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International Immunology, Vol. 12, No. 10, pp. 1397–1408 © 2000 The Japanese Society for Immunology

Identification and characterization of amolecule, BAM11, that associates with thepleckstrin homology domain of mouse Btk

Yuji Kikuchi1,2, Masayuki Hirano1, Masao Seto3 and Kiyoshi Takatsu1

1Department of Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai,Minato-ku, Tokyo 108-8639, Japan2Laboratory of Molecular Immunology, Center for Basic Research, Kitasato Institute, 5-9-1 Shirokane,Minato-ku, Tokyo 108-8642, Japan3Department of Pathology, Aichi Cancer Center, Chikusa-ku, Nagoya 464-8681, Japan

Keywords: IL-5, LTG19/ENL, signal transduction

Abstract

Bruton’s tyrosine kinase (Btk) is required for normal B cell development and signal transductionthrough cell surface molecules, and its defects lead to X-linked immune deficiency in mice andX-linked agammaglobulinemia in humans. In this report, we will describe the identification andcharacterization of a molecule, BAM11, which binds to the pleckstrin homology domain of Btk.A sequence homology search revealed that BAM11 has 89% homology, at the amino acid level, tohuman LTG19/ENL, that was originally identified as one of the fusion partners involved inchromosomal translocations of 11q23, MLL/ALL-1/HRX, in leukemia cells. Deletion mutantsdemonstrated that the region of BAM11 required for binding to Btk was localized between aminoacid residues 240 and 256. Forced expression of a truncated form of BAM11 (amino acids 246–368)inhibited IL-5-induced proliferation by 50%, whereas forced expression of full-length BAM11 in Y16cells did not affect the IL-5 responsiveness. We have also shown that BAM11 (amino acids 246–368) inhibited the kinase activity of Btk. These results suggest that the binding of BAM11 to Btkplays a regulatory role in the Btk signal transduction pathway. A cell fractionation study andanalysis using EGFP-fused Btk protein demonstrated that a proportion of Btk is present within thenucleus.

Introduction

Bruton’s tyrosine kinase (Btk) is a member of the Btk/Tec familyof cytoplasmic tyrosine kinases, and has been implicated incytoplasmic signal transduction through cell surface molec-ules including B cell receptor (BCR) (1,2), high-affinity Ig Ereceptor (FcεRI) (3,4) and cytokine receptors (5). Mutation ofBtk causes X-linked immunodeficiency (Xid) in mice (6,7) andX-linked agammaglobulinemia (XLA) in humans (8,9). B cellsfrom Xid mice carry a single amino acid substitution (Arg28to Cys) in Btk and have defects in their responses to stimulationvia BCR (10), IL-5 receptor (11,12), IL-10 receptor (13), FcεRI(14) and CD38 (15,16). Thus, Btk appears to be critical formultiple signaling pathways that are important for B celldevelopment and activation. It was also recently demonstratedthat FcεRI-induced degranulation was reduced in Xid mastcells (17).

The first two authors contributed equally to this work

Correspondence to: K. Takatsu,

Transmitting editor: D. Kitamura Received 27 March 2000, accepted 16 June 2000

Like other Btk/Tec family kinases, Btk is composed ofpleckstrin homology (PH), and unique Tec homology (TH),SH3, SH2 and kinase domains in this order from the N to Ctermini (18). The catalytic activity of Btk seems to be controlledby regulatory interactions with other molecules (1). The pro-line-rich sequence within the TH domain mediates interactionswith the SH3 domains of Src family kinases (19). It has beenshown that phosphorylation of Tyr551 by a Src family kinaseinduces the autophosphorylation of Tyr223 and augmentedkinase activity (20,21). We previously reported that Lyn isupstream of Btk activation in CD38 signaling (22). Lyn mayinteract with the TH domain of Btk via its SH3 domain andphosphorylate a tyrosine residue in Btk. Recently, a novelBtk–SH3 binding protein, Sab, has been described and itnegatively regulates Btk kinase activity (23,24).

1398 A novel Btk-associated molecule

PH domains have been found in many proteins involved insignal transduction as well as in cytoskeletal proteins (25,26).Several molecules, including βγ complexes of heterotrimericG protein (27), protein kinase C (PKC) (28), phosphatidylinosi-tol[3,4,5]triphosphate (PIP3) (29,30), BAP-135/TFII-I (31,32)and the filamentous form of actin (33), have been shown tointeract with the PH domain of Btk. BCR stimulation inducesinteraction between the PH domain of Btk and PIP3 andrecruits Btk to membrane fraction (34). Btk is able to modulatetyrosine phosphorylation of phopholipase C and sustainedcalcium release and flux in B cells (30,35).

