two different cell types to study the phosphorylation status of key residues and the capacity to phosphorylate exogenous substrate molecules.
BTK–SYK, like ITK–SYK, proved to be constitutively active in transiently transfected COS7 cells (Fig. 2B). In addition to the full-length fusion protein, ITK–SYK produces a very stable and shorter protein in COS7 and 293T cells. This shorter isoform, which can also be phosphorylated, is generated as a result of alternative translation initiation. BTK–SYK also produces a similar isoform which, in contrast with ITK–SYK, is highly unstable as a result of degradation by the ubiquitin–proteasome pathway (A. Hussain et al., unpublished results). In COS7 cells, the fusion protein was highly phosphorylated in the linker region and in the activation loop tyrosines in the absence of any external stimulation. Moreover, BTK–SYK also showed similar phosphorylation when expressed at levels comparable with those of endogenous SYK in 293T cells. The kinase-deficient versions of the fusion proteins were not readily phosphorylated in either cell type.
In particular, the phosphorylation, but also the total protein level, of BTK–SYK was less than that of ITK–SYK in 293T relative to COS7 cells. 293T cells express endogenous SYK, but we do not know whether this kinase influences the behavior of the fusion proteins. It is also possible that the differential expression of SRC family members in these two cell types may influence the phosphorylation levels of BTK–SYK. ITK–SYK was highly phosphorylated in both COS7 and 293T cells and did not vary like BTK–SYK; therefore, the differences in the PH–TH domains remain the decisive factor for this variation.
The B-cell adapter protein BLNK (SLP-65) and its T-cell counterpart SLP-76 are key signaling components downstream of immunoreceptors. ITK–SYK has been reported to potently phosphorylate SLP-76 in the steady state [71,72]. Coexpression of BLNK or SLP-76 with BTK–SYK or ITK–SYK resulted in robust phosphorylation of the two adapter molecules in 293T cells. The phosphorylation levels of BLNK and SLP-76 in cells transfected with BTK–SYK were, however, lower relative to ITK–SYK, consistent with the reduced phosphorylation level of BTK–SYK itself in these cells. In COS7 cells, where BTK–SYK and ITK–SYK are equally phosphorylated, phosphorylation of SLP-76 and BLNK was essentially the same on cotransfection with either of the two fusion proteins (Fig. 2B). In both cell types, kinase-inactive forms of the fusion proteins failed to phosphorylate BLNK and SLP-76.
Thus, we found that BTK–SYK and ITK–SYK were different in terms of their activation and substrate phosphorylation levels in different cell lines. This study shows that seemingly subtle differences in the PH–TH domains of the two fusion proteins play key roles in the activation process and are responsible for variations among different cell types.
In conclusion, TFKs form a family of cytoplasmic enzymes that are important for several aspects of leukocyte biology. Both loss- and gain-of-function mutations in humans have been instrumental in our understanding of their behavior.
This work was supported by the Swedish Science Council, the Stockholm County Council (research grant ALF-projektmedel medicin), the Cancer Foundation, the European Union FP7 grant EURO-PADnet, and the Torsten and Ragnar Söderberg Foundation. Rani Faryal was a recipient of a Postdoctoral Fellowship from the Higher Education Commission (HEC), Pakistan. We are indebted to Dr Jouni Väliaho, University of Tampere, Finland, for modifications to Fig. 1. Dara K. Mohammad was a recipient of a PhD Fellowship from the Ministry of Higher Education and Scientific Research/KRG-Erbil, Iraq.