Tài liệu Báo cáo khoa học: TEC family kinases in health and disease – loss-of-function of BTK and ITK and the gain-of-function fusions ITK–SYK and BTK–SYK pptx

MINIREVIEW
TEC family kinases in health and disease – loss-of-function
of BTK and ITK and the gain-of-function fusions ITK–SYK
and BTK–SYK
Alamdar Hussain
1,2,
*, Liang Yu
1,3,
*, Rani Faryal
1,2
, Dara K. Mohammad
1
, Abdalla J. Mohamed
1,4
and C. I. Edvard Smith
1
1 Clinical Research Center, Department of Laboratory Medicine, Karolinska Institutet, Huddinge University Hospital, Sweden
2 Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan
3 Department of Hematology, Huaian No. 1 Hospital, Nanjing Medical University, Huaian, Jiangsu, China
4 Faculty of Science (Biology), Universiti Brunei Darussalam, Gadong, Brunei Darussalam
Introduction
TEC family kinases (TFKs) evolved 600 million years
ago prior to the existence of metazoans [1] and com-
prise five members in mammals: Bruton’s tyrosine
kinase (BTK), inducible T-cell kinase (ITK), TEC,
BMX [also known as epithelial and endothelial tyro-
sine 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 compro-

mised, a phenotype partially reversed by the expression
of human BTK [2]. Many reviews, mainly concentrat-
ing on intracellular signaling, have been written on
TFKs [3–7]. In this minireview, we focus on human
Keywords
AKT; BMX; BTK; ITK; lymphocyte;
PH domain; RLK; TEC; TXK;
X-linked agammaglobulinemia
Correspondence
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:
*These authors contributed equally to this
work
(Received 31 August 2010, revised 21
March 2011, accepted 20 April 2011)
doi:10.1111/j.1742-4658.2011.08134.x
The TEC family is ancient and constitutes the second largest family of cyto-
plasmic 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 experi-
ments 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 infec-
tion. Both kinases represent interesting targets for inhibition: in the case of

BTK, as an immunosuppressant, whereas there is evidence that the inhibi-
tion of inducible T-cell kinase (ITK) could influence the infectivity of HIV
and also have anti-inflammatory activity. Since 2006, several patients carry-
ing 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.
Abbreviations
AKT, v-akt murine thymoma viral oncogene; BTK, Bruton’s tyrosine kinase; EBV, Epstein–Barr virus; ITK, inducible T-cell kinase; NKT cell,
natural killer T cell; PH, pleckstrin homology; PKB, protein kinase B; R28C, arginine 28 mutated to cysteine; SH2, Src homology 2;
SH3, Src homology 3; SYK, spleen tyrosine kinase; TFK, TEC family kinase; TH, Tec homology; XLA, X-linked agammaglobulinemia.
FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS 2001
disease, in which TFKs are showing increasing impor-
tance, 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 translo-
cation between ITK and spleen tyrosine kinase (SYK).
Very recently, the TXK ⁄ TEC loci have also been asso-
ciated with disease, namely the development of rheu-
matoid arthritis, in a genome-wide screen [8].
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. [1]). Even though informa-
tion 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 recog-
nized 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 sub-
stitution of arginine 28 for cysteine (R28C), in humans
[13] results in classical XLA, clearly demonstrating
that there are species’ differences. Ellmeier et al. [14]
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 [15], was soon engineered and grafted
onto other signaling molecules, such as v-akt murine
thymoma viral oncogene (AKT) [also known as pro-
tein 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, muta-
tions in the BTK gene have contributed to our under-
standing 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
Fig. 1. Structure of PH, SH2 and kinase domains of BTK with color-
ing of residues affected by missense mutations. Top left: locations
of the missense mutation in the BTK PH domain; arginine 28 is in
dark blue, encircled in red. Bottom left: SH2 domain. Right: kinase
domain. The mutated residues are indicated in yellow, a-helices are
in cyan, b-sheets are in magenta and loops are in blue. Modified
from Valiaho et al. [19].
Fig. 2. (A) Schematic representation of BTK, ITK, SYK and the cor-
responding fusion proteins. PH, pleckstrin homology domain; TH,
Tec homology domain; SH3, Src homology 3 domain; SH2, Src
homology 2 domain; Y, linker region tyrosine (Y352); YY, activation
loop tyrosines (Y525 ⁄ Y526). (B) Graphic representation showing
that the PH–TH domain differences between BTK–SYK and ITK–
SYK fusion proteins lead to differential phosphorylation levels of
the fusion proteins themselves, as well as the downstream adapter
proteins SLP76 and BLNK, in 293T and COS7 cells. Size of red
encircled ‘P’ approximately represents the phosphorylation levels.
TEC kinases and disease A. Hussain et al.
2002 FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS
indispensible for ligand binding. Many of the muta-
tions locate to highly structurally conserved regions,
such as a-helices or b-sheets, whereas some are posi-
tioned 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 [18]. This region is