To further elucidate the regulatory mechanism of Btk activityand its signaling pathway, we attempted to identify additionalproteins that interact with Btk and now describe one protein,BAM11, that associates with the Btk PH domain in vivo. Asequence homology search revealed that BAM11 has 89%homology (at the amino acid level) to human LTG19/ENL,which was originally identified as one of the fusion partnersinvolved in the chromosomal translocations of 11q23, MLL/ALL-1/HRX, in infantile leukemias (36,37). We also provideevidence that BAM11 participates in regulation of Btk kin-ase activity.

Methods

Screening and isolation of murine BAM11 cDNA

A cDNA fragment of the Btk PH domain (corresponding toamino acids 1–153) was generated by PCR using murine BtkcDNA as a template and cloned into the bacterial expressionvector pGEX-2a (Amersham Pharmacia Biotech, Uppsala,Sweden). The glutathione S-transferase (GST) fusion proteinwas induced and purified by Bulk GST Purification Module(Amersham Pharmacia Biotech). A λgt11 cDNA library madefrom IL-5-dependent early B cell line, Y16 (38), was screenedusing GST–Btk (PH) as the probe. Phage clones (1.0�106)were plated at a density of 5�104 plaques per 140 mm�100mm agarose plate. After incubation for 4 h at 42°C, the plateswere overlaid with nitrocellulose filters presoaked in 10 mMisopropyl-D-galactopyranoside, as described (39). Incubationwas continued for 4 h at 37°C to induce the protein. Thefilters were then removed, washed with TBST (10 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.05% Tween-20) at 4°C andblocked using TBST containing 5% skim milk for 30 min at4°C. After blocking, the GST–Btk (PH) probes were added ata concentration of 1 µg/ml and the incubation was continuedovernight at 4°C. The filters were washed with TBST 3 timesand incubated with anti-GST antiserum (1:1000 dilution) (39)for 2 h at 4°C. The filters were again washed with TBST 3times, followed by an incubation with alkaline phosphatase-conjugated swine anti-rabbit IgG (Dako, Glostrup, Denmark;1:1000 dilution) for 1 h at 4°C. After washing with TBST, thefilters were incubated with alkaline phosphatase reactionsolution [0.5 mM MgCl2 and 25 mM Na2CO3 (pH 9.8),containing 0.4 mM of nitroblue tetrazolium and 0.4 mM5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt (WakoJunyaku, Tokyo, Japan)]. To obtain specific clones that boundto the PH domain of Btk, the clones were probed with theGST-Btk (PH) or GST respectively at the third screening.

Cell lines and antibodies

Y16 and IL-2-dependent CTLL (40) were maintained inRPMI 1640 supplemented with 8% FCS, 100 U/ml penicillin,100 µg/ml streptomycin and 50 µM 2-mercaptoethanol in thepresence of IL-5 (5 U/ml) and IL-2 (5 U/ml). Rabbit anti-Btkpolyclonal antibody was produced by immunization with apeptide fragment of mouse Btk, amino acid residues 176–193. In some experiments we used goat anti-Btk (C-20) (SantaCruz Biotechnology, Santa Cruz, CA). Anti-T7-tag (Invitrogen,Carlsbad, CA), anti-myc (clone 9E10; ATCC, Rockville, MD),anti-FLAG (Sigma, St Louis, MO), anti-Tec (UpstateBiotechnology, Lake Placid, NY), anti-paxillin (Affiniti ResearchProducts, Nottingham, UK) and anti-phosphotyrosine (clone4G10; Upstate Biotechnology) antibodies were also pur-chased.

