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Phosphorylation of the activation loop tyrosines is required for sustained Syk signaling and growth factor-independent B-cell proliferation

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The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies, considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certain common B-cell malignancies Syk is
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  Phosphorylation of the activation loop tyrosines is required for sustained Syksignaling and growth factor-independent B-cell proliferation Laura Carsetti a , Luca Laurenti b , Stefania Gobessi a , Pablo G. Longo a , Giuseppe Leone b , Dimitar G. Efremov a, ⁎ a ICGEB Molecular Hematology Group, Campus  “   A. Buzzati-Traverso ”  , Rome, 00016, Italy b Department of Hematology, Catholic University Hospital  “   A. Gemelli ”  , Rome, 00168, Italy a b s t r a c ta r t i c l e i n f o  Article history: Received 25 February 2009Accepted 9 March 2009Available online 16 March 2009 Keywords: Syk kinaseB-cell receptorChronic lymphocytic leukemiaB-cell lymphoma The Syk kinase is regarded as a promising target for the treatment of antigen-driven B-cell malignancies,considering its essential role in propagating antigenic stimuli through the B-cell receptor (BCR). In certaincommon B-cell malignancies Syk is activated even in the absence of BCRengagement, suggesting a wider rolefor this kinase in lymphomagenesis. In this paper, we have pro 󿬁 led molecular differences between BCR-induced and constitutive Syk activation in terms of phosphorylation of regulatory tyrosine residues,downstream signaling properties and capacity to sustain B-cell proliferation. Analysis of primary chroniclymphocytic leukemia B-cells and diffuse large B-cell lymphoma cell lines revealed that constitutive and BCR-induced Syk activation differ with respect to the phosphorylation status of the regulatory tyrosines atpositions 352 and 525/526, with only the  󿬁 rst site being phosphorylated in the case of constitutive and bothsites in the case of BCR-induced Syk activation. Syk phosphorylated only on Y352 is capable of downstreamsignaling, as evidenced by experiments with a phosphomimetic mutant in which the activation looptyrosines (YY525/526) were replaced with phenylalanines. However, phosphorylation at YY525/526 wasshown to signi 󿬁 cantly increase the enzymatic activity of Syk and to be required for sustained PLC γ 2, Akt andERK signaling as well as B-cell transformation. These data demonstrate that constitutively active Syk and Sykactivated by BCR crosslinking represent separate stages of Syk activation with distinct signaling propertiesand transforming capacities.© 2009 Elsevier Inc. All rights reserved. 1. Introduction Chronic or recurrent stimulation of the B-cell receptor (BCR) byantigen is considered to play an important role in the developmentand progression of certain common B-cell malignancies [1 – 4]. Thisespecially refers to chronic lymphocytic leukemia (CLL), which ischaracterized by the expansion of B-cells that frequently expresssimilaror identicalBCRs,encodedbya restrictedsetof variableregiongene combinations [5 – 7]. This feature, along with the sharedreactivitytowardcommonforeignandself-antigens,stronglysuggeststhat the initial expansions of the malignant clones are antigen-driven[8 – 11]. In addition, CLL cells typically display an immunophenotypeand a gene expressionpro 󿬁 le of antigen-stimulated B-cells, indicatingthat antigen stimulation continues after the initial transformingeventand possibly contributes to subsequent disease progression [12,13].Other B-cell malignancies that have been linked to chronic antigenstimulation include certain indolent lymphomas that arise in thesetting of chronic Helicobacter Pylori and hepatitis C virus infections[14,15]. In these malignancies the pathogenic role of antigen is furthercorroboratedbyclinicalstudies,whichhaveshownthateliminationof the infectious agent can lead to tumor regression [16 – 18].B-cells respond to antigen stimulation by transmitting a signalfrom the BCR to the interior of the cell. This signal is initiallypropagated by members of the Src