known to bind a Zn
2+
ion, rendering stability to the
adjacent PH domain. The substitution of conserved
Zn
2+
-interacting amino acids results in the formation
of a highly unstable protein, which is essentially unde-
tectable 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. [19].
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://bio-
inf.uta.fi/BTKbase/, contains more than 1100 entries
[19–21]. This represents in excess of 970 unrelated fam-
ilies 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 muta-
tions increases. Of the residues affected by missense
mutations, proline residues are over-represented, pre-
sumably secondary to the strong influence of prolines
on peptide folding [21]. 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
[21]. Owing to the high frequency of CpG dinucleo-
tides in arginine codons, the mutation spectrum pro-
vides 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 [21]. Conversely, for other
arginine codons, corresponding to, for example, R520
and R525, located in the kinase domain, both non-
sense and missense mutations cause XLA (P < 0.001).
This provides immediate insight into potential con-
formational restrictions, as ‘tolerated’ BTK substitu-
tions, 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 sub-
stitution, was reported [23]. Structural analysis shows
that this residue is located in the RT loop of the SH3
domain, which is involved in the recognition of inter-
acting partners [24].

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 rela-
tive 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 result-
ing in an almost complete absence of B lymphocytes
and plasma cells and very low levels of immunoglobu-
lins 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. [28]). 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 [29]. Prophylaxis in the form of c-globulin
replacement is standard for all patients [30,31].

Over the last few years, several companies have devel-
oped small-molecule inhibitors for BTK [32] and ITK
[33,34]. ITK inhibitors may potentially be used for the
treatment of inflammatory diseases [34] and, as discussed
below, may also become part of the anti-HIV therapeu-
tic 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
A. Hussain et al. TEC kinases and disease
FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS 2003
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 [35]. 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 disor-
ders, 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. [35] identified two sisters

from a consanguineous Turkish family who both died
after developing severe immune dysregulation follow-
ing 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 dis-
ease. 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 [35]. In 2011, Stepensky
et al. [42] reported three cases from a single Arab fam-
ily with a biallelic, nonsense mutation in the kinase
domain. The nonsense mutation, C1764G, was pre-
dicted 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. Follow-
ing chemotherapy, one patient went into stable remis-
sion 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 pro-
tein 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 nec-
essary for enzymatic activity, it is likely that kinase
activity is also compromised in certain mutants desta-
bilizing 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 [45]. 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 Hodg-
kin’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 [48]. 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 func-
tions; in ITK-deficient mice, the overall NKT percent-
age 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 num-
ber 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 [35] 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 pheno-
type [54,55]. Additional patients with other mutations
were recently presented at the XIVth Meeting of the
European Society for Immunodeficiencies, where Huck
TEC kinases and disease A. Hussain et al.
2004 FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS
et al. [56] reported two new missense mutations and
one family with a deletion in the ITK gene. EBV-asso-
ciated 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 cur-
rently 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 [57]. Although the introduc-
tion 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 vac-
cine. Therefore, the development of novel antiviral
drugs should be a priority. Notably, the tools of mod-
ern molecular biology have enabled the design of
nucleic acid analogs that could modulate gene expres-
sion in mammalian cells. Small interfering RNA is a

