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ABSTRACT ITK-SYK and TEL-SYK (also known as ETV6-SYK) are human tumor-causing chimeric proteins containing the kinase region of SYK, and the membrane-targeting, N-terminal, PH-TH

domain-doublet of ITK or the dimerizing SAM-PNT domain of TEL, respectively. ITK-SYK causes peripheral T cell lymphoma, while TEL-SYK was reported in myelodysplastic syndrome. BTK is a

kinase highly related to ITK and to further delineate the role of the N-terminus, we generated the corresponding fusion-kinase BTK-SYK. By generating and analyzing these fusion kinases, we

aim to understand the contribution of N-terminal domains to their distinct cellular behavior and oncogenic properties. The fusion kinases were found to behave differently. TEL-SYK showed

stronger oncogenic capacity when compared with ITK-SYK and BTK-SYK. Furthermore, ITK-SYK and BTK-SYK triggered IL-3-independent growth of BAF3 pro-B cells. In contrast to BTK-SYK and

TEL-SYK, which predominantly localized in perinuclear region and cytoplasm respectively, ITK-SYK exhibits a more diverse cellular distribution, being present in the nucleus, cytoplasm and

membrane-bound compartments. Notably, we observed that ITK-SYK undergoes activation-mediated nuclear translocation, a phenomenon that is uncommon among kinases. This unique feature of

ITK-SYK is therefore of particular interest due to its potential connection to its transforming capability. SIMILAR CONTENT BEING VIEWED BY OTHERS FUNCTIONAL CHARACTERIZATION OF NPM1–TYK2

FUSION ONCOGENE Article Open access 18 January 2022 AN HUR MUTANT, HUR-V225I, IDENTIFIED IN ADULT T-CELL LEUKEMIA/LYMPHOMA, ALTERS THE PRO-APOPTOTIC FUNCTION OF HUR Article Open access 18

December 2024 TNK1 IS A UBIQUITIN-BINDING AND 14-3-3-REGULATED KINASE THAT CAN BE TARGETED TO BLOCK TUMOR GROWTH Article Open access 09 September 2021 INTRODUCTION Chromosomal alterations

have often resulted in the chimeragenesis and deregulation of various proteins culminating in cancer1,2. Until now, spleen tyrosine kinase (SYK) has been described in two different

chromosomal translocation events that give rise to chimeric oncogenes, TEL-SYK and ITK-SYK3,4. ITK-SYK was recurrently identified in a subset of peripheral T cell lymphomas, while

translocation leading to TEL-SYK has been documented in 2 patients3,5 with myelodysplastic syndrome. Both fusion kinases are potent oncogenes3,4,6,7,8,9,10. Moreover, aberrant/over

expression of SYK has also been reported in both T and B cell lymphomas11,12,13,14. SYK comprises N-terminal, tandem SRC homology 2 (SH2) domains connected via the linker-A region, while the

linker-B region joins them with the C-terminal kinase domain15,16. SYK is expressed in a wide variety of cells including T and B cells and plays critical roles in immune receptor

signaling17,18,19. Following B and T cell receptor ligation, SYK can be activated either by phosphorylation at key tyrosine residues in the linker-regions or by binding to immune receptor

tyrosine based activation motifs, ITAMs18,20,21. In resting cells, SYK retains a “closed” conformation, where the tandem SH2 domains fold together with the kinase domain adapting a so-called

“linker-kinase sandwich”15,18,20. Fusion-kinases of SYK on the other hand lack tandem SH2 domains altering intramolecular SYK regulatory mechanisms. Thus, in ITK-SYK, the tandem SH2 domains

are replaced by the Pleckstrin homology, Tec homology (PH-TH) domain-doublet from ITK. The PH-TH doublet is fused to the major part of the linker-B region4. Based on cell lines and

transgenic animal studies, the ITK-SYK chimera is known to be non-auto inhibited and therefore constitutively active4,6,7,8,9,22,23. It has a highly efficient phosphorylation capacity for

the B- and T cell adapter proteins BLNK (B cell linker), also known as SLP-65, and for SLP-76 (SH2 domain containing leukocyte protein of 76 kDa), respectively. Initially, it was thought

that, like TEC family kinases, membrane localization of ITK-SYK leads to phosphorylation and, thus, constitutive kinase activation culminating in oncogenesis. However, intriguingly, a PH

domain mutant that lacks membrane-tethering still causes T cell lymphomas in an animal model6. Interestingly, a study in transgenic mice showed that CD19 promoter-driven B cell expression of

