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

1 Hospital, Nanjing Medical University, Huaian, Jiangsu, China 4 Faculty of Science Biology, Universiti Brunei Darussalam, Gadong, Brunei Darussalam Introduction TEC family kinases TFKs

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 Hussain1,2,*, Liang Yu1,3,*, Rani Faryal1,2, Dara K Mohammad1, Abdalla J Mohamed1,4 and C I Edvard Smith1

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 signalconcentrat-ing, 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: liang.yu@ki.se

*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, originatcarry-ing from a translocation joincarry-ing genes encodcarry-ing 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.

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 funcinforma-tion, 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 cyspro-teine 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.

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 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

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 misnon-sense 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

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

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,

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

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

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