case in point [58]. To this end, we and other research
groups have investigated RNA interference as a treat-
ment regimen for HIV ⁄ AIDS [59,60]. By employing
this approach, close to 70% inhibition of viral infec-
tion 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 [60].
More recently, we have demonstrated that protea-
some inhibitors reduce the steady-state levels of TFKs
in hematopoietic cell lines [61]. 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 sus-
ceptible cells at multiple levels [62]. 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. Accord-
ingly, the inhibition of ITK activity compromises HIV
infection, gene expression and replication [62].
Our group has recently evaluated the effect of

proteasome inhibitors on HIV infection and ⁄ or replica-
tion. To determine whether the depletion of ITK could
affect HIV replication, we treated activated periph-
eral blood mononuclear cells with the clinically
approved proteasome inhibitor bortezomib (Velcade)
and challenged the cells with a strain of HIV. Surpris-
ingly, HIV replication was dramatically blocked [63].
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 cur-
rently in the pipeline for clinical approval [64], 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 tyro-
sine-based activation motifs fixes the molecule in an
extended configuration, thereby stabilizing the nonin-
hibited 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 main-
tenance of hematological malignancies [67]. 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 [68].
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 [69]. Conse-
quently, 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 phosphatidylino-
sitol-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 transphosph-
orylation, and that mediate interaction with Vav,
c-Cbl and the p85a 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,
A. Hussain et al. TEC kinases and disease
FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS 2005
ITK–SYK, but not ITK or SYK themselves, is capable
of transforming NIH-3T3 cells [70]. In addition, we

and others have demonstrated that the activation and
plasma membrane localization of the fusion construct
are dependent on phosphatidylinositol 3-kinase signal-
ing, 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 [72], as well as another mouse model in
which bone marrow cells were transduced with a vec-
tor expressing ITK–SYK [73], 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 recep-
tor-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 lym-
phoma but, instead, yielded T-cell tumors, albeit with
considerable delay, probably caused by promoter leaki-
ness [72]. 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 surpris-
ingly 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 constitu-
tively 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 pro-
duces 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 lev-
els 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 compo-
nents 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 phos-
phorylation 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 cotransfec-
tion 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 varia-
tions among different cell types.
TEC kinases and disease A. Hussain et al.
2006 FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS
In conclusion, TFKs form a family of cytoplasmic
enzymes that are important for several aspects of leu-
kocyte biology. Both loss- and gain-of-function muta-
tions in humans have been instrumental in our
understanding of their behavior.
Acknowledgements
This work was supported by the Swedish Science
Council, the Stockholm County Council (research
grant ALF-projektmedel medicin), the Cancer Founda-
tion, the European Union FP7 grant EURO-PADnet,
and the Torsten and Ragnar So
¨
derberg Foundation.
Rani Faryal was a recipient of a Postdoctoral Fellow-
ship from the Higher Education Commission (HEC),
Pakistan. We are indebted to Dr Jouni Va

¨
liaho, Uni-
versity 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.
References
1 Ortutay C, Nore BF, Vihinen M & Smith CI (2008)
Phylogeny of Tec family kinases: identification of a
premetazoan origin of Btk, Bmx, Itk, Tec, Txk, and the
Btk regulator SH3BP5. Adv Genet 64, 51–80.
2 Hamada N, Backesjo CM, Smith CI & Yamamoto D
(2005) Functional replacement of Drosophila Btk29A
with human Btk in male genital development and
survival. FEBS Lett 579, 4131–4137.
3 Andreotti AH, Schwartzberg PL, Joseph RE & Berg LJ
(2010) T-cell signaling regulated by the Tec family
kinase, Itk. Cold Spring Harb Perspect Biol
doi:10.1101/cshperspect.a002287.
4 Koprulu AD & Ellmeier W (2009) The role of Tec
family kinases in mononuclear phagocytes. Crit Rev
Immunol 29, 317–333.
5 Mohamed AJ, Yu L, Backesjo CM, Vargas L, Faryal
R, Aints A, Christensson B, Berglof A, Vihinen M,
Nore BF et al. (2009) Bruton’s tyrosine kinase (Btk):
function, regulation, and transformation with special
emphasis on the PH domain. Immunol Rev 228, 58–73.
6 Readinger JA, Mueller KL, Venegas AM, Horai R &
Schwartzberg PL (2009) Tec kinases regulate T-lympho-
cyte development and function: new insights into the