ITK-SYK caused T cell lymphoma, but no B cell malignancies, presumably through leaky expression in T-cells8. To replicate this approach in B cells, we replaced the PH-TH domain doublet of

ITK-SYK with the corresponding part of BTK, a kinase highly related to ITK, but whose function instead is crucial for B cell development24. The aim of the current study is to determine

whether the BTK-SYK fusion would behave differently from ITK-SYK in terms of transforming capacity, localization and phosphorylation of key tyrosines. The PH-TH domain-doublet of BTK

comprises 196 residues and contains two proline-rich regions (PRRs), compared to that of ITK with a single PRR, resulting in a shorter length of 165 amino acids. ITK-SYK and TEL-SYK fusions

share most of the SYK linker-B region and the entire kinase domain. However, the linker-B region is 40 amino acids shorter in ITK-SYK. N-terminal regions in these chimeras are entirely

different. In TEL-SYK, the N-terminus is formed by the dimerizing SAM-PNT domain, which is derived from TEL/ETV63,10. TEL-SYK acquires constitutive activation due to SAM-PNT domain-mediated

dimerization and subsequent auto-phosphorylation. STAT5 is the main downstream target of TEL-SYK25,26, which also causes constitutive activation of PI3-kinase and AKT3,10. Furthermore,

TEL-SYK can transform pre-B cells resulting in IL-3 independent growth, in vitro13. For ITK-SYK downstream signaling comprises of STAT327,28 and in the tumor microenvironment IL-6 seems to

play a crucial role28. In this study, we have compared the role of N-terminal region domains in regulating SYK fusion kinases in terms of phosphorylation, activation, stability and

localization. Unexpectedly, we found that ITK-SYK has the unique capacity to translocate to the nucleus upon activation. MATERIALS AND METHODS PLASMID CONSTRUCTS The following fusion kinase

constructs were analyzed in this study; BTK-SYK, BTK-SYK-KD, BTK-SYK R28C, ITK-SYK, ITK-SYK-KD, ITK-SYK R29C, TEL-SYK, Δ-TEL-SYK and SLP-76. ITK-SYK, ITK-SYK-KD, and SLP-76 have been

previously described7,23. BTK-SYK, BTK-SYK-KD and PH-TH domains mutants ITK-SYK-R29C and BTK-SYK-R28C were created by site-directed mutagenesis and cloned in the pCDNA3 mammalian expression

vector. The TEL-SYK and Δ-TEL-SYK were kind gifts of Dr. Akihiro Abe, Nagoya University School of Medicine, Japan. All constructs were sequenced and the presence of the corresponding protein

was verified. CELL LINES, REAGENTS AND TRANSFECTION COS-7 (African green monkey fibroblast-like kidney), HEK-293T (Human embryonic kidney cells), NIH3T3 (mouse embryonic fibroblast), SYF

(mouse embryonic fibroblast cells deficient in SRC, YES and FYN kinases), Jurkat (human T-cell leukemia cell line) and BAF3 (murine interleukin-3 dependent pro-B cell line) cells were

obtained from the American Type Culture Collection (ATCC). COS-7, HEK-293T, SYF and NIH3T3 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum. BAF3 and

Jurkat cells were cultured in RPMI 1640 medium with supplements. All cells were cultivated at 37 °C in a humidified 5% CO2 incubator. COS-7, HEK-293T, NIH3T3 and SYF cells were transfected

using Polyethylene imine (PEI) as previously described5,23. BAF3 and Jurkat cells were transfected using the Neon electroporation system23. IL-3-independent growth of BAF3 cells was

evaluated following transfection with ITK-SYK, BTK-SYK or mock pEGFP as control. Cells were washed with cell culture medium, free from IL-3 and electroporated. Nuclear and cytoplasmic

fractionation was done by using a fractionation reagent from Thermo Scientific. ANTIBODIES Monoclonal mouse anti-SYK (4D10) from Santa Cruz Biotechnology. The phospho-SYK antibodies Tyr-352,

and Tyr-525/526, SRC antibody and SLP-76 antibody were purchased from Cell Signaling Technology. Anti-SLP-76 pY128 was from BD Biosciences Pharmingen. Secondary antibodies goat

anti-mouse-800CW, goat anti-rabbit-800CW, goat anti-mouse-680LT, and goat anti-rabbit- 680 were from LI-COR Biosciences. Membranes were scanned using Odyssey Imager (LI-COR Biosciences).

