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• W2002246336 abstract "ITK-SYK, a novel fusion tyrosine kinase (FTK) resulting from a recurrent t(5;9)(q33;q22), was recently identified in a poorly understood subset of peripheral T-cell lymphomas. However, the biochemical and functional properties of ITK-SYK are unknown. Here we demonstrate that ITK-SYK is a catalytically active tyrosine kinase that is sensitive to an established inhibitor of SYK. The expression of ITK-SYK, but not SYK, transformed NIH3T3 cells, inducing loss of contact inhibition and formation of anchorage-independent colonies in soft agar, in a kinase activity-dependent manner. ITK-SYK is unusual among FTKs in having an N-terminal phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology (PH) domain. Introduction of a well characterized loss-of-function mutation (R29C) into the PH domain of ITK-SYK inhibited its phosphorylation, markedly reduced its catalytic activity, and abrogated its ability to activate the ERK signaling pathway and to transform NIH3T3 cells. Although ITK-SYK was membrane-associated, ITK-SYK-R29C was not. However, each of these properties could be recovered by retargeting ITK-SYK-R29C back to the plasma membrane by the addition of an N-terminal myristylation sequence. Consistent with a model in which ITK-SYK requires PH domain-mediated binding to phosphatidylinositol 3,4,5-trisphosphate generated by phosphatidylinositol 3-kinase (PI3K), ITK-SYK activity was reduced by pharmacological inhibition of PI3K and increased by co-expression with a constitutively active form of PI3K. Together, these findings identify ITK-SYK as an active, transforming FTK dependent upon PH domain-mediated membrane localization, identify a novel mechanism for activation of an oncogenic FTK, and suggest ITK-SYK as a rational therapeutic target for t(5;9)(q33;q22)-positive lymphomas. ITK-SYK, a novel fusion tyrosine kinase (FTK) resulting from a recurrent t(5;9)(q33;q22), was recently identified in a poorly understood subset of peripheral T-cell lymphomas. However, the biochemical and functional properties of ITK-SYK are unknown. Here we demonstrate that ITK-SYK is a catalytically active tyrosine kinase that is sensitive to an established inhibitor of SYK. The expression of ITK-SYK, but not SYK, transformed NIH3T3 cells, inducing loss of contact inhibition and formation of anchorage-independent colonies in soft agar, in a kinase activity-dependent manner. ITK-SYK is unusual among FTKs in having an N-terminal phosphatidylinositol 3,4,5-trisphosphate-binding pleckstrin homology (PH) domain. Introduction of a well characterized loss-of-function mutation (R29C) into the PH domain of ITK-SYK inhibited its phosphorylation, markedly reduced its catalytic activity, and abrogated its ability to activate the ERK signaling pathway and to transform NIH3T3 cells. Although ITK-SYK was membrane-associated, ITK-SYK-R29C was not. However, each of these properties could be recovered by retargeting ITK-SYK-R29C back to the plasma membrane by the addition of an N-terminal myristylation sequence. Consistent with a model in which ITK-SYK requires PH domain-mediated binding to phosphatidylinositol 3,4,5-trisphosphate generated by phosphatidylinositol 3-kinase (PI3K), ITK-SYK activity was reduced by pharmacological inhibition of PI3K and increased by co-expression with a constitutively active form of PI3K. Together, these findings identify ITK-SYK as an active, transforming FTK dependent upon PH domain-mediated membrane localization, identify a novel mechanism for activation of an oncogenic FTK, and suggest ITK-SYK as a rational therapeutic target for t(5;9)(q33;q22)-positive lymphomas. The generation of constitutively active fusion tyrosine kinases (FTKs) 2The abbreviations used are: FTKfusion tyrosine kinasePHpleckstrin homologyPIP3phosphatidylinositol 3,4,5-trisphosphatePI3Kphosphatidylinositol 3-kinasePTCLperipheral T-cell lymphoma(s)BCRB-cell receptorDMEMDulbecco's modified Eagle's mediumMAPmitogen-activated proteinHAhemagglutininERKextracellular signal-regulated kinasePVpervanadateNBCSnewborn calf serum. 