roles of Itk and Rlk ⁄ Txk. Immunol Rev 228, 93–114.
7 Smith CI, Islam TC, Mattsson PT, Mohamed AJ, Nore
BF & Vihinen M (2001) The Tec family of cytoplasmic
tyrosine kinases: mammalian Btk, Bmx, Itk, Tec, Txk
and homologs in other species. Bioessays 23, 436–446.
8 Freudenberg J, Lee AT, Siminovitch KA, Amos CI,
Ballard D, Li W & Gregersen PK (2010) Locus cate-
gory based analysis of a large genome-wide association
study of rheumatoid arthritis. Hum Mol Genet 19,
3863–3872.
9 Tsukada S, Saffran DC, Rawlings DJ, Parolini O, Allen
RC, Klisak I, Sparkes RS, Kubagawa H, Mohandas T,
Quan S et al. (1993) Deficient expression of a B cell
cytoplasmic tyrosine kinase in human X-linked
agammaglobulinemia. Cell 72, 279–290.
10 Vetrie D, Vorechovsky I, Sideras P, Holland J, Davies
A, Flinter F, Hammarstrom L, Kinnon C, Levinsky
R, Bobrow M et al. (1993) The gene involved in
X-linked agammaglobulinaemia is a member of the src
family of protein-tyrosine kinases. Nature 361,
226–233.
11 Thomas JD, Sideras P, Smith CIE, Vorechovsky I,
Chapman V & Paul WE (1993) Colocalization of
X-linked agammaglobulinemia and X-linked immuno-
deficiency genes. Science 261, 355–358.
12 Rawlings DJ, Saffran DC, Tsukada S, Largaespada
DA, Grimaldi JC, Cohen L, Mohr RN, Bazan JF,
Howard M, Copeland NG et al. (1993) Mutation of
unique region of Bruton’s tyrosine kinase in immuno-
deficient XID mice. Science 261, 358–361.

13 Vihinen M, Belohradsky BH, Haire RN, Holinski-Feder
E, Kwan SP, Lappalainen I, Lehvaslaiho H, Lester T,
Meindl A, Ochs HD et al. (1997) BTKbase, mutation
database for X-linked agammaglobulinemia (XLA).
Nucleic Acids Res 25, 166–171.
14 Ellmeier W, Jung S, Sunshine MJ, Hatam F, Xu Y,
Baltimore D, Mano H & Littman DR (2000) Severe
B cell deficiency in mice lacking the tec kinase family
members Tec and Btk. J Exp Med 192, 1611–1624.
15 Salim K, Bottomley MJ, Querfurth E, Zvelebil MJ,
Gout I, Scaife R, Margolis RL, Gigg R, Smith CI,
Driscoll PC et al. (1996) Distinct specificity in the
recognition of phosphoinositides by the pleckstrin
homology domains of dynamin and Bruton’s tyrosine
kinase. EMBO J 15
, 6241–6250.
16 Franke TF, Kaplan DR, Cantley LC & Toker A (1997)
Direct regulation of the Akt proto-oncogene product by
phosphatidylinositol-3,4-bisphosphate. Science 275 ,
665–668.
17 Sable CL, Filippa N, Filloux C, Hemmings BA &
Van Obberghen E (1998) Involvement of the pleckstrin
homology domain in the insulin-stimulated activation
of protein kinase B. J Biol Chem 273, 29600–29606.
18 Vihinen M, Nore BF, Mattsson PT, Backesjo CM, Nars
M, Koutaniemi S, Watanabe C, Lester T, Jones A,
Ochs HD et al. (1997) Missense mutations affecting a
conserved cysteine pair in the TH domain of Btk. FEBS
Lett 413, 205–210.
19 Valiaho J, Smith CI & Vihinen M (2006) BTKbase: the