IMMUNOFLUORESCENCE AND MICROSCOPY Cells were seeded overnight at 50% confluence in six-well dishes. The next day, cells were transfected with plasmids and incubated for 48 h. Cells were

fixed and permeabilized and immunofluorescence staining was performed as previously described29. IMMUNOBLOTTING Samples were prepared for Western blotting as described previously30. Briefly,

48 h post-transfection, cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, separated by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose

membranes, followed by incubation with primary and secondary antibodies. The membranes were scanned using Odyssey Imager from LI-COR Biosciences GmbH. HEALING ASSAY We used µ-Dish 35 mm,

high, with a Culture Insert (from ibidi) for wound healing assay. Cells were transfected with the plasmids, and after 24 h, a suspension of the transfected cells was placed in both wells of

the silicon-insert, and incubated for additional 24 h at 37 °C and 5% CO2. Then the silicone-insert was removed using sterile tweezers, and a cell free gap of 500 µm was created, and cell

images were captured at regular intervals using 4X dry objective under a fluorescence microscope. STATISTICS Statistical analysis was performed using GraphPad Prism10 Software. Statistical

significance was determined using 1-way ANOVA, followed by the Duncan comparison test. _P_ ≤ 0.05 was considered statistically significant. RESULTS SYK FUSION KINASES SHOW EQUAL STABILITY

AND ACTIVATION ITK-SYK and BTK-SYK both acquire their N-terminal PH-TH domain from the TEC family kinases (TFKs) ITK and BTK, respectively, while dimerizing SAM-PNT domain forms the

N-terminus of TEL-SYK. The C-terminal catalytic domain and the adjacent linker-B region of the fusion kinases are derived from SYK (Fig. 1A). The PH domain is known to play a key role in the

activation of TFKs by tethering them onto plasma membrane phospholipids31,32,33,34. Despite a high degree of conservation between the N-terminal region of ITK and BTK, subtle

characteristics may still result in functional differences22,35,36,37. To compare the attributes conferred by the PH-TH domains of ITK and BTK, and the SAM-PNT domain of TEL-SYK in fusion

kinases, constructs encoding ITK-SYK, BTK-SYK and TEL-SYK were co-expressed with the adapter protein SLP-76. SYK fusion kinases were equally phosphorylated at Tyrosine 352 and 525/526 in the

linker-region and activation-loop that play a key role in the activation of SYK, and they potently trans-phosphorylated the SLP-76 substrate in COS-7 cells (Fig. 1B, Supplementary Figure

S1A). Moreover, for ITK-SYK, in addition to the 55 kDa full length protein, a shorter, presumably truncated version corresponding to 45 kDa, is generated. In contrast, the expression of

BTK-SYK leads to the production of a single detectable molecule, which, as expected, has a slightly higher molecular weight than ITK-SYK. Since SYK has been shown to produce a shorter

isoform of 40 kDa, we set out to investigate whether the shorter band of ITK-SYK is due to a second translation initiation site or to a proteolytic event38. Inspection of the primary

sequence of ITK-SYK reveals an additional in-frame translation initiation codon in the PH-TH doublet. Substitution of the corresponding methionine for leucine in the form of the

ITK-SYK-M126L mutant was generated to abolish this second translation initiation site. Expression of ITK-SYK-M126L in COS-7 cells resulted in the production of only the predicted longer