2The abbreviations used are: FTKfusion tyrosine kinasePHpleckstrin homologyPIP3phosphatidylinositol 3,4,5-trisphosphatePI3Kphosphatidylinositol 3-kinasePTCLperipheral T-cell lymphoma(s)BCRB-cell receptorDMEMDulbecco's modified Eagle's mediumMAPmitogen-activated proteinHAhemagglutininERKextracellular signal-regulated kinasePVpervanadateNBCSnewborn calf serum. by chromosomal translocation is a recurrent mechanism of oncogenesis in hematological neoplasms (1Chalandon Y. Schwaller J. Haematologica. 2005; 90: 949-968PubMed Google Scholar, 2Turner S.D. Alexander D.R. Leukemia. 2006; 20: 572-582Crossref PubMed Scopus (26) Google Scholar). In each case, FTKs comprise the C-terminal region of a tyrosine kinase, invariably including its catalytic domain, coupled to the N-terminal domains of a distinct “partner” protein. Well characterized examples include those involving the nonreceptor tyrosine kinase ABL and various N-terminal partners (most often BCR-ABL) in both chronic and acute leukemias (3Wong S. Witte O.N. Annu. Rev. Immunol. 2004; 22: 247-306Crossref PubMed Scopus (315) Google Scholar) and those involving the receptor tyrosine kinase ALK and one of several N-terminal partners (most frequently NPM-ALK) in ALK-positive anaplastic large cell lymphoma (4Chiarle R. Voena C. Ambrogio C. Piva R. Inghirami G. Nat. Rev. Cancer. 2008; 8: 11-23Crossref PubMed Scopus (675) Google Scholar). Understanding how such active FTKs are generated by chimerization of tyrosine kinases has shed considerable light on the molecular basis of these malignancies, and, as illustrated by the remarkable clinical activity of the ABL inhibitor imatinib, FTKs represent attractive targets for pharmacological intervention (3Wong S. Witte O.N. Annu. Rev. Immunol. 2004; 22: 247-306Crossref PubMed Scopus (315) Google Scholar). fusion tyrosine kinase pleckstrin homology phosphatidylinositol 3,4,5-trisphosphate phosphatidylinositol 3-kinase peripheral T-cell lymphoma(s) B-cell receptor Dulbecco's modified Eagle's medium mitogen-activated protein hemagglutinin extracellular signal-regulated kinase pervanadate newborn calf serum. fusion tyrosine kinase pleckstrin homology phosphatidylinositol 3,4,5-trisphosphate phosphatidylinositol 3-kinase peripheral T-cell lymphoma(s) B-cell receptor Dulbecco's modified Eagle's medium mitogen-activated protein hemagglutinin extracellular signal-regulated kinase pervanadate newborn calf serum. Peripheral T-cell lymphomas (PTCL) are clinically aggressive, often fatal, tumors arising from mature T-cells, for which current treatment strategies are inadequate (5Jaffe E.S. Harris N.L. Stein H. Vardiman J.W. World Health Organisation Classification of Tumours: Pathology and Genetics of Tumours of Haematopoietic and Lymphoid Tissues.. IARC Press, Lyon2001Google Scholar, 6Savage K.J. Blood Rev. 2007; 21: 201-216Crossref PubMed Scopus (78) Google Scholar). This is the result, at least in part, of a very limited understanding of their genetic and biochemical pathogenesis and a consequent paucity of rational therapeutic targets. Recently, Streubel et al. (7Streubel B. Vinatzer U. Willheim M. Raderer M. Chott A. Leukemia. 2006; 20: 313-318Crossref PubMed Scopus (209) Google Scholar, 8Huang Y. Moreau A. Dupuis J. Streubel B. Petit B. Le Gouill S. Martin-Garcia N. Copie-Bergman C. Gaillard F. Qubaja M. Fabiani B. Roncador G. Haioun C. Delfau-Larue M.