redistribution of bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and...

0014-2980/00/0101-145$17.50+.50/0© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000

Redistribution of Bruton’s tyrosine kinase byactivation of phosphatidylinositol 3-kinase andRho-family GTPases

Beston F. Nore1, 3, Leonardo Vargas1, 3, Abdalla J. Mohamed1, 3, Lars J. Branden1, 3,Carl-Magnus Bäckesjö1, 3, Tahmina C. Islam1, 3, Pekka T. Mattsson1, 2, 3, Kjell Hultenby3,Birger Christensson3 and C. I. Edvard Smith1, 3

1 Department of Biosciences at Novum, Karolinska Institutet, SE-141 57 Huddinge, Sweden2 Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku,

Finland3 Department of Immunology, Microbiology, Pathology, and Infectious Diseases (IMPI),

Karolinska Institute, Huddinge University Hospital, SE-141 86 Huddinge, Sweden

Bruton’s tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK)characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interactwith phosphoinositides. We report here that wild-type (wt) and also a gain-of-functionmutant of Btk are redistributed following a wide range of receptor-mediated stimuli throughphosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluores-cent protein in transient transfections resulted in Btk translocation to the cytoplasmic mem-brane of live cells through various forms of upstream PI 3-K activation. The redistributionwas blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-functionmutant of Btk was found to be a potent inducer of lamellipodia and/or membrane ruffle for-mation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localizedwith actin in these regions. Formation of the membrane structures was blocked by the domi-nant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cellsurface receptors activating PI 3-K and certain members of the Rho-family of small GTPa-ses. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pat-tern and showed decreased capacity to form aggregates, further suggesting that cytoskele-tal regulation mediated by Btk may be of physiological relevance.

Key words: Bruton’s tyrosine kinase / Tec tyrosine kinase / Green fluorescent protein / Phosphati-dylinositol 3-kinase / Rho-family GTPase

Received 10/6/99Revised 6/9/99Accepted 24/9/99

[I 19700]

The first three authors contributed equally to this work.

Abbreviations: Btk: Bruton’s tyrosine kinase PTK: Proteintyrosine kinase PI 3-K: Phosphatidylinositol 3-kinase BCR:B cell receptor GFP: Green fluorescent protein HA: Hem-agglutinin

1 Introduction

X-linked agammaglobulinemia (XLA) is an inheritedgenetic disease characterized by a lack of mature B cells(for a recent review see [1]). The gene which is responsi-ble for XLA encodes the cytoplasmic protein tyrosinekinase (PTK), Bruton agammaglobulinemia tyrosinekinase, Btk [2, 3]. Btk belongs to the Tec family of PTK,which also includes Itk (Emt/Tsk), Tec, Bmx and Txk(Rlk).

Other cytoplasmic PTK have also been found to playimportant roles in lymphocyte development. Lck, Fynand Zap 70 are essential for T cell maturation, whereasdefects in Lyn and Syk affect B cell development. TheSrc and Syk/Zap kinases seem to convey signals origi-nating from immunoreceptors. Although the Btk/Tecfamily of kinases have also been implicated in immuno-receptor signaling, the picture is still only fragmentarywith evidence of many pathways being affected.

More than 500 XLA-causing loss-of-function mutationshave been identified. They are scattered throughout theBTK gene [2, 4]. Btk is composed of five domains, a C-terminal catalytic region and four N-terminal domainshaving localizing and structural functions [5, 6]. Follow-ing a random mutagenesis scheme, a particular pleck-strin homology (PH domain missense mutation (gluta-mate 41 1 lysine), designated Btk*, was found to result

Eur. J. Immunol. 2000. 30: 145–154 Btk links PI 3-K to small GTPases 145

in gain-of-function instead [7]. Like in other cytoplasmicPTK, phosphorylation of an invariant tyrosine residue inthe kinase domain enhances the catalytic activity. Sub-sequently, tyrosine phosphorylation takes place in theSrc-homology 3 (SH3) domain, the function of which isnot well understood [8]. The SH2 domain of Btk interactswith tyrosine phosphorylated proteins, although the cor-responding partner(s) remains elusive. Finally, the Techomology domain binds a Zn2+ ion and has a structuralfunction [9, 10].