and Syk kinase families. The  󿬁 rstevent involves phosphorylation of the immunoreceptor tyrosine-basedactivationmotifs(ITAMs)intheIg- α andIg- β chainsof theBCR,which serve as docking sites for Syk. Binding to the ITAMs induces aconformational change in Syk that exposes a conserved tyrosineresidue at position 352. This tyrosine residue is then phosphorylatedbyLyn or other Src-family kinases, leading to release of the Syk kinasedomain from the closed, autoinhibitory con 󿬁 guration. Syk then trans-autophosphorylates the activation loop tyrosines at position 525/526and further propagates the BCR signal by associating with adaptorproteinsandphosphorylatingimportantsignalingintermediates,suchas BLNK (B cell linker protein), PI3K (phosphatidylinositol 3-kinase)and PLC γ 2 (phospholipase C γ 2) [19 – 24]. The signaling cascade nextproceeds with activation of signaling molecules that regulate thecellular response, such as the Akt, ERK (extracellular signal-regulatedkinase) and JNK (c-JUN NH 2 -terminal kinase) kinases and the NF- κ B(nuclear factor- κ B) transcription factor [24]. Cellular Signalling 21 (2009) 1187 – 1194 ⁎  Correspondingauthor.ICGEBOutstation-Monterotondo,CNRCampus “ AdrianoBuzzati-Traverso ” , Via E. Ramarini 32, I-00016 Monterotondo Scalo (Rome), Italy. Tel.: +39 0690091300; fax: +39 06 90091260. E-mail address:  efremov@icgeb.org (D.G. Efremov).0898-6568/$  –  see front matter © 2009 Elsevier Inc. All rights reserved.doi:10.1016/j.cellsig.2009.03.007 Contents lists available at ScienceDirect Cellular Signalling  journal homepage: www.elsevier.com/locate/cellsig  The Syk kinase has recently received additional attention becauseof observations that in certain common B-cell malignancies it isactivated even in the absence of BCR engagement. Thus, constitutiveor antigen-independent activation of Syk has been reported infollicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL),mantle cell lymphoma and more recently in CLL  [25 – 29]. Inhibitionordownregulation of Syk was shown to induce apoptosis in primary CLL cells and lymphoma cell lines  in vitro  and to delay tumor progressionin mouse lymphoma models  in vivo , suggesting that deregulated Sykactivity may playa role in the developmentor maintenance of variousB-cell neoplasms [25,26,28 – 31].The possibility that Syk may function as an oncogene is furthersupported by the identi 󿬁 cation of translocations involving Sykin patients with MDS and peripheral T-cell lymphoma [32,33].These translocations generate fusion proteins in which the tyrosinekinase domain of Syk is joined to the dimerization domain of thetranscription factor TEL or to the N-terminal pleckstrin homologydomain of the ITK kinase. Importantly, functional studies with TEL-Syk have been performed and shown that this dimeric protein isconstitutively active and capable of transforming mouse B cells[32,34].In this paper we attempted to de 󿬁 ne the molecular differencesbetween antigen-dependent and antigen-independent Syk activation,in terms of the phosphorylationstatus of regulatory tyrosine residues,capacity to activate downstream signaling pathways and capacity toinduce B-cell transformation. Experiments with DLBCL cell lines andprimary CLL cells revealed that constitutively active Syk and Sykactivated by BCR crosslinking differ with respect to the phosphoryla-tion status of YY525/526 in the activation loop of the kinase domain.To determine how this phosphorylation affects the signaling andtransforming capacity of Syk, we generated a series of Syk mutantsthat mimic various stages of Syk activation. These phosphomimeticswere then introduced into the IL-3 dependent B-cell line BaF3 toevaluate their capacity to activate downstream signaling pathwaysand sustain growth-factor independent B-cell proliferation. Theobtained data con 󿬁 rm that Syk phosphorylated only at Y352 isenzymatically active and capable of downstream signaling, but alsoshow that the signaling and transforming capacity of Syk is greatlyenhancedthroughdimerizationandphosphorylationof theactivationloop tyrosines, as occurs following BCR crosslinking by antigen. 