ITK-SYK, suggesting that alternative initiation and not proteolysis is the underlying mechanism (Fig. 1C). While inspection of the BTK-SYK sequence also revealed potential additional

translation initiation codons, we were unable to see shorter isoforms following the expression of the constructs in all tested cell types. Although, we envisaged that a shorter isoform is

also produced in cells expressing BTK-SYK, it might be subject to rapid degradation making its detection difficult. To determine whether a shortened isoform is degraded by the ubiquitin

proteasome pathway, we expressed constructs encoding ITK-SYK and BTK-SYK in COS7 cells, in the presence, or absence, of the proteasome inhibitor MG132. The two shorter isoforms of BTK-SYK

were detected in the presence of MG132, thus suggesting their rapid degradation in the absence of proteasome inhibition (Fig. 1D). SYK FUSION KINASES ARE PHOSPHORYLATED BY SRC AND INHIBITED

BY SYK AND PI3-KINASE INHIBITORS SRC family kinases (SFK) initiate intracellular signaling in response to different antigen receptor ligation. This is followed by recruitment and binding of

SYK to the phosphorylated tyrosine (pY) on ITAM residues, and the activated SYK will subsequently phosphorylate downstream substrates. To investigate the role of SFK in the phosphorylation

and activation of ITK-SYK, BTK-SYK and TEL-SYK, we used an SFK-deficient cell line, SYF. In SYF cells, ITK-SYK and BTK-SYK did not show any detectable phosphorylation, while TEL-SYK showed

some phosphorylation. However, when expression plasmids were co-transfected with plasmids expressing SRC, cells showed detectable phosphorylation of the fusion constructs. Interestingly,

phosphorylation of SLP-76 was seen in the presence or absence of SRC (Fig. 2A, Supplementary Figure S1B). In the next set of experiments, we compared the phosphorylation capacity of the

kinases in the presence or absence of different inhibitors. R406 (the active metabolite of fostamatinib) is a competitive inhibitor of ATP for the SYK kinase. All chimeras were expressed in

COS7 cells and R406 was added to compare its potential suppressive effect for SYK fusion kinase activity. Upon the addition of R406, the phosphorylation of the fusion kinases at Y525/526,

but not at Y352, was reduced (Fig. 2B, Supplementary Figure S1C). ITK-SYK is believed, like the native TFKs, to be regulated by the PI3-kinase signaling pathway (5,7,22). To determine

whether activation of the BTK-SYK fusion is also PI3-kinase dependent, we employed the PI3-kinase inhibitor LY294002. PI3-kinase inhibition minimized ITK-SYK, BTK-SYK, and to much lower

extent, TEL-SYK phosphorylation at Y525/526 (Fig. 2B, Supplementary Figure S1C). Furthermore, we compared the oncogenic potential of the fusion kinases by using wound-healing assay. Cells

expressing TEL-SYK showed stronger oncogenic capacity, as measured by rapid closing of the gap and greater proliferation and migration rate compared to ITK-SYK and BTK-SYK (Supplementary

Figure S2A,B). The BAF3 cell line is often exploited for its property of IL-3-dependent growth39. Several proteins with oncogenic potential have been analyzed using BAF3 as an in vitro

model40, and many oncogenes induce IL-3-independent growth of these cells. Expression of both ITK-SYK and BTK-SYK rendered proliferation of BAF3 cells IL-3-independent. However,

IL-3-independent growth was more pronounced in cells transfected with ITK-SYK (Supplementary Figure S2C). ITK-SYK SHOWS ROBUST STIMULATION-MEDIATED NUCLEAR TRANSLOCATION To further

investigate the role of N-terminal domains of the fusion kinases, we looked into their subcellular localization. We used fluorescence microscopy to study the localization of these proteins

in COS-7 cells both in steady state and after activation. In a steady state, ITK-SYK was predominantly in the cytoplasm, and only a small fraction was localized to the nucleus or the

membrane (Fig. 3A). Most BTK-SYK appeared perinuclearly, whereas TEL-SYK was predominantly cytoplasmic (Fig. 3A), as previously reported10. Upon activation of the cells, although, BTK-SYK

and TEL-SYK distribution largely remained unaffected, ITK-SYK showed robust nuclear translocation (Fig. 3B). We also found ITK-SYK in the membrane in a subset of cells, but the main part

accumulated in the nucleus (Table 1). In contrast, when we used SYF cells that completely lack SFKs, we found that all the fusion kinases were localized in the cytoplasm, both in steady

state and after activation (Fig. 3A,B). Upon nuclear-cytoplasmic fractionation for Jurkat cells transfected with the fusion kinases, again we found that only ITK-SYK was localized in the

nucleus and this nuclear localization increased after activation of the cells (Fig. 3C). Collectively these findings demonstrate that the ITK-SYK-fusion behaves differently compared to both