Following the initial demonstration that PH domains maybind phosphoinositides in the cell membrane [11], it wasfound that Btk interacts with this family of molecules,phosphatidylinositol 3,4,5-trisphosphate (PIP3) in partic-ular [12, 13]. This indicated that activation of Btk occursdownstream of phosphatidylinositol 3-kinase (PI 3-K)and recent evidence supports this idea [14, 15]. In viewof the fact that translocation to the cell surface is essen-tial for the function of cytoplasmic PTK belonging toother families, it could be anticipated that a similar acti-vation scheme would occur for members of the Tec fam-ily. However, substrates for Btk and the downstream sig-naling of this kinase still remain elusive, although inacti-vation of the gene encoding Btk in the chicken DT40 Bcell line rendered these cells defective in Ca2+ flux [16] inresponse to Ig-receptor ligation.

2 Results and discussion

2.1 Btk-GFP fusion proteins have intact Btkactivity

To investigate the signaling function of Tec-familykinases, fusion genes containing Btk and green fluores-cent protein (GFP) were constructed to study Btk-GFPdistribution in living cells, in real time, including the anal-ysis of the anticipated translocation following activation.All plasmid transfections were transient to avoid the pos-sibility of selection of non-representative clones duringthe procedure of establishing stable transfectants. In afirst set of experiments, various cell lines were trans-fected with human Btk and Btk-GFP expression plas-mids. In these experiments a number of different non-lymphoid cell lines were also tested, since many adher-ent cell lines have a comparatively large cytoplasm andthe distribution of cytoplasmic proteins would, therefore,be better visualized as compared to lymphoid cells.However, B cell lines were always tested in a similarexperimental set-up to verify that the distribution wasnot unique to non-lymphoid cells.

The fusion protein obtained from transiently transfectedcells was subjected to immunoprecipitation and subse-

quently enzymatic activity was monitored using an invitro kinase assay measuring the capacity of Btk to elicitauto-phosphorylation (Fig. 1 a). These experiments werecarried out in non-hematopoietic cells to avoid interfer-ence from endogenous Btk. The catalytic activity of Btk-GFP and native Btk was indistinguishable, and did notdiffer from endogenous Btk isolated from hematopoieticcells, indicating that GFP did not interfere with the func-tion of Btk (Fig. 1 a). Similar results were obtained usinga hemagglutinin (HA) tag in the C terminus of native Btk(Fig. 1 a).

2.2 Stimulation of PI 3-K causes activation andmembrane translocation of Btk

Enzymes belonging to the PI 3-K family are generators ofPIP3 and contribute to the signaling from a multitudeof cell surface molecules, including immunoreceptors.Other enzymes will degrade PIP3 making this a highlyregulated biological system. Since biochemical investi-gations had demonstrated selective binding of Btk toPIP3 via the PH domain [12, 13], we sought to determinewhether PI 3-K was implicated in this pathway in intact,live cells and, furthermore, whether any form of PI 3-Kstimulation could activate Btk. The stimulation of manyprototype cell surface receptors, such as the insulinreceptor, is known to result in the subsequent activationof PI 3-K. In our initial studies we investigated the adher-ent CHO cell line, known to express endogenous insulinreceptors. Stimulation of transfected cells resulted intyrosine phosphorylation of Btk and redistribution of thekinase from the cytoplasm to the cell surface (Fig. 1 b, c).These studies were performed not only in CHO cellsexpressing endogenous insulin receptor, but also in cellsco-transfected with insulin receptor-expressing plas-mids. Increased receptor levels enhanced Btk-GFPmembrane translocation following stimulation (notshown).