Vectors and constructs

pME18S-myc mammalian expression vector was provided byTadashi Yamamoto (University of Tokyo). The DNA encodingthe full-length BAM11 was amplified by PCR. The amplifiedproducts were cloned into the EcoRI sites of pME18S-myc toproduce the plasmid pME18S-myc-BAM11. To produceFLAG-tagged Btk, DNA fragments encoding the full length ofBtk were amplified by PCR. The amplified products werecloned between the BamHI and EcoRI sites of the pFLAG-mac vector (Sigma) to produce the plasmid pFLAG-Btk. Forconstruction of GST fusion proteins, DNA fragments encodingthe truncated form of BAM11, #11-1 (amino acids 1-368),#11-2 (amino acids 131–256), #11-3 (amino acids 240–368)or #11-4 (amino acids 363–547) were amplified by PCR. Theamplified products were cloned into EcoRI sites of pGEX-4T(Amersham Pharmacia Biotech). To generate T7 epitope-tagged Btk, the DNA fragments encoding full-length Btkwere amplified by PCR using forward and reverse primersincorporated EcoRI sites at their 5� ends. The reverse primeralso contained a sequence encoding T7 sequences(MASMTGGQQMG). The amplified products were cloned intothe EcoRI sites of pApuro vector, provided by TomohiroKurosaki (Kansai Medical University, Moriguchi, Osaka,Japan). To produce Btk mutant lacking the PH domain, theDNA fragments encoding amino acids 138–659 of Btk wereamplified by PCR using forward (AGAGCTCGAGCCAT-GCTGGTACAGAAATACCAT) and reverse (AGAGCGGCCG-CTCAGGATTCTTCATCCATC) primers, and the amplifiedproducts were cloned between XhoI and NotI sites of thepME18S vector. To produce EGFP fusion constructs, the DNAfragments encoding Btk, Btk (K430R) and GAPDH wereamplified by PCR. These amplified products were cloned intopEGFP-N1 vectors (Clontech, Palo Alto, CA). All constructionswere verified by nucleotide sequencing.

Preparation of cell lysates of IL-5-stimulated Y16 transfectants

Y16, Y16/BAM11 (full), Y16/BAM11 (amino acids 1–186) andY16/BAM11 (amino acids 240–368) cells were deprived ofIL-5 for 15 h of incubation before stimulation. Subsequently,cells were cultured at 107 cells/ml with 2000 U/ml of IL-5 for5 min at 37°C. They were then harvested by centrifugationand lysed in ice-cold lysis buffer (2�107 cells/ml) containing20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10%

A novel Btk-associated molecule 1399

glycerol, 2 mM EDTA, 100 U/ml aprotinin, 1 mM NaF, 1 mMNa3VO4 and 10 µg/ml leupeptin. Unsolubilized materials wereremoved by centrifugation for 15 min at 12 000 g.

Immunoprecipitation and Western blot analysis

Cell lysates were prepared and subjected to immunoprecipi-tation as previously described (5). Briefly, cell lysates of Y16transfectants and Cos7 cells expressing Btk/myc-BAM11 orBtk (amino acids 138–659)/myc-BAM11 were precleared withProtein G–Sepharose 4B and incubated at 4°C overnight with10 µg of anti-Btk antibody or anti-Tec antibody. Immunecomplexes were collected on Protein G–Sepharose during a60 min incubation at 4°C, washed 5 times with lysis bufferand boiled for 5 min with 2�Laemmli’s sample buffer. Forthe Western blot, samples were electrophoresed on SDS–polyacrylamide gels (8%) and transferred to an Immobilon-Pmembrane (Nihon Millipore, Tokyo, Japan). After blockingwith TBS containing 5% BSA the membranes were incubatedwith the appropriate primary antibody and washed in TBScontaining 0.1% Tween 20 (TBS-T). After incubation with goatanti-mouse IgG or goat anti-rabbit IgG secondary antibodiescoupled to horseradish peroxidase, the membranes werewashed 4 times with TBS-T and subjected to an ECL detectionsystem (Amersham Pharmacia Biotech).

Binding assay for GST fusion proteins with Btk

Interaction of the GST fusion proteins made from truncatedBAM11 attached to Btk was examined as follows. Equalamounts of GST fusion proteins were incubated with bacterialcell lysates expressing FLAG-Btk for 4 h at 4°C and washedwith 0.1% of Triton X-100. After adding the SDS–PAGE samplebuffer, the precipitates bound to GST fusion proteins wereeluted by boiling and examined by immunoblotting with theanti-FLAG mAb.

Proliferation assay

Y16, CTLL or their transfectants (1�104/well) were culturedfor 48 h with various concentrations of IL-5 or IL-2. The cellswere pulse labeled with [3H]thymidine (0.2 µCi/well) duringthe last 6 h of their 48 h culture period and the incorporationof radioactivity was measured.