-H. Marafioti T. Chott A. Gaulard P. Am. J. Surg. Pathol. 2009; 33: 682-690Crossref PubMed Scopus (162) Google Scholar) described a novel, recurrent t(5;9)(q33;q22) in a subset of PTCL derived from follicular helper T-cells and showed this to involve ITK and SYK, genes encoding two nonreceptor tyrosine kinases. In this study, all PTCL carrying an ITK-SYK translocation contained identical ITK-SYK transcripts in which the 5′ coding sequence of ITK was joined to the 3′ coding sequence of SYK, predicting a chimeric protein in which the N-terminal pleckstrin homology (PH) and TEC homology domains of ITK (amino acids 1–165) are fused to much of interdomain B and the complete C-terminal kinase domain of SYK (amino acids 306–635) (see Fig. 1). Immunohistochemical staining for SYK was consistent with expression of this fusion protein in such cases; however, whether ITK-SYK is capable of cellular transformation, whether it is an active tyrosine kinase, and the biochemical mechanisms of such activity have not been established. ITK and SYK both play key roles in lymphocyte antigen receptor signaling. ITK, a TEC family kinase, regulates several aspects of T-cell development, differentiation, and effector function (9Berg L.J. Finkelstein L.D. Lucas J.A. Schwartzberg P.L. Annu. Rev. Immunol. 2005; 23: 549-600Crossref PubMed Scopus (257) Google Scholar, 10Felices M. Falk M. Kosaka Y. Berg L.J. Adv. Immunol. 2007; 93: 145-184Crossref PubMed Scopus (50) Google Scholar). Upon T-cell receptor ligation, ITK is recruited to and activated at the immunological synapse. This requires binding of its PH domain to membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PIP3) generated by phosphatidylinositol 3-kinase (PI3K) (11August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar, 12Ching K.A. Kawakami Y. Kawakami T. Tsoukas C.D. J. Immunol. 1999; 163: 6006-6013PubMed Google Scholar, 13Woods M.L. Kivens W.J. Adelsman M.A. Qiu Y. August A. Shimizu Y. EMBO J. 2001; 20: 1232-1244Crossref PubMed Scopus (88) Google Scholar), association with the SLP-76/LAT-nucleated adaptor complex (14Bogin Y. Ainey C. Beach D. Yablonski D. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 6638-6643Crossref PubMed Scopus (64) Google Scholar), and phosphorylation by SRC family kinases such as LCK (15Heyeck S.D. Wilcox H.M. Bunnell S.C. Berg L.J. J. Biol. Chem. 1997; 272: 25401-25408Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). ITK subsequently plays important roles in calcium signaling and actin reorganization, the former through phosphorylation and activation of phospholipase Cγ (9Berg L.J. Finkelstein L.D. Lucas J.A. Schwartzberg P.L. Annu. Rev. Immunol. 2005; 23: 549-600Crossref PubMed Scopus (257) Google Scholar, 16Gomez-Rodriguez J. Readinger J.A. Viorritto I.C. Mueller K.L. Houghtling R.A. Schwartzberg P.L. Immunol. Rev. 2007; 218: 45-64Crossref PubMed Scopus (46) Google Scholar). In lymphocytes, SYK is expressed at low levels in T-cells but at high levels in developing and mature B-cells, in which it is required for pre-BCR and BCR signaling (17Chan A.C. van Oers N.S. Tran A. Turka L. Law C.L. Ryan J.C. Clark E.A. Weiss A. J. Immunol. 1994; 152: 4758-4766PubMed Google Scholar, 18Turner M. Schweighoffer E. Colucci F. Di Santo J.P. Tybulewicz V.L. Immunol. Today. 