mutation database for X-linked agammaglobulinemia.
Hum Mutat 27, 1209–1217.
20 Conley ME, Dobbs AK, Farmer DM, Kilic S, Paris K,
Grigoriadou S, Coustan-Smith E, Howard V &
A. Hussain et al. TEC kinases and disease
FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS 2007
Campana D (2009) Primary B cell immunodeficiencies:
comparisons and contrasts. Annu Rev Immunol 27,
199–227.
21 Lindvall JM, Blomberg KE, Valiaho J, Vargas L, Hei-
nonen JE, Berglof A, Mohamed AJ, Nore BF, Vihinen
M & Smith CI (2005) Bruton’s tyrosine kinase: cell biol-
ogy, sequence conservation, mutation spectrum, siRNA
modifications, and expression profiling. Immunol Rev
203, 200–215.
22 Holinski-Feder E, Weiss M, Brandau O, Jedele KB,
Nore B, Backesjo CM, Vihinen M, Hubbard SR, Beloh-
radsky BH, Smith CI et al. (1998) Mutation screening
of the BTK gene in 56 families with X-linked agamma-
globulinemia (XLA): 47 unique mutations without cor-
relation to clinical course. Pediatrics 101, 276–284.
23 Perez de Diego R, Bravo J, Allende LM, Lopez-Grana-
dos E, Rivera J, Ferreira A, Fontan G & Garcia Rodri-
guez MC (2008) Identification of novel non-pathogenic
mutation in SH3 domain of Btk in an XLA patient.
Mol Immunol 45, 301–303.
24 Hansson H, Mattsson PT, Allard P, Haapaniemi P,
Vihinen M, Smith CI & Hard T (1998) Solution struc-
ture of the SH3 domain from Bruton’s tyrosine kinase.
Biochemistry 37, 2912–2924.

25 Wood PM, Mayne A, Joyce H, Smith CI, Granoff DM
& Kumararatne DS (2001) A mutation in Bruton’s
tyrosine kinase as a cause of selective anti-polysaccha-
ride antibody deficiency. J Pediatr 139, 148–151.
26 Ochs HD & Smith CI (1996) X-linked agammaglobulin-
emia. A clinical and molecular analysis. Medicine
(Baltimore) 75, 287–299.
27 Plebani A, Soresina A, Rondelli R, Amato GM, Azzari
C, Cardinale F, Cazzola G, Consolini R, De Mattia D,
Dell’Erba G et al. (2002) Clinical, immunological, and
molecular analysis in a large cohort of patients with
X-linked agammaglobulinemia: an Italian multicenter
study. Clin Immunol 104, 221–230.
28 Ellmeier W, Abramova A & Schebesta A (2011) Tec
family kinases: regulation of FcepsilonRI-mediated
mast cell activation. FEBS J 278, 1990–2000.
29 Quartier P, Debre M, De Blic J, de Sauverzac R, Say-
egh N, Jabado N, Haddad E, Blanche S, Casanova JL,
Smith CI et al. (1999) Early and prolonged intravenous
immunoglobulin replacement therapy in childhood
agammaglobulinemia: a retrospective survey of 31
patients. J Pediatr 134, 589–596.
30 Gardulf A, Andersen V, Bjorkander J, Ericson D,
Froland SS, Gustafson R, Hammarstrom L, Jacobsen
MB, Jonsson E, Moller G et al. (1995) Subcutaneous
immunoglobulin replacement in patients with primary
antibody deficiencies: safety and costs. Lancet 345,
365–369.
31 Misbah S, Sturzenegger MH, Borte M, Shapiro RS,
Wasserman RL, Berger M & Ochs HD (2009) Subcuta-