2. Materials and methods  2.1. Patient samples Blood samples were collected from patients that satis 󿬁 ed standardmorphologic and immunophenotypic criteria for B-cell CLL. Informedconsent was obtainedfromallpatientsaccording tothe Declarationof Helsinki and approval for the study was obtained from the institu-tional human research committee at the Catholic University Hospital “ A. Gemelli ” . Peripheral blood mononuclear cells were separated byFicoll gradient centrifugation and CLL B-cells were puri 󿬁 ed bynegative selection with anti-CD3, anti-CD14 and anti-CD16 mousemonoclonal antibodies (kindly provided by Prof. Fabio Malavasi,University of Turin, Italy) and Dynabeads coated with anti-mouse IgG(Dynal Biotech, Oslo, Norway). The purity of the selected B-cellpopulations was evaluated by stainingwith anti-CD5 R-phycoerythrin(R-PE)-conjugated and anti-CD19  󿬂 uorescein (FITC)-conjugated anti-bodies (BD Biosciences, Franklin Lakes, NJ), followed by  󿬂 owcytometricanalysisonaFACSCalibur 󿬂 owcytometer(BDBiosciences).In all cases the purity of the CLL B-cell samples exceeded 98%.  2.2. Cell lines and culture conditions Freshly isolated CLL B-cells, the human lymphoma B-cell linesBJAB, B104, DHL-4, DHL-6, DHL-8, DHL-10, Toledo and WSU, and themouse lymphoma B-cell line WEHI-231 were cultured in RPMI-1640supplemented with 10% heat-inactivated fetal bovine serum (FBS),100 U/ml penicillin, 0.1 mg/ml streptomycin, 2 mM  L  -glutamine and1 mM sodium pyruvate (Invitrogen, Carlsbad-CA), at 37 °C in thepresence of 5% CO 2 . The IL-3 dependent mouse pro-B cell line BaF3was maintained under the same conditions except for the presence of 5 ng/ml recombinant mouse IL-3 (R&D Systems, Minneapolis, MN).CLL and lymphoma B-cells (1×10 7 /mL) were stimulated at 37 °C forthe indicated times with 10  μ  g/ml goat F(ab') 2  anti-human IgM oranti-human IgG (Southern Biotechnology Associates, Birmingham,AL), as appropriate.  2.3. Vectors and transfections Cloned human Syk cDNA (pCMVXL5-Syk) was purchased fromOriGene Technologies, Inc. (Rockville, MD) and subcloned intopcDNA3.1(+) (Invitrogen). Subsequently, the FLAG-epitope tag wasadded to the C-terminus of Syk. The Syk phosphomimetics Y352D,Y352D-YY525/526FF and Y352D-YY525/526DD were generated byPCR mutagenesis using the QuickChange II site-directed mutagenesiskit (Stratagene, La Jolla, CA). These plasmids were also used to pro-duce myristoylated variants, by cloning the Lck myristoylation signalsequence at the N-terminus. The dimerization PNT domain of thetranscription factor TEL was ampli 󿬁 ed by RT/PCR and subcloned intothe XhoI and XbaI sites of the corresponding pcDNA3.1(+)-Syk-FLAGvectors to generate TEL-Syk, TEL-Syk Y352D and TEL-Syk Y352D-YY525/526FF.Plasmid DNA for transfection was puri 󿬁 ed with the EndoFreePlasmid Maxi Kit (Qiagen, Hilden, Germany). The various plasmidswere linearized with PvuI and transfected by nucleofection into8×10 6 BaF3 cells using Nucleofector Solution V and the T20 program(Amaxa Biosystems GmbH, Cologne-Germany). Stable transfectantclones were obtained by limiting dilution and selection in Geneticin/G418 (Invitrogen). Expression of TEL-Syk protein was veri 󿬁 ed byimmunoblotting analysis using antibodies against Syk and FLAG. Fortransient transfection experiments, BaF3 cells were washed twicebefore nucleofection in PBS with 0.1% BSA to eliminate residual IL-3.Following nucleofection the cells were placed in IL-3-free mediumand collected after 6 h for further analysis.  2.4. Cell proliferation assay G418-resistant BaF3 cells expressing the indicated proteins andcontrol cells were grown in RPMI 1640 supplemented with 10% FCSand 5 ng/mL recombinant murine IL-3. Prior to the experiment cellswerewashedtwiceinIL-3-freemediumandseededin6-wellplatesata concentration of 2×10 5 cells/ml. The number of viable cells in thepresence or absence of IL-3 was counted by trypan blue exclusion(Sigma-Aldrich, St Louis, MO) at the indicated days.  2.5. Immunoblotting analysis Cell pellets were lysed in ice-cold RIPA lysis buffer (10 mM Tris – HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate[SDS], 0.1% sodium deoxycholate), containing 1:30 dilution of protease and 1:75 dilution of phosphatase inhibitor cocktail formammalian cells (Sigma-Aldrich). The protein