BTK-SYK and TEL-SYK. Since both the PH and the TH domains of BTK and ITK are unique, it is impossible to determine the origin of these differences conclusively. However, given that the

native SYK protein has a complex mode of regulation it is not surprising that varying the N-terminal portion of a SYK fusion protein could dramatically affect its function. NON-MEMBRANE

BINDING AND NON-DIMERIZING MUTANTS OF SYK FUSION KINASES RETAIN CATALYTIC ACTIVITY Idiosyncratic behavior of fusion-kinases like differential phosphorylation, and inhibition upon use of

PI3-kinase inhibitors or localization, prompted us to investigate the impact of loss-of-function mutations in the PH-domain (ITK-SYK-R29C and BTK-SYK-R28C), where point mutations into the

pleckstrin homology domain could alter the membrane-binding ability. The PH-domain plays a key role in activating TFKs22,41,42,43. Although a similar loss-of-function mechanism was thought

to operate also in the ITK-SYK activation, unexpectedly, ITK-SYK-R29C induced T cell lymphomas in a mouse model6. Despite cytoplasmic localization, we hypothesized that the loss-of-function

mutant ITK-SYK-R29C may sustain substrate phosphorylation. Likewise, it seemed possible that BTK-SYK-R28C could also be active since even if it did not translocate to the membrane upon

activation, it was equally phosphorylated, similar to the non-mutated form. Both fusion-kinases were co-expressed with the adapter protein SLP-76 in COS-7 cells. ITK-SYK-R29C as well as

BTK-SYK-R28C potently phosphorylated SLP-76 (Fig. 4). Moreover, the ΔTEL-SYK mutant, in which the dimerization domain was deleted, also strongly phosphorylated SLP-76 (Fig. 4). DISCUSSION

The non-receptor tyrosine kinase SYK is considered an “OR” switch, since it can be activated by phosphorylation at key tyrosines or ITAM binding20. Tec family kinases (TFK), on the other

hand, have been designated “AND” switches because they are activated in two steps; by PH-TH domain-mediated membrane binding and by SFK-mediated trans-phosphorylation20. Upon chromosomal

translocation, SYK contributes its kinase domain into two known fusion-proteins, ITK-SYK and TEL-SYK3,4. Constitutive/deregulated activation of both chimeras leads to oncogenesis.

Unexpectedly, B cell expression of ITK-SYK resulted in delayed development of T cell lymphomas instead of B cell malignancies8. This observation underlines the uniqueness of transforming

events. In order to study this further, we cloned BTK-SYK, as the corresponding fusion protein for B cells and compared it with ITK-SYK. Domains are functional and structural units of the

protein. These fusion kinases possess the same catalytic domain but different N-terminal region domains. In this study, we compared these fusion kinases in order to estimate what influence

these N-terminal domains will have when they are introduced in these kinases, also to find out if these domains keep and demonstrate the same characteristics as in their native forms. We

have compared the oncogenic potential of ITK-SYK, BTK-SYK and TEL-SYK by measuring the proliferation and migration rate, using a wound-healing assay. All fusion kinases induced the healing

process, and the wound gap filling. The oncogenic capacity was significantly stronger upon TEL-SYK expression compared to ITK-SYK or BTK-SYK expression. This finding, together with the other

observations on the activity of TEL-SYK, is of particular interest given the few reports of TEL-SYK mutations that have been reported to date. Thus, in 2001 Japanese researchers presented

the first case of TEL-SYK, as a Driver mutation for the Myelodysplastic Syndrome (MDS)4. Recently, a second patient was documented5. Given the enormous amount of sequenced patient samples to

date, it is remarkable that only 2 known MDS patients are carrying this Driver mutation. To this end, TEL(ETV6) can fuse with a number of partners; for many of these, there are numerous