neous immunoglobulin: opportunities and outlook. Clin
Exp Immunol 158(Suppl 1), 51–59.
32 Honigberg LA, Smith AM, Sirisawad M, Verner E,
Loury D, Chang B, Li S, Pan Z, Thamm DH, Miller
RA et al. (2010) The Bruton tyrosine kinase inhibitor
PCI-32765 blocks B-cell activation and is efficacious in
models of autoimmune disease and B-cell malignancy.
Proc Natl Acad Sci USA 107, 13075–13080.
33 Lo HY (2010) Itk inhibitors: a patent review. Expert
Opin Ther Pat 20, 459–469.
34 Sahu N & August A (2009) ITK inhibitors in inflamma-
tion and immune-mediated disorders. Curr Top Med
Chem 9, 690–703.
35 Huck K, Feyen O, Niehues T, Ruschendorf F, Hubner
N, Laws HJ, Telieps T, Knapp S, Wacker HH, Meindl
A et al. (2009) Girls homozygous for an IL-2-inducible
T cell kinase mutation that leads to protein deficiency
develop fatal EBV-associated lymphoproliferation.
J Clin Invest 119, 1350–1358.
36 Siliciano JD, Morrow TA & Desiderio SV (1992) itk, a
T-cell-specific tyrosine kinase gene inducible by interleu-
kin 2. Proc Natl Acad Sci USA 89, 11194–11198.
37 Gibson S, Leung B, Squire JA, Hill M, Arima N, Goss
P, Hogg D & Mills GB (1993) Identification, cloning,
and characterization of a novel human T-cell-specific
tyrosine kinase located at the hematopoietin complex
on chromosome 5q. Blood 82, 1561–1572.
38 Mueller C & August A (2003) Attenuation of
immunological symptoms of allergic asthma in mice
lacking the tyrosine kinase ITK. J Immunol 170,

5056–5063.
39 Matsumoto Y, Oshida T, Obayashi I, Imai Y, Matsui
K, Yoshida NL, Nagata N, Ogawa K, Obayashi M,
Kashiwabara T et al. (2002) Identification of highly
expressed genes in peripheral blood T cells from
patients with atopic dermatitis. Int Arch Allergy
Immunol 129, 327–340.
40 Gomez-Rodriguez J, Kraus ZJ & Schwartzberg PL
(2011) Tec family kinases Itk and Rlk ⁄ Txk in T lym-
phocytes: cross-regulation of cytokine production and
T cell fates. FEBS J 278, 1980–1989.
41 Qi Q, Kannan AK & August A (2011) Tec family
kinases: Itk signaling and the development of NKT
alphabeta and gammadelta T cells. FEBS J 278,
1970–1979.
42 Stepensky P, Weintraub M, Yanir A, Revel-Vilk S,
Krux F, Huck K, Linka RM, Shaag A, Elpeleg O,
Borkhardt A et al. (2011) IL-2-inducible T-cell kinase
deficiency: clinical presentation and therapeutic
approach. Haematologica 96, 472–476.
43 Joseph RE, Min L & Andreotti AH (2007) The linker
between SH2 and kinase domains positively regulates
catalysis of the Tec family kinases. Biochemistry 46,
5455–5462.
TEC kinases and disease A. Hussain et al.
2008 FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS
44 Guo S, Wahl MI & Witte ON (2006) Mutational analy-
sis of the SH2-kinase linker region of Bruton’s tyrosine
kinase defines alternative modes of regulation for cyto-
plasmic tyrosine kinase families. Int Immunol 18, 79–87.