concentration of eachcell lysate was determined with the RC DC Protein Assay (Bio-RadLaboratories). Proteins were separated by SDS-polyacrylamidegel electrophoresis (SDS-PAGE) and transferred on Immobilon-Ppolyvinylidene di 󿬂 uoride membranes (Millipore, Bedford, MA).Membranes were blotted at 4 °C with the following antibodies:phospho-Syk Y352 , phospho-Syk YY525/526 , phospho-ERK T202/Y204 ,phospho-Akt S473 , phospho-FoxO1 T24 /FoxO3a T32 , phospho-4E-BP1 S65 , ERK, Syk, PLC γ 2, rabbit IgG HRP-linked, mouse IgG HRP-linked (Cell Signaling Technology, Danvers, MA), phospho-Cbl Y700 , 1188  L. Carsetti et al. / Cellular Signalling 21 (2009) 1187  – 1194  phospho-Cbl Y774 , phospho-PLC γ 2 Y759 (BD Biosciences, Franklin-Lakes,NJ),Cbl(SantaCruzBiotechnology,SantaCruz,CA),FLAGandbeta-actin(Sigma-Aldrich). Immunodetection was performed using ECL Plusenhanced-chemiluminescence (Amersham Biosciences, Buckingham-shire, United Kingdom) and the Gel Logic 2200 Imaging System(Eastman Kodak, Rochester, NY). 3. Results  3.1. Constitutively active Syk is characterized by Y352 phosphorylationand lack of elevated YY525/526 phosphorylation A panel of DLBCL cell lines and primary CLL B-cells were usedto investigate whether constitutively active Syk and Syk activatedby BCR crosslinking differ with respect to the phosphorylation statusof the tyrosine residues that are involved in Syk kinase activation(Fig. 1). Analysis of Syk phosphorylation status was performed incellular extracts from unstimulated and anti-IgM or anti-IgG stimu-latedcells,usingphospho-speci 󿬁 cantibodiesthatrecognizeSykwhenphosphorylated at Y352 or YY525/526. Immunoblotting analysis of unstimulated cells revealed constitutive phosphorylation of Syk atY352 in most CLL B-cell samples and in a subset of the DLBCL cell lines, consistent with previously published data [28,29]. Surpris-ingly, phosphorylation at YY525/526 could not be detected inunstimulated cells, but could be ef  󿬁 ciently induced in the same cellsby BCR crosslinking with anti-IgM or anti-IgG antibodies. BCR crosslinking also induced a substantial increase in the amount of Y352-phosphorylated Syk protein.  3.2. Syk can activate downstream signaling pathways in the absence of YY525/526 phosphorylation The CLL samples and DLBCL cell lines with Y352-phosphorylatedSyk that were used in the experiment in Fig. 1 had previously beenshown to express an enzymatically active Syk protein [28,29].Therefore, the absence of YY525/526-phosphorylated Syk in thesecells suggested that phosphorylation of YY525/526 may not beabsolutely required for Syk activation, in contrast to what is generallybelieved. To further address this possibility, we decided to investigatewhether Syk molecules that cannot be phosphorylated at YY525/526are capable of downstream signaling. For this purpose we produced aSyk phosphomimetic in which Y352 was substituted with asparticacid (Y352D) and YY525/526 were substituted with phenylalanines(YY525/526FF).ThisproteinshouldmimicSykthatisphosphorylatedat Y352 but is not phosphorylated at YY525/526, as phenylalaninesubstitutions prevent phosphorylation at position 525/526, whereasthe aspartic acid substitution at position 352 brings a negative chargethat mimics phosphorylation. A similar substitution in the corre-sponding Y319 residue in the homologous kinase ZAP-70 (Y319E) hadpreviously been shown to be suf  󿬁 cient for ZAP-70 activation [35].TheY352D-YY525/6FFphosphomimeticwastransientlyexpressedintheIL-3dependentB-celllineBaF3anditsactivitywasevaluatedbyanalyzing phosphorylation of downstream signaling molecules fol-lowing IL-3 withdrawal. As shown in Fig. 2, the Y352D-YY525/6FFphosphomimetic induced the phosphorylation of all investigateddownstream substrates, including PLC γ 2, Cbl, Akt, 4E-BP1, FoxO1/3aand ERK, whereas wild type Syk was completely inactive (Fig. 2B,lanes 4 and 1, respectively). Thus, this experiment demonstrated thatSyk can activate downstream signaling pathways even when notphosphorylated at YY525/526.TounderstandwhatisthefunctionofYY525/526phosphorylation,we tried to generate a phosphomimetic that would mimic Syk whenphosphorylated at both Y352 and YY525/526. This was  󿬁 rstattempted by replacing