This redistribution was demonstrated at higher resolu-tion by immunoelectron microscopy (Fig. 2 a, c). Theconcentration of gold markers for GFP, reacting with theBtk-GFP fusion protein, was higher at the cell bordercompared to the central region of the cytoplasm. Theratio of markers (gold markers at the border within0.1 ? m of the plasma membrane versus gold markers inthe central region of the cell cytoplasm) was 7.9 goldparticles/ ? m2 in stimulated cells compared to 2.9 goldparticles/ ? m2 in controls, clearly showing a shift in locali-zation of receptors to the plasma membrane.

Similar results were obtained following insulin-likegrowth factor-1 (IGF-1) and epidermal growth fctor (EGF)stimulation of PI 3-K in cell lines expressing the corre-

146 B. F. Nore et al. Eur. J. Immunol. 2000. 30: 145–154

Fig. 1. Btk activation and membrane association. (a) Activity of Btk fusions with GFP and HA measured by in vitro kinaseassay in HEK 293 cells. (b) Btk phosphorylation in CHO cells upon insulin (1 ? g/ml) stimulation for 10 min on ice. (c) Time-lapseconfocal images of Btk-GFP from a mid-section segment of a living CHO cell after activation with 1 ? g/ml insulin at 25 °C. Thecytoplasmic localization of Btk-GFP at onset was similar to the localization after 10 min. Accumulation of Btk-GFP in the mem-brane was complete within 30 min after insulin treatment. As with insulin (c), treatment with 100 ng IGF-1 (10 min) (d) or 30 ngEGF (10 min) (e) induced the membrane-association of Btk-GFP.

sponding receptors (Fig. 1 d, e). Using the GFP vectoralone, no response to growth factor stimulation wasseen (not shown). Receptor tyrosine kinases activate PI3-K directly by recruiting the p85 subunit of PI 3-K, orthrough the stimulation of the small GTPase, Ras, whichcan also bind and activate PI 3-K [17]. To study lympho-cytes, the effect of cross-linking of the B cell antigenreceptor (BCR) in the Ramos B cell line was investigated.The addition of PI 3-K inhibitors wortmannin orLY294002, to BCR-stimulated Ramos or to insulin-stimulated HEK 293 cells, respectively, resulted in inhibi-tion of Btk phosphorylation (Fig. 3 a, b). Cross-linking ofimmunoreceptors through Fc 4 R1 and BCR in RBL-2H3mast cells and chicken DT40 B cells, respectively,induced pronounced redistribution of Btk to the cyto-plasmic membrane (Fig. 4 a, c). Moreover, cross-linking

of the BCR causes activation of ribosomal p70S6 serine/threonine kinase (p70S6K) in chicken DT40 cells [18]. Inour studies Btk activation was not affected by rapamy-cin, a specific inhibitor of p70S6K, suggesting that Btkstimulation represents a discrete pathway independentof p70S6K activation (Fig. 3 a, b).

Recently, SH2-containing inositol-5'-phosphatase (SHIP)was identified as an enzyme catalyzing the conversion ofPIP3 to bisphosphate by removing the phosphate at the5' position [19]. PTEN (phosphatase and tensin homol-ogy deleted on chromosome 10) was identified [20] as atumor suppressor. The biologically relevant targets forPTEN are phosphatidylinositol phosphate at 3' positionboth in vitro and in vivo [21]. Co-transfection of eitherSHIP or PTEN (Fig. 4 c) impaired translocation of Btk-


Fig. 2. Immunoelectron microscopic localization of Btk-GFP. Labeling of gold markers (arrowheads) for Btk-GFP tothe plasma membrane border (a) and also localization in themembrane (b) and (c) of HEK 293 cells. (a) × 56 000, (b)× 99 000, (c) × 180 000.

Fig. 3. PI 3-K-dependent activation of Btk following insulin and BCR cross-linking in HEK 293 and Ramos B cells, respectively.Activation of Btk by insulin (1 ? g/ml) in Btk transfected HEK 293 cells (a) and by anti-human IgM, F(ab')2 fragment (20 ? g/ml) inRamos B-cells (b), respectively. Stimulations were performed for 10 min on ice. Equal amounts of cell lysates (800 ? g) wereimmunoprecipitated with polyclonal anti-Btk Ab. PI 3-K inhibitors LY294002 (30 ? M), wortmannin (100 nM) and p70S6K inhibitorrapamycin (20 nM) were pre-incubated at 37 °C for 30 min prior to stimulation.