45 Lappalainen I, Thusberg J, Shen B & Vihinen M (2008)
Genome wide analysis of pathogenic SH2 domain
mutations. Proteins 72, 779–792.
46 Rigaud S, Fondaneche MC, Lambert N, Pasquier B,
Mateo V, Soulas P, Galicier L, Le Deist F, Rieux-Lau-
cat F, Revy P et al. (2006) XIAP deficiency in humans
causes an X-linked lymphoproliferative syndrome.
Nature 444, 110–114.
47 Pasquier B, Yin L, Fondaneche MC, Relouzat F,
Bloch-Queyrat C, Lambert N, Fischer A, de Saint-
Basile G & Latour S (2005) Defective NKT cell devel-
opment in mice and humans lacking the adapter SAP,
the X-linked lymphoproliferative syndrome gene
product. J Exp Med 201, 695–701.
48 Kakimi K, Guidotti LG, Koezuka Y & Chisari FV
(2000) Natural killer T cell activation inhibits hepati-
tis B virus replication in vivo. J Exp Med 192 , 921–930.
49 Felices M & Berg LJ (2008) The Tec kinases Itk and
Rlk regulate NKT cell maturation, cytokine production,
and survival. J Immunol 180, 3007–3018.
50 Au-Yeung BB & Fowell DJ (2007) A key role for Itk in
both IFN gamma and IL-4 production by NKT cells.
J Immunol 179, 111–119.
51 Atherly LO, Lucas JA, Felices M, Yin CC, Reiner SL
& Berg LJ (2006) The Tec family tyrosine kinases Itk
and Rlk regulate the development of conventional
CD8+ T cells. Immunity 25, 79–91.
52 Broussard C, Fleischacker C, Horai R, Chetana M,
Venegas AM, Sharp LL, Hedrick SM, Fowlkes BJ &
Schwartzberg PL (2006) Altered development of CD8+

T cell lineages in mice deficient for the Tec kinases Itk
and Rlk. Immunity 25, 93–104.
53 Blomberg KE, Boucheron N, Lindvall JM, Yu L,
Raberger J, Berglof A, Ellmeier W & Smith CE (2009)
Transcriptional signatures of Itk-deficient CD3+,
CD4+ and CD8+ T-cells. BMC Genomics 10, 233.
54 Raberger J, Schebesta A, Sakaguchi S, Boucheron N,
Blomberg KE, Berglof A, Kolbe T, Smith CI, Rulicke
T & Ellmeier W (2008) The transcriptional regulator
PLZF induces the development of CD44 high memory
phenotype T cells. Proc Natl Acad Sci USA 105,
17919–17924.
55 Weinreich MA, Odumade OA, Jameson SC & Hogquist
KA (2010) T cells expressing the transcription factor
PLZF regulate the development of memory-like CD8+
T cells. Nat Immunol 11, 709–716.
56 Huck K, Feyen O, Ruschendorf F, Knapp S, Niehues
T, Synaeve C, Latour S, Vettenranta K, Risse SL, Krux
F et al. (2010) A novel immunodeficiency due to muta-
tions in ITK causes an EBV-associated lymphoprolifer-
ative disease in children. In XIVth Meeting of the
European Society for Immunodeficiencies (Casanova JL
& Kutukculer N, eds), p 56. Topkon Congress Services,
Kadikoy-Istanbul, Turkey.
57 Piot P, Bartos M, Ghys PD, Walker N & Schwartland-
er B (2001) The global impact of HIV ⁄ AIDS. Nature
410, 968–973.
58 Elbashir SM, Harborth J, Lendeckel W, Yalcin A,
Weber K & Tuschl T (2001) Duplexes of 21-nucleotide
RNAs mediate RNA interference in cultured mamma-