reports of affected patients44,45. Considering that ITK-SYK failed to cause lymphomas in B cells in transgenic mice, we hypothesized that the BTK-SYK fusion may show stronger oncogenic

potential in a B cell milieu instead. The bone marrow-derived pro-B cell line BAF3 was considered a good candidate for this experiment. These cells are strictly dependent on IL-3 for their

survival and proliferation. However, following the expression of many oncogenes, BAF3 cells can grow readily in the absence of IL-3. Both ITK-SYK- and BTK-SYK-encoding plasmids rendered BAF3

cells IL-3-independent. ITK-SYK induced stronger IL-3-independent proliferation, suggesting that BTK-SYK might be less oncogenic even in a B cell setting. We then compared the inhibitor

sensitivity, and the SYK inhibitor (R406) was used to block the kinase activity of the fusion kinases. Although the phosphorylation at Y525/526 was inhibited, R406 did not block Y352

phosphorylation. The insensitivity of Y352 phosphorylation towards the SYK inhibitor could be due to trans-phosphorylation by SRC family kinases, which are insensitive to the SYK inhibitor.

Activation of many different receptors, with PI3-kinase as a common downstream denominator, results in PH-domain-dependent membrane localization of TFKs43. The well-known PI3-kinase

inhibitor LY294002 was tested to study the effects of PI3-kinase inhibition on the fusion kinases. The PI3-kinase inhibitor did not completely block the TEL-SYK phosphorylation, although the

drug potently inactivated ITK-SYK and BTK-SYK under the same conditions. These observations prompted us to compare the subcellular localization of the fusion kinases. Unlike BTK-SYK and

TEL-SYK, which localized predominantly in the perinuclear region and cytoplasm, respectively, ITK-SYK showed differential cellular localization, and was found in various cellular

compartments (nucleus, cytoplasm and membrane) (Table 1). Here, we report the activation- mediated nuclear translocation of ITK-SYK in COS-7 and Jurkat cells, using two different methods,

Western blotting and fluorescence microscopy. The robust nuclear translocation of ITK-SYK suggests a specific mechanism for this activation-induced translocation. There are significant

numbers of signaling proteins whose nuclear shuttling is induced by activation, like ERK, SMAD3, MEK, and Tet246,47. Upon stimulation these kinases translocate from the cytoplasm to the

nucleus. Importins also have a crucial role in nuclear translocation of proteins that do not contain a bona fide nuclear localization signal (NLS). Therefore we analyzed importin-7, and we

found that there is an interaction between ITK-SYK and Importin-7, but when we knocked down importin-7 expression by using specific siRNA, we did not find any detectable effect on ITK-SYK

nuclear translocation (data not shown). SFKs have been implicated in stimulation-mediated nuclear translocation of numerous receptor and non-receptor tyrosine kinases and transcription

factors48,49,50,51. Interestingly, when we used SYF cells for localization study, ITK-SYK was mainly found in the cytoplasm, even after activation. This strongly suggests that SFKs play a

role in regulating the stimulated nuclear translocation of ITK-SYK. This was intriguing, and we therefore decided to investigate the activity of the loss of function PH-domain mutant

ITK-SYK-R29C that despite its cytoplasmic localization in vitro, leads to lymphomas in a transgenic mouse model. Along with ITK-SYK-R29C, we also generated its counterpart BTK-SYK-R28C. We

further investigated the activity of the non-dimerizing ΔTEL-SYK mutant. All fusion kinase mutants potently phosphorylated the adapter protein SLP-76. Overall, in our study, we found that

ITK-SYK, BTK-SYK and TEL-SYK are constitutively active kinases that behave differently from each other in terms of phosphorylation, localization and transformation potential, seemingly

reflecting the differences of the N-terminal region, PH-TH domain-doublets of BTK and ITK and the SAM-PNT domain of TEL. Another finding is that the activation of SYK fusion-kinases is not

only dependent on the presence of intact PH-TH or SAM-PNT domains, but that the absence of an auto-inhibitory conformation may also be involved in activating such deregulated kinases. While

our study provides important insight into the constitutive activity and distinct behavior of SYK fusion kinases, a limitation of the data presented is that they primarily are derived from

over expression of these kinases in non-lymphoid cell lines. Hence, while these cell models are potentially valuable for dissecting molecular mechanisms; they may not fully recapitulate the

cellular context in which these kinases naturally function. Further studies using physiological expression and relevant models are needed to validate and extend our findings. DATA