all three regulatory tyrosines in Syk withaspartic acid residues. However, the Y352D-YY525/6DD phosphomi-metic did not show any activity in BaF3 cells, presumably because the525/526 substitutions induced structural changes in the activationloop of Syk that were incompatible with an active conformation(lane 3, Fig. 2B).Inthis experiment we alsotested a third phosphomimetic, inwhichonlyY352wassubstitutedwithasparticacid,whereasYY525/526wereleft intact. Interestingly, this phosphomimetic displayed identicalactivity and substrate speci 󿬁 city as the Y352D-YY525/526FF phospho-mimetic, despite being phosphorylated at YY525/6 (lane 2, Fig. 2B).  3.3. Phosphorylation at YY525/526 signi  󿬁 cantly enhances downstreamsignaling  The lack of any differences between the activity of the Y352D andtheY352D-YY525/526FFphosphomimeticindicatedthateitherYY525/ Fig.1.  Analysis of Syk phosphorylation in unstimulated and anti-IgM or anti-IgG stimulated CLL and lymphoma B-cells. A) Cellular extracts were prepared from freshly isolated andpuri 󿬁 ed CLL B-cells and from the indicated lymphoma cell lines. Phosphorylation at Y352 and YY525/526 in Syk was evaluated by Western blotting. BJAB B-cells, unstimulated orstimulated for 5 minwith anti-IgM (aIgM), were used as a negative and positive control for Syk phosphorylation, respectively. Total Syk was used as a loading control. B) CLL, DHL-6andBJABcellswerestimulatedfortheindicatedtimeswithanti-IgMantibody;DHL-4cellswerestimulatedwithanti-IgG.CellswerelysedandanalyzedbyWesternblottingwiththepSyk-Y352 and pSyk-YY525/526 antibody.1189 L. Carsetti et al. / Cellular Signalling 21 (2009) 1187  – 1194  526 play a minor role in Syk activation and signaling, or that phos-phorylation of these tyrosines in the Y352D phosphomimetic isnotef  󿬁 cientenoughforadiscernableeffect.Inef  󿬁 cientphosphorylationof the Y352D protein could have been due to lack of phosphorylatedITAMs in BaF3 cells,whichmay be requiredto providedocking supportfor trans-autophosphorylation. The latter possibility was supported bycomparing the levels of YY525/526 phosphorylation in the Y352Dphosphomimetic with Syk that was activated by BCR crosslinking(Fig. 3A). After adjusting for Syk protein levels, the phosphospeci 󿬁 csignal in Syk Y352D was more than 15 times lower than the signalinduced by anti-IgM treatment. Phosphorylation at YY525/526 didnot increase even when the Syk Y352D protein was targeted to thecellular membrane by the addition of a myristoylation signal sequenceto the N-terminus (Fig. 3A). Fig. 3.  Analysis of YY525/526 phosphorylation in Syk phosphomimetics and TEL-Syk fusion proteins. A) Analysis of YY525/526 phosphorylation in the constitutively active Sykphosphomimetics Y352D (Syk Y352D) and myristoylated-Y352D (myr.Syk Y352D) after transient transfection in BaF3 cells. The phosphospeci 󿬁 c signal was adjusted for differencesin Syk proteinlevelsandcomparedwith Syk activatedbyBCR crosslinking in WEHI B-cells.B) Schematic representation of the threeTEL-Syk constructs indicating the location of theTEL, SH2 and kinase domains (dom.). C) Comparison of YY525/526 phosphorylation in Syk Y352D, TEL-Syk and TEL-Syk Y352D following transient transfection in BaF3 cells. Thephosphospeci 󿬁 c signal was quanti 󿬁 ed and adjusted for differences in Syk protein levels. The transiently expressed proteins were distinguished from endogenous Syk by thedifference in molecular weight. Fig. 2.  Analysis of downstream signaling induced by Syk phosphomimetics with mutations in Y352 and YY525/526. A) Schematic representation of wild type (w.t.) Syk and thephosphomimetics used in this experiment. B) BaF3 cells were washed twice to eliminate residual IL-3 and then transfected by nucleofection with the indicated vectors. FollowingnucleofectionthecellswereplacedinIL-3-freemediumandcollected6hlaterforimmunoblottinganalysisofdownstreamsignalingevents.FLAG-taggedSykwasdistinguishedfromendogenous Syk by the difference in molecular weight. ERK was used as a loading control.1190  L. Carsetti et al. / Cellular Signalling 21 (2009) 1187  – 1194  The previous experiments suggested that phosphorylation atYY525/526isavery inef  󿬁 