GFP to the cell membrane, compatible with PIP3 beinginvolved in membrane tethering.

2.3 The chemokine SDF-1 > activates Btkthrough PI 3-K

It has been reported recently that G-proteins can stimu-late Btk activity [22]. The fact that the G-protein-dependent PI 3-K + isoform [14] activates Btk may sug-gest that this phenomenon is also related to Btk beingdownstream of PI 3-K. To further study the relationshipbetween various forms of PI 3-K activation and the stim-ulation of Btk, we investigated the effect of a G-proteinligand known to affect B cell development [23]. Due to

the advantage of studying adherent cells with a largecytoplasm, HeLa cells, which endogenously express thecorresponding G-protein-coupled CXCR4 receptor, wereemployed. Addition of the lymphocyte chemokine, stro-mal cell-derived factor (SDF)-1 § , to HeLa cells, resultedin Btk membrane translocation (Fig. 4 d). This effectcould be inhibited by LY294002, which further supportsthe idea that all forms of PI 3-K activation result in Btkmembrane translocation (Fig. 4). Following SDF-1 § stim-ulation, nuclear localization of Btk was also observed, aphenomenon presently under investigation (Fig. 4 d). Inkeeping with this concept, a constitutively activated,membrane-targeted form of PI 3-K, designated p110*,also increased Btk membrane localization (Fig. 4 e). Thegain-of function mutation of Btk, Btk*, also enhancedmembrane localization in a similar fashion to p110*(Fig. 4 e). However, without induction, native Btkexpressed in different cell lines was localized predomi-nantly in the cytoplasm. Collectively, this extends previ-ous observations by demonstrating PI 3-K-dependentreceptor tyrosine kinase-, G-protein-coupled receptor-,and immunoreceptor-induced activation of Btk. In Bcells, Btk is mainly thought to function as a transducer inthe BCR signaling pathway. However, our findings alsoimplicate other membrane proteins, such as the CXCR4G-protein-coupled receptor as potential physiologicalupstream partners of Btk, and it is notable that inactiva-tion of the mouse SDF-1 § gene results in a profound Blymphocyte deficiency [23].

2.4 Src-family kinases enhance activation andmembrane translocation of Btk

Src-family kinases have been reported to phosphorylateTec-family members on an activating tyrosine (Y551 inBtk) in the kinase domain [14, 15, 24]. We found that co-


Fig. 4. PI 3-K activity is required for membrane translocation of activated Btk-GFP. Cells were exposed to various ligandsknown to activate the corresponding cell line, as specified in Sect. 3.1, and inhibited with pharmacological (LY294002, 30 ? M for30 min at 37 °C) or biological (SHIP, PTEN; the corresponding cDNA clones were co-transfected) inhibitors of PI 3-K (a–d). Thefollowing cell lines were used RBL-2H3 mast cells (a), DT40 B cells (b), HEK 293 cells (c), and HeLa cells (d). Co-transfection withBtk*-GFP alone induced membrane localization in COS-7 cells (e). Membrane localization and membrane reorganization wasinduced when Btk-GFP was expressed with p110* or with c-Src (e).

transfection of Src and Btk resulted in increased mem-brane localization of Btk (Fig. 4 e), a phenomenon thatcould also be inhibited by LY294002 (not shown). Fur-thermore, addition of the Src tyrosine kinase inhibitorPP1 impaired the activity of Src on Btk for both translo-

cation and phosphorylation (not shown). This indicatesthat Src kinases have a dual function in Btk stimulation,i.e. activation of PI 3-K, with subsequent membranetranslocation, and phosphorylation of the auto-regulatory, activating tyrosine in the kinase domain.