2000; 21: 148-154Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). Upon receptor ligation, SYK is activated by binding of its tandem N-terminal SH2 domains to diphosphorylated immunoreceptor tyrosine-based activation motifs, by subsequent phosphorylation (predominantly autophosphorylation) of tyrosine residues within the activation loop of the kinase domain, and by phosphorylation of additional tyrosine residues by SRC family kinases (19Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (222) Google Scholar, 20Rowley R.B. Burkhardt A.L. Chao H.G. Matsueda G.R. Bolen J.B. J. Biol. Chem. 1995; 270: 11590-11594Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 21Keshvara L.M. Isaacson C.C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar). SYK then binds and phosphorylates adaptor proteins and enzymes such as BLNK, VAV, phospholipase Cγ, and BTK to activate diverse downstream signaling pathways (22Deckert M. Tartare-Deckert S. Couture C. Mustelin T. Altman A. Immunity. 1996; 5: 591-604Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 23Law C.L. Chandran K.A. Sidorenko S.P. Clark E.A. Mol. Cell. Biol. 1996; 16: 1305-1315Crossref PubMed Google Scholar, 24Fu C. Turck C.W. Kurosaki T. Chan A.C. Immunity. 1998; 9: 93-103Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar, 25Baba Y. Hashimoto S. Matsushita M. Watanabe D. Kishimoto T. Kurosaki T. Tsukada S. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 2582-2586Crossref PubMed Scopus (107) Google Scholar). The discovery of an FTK involving SYK is not without precedent, because a TEL-SYK chimera resulting from a t(9;12)(q22;p12) in a patient with myelodysplastic syndrome/acute myeloid leukemia has been described (26Kuno Y. Abe A. Emi N. Iida M. Yokozawa T. Towatari M. Tanimoto M. Saito H. Blood. 2001; 97: 1050-1055Crossref PubMed Scopus (107) Google Scholar). This FTK contains part of the C-terminal SH2 domain and the complete kinase domain of SYK fused to the N terminus of TEL/ETV6, an ets family transcription factor that is the N-terminal fusion partner in various FTKs found in several types of hematopoietic neoplasm (27Bohlander S.K. Semin. Cancer Biol. 2005; 15: 162-174Crossref PubMed Scopus (140) Google Scholar). TEL-SYK is a constitutively active tyrosine kinase that induces cytokine independence when expressed in BaF3 cells and primary pre-B cells in vitro, the latter able to induce leukemia when subsequently injected into mice (26Kuno Y. Abe A. Emi N. Iida M. Yokozawa T. Towatari M. Tanimoto M. Saito H. Blood. 2001; 97: 1050-1055Crossref PubMed Scopus (107) Google Scholar, 28Wossning T. Herzog S. Köhler F. Meixlsperger S. Kulathu Y. Mittler G. Abe A. Fuchs U. Borkhardt A. Jumaa H. J. Exp. Med. 2006; 203: 2829-2840Crossref PubMed Scopus (57) Google Scholar). Like other FTKs containing TEL, the tyrosine phosphorylation and transforming ability of TEL-SYK requires homodimerization mediated by the conserved N-terminal helix-loop-helix pointed domain of TEL (26Kuno Y. Abe A. Emi N. Iida M. Yokozawa T. Towatari M. Tanimoto M. Saito H. Blood. 2001; 97: 1050-1055Crossref PubMed Scopus (107) Google Scholar). Indeed, the majority of oncogenic FTKs are activated by a common mechanism involving oligomerization via motifs in the N-terminal fusion partner and consequent autophosphorylation and constitutive activity, predominantly within the cytoplasm (1Chalandon Y. Schwaller J. Haematologica. 2005; 90: 949-968PubMed Google Scholar, 2Turner S.D. Alexander D.R. Leukemia. 2006; 20: 572-582Crossref PubMed Scopus (26) Google Scholar, 3Wong S. Witte O.N. Annu. Rev. Immunol. 