lian cells. Nature 411, 494–498.
59 Novina CD, Murray MF, Dykxhoorn DM, Beresford
PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shan-
kar P & Sharp PA (2002) siRNA-directed inhibition of
HIV-1 infection. Nat Med 8, 681–686.
60 Arteaga HJ, Hinkula J, van Dijk-Hard I, Dilber MS,
Wahren B, Christensson B, Mohamed AJ & Smith CI
(2003) Choosing CCR5 or Rev siRNA in HIV-1.
Nat Biotechnol 21, 230–231.
61 Yu L, Mohamed AJ, Simonson OE, Vargas L,
Blomberg KE, Bjorkstrand B, Arteaga HJ, Nore BF &
Smith CI (2008) Proteasome-dependent autoregulation
of Bruton tyrosine kinase (Btk) promoter via
NF-kappaB. Blood 111, 4617–4626.
62 Readinger JA, Schiralli GM, Jiang JK, Thomas CJ,
August A, Henderson AJ & Schwartzberg PL (2008)
Selective targeting of ITK blocks multiple steps of HIV
replication. Proc Natl Acad Sci USA 105, 6684–6689.
63 Yu L, Mohanram V, Simonson OE, Smith CI, Spetz
AL & Mohamed AJ (2009) Proteasome inhibitors block
HIV-1 replication by affecting both cellular and viral
targets. Biochem Biophys Res Commun 385, 100–105.
64 Yang H, Zonder JA & Dou QP (2009) Clinical develop-
ment of novel proteasome inhibitors for cancer treat-
ment. Expert Opin Investig Drugs 18, 957–971.
65 Kulathu Y, Grothe G & Reth M (2009) Autoinhibition
and adapter function of Syk. Immunol Rev 232, 286–
299.
66 Geijtenbeek TB & Gringhuis SI (2009) Signalling
through C-type lectin receptors: shaping immune

responses. Nat Rev Immunol 9, 465–479.
67 Mocsai A, Ruland J & Tybulewicz VL (2010) The SYK
tyrosine kinase: a crucial player in diverse biological
functions. Nat Rev Immunol 10, 387–402.
68 Kuno Y, Abe A, Emi N, Iida M, Yokozawa T, Towa-
tari M, Tanimoto M & Saito H (2001) Constitutive
kinase activation of the TEL–Syk fusion gene in myelo-
dysplastic syndrome with t(9;12)(q22;p12). Blood 97,
1050–1055.
69 Streubel B, Vinatzer U, Willheim M, Raderer M &
Chott A (2006) Novel t(5;9)(q33;q22) fuses ITK to
SYK in unspecified peripheral T-cell lymphoma. Leuke-
mia 20, 313–318.
70 Rigby S, Huang Y, Streubel B, Chott A, Du MQ,
Turner SD & Bacon CM (2009) The lymphoma-associ-
ated fusion tyrosine kinase ITK–SYK requires pleck-
A. Hussain et al. TEC kinases and disease
FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS 2009
strin homology domain-mediated membrane localiza-
tion for activation and cellular transformation. J Biol
Chem 284, 26871–26881.
71 Hussain A, Faryal R, Nore BF, Mohamed AJ & Smith
CI (2009) Phosphatidylinositol-3-kinase-dependent
phosphorylation of SLP-76 by the lymphoma-associated
ITK–SYK fusion-protein. Biochem Biophys Res
Commun 390, 892–896.
72 Pechloff K, Holch J, Ferch U, Schweneker M, Brunner
K, Kremer M, Sparwasser T, Quintanilla-Martinez L,
Zimber-Strobl U, Streubel B et al. (2010) The fusion
kinase ITK–SYK mimics a T cell receptor signal and

drives oncogenesis in conditional mouse models of
peripheral T cell lymphoma. J Exp Med 207, 1031–
1044.
73 Dierks C, Adrian F, Fisch P, Ma H, Maurer H, Her-
chenbach D, Forster CU, Sprissler C, Liu G, Rottmann
S et al. (2010) The ITK–SYK fusion oncogene induces
a T-cell lymphoproliferative disease in mice mimicking
human disease. Cancer Res 70, 6193–6204.
TEC kinases and disease A. Hussain et al.
2010 FEBS Journal 278 (2011) 2001–2010 ª 2011 The Authors Journal compilation ª 2011 FEBS