In vitro kinase assay

The T7-Btk pApuro vector was transfected into Cos7 cells. After48 h, the cells were lysed with a kinase lysis buffer (1% TritonX-100, 10 mM NaH2PO4/Na2HPO4, pH 7.0, 150 mM NaCl, 5mM EDTA, 1 mM PMSF and 10 µg/ml leupeptin) and the T7epitope-tagged Btk protein was immunopurified using anti-T7-tag antibody and Protein G–Sepharose beads (AmershamPharmacia Biotech). The purified GST–Btk protein expressedby insect cells using the baculovirus expression system wasprovided by Keisuke Horikawa (University of Tokyo). The T7–or GST–Btk was dissolved in kinase buffer and the appropriateamount of GST fusion proteins and 2 µg of a peptide, containingthe Btk autophosphorylation site (KKVVALYDYMPMN), wereadded. The reaction was initiated by addition of 20 µCi of[γ-32P]ATP (Amersham Pharmacia Biotech) and 20 pmol ofunlabeled ATP, and then allowed to proceed for 5 min at 25°C,and finally stopped by adding cold ATP and EDTA. The mixturewas then transferred onto P81 phosphocellulose paper. After

washing, the amount of incorporated [γ-32P]ATP was measuredusing a scintillation counter.

Subcellular fractionation

The cells were washed twice with SET (150 mM NaCl, 50 mMTris–HCl and 1 mM EDTA, pH 7.2) and scraped into hypo-tonic buffer (1 mM EGTA, 1 mM EDTA, 1 mM Na3VO4,2 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 40 µg/ml PMSF,10 µg/ml pepstain and 10 µg/ml leupeptin) (41). The cellswere incubated for 30 min on ice and then dounce-homogen-ized. The homogenate was loaded onto 1 ml of 1 M sucrosecushion and centrifuged at 1600 g for 10 min to pelletthe nuclei. The supernatants were fractionated into pellets(membrane fraction) and supernatants (cytosol fraction) bycentrifugation at 100,000 g for 30 min at 4°C. The nuclearpellet was solubilized in hypotonic lysis buffer containing0.5% Nonidet P-40, 0.1% deoxycholate and 0.1% Brij-35, andthen centrifuged at 12,000 g for 15 min to remove anyinsoluble material. We monitored the purity of each fractionby measuring the activity of cytosol marker enzyme, lactatedehydrogenase, as previously described (42).

Histochemistry

The Btk–EGFP, Btk (K430R)–EGFP, GAPDH–EGFP and myc-BAM11 constructs were introduced into Cos7 cells usingelectropolation. After 48 h, the cells were collected and fixedonto 0.1% poly-L-lysine-treated glass slides. For the stainingof myc-BAM11, glass slides were incubated with mouse anti-myc antibody (9E10) followed by FITC-conjugated rabbitanti-mouse IgG. All samples were incubated with 0.1 mg/mlof propidium iodide (PI) for 15 min at room temperature. Thelocalization of the EGFP fusion proteins and BAM11 wasmonitored by confocal laser scanning microscopy (CLSM)(BioRad, Hercules, CA)

Results

Isolation of a molecule which binds to the PH domain of Btk

We used an expression cloning technique to isolate a moleculethat associates with the PH domain of Btk. A fusion proteinmade from GST and the Btk PH domain (GST–BTK-PH) wasused as a probe to screen a λgt11 cDNA expression libraryoriginating from the murine early B cell line Y16. Inducedλgt11 recombinant proteins from the cDNA library weretransferred to nitrocellulose filters and the filters were hybrid-ized with GST–Btk-PH. By the third round of screening with1.0�106 independent phage clones, three clones demon-strated prominent binding to GST–Btk-PH, but not to GSTprotein alone. The nucleotide sequence of these clonesindicated that each clone originated from different mRNAs.One of these clones (clone #11) was selected for isolation offull-length cDNA and further analysis. Using the 5�- and3�-RACE method, a full-length cDNA was obtained. Sequenceanalysis of this full-length cDNA revealed that the predictedopen reading frame is 1641 nucleotides, which encodes 547amino acid residues (Fig. 1). This protein was termed BAM11(Btk Associated Molecule-11). Northern blotting analysis dem-onstrated that the transcripts of this gene are expressed in avariety of murine tissues (brain, heart, thymus, liver, spleen,


Fig. 1. Nucleotide sequence and deduced amino acid sequence of BAM11. The nucleotide sequence is shown on the upper line; the deducedamino acid sequence is shown on the lower line. The numerical positions of the nucleotides are also shown.

lymph node, bone marrow, intestine, muscle and lung) (datanot shown). A sequence homology search revealed thatBAM11 has 89% homology to human LTG19/ENL at the aminoacid level (Fig. 2).