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Swedish Cancer Society (CAN2013/389; 22 2361 Pj 01 H), the Swedish Medical Research Council (K2015-68X-11247-21-3) and the Swedish County Council (ALF-project 2012006; FoUI97465) and Center

for Innovative Medicine (CIMED). FUNDING Open access funding provided by Karolinska Institute. AUTHOR INFORMATION Author notes * Abdulrahman Hamasy and Alamdar Hussain contributed equally to

this work. AUTHORS AND AFFILIATIONS * Department of Laboratory Medicine, Karolinska Institutet, ANA Futura, Alfred Nobels Allé 8, Floor 8, 14152, Huddinge, Sweden Abdulrahman Hamasy, Qing

Wang, Manuela Gustafsson Sfetcovici, Beston F. Nore, Abdalla J. Mohamed, Rula Zain & C. I. Edvard Smith * Department of Clinical Analysis, College of Pharmacy, Hawler Medical University,

Erbil, Kurdistan Region, Iraq Abdulrahman Hamasy * Center for Hematology and Regenerative Medicine (HERM), Department of Medicine Huddinge, Karolinska Institutet, 141 83, Stockholm, Sweden

Alamdar Hussain & Dara K. Mohammad * College of Agricultural Engineering Sciences, Salahaddin University-Erbil, Erbil, Kurdistan Region, 44002, Iraq Dara K. Mohammad * Department of

Biomedical Science, Komar University of Science and Technology (KUST), Qliasan St, Sulaymaniyah City, Kurdistan Region, 46002, Iraq Beston F. Nore * Department of Basic Medical and Dental

Sciences, Faculty of Dentistry, Zarqa University, Zarqa, Jordan Abdalla J. Mohamed * Karolinska ATMP Center, Karolinska Institutet, Karolinska University Hospital, 171 76, Stockholm, Sweden

Rula Zain & C. I. Edvard Smith * Centre for Rare Diseases, Department of Clinical Genetics and Genomics, Karolinska University Hospital, Stockholm, Sweden Rula Zain Authors * Abdulrahman

Hamasy View author publications You can also search for this author inPubMed Google Scholar * Alamdar Hussain View author publications You can also search for this author inPubMed Google

Scholar * Dara K. Mohammad View author publications You can also search for this author inPubMed Google Scholar * Qing Wang View author publications You can also search for this author

inPubMed Google Scholar * Manuela Gustafsson Sfetcovici View author publications You can also search for this author inPubMed Google Scholar * Beston F. Nore View author publications You can

also search for this author inPubMed Google Scholar * Abdalla J. Mohamed View author publications You can also search for this author inPubMed Google Scholar * Rula Zain View author

publications You can also search for this author inPubMed Google Scholar * C. I. Edvard Smith View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS A.H., AL.H. and D.K.M. performed most of the experiments, analyzed data, and wrote the manuscript; Q.W., and M.O.G. performed some experiments and edited the manuscript;

B.F.N., A.J.M., and R.Z. were involved in planning the research and contributed to writing and editing the manuscript; and CIES conceived the project, designed the experiments, interpreted

data, revised the manuscript, and obtained research funding. CORRESPONDING AUTHORS Correspondence to Abdulrahman Hamasy or C. I. Edvard Smith. ETHICS DECLARATIONS COMPETING INTERESTS The

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http://creativecommons.org/licenses/by-nc-nd/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Hamasy, A., Hussain, A., Mohammad, D.K. _et al._ Differential regulatory

effects of the N-terminal region in SYK-fusion kinases reveal unique activation-inducible nuclear translocation of ITK-SYK. _Sci Rep_ 15, 814 (2025).

https://doi.org/10.1038/s41598-024-83962-8 Download citation * Received: 11 June 2024 * Accepted: 18 December 2024 * Published: 04 January 2025 * DOI:

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