2004; 22: 247-306Crossref PubMed Scopus (315) Google Scholar). Notably, however, ITK-SYK does not contain a recognized dimerization motif and instead is unusual among FTKs in containing a PH domain, presenting the possibility of a novel mechanism of FTK activation. In this study we have investigated the catalytic and transforming properties of ITK-SYK and examined the hypothesis that the PH domain might be an important determinant of the activation and downstream functions of this structurally unique FTK. Jurkat E6.1 T-cells and HEK-293T cells were grown in RPMI 1640, 10% fetal bovine serum. NIH3T3 cells were grown in DMEM, 10% NBCS. Anti-phospho-SYK (Tyr525/Tyr526) rabbit polyclonal, anti-phospho-p44/42 MAP kinase (Thr202/Tyr204) rabbit polyclonal, anti-p44/42 MAP kinase rabbit polyclonal, anti-phospho-AKT (Thr308) 244F9 rabbit monoclonal, and anti-AKT rabbit polyclonal antibodies were from Cell Signaling Technology/NEB (Hitchin, UK). Anti-SYK mouse monoclonal antibody 4D10 was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-HA mouse monoclonal antibody HA-7 was from Sigma-Aldrich (Gillingham, UK). Horseradish peroxidase-labeled rabbit anti-mouse and swine anti-rabbit monoclonal antibodies for Western blotting were from Dako (Ely, UK). Alexa Fluor 488-conjugated goat anti-mouse antibody for immunofluorescent staining was from Invitrogen. Sodium pervanadate was generated by adding 50 μl of 10 mm sodium orthovanadate to 450 μl of 10 mm H2O2. The PI3K inhibitor LY294002 and the SYK inhibitor Piceatannol (both from Calbiochem/Merck) were dissolved in Me2SO/phosphate-buffered saline as directed. The ITK-SYK plasmid constructs used in this study are depicted in Fig. 1. The full ITK-SYK cDNA sequence was cloned into pIRESpuro2 (Clontech Laboratories/Takara Bio Europe, Saint-Germain-en-Laye, France) by reverse transcription-PCR of RNA from a PTCL carrying a t(5;9)(q33;q22)/ITK-SYK (the index case of Streubel et al. (7Streubel B. Vinatzer U. Willheim M. Raderer M. Chott A. Leukemia. 2006; 20: 313-318Crossref PubMed Scopus (209) Google Scholar)) to create pIRESpuro2-ITK-SYK. pIRESpuro2-HA-ITK-SYK was generated by subcloning ITK-SYK into a pIRESpuro2 derivative containing an N-terminal HA tag (kindly provided by Dr. Alex Appert, University of Cambridge, Cambridge, UK). pIRESpuro2-HA-ITK-SYK-K262R and pIRESpuro2-HA-ITK-SYK-R29C were generated using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). pIRESpuro2-Myr-HA-ITK-SYK-R29C was generated by adding sequence encoding the first 12 amino acids (myristylation/palmitylation sequence, MGCVCSSNPEDDWMENIDVC) of chicken LCK to the 5′ end of HA-ITK-SYK-R29C by PCR. pMSCVpuro-HA-ITK-SYK, pMSCV-puro-HA-ITK-SYK-R29C, and pMSCVpuro-Myr-HA-ITK-SYK-R29C were generated by subcloning the respective inserts into pMSCVpuro (Clontech). Full-length human SYK cDNA (IMAGE clone 3542895) was obtained from Geneservice (Cambridge, UK) and subcloned into pMSCVpuro. pSG5-p110α-CAAX (29Wennström S. Downward J. Mol. Cell. Biol. 1999; 19: 4279-4288Crossref PubMed Scopus (254) Google Scholar) was generated by Dr. Stefan Wennstrom and used with the kind permission of Dr. Julian Downward (CRUK-LRI, London, UK). 293T cells were transiently transfected with pIRESpuro2-derived or pMSCVpuro-derived plasmids using Lipofectamine 2000 (Invitrogen). Oligoclonal NIH3T3 cell lines stably expressing HA-ITK-SYK, HA-ITK-SYK-R29C, Myr-HA-ITK-SYK-R29C, or SYK (or carrying empty pMSCVpuro) were generated by retroviral transduction using the Phoenix ecotropic helper-free producer line followed by selection in puromycin. Jurkat E6.1 cells stably expressing ITK-SYK were generated by electroporation with pIRESpuro2-ITK-SYK using a Bio-Rad GenePulser II at 250 V, 950 microfarads, followed by puromycin selection and cloning by limiting dilution. Cell lysates were prepared using Triton X-100 lysis buffer (1% Triton X-100, 50 mm Tris, pH 7.4, 300 mm NaCl, 2 mm EDTA, 1 μg/ml each of aprotinin, leupeptin, and pepstatin, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 20 mm NaFl). For immunoprecipitation, Triton X-100 lysates were incubated at 4 °C with antibody preadsorbed onto protein G-Sepharose beads (Amersham Biosciences). Western blotting of immunoprecipitated proteins or cell lysates resolved by SDS-PAGE and transferred