cientprocessintheabsenceofamechanismthatwoulddrivetwoSykmoleculesincloseproximity.To favorsuchaprocess, we decided to produce dimeric Syk proteins that wouldresemble the TEL-Syk fusion protein that was identi 󿬁 ed in the MDStranslocation and was shown to be constitutively active in BaF3 cells[32,36]. These dimeric Syk proteins were generated by adding thedimerization domain of TEL to the N-terminus of wild type Syk, SykY352D and Syk Y352D-YY525/526FF (Fig. 3B). In comparison to theSykY352Dphosphomimetic,theTEL-Sykprotein showedamorethan400 fold greater phosphorylation at YY525/526, con 󿬁 rming that onlya small fraction of Syk Y352D molecules become phosphorylated atthis site by virtue of random encounter (Fig. 3C). A similar level of YY525/526phosphorylationwas detectedinTEL-Syk Y352D, whereasnophosphorylationwasdetectedintheTEL-SykproteinwithmutatedYY525/526 residues (TEL-Syk Y352D-YY525/526FF).We next compared the capacity of Syk Y352D, TEL-Syk, TEL-SykY352D and TEL-Syk Y352D-YY525/526FF to induce the phosphoryla-tion and activation of downstream signaling molecules (Fig. 4). BothTEL-Syk and TEL-Syk Y352D were 7 to 70 fold more potent than SykY352D in inducing phosphorylation of Akt, ERK, PLC γ 2 and Cbl.Substitution of YY525/526 with phenylalanines in TEL-Syk Y352Dreducedthekinaseactivityof thisenzymetoalmostthesamelevelsasin Syk Y352D. Together, these data demonstrate that phosphorylationat YY525/526 leads to a substantial increase in the kinase activity of Syk.  3.4. Syk dimerization and phosphorylation at YY525/6 is required for  growth factor independent B-cell proliferation and sustaineddownstream signaling  Previous studies had shown that the TEL-Syk fusion protein cantransformBaF3B-cellsandsustaintheirproliferationintheabsenceof IL-3 [32]. To more precisely de 󿬁 ne the molecular requirements forSyk-mediated B-cell transformation, we generated stable BaF3 clonesthat expressed the various Syk phosphomimetics. Withdrawal of IL-3 from the culture medium led to rapid cell death in clones trans-fected with wild-type Syk, Syk Y352D, Syk Y352D-YY525/526FF orempty vector, withasomewhatprolongedsurvivalof cellstransfectedwith TEL-Syk Y352D-YY525/526FF (Fig. 5). In contrast, cells expres-sing TEL-Syk and TEL-Syk Y352D continued to proliferate at a ratesimilar to control cells grown in the presence of IL-3. Collectively,these data show that phosphorylation at YY525/526 is required forSyk-mediated B-cell transformation.ThecapacityofTEL-SykandTEL-SykY352DtotransformBaF3cellswas associated with sustained signaling through PLC γ 2, Akt and ERK(Fig. 6). In contrast, these signaling molecules were no longer acti-vated in BaF3 clones with stable expression of Syk Y352D and Y352D-YY525/526FF, although phosphorylation of Cbl was unaffected. TheTEL-Syk Y352D-YY525/526FF protein, which was also incapable of supporting IL-3 independent proliferation of BaF3 cells, showed only Fig. 4.  Analysis of downstream signaling events in the presence or absence of YY525/526 phosphorylation. A) The indicated vectors were transiently transfected in IL-3-deprivedBaF3 cells and collected after 6 h for immunoblotting analysis with phospho-speci 󿬁 c antibodies. B) To calculate the relative phospho-speci 󿬁 c signal, we  󿬁 rst normalized the signalobtained with each phospho-speci 󿬁 c antibodyagainst the amount of transgenic Syk protein. The obtained values were then expressed as fold change relative tothe values observedwith Syk Y352D, which were arbitrarily set to 1. The following formula was used for this calculation: (phospho-speci 󿬁 c signal/Syk)/(phospho-speci 󿬁 c signal Syk-Y352D /Syk Syk-Y352D ).Values represent means from two independent experiments. Fig. 5.  Analysis of the capacity of various Syk and TEL-Syk phosphomimetics to supportgrowth-factor independent proliferation in stable BaF3 transfectants. Stable BaF3transfectants expressing the indicated proteins and vector control cells were grown inthe presence or absence of IL-3. The number of viable cells at each time point wasdetermined by trypan blue exclusion. Each point indicates mean of three independentexperiments.1191 L. Carsetti et al. / Cellular Signalling 21 (2009) 1187  – 1194
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