Btk association with BAM11 in cells

To analyze the in vivo association between Btk and BAM11,human myc epitope-tagged BAM11 was expressed in Y16


Fig. 2. Sequence homology of BAM11 with human LTG19/ENL. The deduced amino acid sequences from BAM11 cDNA and human LTG19/ENL are aligned using GENETYX-MAC software. Identical amino acids are boxed. The possible tyrosine kinase phosphorylation motifs of [R/K]-[X2/X3]-[D/E]-[X2/X3]-Y are underlined. The nuclear localization sequences in the BAM11 are depicted as overlined.

cells. The cell lysates were immunoprecipitated with anti-Btkantibody and co-precipitated myc-BAM11 was detected byimmunoblotting with monoclonal anti-human myc antibody. A78–80 kDa band was found in immunoprecipitates with anti-Btk antibody of myc-BAM11-transfected Y16 cells, but not inimmunoprecipitates from non-transfected Y16 cells (Fig. 3A,lane 3 versus lane 1). To clarify the binding specificity ofBAM11 to Btk, the association of BAM11 with Tec wasexamined using anti-Tec antibody for immunoprecipitation.BAM11 protein was not detected in the immunoprecipitatesfollowing anti-Tec antibody treatment (Fig. 3A, lane 4). Involve-ment of the PH domain of Btk in the in vivo interaction betweenBtk and BAM11 was confirmed by using Cos7 cells expressingwild-type Btk or Btk lacking the PH domain. As shown in Fig.

3(B), association of Btk with BAM11 was detected when wild-type Btk and BAM11 were co-expressed in Cos7 cells, butwas not detected when Btk lacking the PH domain wasexpressed (Fig. 3B, lane 2 versus lane 3). These resultssuggest that Btk interacts with BAM11 via the PH domainin vivo and demonstrate that BAM11 associates with the PHdomain of Btk in cells, but not with the PH domain of Tec kinase.

Determination of the region in BAM11 responsible for theassociation with Btk

In order to determine the BAM11 Btk-binding region, wegenerated GST fusion proteins containing full-length BAM11and various truncated mutants (Fig. 4). By using the sameamounts of GST fusion protein conjugated onto glutathione-


Fig. 3. Associations between BAM11 and Btk in cells. (A) Y16 cells or myc epitope-tagged BAM11-expressing Y16 transfectants were lysedand immunoprecipitated with anti-Btk (lanes 1 and 3) or anti-Tec (lane 4) antibody. The precipitates were separated using SDS–PAGE,transferred to membranes and blotted with anti-myc antibody. Preimmune rabbit sera were used as a control (lane 2) (upper panel). The blotwas stripped and reprobed with anti-Btk or anti-Tec antibodies (lower panel). (B) Cos7 cells transfected with Btk (wild-type) together with myc-BAM11 (lane 2) or Btk lacking the PH domain (amino acids 138–659) together with myc-BAM11 (lane 3) or non-transfected Cos7 cells (lane1) were lysed and immunoprecipitated with anti-Btk antibody. The precipitates were separated using SDS–PAGE, transferred to membranesand blotted with anti-myc antibody (upper panel). The blot was stripped and reprobed with anti-Btk antibody (lower panel).

Fig. 4. Location of the Btk binding site in BAM11. Upper panel shows the schematic representation of the deletion mutants of GST–BAM11fusion proteins used for the binding assay (upper panel). Equal amounts of the deletion mutants of GST–BAM11 fusion proteins were boundto glutathione-coupled Sepharose beads and incubated with bacterial cell lysates expressing FLAG–Btk for 4 h at 4°C. The precipitatedcomplexes were then separated using SDS–PAGE, transferred to membranes and blotted with an anti-FLAG antibody (lower panel).