to Immobilon P polyvinylidene difluoride membrane (Millipore, Billerica, MA) was performed using antibodies diluted in Tris-buffered saline, 0.1% Tween 20, 5% powdered nonfat milk and ECL (Amersham Biosciences). For in vitro kinase assays, post-immunoprecipitation Sepharose beads were used in a protein-tyrosine kinase assay kit (Sigma-Aldrich) in which phosphorylation of plate-bound poly-Glu-Tyr substrate was detected by horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody PT-66 using a PerkinElmer Victor plate reader at 490 nm. Transfected 293T cells, grown on poly-l-lysine-coated coverslips (BD Laboratories) were fixed with 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, blocked with bovine serum albumin, sequentially incubated with anti-HA primary and goat anti-mouse Alexa Fluor 488 secondary antibodies, washed, and mounted with 4′,6-diamidino-2-phenylindole 1 mounting medium (Abbott, Maidenhead, UK). The cells were visualized using a Leica TCS SP laser scanning confocal microscope. For focus formation assays, stably transfected NIH3T3 cell lines were plated at 80% density and cultured for 2 weeks with fresh medium every 2–3 days. Subsequently, the cells were fixed in 1% glutaraldehyde and stained with 0.5% Crystal Violet, 25% methanol. For soft agar colony formation assays, stably transfected NIH3T3 cell lines were seeded at 50,000 cells/well in 2 ml of DMEM, 10% NBCS, 0.35% agarose over a base of 2 ml of DMEM, 10% NBCS, 0.6% agarose in 6-well plates. 2 ml of DMEM, 10% NBCS was added on top of the agar, and the cells were cultured for 3 weeks, with replacement of covering medium every 2–3 days. Phosphorylation of paired, conserved tyrosine residues (Tyr525/Tyr526) in the activation loop of the kinase domain of SYK occurs predominantly by autophosphorylation, is required for maximal catalytic activity and for downstream signaling activity, and is considered a good marker of active SYK (19Kurosaki T. Johnson S.A. Pao L. Sada K. Yamamura H. Cambier J.C. J. Exp. Med. 1995; 182: 1815-1823Crossref PubMed Scopus (222) Google Scholar, 21Keshvara L.M. Isaacson C.C. Yankee T.M. Sarac R. Harrison M.L. Geahlen R.L. J. Immunol. 1998; 161: 5276-5283PubMed Google Scholar, 30Couture C. Williams S. Gauthier N. Tailor P. Mustelin T. Eur. J. Biochem. 1997; 246: 447-451Crossref PubMed Scopus (30) Google Scholar, 31El-Hillal O. Kurosaki T. Yamamura H. Kinet J.P. Scharenberg A.M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1919-1924Crossref PubMed Scopus (108) Google Scholar, 32Zhang J. Billingsley M.L. Kincaid R.L. Siraganian R.P. J. Biol. Chem. 2000; 275: 35442-35447Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Therefore, to begin to determine whether ITK-SYK is a functional tyrosine kinase, we examined the phosphorylation of these residues (corresponding to Tyr385/Tyr386 of ITK-SYK) using a phosphospecific antibody. ITK-SYK was transiently transfected into 293T cells (Fig. 2a) and stably expressed in the Jurkat T-cell line (Fig. 2b) with similar results. ITK-SYK immunoprecipitated from either cell line was constitutively phosphorylated on Tyr385/Tyr386 in the absence of an exogenous stimulus, suggesting that it is present in an active state in these cells. Treatment of cells with the broad tyrosine phosphatase inhibitor pervanadate, known to activate many cellular tyrosine kinases, resulted in increased phosphorylation at these residues. Overexpression of SYK in 293T cells resulted in a basal level of phosphorylation at Tyr525/Tyr526, which could be enhanced by pervanadate treatment (Fig. 2c). To further examine whether the catalytic activity of ITK-SYK itself could mediate phosphorylation of Tyr385/Tyr386, cells were transfected with either ITK-SYK or a “kinase dead” mutant (ITK-SYK-K262R; Fig. 1) in which an essential, conserved lysine residue in the ATP-binding pocket of the SYK kinase domain was mutated to arginine (Fig. 2d) (31El-Hillal O. Kurosaki T. Yamamura H. Kinet J.P. Scharenberg A.M. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1919-1924Crossref PubMed Scopus (108) Google Scholar, 33Hanks S.K. Hunter T. FASEB J. 1995; 9: 576-596Crossref PubMed Scopus (2252) Google Scholar). Unlike ITK-SYK, in the absence of pervanadate treatment ITK-SYK-K262R was not phosphorylated on Tyr385/Tyr386, suggesting that these residues represent target sites of autocatalytic activity in ITK-SYK as in SYK. Nevertheless, in the presence of pervanadate, ITK-SYK-K262R was phosphorylated at Tyr385/Tyr386, suggesting that these residues could be phosphorylated by other tyrosine kinases under these conditions. To confirm that ITK-SYK is an active tyrosine kinase, its catalytic activity was examined using an in vitro kinase assay. As expected, ITK-SYK immunoprecipitated from pervanadate-treated cells (Fig. 2e) or from untreated cells (see Fig. 4c) showed readily detectable phosphorylation of an exogenous substrate, whereas ITK-SYK-K262R consistently showed no significant activity above control immunoprecipitates. In keeping with the catalytic activity of ITK-SYK, treatment of ITK-SYK-transfected cells with the well characterized, selective SYK inhibitor Piceatannol inhibited the autophosphorylation of Tyr385/Tyr386 in a concentration-dependent manner (Fig. 2f). Piceatannol reduced the phosphorylation of Tyr385/Tyr386 at a concentration of 10 μm, the approximate IC50 for inhibition of SYK, with maximal inhibition at 30–50 μm (34Oliver J.M. Burg D.L. Wilson B.S. McLaughlin J.L. Geahlen R.L. J. Biol. Chem. 1994; 269: 29697-29703Abstract Full Text PDF PubMed Google Scholar, 35Yamamoto N. Hasegawa H. Seki H. Ziegelbauer K. Yasuda T. Anal. Biochem. 2003; 315: 256-261Crossref PubMed Scopus (14) Google Scholar). Although the recurrent detection of the ITK-SYK translocation in a subset of PTCL suggests a role for ITK-SYK in oncogenesis, whether ITK-SYK does indeed have transforming ability is unknown. To address this and to determine the role of the kinase activity of ITK-SYK in any transforming activity, we stably expressed ITK-SYK, ITK-SYK-K262R, or SYK in NIH3T3 cells and subjected these cell lines to standard in vitro transformation assays. Although cells carrying only empty vector grew to confluence and thereafter showed contact inhibition of growth, cells expressing ITK-SYK grew beyond confluence, without contact inhibition, forming numerous multilayered foci (Fig. 3a, top panels). The morphology of the transformed cells within these foci also became altered, the cells adopting a more rounded appearance (Fig. 3a, bottom panels). We also asked whether ITK-SYK could induce anchorage-independent growth in soft agar. Consistent with a transformed phenotype, cells expressing ITK-SYK were able to form large, anchorage-independent, multicellular colonies, whereas empty vector-transfected cells could not (Fig. 3b). Importantly, NIH3T3 cells expressing either SYK or ITK-SYK-K262R grew in a similar manner to empty vector-transfected cells, were unable to form foci in the focus formation assay (Fig. 3a), and did not form colonies in soft agar (Fig. 3b). These results show that ITK-SYK, but not SYK, has transforming activity in this in vitro system and indicate a requirement for catalytic activity in ITK-SYK-mediated transformation. In the majority of FTKs the N-terminal fusion partner functions mainly to induce homo-oligomerization, enabling autophosphorylation and subsequent constitutive kinase activity (1Chalandon Y. Schwaller J. Haematologica. 