Fig. 5. Co-localization of activated Btk with F-actin. Fluorescence images of total F-actin labeled with rhodamine-phalloidin (red)[37], GFP (green), nucleus staining DAPI (blue); co-localization of F-actin with Btk-GFP (yellow). Transient transfection in COS-7 withmock vector pSVK3 (a), wt Btk (b), Btk* (c), wt Btk-GFP (d), Btk*-GFP (e), Btk-GFP + N17Rac1 (1:9 ratio) (f), Btk*-GFP + N17Rac1(1:9 ratio) (g). Before fixation, cells were starved for 24 h prior to stimulation with 10 % serum for 1 h. (a)–(g) × 63.

2.5 Btk and a Btk gain-of-function mutationinduces cytoskeletal reorganization

When transfected cells were activated following serumdeprivation, we observed that Btk was translocated tothe membrane and was found predominantly in areasreferred to as membrane ruffles or lamellipodia (Fig. 5),

structures known to be caused by polymerization ofactin [25]. This finding was of interest given the crucialrole that cytoskeletal changes play during lymphocytedifferentiation and activation [26]. To see whether Btkactivity contributed to the formation of these cytoskeletalalterations, Btk* was employed. Interestingly, transfec-tion of Btk* and Btk*-GFP expression plasmids resulted


Fig. 6. Scanning electron microscopy of Btk*-GFP. Pro-nounced induction of lamellipodia and surface protrusionsby Btk*-GFP in COS-7 cells (a), higher magnification oflamellipodia (b). Before fixation, cells were starved for 24 hprior to stimulation with 10 % serum for 1 h. (a) × 1400,(b) × 5000.

in pronounced induction of lamellipodia, with some for-mation of filopodia (Fig. 5 c, e), compared to mock andBtk wild-type (wt) transfected cells (Fig. 5 a, b). Theeffect varied between different cell lines, with expressionof filopodia being predominant in transfected Hek 293cells (not shown). Upon serum activation, membrane ruf-fling was also noticed following expression of wt Btk, aswell as of Tec (not shown), although the extent wasreduced as compared to Btk* (Fig. 5 c, e). This processwas inhibited by LY294002, indicating that membranetranslocation of Btk is crucial for this phenomenon (notshown). Similar results were obtained using scanningelectron microscopy (SEM), which clearly showed induc-tion of membrane ruffling and surface protrusions byBtk* (Fig. 6).

2.6 Btk links PI 3-K to the Rho familyGTPases

As formation of lamellipodia, membrane ruffling, and filo-podia are known to be induced by small Ras-like GTP-ases [25], especially Rac1 and Cdc42, this implicated theTec family in this signaling pathway. Transient transfec-tion with activated forms of these GTPases, V12Rac1and to a lesser degree V12Cdc42, also caused theappearance of ruffles. However, of the correspondingdominant negative forms, N17Rac1 and N17Cdc42, onlyN17Rac1 blocked the formation of ruffles induced by Btk(Fig. 5 f). Induction of membrane ruffling by Btk* wasinhibited by N17Rac1, even when the ratio was 1:9(N17Rac1:Btk*) (Fig. 5 g), whereas the correspondingN19 mutation of the small GTPase Rho A had no effecton the formation of lamellipodia elicited by either wt Btkor Btk* (not shown). Taken together, these findings sup-

port the idea that constitutively active Cdc42 actsupstream of Rac [25], whereas Btk-stimulated ruffle for-mation is mediated mainly through Rac. It is noteworthythat both Btk (Fig. 5) and Tec (not shown) not onlyinduced ruffle formation, but also co-localized with actinin these membrane regions (Fig. 5). This is similar toother molecules such as Rac and IQGAP, both of whichare known to regulate actin organization. It is worthwhilementioning that both Rac1- and Cdc42-induced motilityand invasiveness require PI 3-K stimulation [27]. Further-more, cross-linking of the BCR has recently been shownto activate p70S6K in a PI 3-K-dependent fashion [28].Moreover, although p70S6K has been implicated in thereorganization of the actin cytoskeleton [28], our findingthat the PI 3-K-induced activation of Btk is independentof p70S6K (Fig. 3 a, b) places Btk upstream or in parallel tothe p70S6K pathway.