coupled Sepharose beads, we carried out binding assays forassociation with Btk. When bacterial cell lysates expressingFLAG-tagged Btk were incubated with GST alone, no Btkprotein was detected by immunoblotting (Fig. 4). However,the GST fusion proteins containing full-length BAM11 (GST-#11-full) did bind Btk. Furthermore, the GST fusion protein,

GST-#11-1 (containing amino acids 1-368), GST-#11-2 (aminoacids 131–256) and GST-#11-3 (amino acids 240–368) wasable to bind Btk, but GST-#11-4 (amino acids 363–547) wasobserved not to bind Btk. These findings indicate that theregion of BAM11 responsible for binding to Btk lies betweenamino acid residues 240 and 256. It should be noted that the


Fig. 5. Effects of BAM11 protein overexpression on IL-5- or IL-2-induced proliferation and Btk activation. BAM11/full, BAM11/aa1–186 andBAM11/aa240–368 proteins were stably overexpressed in Y16 cells (A) or CTLL cells (B). Parental cells (u), BAM11/full (d), BAM11/aa1–186(s) and BAM11/aa240–368 (m) transfectants (1�104/well/0.2 ml) were cultured with various concentrations of IL-5 (A) or IL-2 (B). The cellswere pulse labeled with [3H]thymidine for the last 6 h of 48 h cultures. Clones expressing different amounts of BAM11/aa240–368 (d, j andm) and parental Y16 cells (u) were cultured with various concentrations of IL-5 (C, upper). Level of expressed BAM11/aa240–368 in eachtransfectant was estimated by Western blot (C, lower). Results represent the mean � SD of triplicate determinations. (D): Y16, Y16/BAM11(full),Y16/BAM11(amino acids 1–186) and Y16/BAM11(amino acids 240–368) cells were stimulated with 2000 U/ml mIL-5 for 5 min. Cells were lysedand immunoprecipitated with anti-Btk antibody. Immune complexes were subjected to Western blotting using anti-phosphotyrosine mAb (4G10)and tyrosine-phosphorylated Btk were detected by the ECL assay. There were two tyrosine-phosphorylated bands around 78 kDa, the lowerband corresponded to Btk (upper). The same blot was stripped and reprobed with anti-Btk antibody (lower panel).

other regions of BAM11 may also contribute to certain extentto the binding of Btk, since the amounts of Btk bound to eachBAM11 truncated protein differed.

Effect of the overexpression of BAM11 protein on IL-5 signaling

As we reported (5), Y16 exhibits not only IL-5-dependentproliferation but also enhanced Btk activity upon IL-5 stimula-tion. So we examined whether forced expression of BAM11proteins affected IL-5-induced proliferation in Y16. cDNA forBAM11/full-length, BAM11/aa1–186 and BAM11/aa240–368were stably transfected into Y16 or CTLL. Clones thus estab-lished were stimulated with IL-5 or IL-2 and the uptake of[3H]thymidine was determined. Three independent clonesexpressing the same levels of transgene-derived BAM11proteins were examined for each transfectant and similarresults were obtained. The representative results are shownin Fig. 5. As shown in Fig. 5(A), forced expression of BAM11/fulland BAM11/aa1–186 did not affect IL-5-induced proliferation,while the proliferative response was reduced by 50% in theclones that overexpressed BAM11/aa240–368. The degree ofthe reduction was dependent on the expression levels of theBAM11/aa240–368 (Fig. 5C). CTLL clones expressing either

BAM11/full, BAM11/aa1–186 or BAM11/aa240–368 did notshow any reduction in IL-2 responsiveness (Fig. 5B).

Inhibition of Btk kinase activity by BAM11

The inhibitory effect of BAM11/aa240–368 overexpression onthe IL-5-induced proliferation described above led us toevaluate whether the BAM11 protein regulates Btk kinaseactivity. Then, we examined Btk activities after IL-5 stimulationin Y16 transfectants. In contrast to the marked enhancementof autophosphorylation of Btk in parental Y16 cells, BAM11/full and BAM11/aa1–186 transfectants, enhancement of Btkautophosphorylation in the BAM11/aa240–368 transfectantwas weak (Fig. 5D). The analysis by densitometery revealedthat the extent of the enhancement of Btk autophosphorylationin BAM11/full and BAM11/aa1–186 transfectants were com-parable with that of Y16, whereas the enhancement of Btkautophosphorylation in the BAM11/aa240–368 transfectantwas reduced by 60% (data not shown). Next, to examinedirect effects of BAM11 on Btk kinase activity, we carried outan in vitro kinase assay on Btk in the presence of BAM11proteins. T7 epitope-tagged murine Btk was immunopurifiedfrom the lysate of Cos7 cells transfected with T7 epitope-