2005; 90: 949-968PubMed Google Scholar, 2Turner S.D. Alexander D.R. Leukemia. 2006; 20: 572-582Crossref PubMed Scopus (26) Google Scholar, 3Wong S. Witte O.N. Annu. Rev. Immunol. 2004; 22: 247-306Crossref PubMed Scopus (315) Google Scholar). However, as noted above, ITK-SYK does not contain a recognized dimerization motif but does harbor an N-terminal PH domain that, in ITK and other TEC family kinases, mediates the binding to membrane-associated PIP3 required for kinase activation (11August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar, 12Ching K.A. Kawakami Y. Kawakami T. Tsoukas C.D. J. Immunol. 1999; 163: 6006-6013PubMed Google Scholar, 13Woods M.L. Kivens W.J. Adelsman M.A. Qiu Y. August A. Shimizu Y. EMBO J. 2001; 20: 1232-1244Crossref PubMed Scopus (88) Google Scholar). To investigate whether a competent PH domain is required for the function of ITK-SYK, we introduced a point mutation, R29C, into the lipid-binding pocket of the PH domain (Fig. 1). This mutation abrogates both the binding of TEC family kinases to PIP3 and their associated membrane localization (36Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (487) Google Scholar, 37Bunnell S.C. Diehn M. Yaffe M.B. Findell P.R. Cantley L.C. Berg L.J. J. Biol. Chem. 2000; 275: 2219-2230Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 38Várnai P. Rother K.I. Balla T. J. Biol. Chem. 1999; 274: 10983-10989Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). As shown in Fig. 4a, whereas ITK-SYK was phosphorylated on Tyr385/Tyr386 in untreated cells, ITK-SYK-R29C, like the “kinase-dead” ITK-SYK-K262R, was not. Furthermore, the R29C mutation markedly reduced the phosphorylation of Tyr385/Tyr386 in cells treated with pervanadate, exerting a much greater effect than the K262R mutation under these conditions. Together, these results suggest that both the constitutive autophosphorylation of ITK-SYK and its maximal transphosphorylation at Tyr385/Tyr386 by other kinases require a PH domain able to bind PIP3. To confirm the requirement for a functionally intact PH domain in the activation of ITK-SYK, we compared ITK-SYK, ITK-SYK-R29C, and ITK-SYK-K262R in the in vitro kinase assay in both the presence (Fig. 4b) and absence (Fig. 4c) of pervanadate. Consistent with such a role, although ITK-SYK-R29C showed some enzymatic activity compared with untransfected cells or to ITK-SYK-K262R in the presence of pervanadate, there was a marked reduction in activity compared with ITK-SYK. Because cellular PIP3 levels are controlled principally by PI3K and the activation of TEC family kinases requires PI3K activity (11August A. Sadra A. Dupont B. Hanafusa H. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 11227-11232Crossref PubMed Scopus (150) Google Scholar, 38Várnai P. Rother K.I. Balla T. J. Biol. Chem. 1999; 274: 10983-10989Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar), we tested whether PI3K activity was required for the phosphorylation of ITK-SYK Tyr385/Tyr386. Cells transfected with ITK-SYK were treated with the PI3K inhibitor LY294002 prior to immunoprecipitation of ITK-SYK and assessment of Tyr385/Tyr386 phosphorylation (Fig. 4d). Treatment with LY294002 reduced the basal phosphorylation of the PI3K effector AKT, confirming effective inhibition of PI3K activity in these experiments. In the same experiments, LY" @default.
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• W2002246336 date "2009-09-01" @default.
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• W2002246336 title "The Lymphoma-associated Fusion Tyrosine Kinase ITK-SYK Requires Pleckstrin Homology Domain-mediated Membrane Localization for Activation and Cellular Transformation" @default.
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