Small GTPases are activated by guanine nucleotide-exchange factors, whereas GTPase-activating proteinsand guanine-nucleotide dissociation inhibitors inhibittheir activity. It seems likely that members of at least oneof these families are targets for Tec family kinases. So farwe haven been unable to detect any activation of thehematopoietic guanine-nucleotide exchange factor Vavby Btk in transfected cells (not shown). It is interesting tonote that the recent demonstration of binding betweensmall GTPases and domains with a PH fold is compatiblewith a direct interaction between Btk and Rho-familymembers [29].

2.7 Btk induces aggregate formation in DT40cells

To study whether Btk also influences actin organizationin B lymphocytes, the chicken cell line DT40 wasemployed both as wt and as deletion mutant lacking Btk[14]. The actin organization in DT40 cells, as revealed byrhodamine-phalloidin staining, clearly showed differentpatterns in wt DT40 and the mutant lacking Btk (Fig. 7 a,b). Thus, in wt DT40 cells pronounced actin bundleswere formed toward the periphery of the cell, suggestinga Btk requirement for the rearrangement of actin bundlesas surface membrane projections (Fig. 7 a). We have alsonoticed that wt DT40 differs from the Btk deletion mutantwith regard to growth characteristics. As shown inFig. 7 c and d, wt DT40 cells form large aggregates,whereas the Btk mutant cultures display considerablysmaller and fewer aggregations. This phenomenon wasobserved under various degrees of confluency. Similarly,a cell line from an XLA-patient carrying a frameshiftmutation showed smaller and fewer aggregates as com-pared to control cells (not shown). As small GTPaseshave been found to induce the assembly of integrin com-


Fig. 7. Phenotype of the chicken B cell line wt DT40 anda Btk-deficient mutant. (a, b) Fluorescence images of total F-actin distribution; (a) wt, (b) Btk-deficient mutant, labeledwith rhodamine-phalloidin (red) [37], and nucleus stainingDAPI (blue). (c, d) Micrographs of the tissue culture aggre-gates formed in wt DT40 (c) and in Btk mutant (d). (a, b) ×100; (c, d) × 10.

Fig. 8. Schematic representation of the tentative path-way of Btk activation as analyzed in this report. ( 1 ) denotesa direct stimulatory effect, ( 1 1 ) and effector likely to involvetwo, or multiple components, (¯ ) denotes inhibition.

plexes, this may suggest the involvement of Rac/Cdc42in this process [25, 30–33]. In Fig. 8, we present a sche-matic representation of the Btk pathways delineated inthis report. Moreover, XLA-patient platelets have beenreported to be less prone to aggregation following recep-tor stimulation [34]. The differential patterns for actin areconsistent with a role for Btk in the control of cytoskeletalorganization. Interestingly, in two recent publicationsSrc64 was shown to act upstream of Tec29, regulatingring canal formation during drosophila development [35,36]. Mutants lacking either of these kinases showed ringcanal growth arrest and it was suggested that they mightregulate actin bundling proteins. It is noteworthy that,similar to our analysis of Btk, Tec29 was reported to co-localize with actin [36]. Collectively, these findings impli-cate Btk in the physiological regulation of the cytoskele-ton.

3 Materials and methods

3.1 Plasmid constructs, cell transfections and cellstimulations

To express a Btk-GFP fusion protein, the Btk cDNA wascloned in-frame into the unique Eco47III site of pEGFP-N3(Clontech). Rat basophilic leukemia (RBL-2H3), HEK 293,HeLa, CHO and COS-7 cells were cultured in DMEM supple-mented with 10 % fetal calf serum and transient trans-fections were carried out using FuGeneTM 6 (BoehringerMannheim). The Ramos and DT40 cell lines were cultured inRPMI 1640 supplemented with 10 % fetal calf serum (LifeTechnologies). In addition, for DT40 cells 1 % chicken serumwas added (Sigma). Transient transfections of DT40 cellswas carried out using DMRIE-C Reagent (Life Technologies).In co-transfections, the DNA level of either Btk-GFP or Btk*-GFP constructs to the DNA of other expression plasmids(p110*, SHIP, PTEN, c-Src) were normally at a 1:9 ratio, toassure expression of the co-transfectant. However, for theRho-family small GTPases N17Rac1 and N17 Cdc42 theratio was also reversed to show the high specificity of theinhibition. Western blotting was done 48 h post-transfection


to ascertain that the transfections worked. RBL-2H3 mastcells (1 × 106) transfected with Btk-GFP were sensitized by1 ? g/ml rat anti-DNP IgE for 45 min at 37 °C. IgE-sensitizedRBL cells were stimulated with 10 ? g/ml DNP-BSA for 5 minat 37 °C. Chicken DT40 B cells (2 × 106) were stimulated with10 ? g/ml anti-chicken IgM mAb (M4) for 10 min at 37 °C.HeLa cells transfected with the Btk-GFP were stimulatedwith 50 ng/ml recombinant human SDF-1 § (R & D Systems)for 10 min at 37 °C.

3.2 Immunoprecipitation and immunoblot analysis

Immunoprecipitations were performed as described in [10].SDS-PAGE and immunoblotting was performed using stan-dard procedures. In vitro kinase assay was performed onimmunoprecipitated Btk beads in the presence of 20 ? lkinase buffer containing 10 mM MgCl2, 5 mM MnCl2 and200 ? M ATP for 10 min at room temperature. The reactionwas stopped by addition of 10 ? l 2 × sample buffer. Mono-clonal phosphotyrosine antibody 4G10 (Upstate Biotechnol-ogy) was used for detection of phosphorylated Btk usingECL detection system (Pierce).

3.3 Cross-linking and fluorescence labeling ofcytoskeleton

Cells were seeded on coverslips and prepared for labelingaccording to [37]. Total F-actin was labeled with 2 ? M phal-loidin conjugated to rhodamine (Molecular Probes) for30 min. Images were captured on a Leica DMRXA micro-scope equipped with a 3D digital microscopy workstation(Intelligent Imaging Innovations).

3.4 Immunoelectron microscopy

Cells were fixed in a mixture of 3 % paraformaldehyde and0.3 % glutaraldehyde in 0.1 M cacodylate buffer and 0.1 Msucrose containing 3 mmol CaCl2, pH 7.4, for 30 min. Cellswere then washed in buffer, infiltrated with 10 % gelatin,fixed in the same fixative and infiltrated with 2.3 M sucrose,and frozen in liquid nitrogen. Specimens were sectionedaccording to Tokuyasu [38]. Polyclonal antibodies againstGFP (1:300) were detected by protein A conjugated with 10-nm gold (Amersham). The sections were analyzed in a Leo906 microscope at 80 kV.

3.5 Scanning electron microscopy

Cells were grown on coverslips and fixed for 30 min (seeSect. 3.4). The cells were dried in a critical point dryer (Bal-zer, CPD 010) and coated in a sputter with 15-nm platinum(Polaron). The cells were examined in a Jeol JSM-820 scan-ning electron microscope at 15 kV.

Acknowledgements: This work was supported by theSwedish Cancer Society, the Swedish Medical ResearchCouncil and the European Union BIOTECH grant BIO4-98-0142. We are indebted to Drs. J. Collard, Dr. L. Williams, Dr.F. van Leeuwen, Dr. W. H. Moolenaar, Dr. G. Superti-Furga,Dr. T. Pawson and J. E. Dixon for providing cloned materials.We are grateful to Dr. T. Kurosaki for the DT40 cell lines.

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Correspondence: C. I. Edvard Smith, Department of Bio-sciences at Novum, CBT-Novum, Karolinska Institutet,SE-141 57 Huddinge, SwedenFax: +46-8-774-5538e-mail: edvard.smith — cbt.ki.se


redistribution of bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and...

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