Fig. 6. Effects of BAM11 protein on the kinase activity of Btk.Immunoprecipitated Btk expressed by Cos7 cells (A) or insect cells(B) was mixed with the indicated concentrations of GST (u), GST-BAM11/full (d), GST-BAM11/aa1–186 (s) or GST-BAM11/aa240–368(m) proteins. The in vitro kinase assay was carried out using thepeptide substrate as described in Methods.

tagged murine Btk cDNA as described in Methods. Thekinase activity of the immunopurified Btk expressed by Cos7cells was assayed using a peptide substrate with thesequence KKVVALYDYMPMN, which corresponded to theamino acid residues 217–229 of murine Btk. The tyrosine atresidue 223 in this sequence is the Btk autophosphorylationsite. This peptide does not contain any serine or threonineresidues and has been shown to be a good substrate forBtk (24,43). As shown in Fig. 6(A), the kinase activity ofimmunopurified Btk was inhibited by the addition of GST-BAM11/aa240–368 protein to the reaction in a dose-depend-ent manner. In contrast, no inhibition of Btk kinase activitywas observed following the addition of GST protein aloneor GST-BAM11/aa1–186 protein. These results suggest thatBAM11/aa240–368 acts as an inhibitor of Btk kinase activity.To exclude the effects of other cellular protein which couldbe co-immunoprecipitated with Btk from the mammalian cells,we also used recombinant GST–Btk which was expressed byinsect cells. Like Btk expressed by Cos7 cells, the kinaseactivity of Btk expressed by insect cells was inhibited by GST-BAM11/aa240–368 protein (Fig. 6B). We infer from theseresults that the reduction of IL-5 responsiveness by theoverexpression of BAM11/aa240–368 in Y16 cells might bedue to inhibition of the IL-5-enhanced Btk activity.

Subcellular localization of Btk

As described above, BAM11 has 89% homology to humanLTG19/ENL at the amino acid level. It has been shown thatLTG19/ENL localizes in the nucleus (44) and BAM11 proteinalso contains nuclear targeting sequences (Fig. 2). In orderto obtain further clues to the association pattern of Btkand BAM11, the subcellular localization of Btk in Y16 wasdetermined. Cell lysates of Y16 were fractionated into nuclearand cytoplasmic fractions, and amounts of Btk protein in eachfraction were detected by immunoblotting. We also used CTLLand Btk cDNA transfected CTLL as controls. The purity ofeach fraction was assessed by reprobing the same blotwith anti-paxillin antibody that recognize paxillin, which is acytoskeletal protein. Results revealed that contamination ofcytoplasmic fractions into nuclear fractions were �10%. Wealso monitored lactate dehydrogenase activity that is also a

Fig. 7. Subcellular localization of Btk. Y16, CTLL or CTLL-mBtktransfectants were homogenized in a hypotonic buffer. The nucleiwere pelleted using a 1 M sucrose cushion. The supernatants thesucrose cushion were then centrifuged (150,000 g, 30 min) and usedas the cytosol fraction. A 10 µg sample of proteins from each fractionwas separated on SDS–PAGE, transferred to a PVDF membrane andblotted with anti-Btk antibody (upper panel). The blot was strippedand reprobed with an anti-paxillin antibody to examine the purity ofeach fraction (lower panel)

cytoplasmic enzyme (42). The distribution of enzyme activityin each fraction from the Y16 cells was 91% cytosolic and9% nuclear (data not shown). Btk protein (78 kDa) was foundat a similar extent in both the nuclear and cytoplasmicfractions of Y16 and CTLL transfectants (Fig. 7, lanes 1–4),but not in the fractions from untransfected CTLL cells (Fig. 7,lanes 5 and 6). In this experiment, we used 10 µg of proteinfrom each fraction. It should be noted that when we adjustedthe amount of protein based on cell number (1.5�106), Btkproteins were detected only in the cytoplasmic fractions (datanot shown).

We then took a different approach to assess the subcellularlocalization of Btk. Btk–EGFP fusion protein was transientlyexpressed in Cos7 cells and analyzed by CLSM. Cos7transfectants were fixed on glass slides and their nuclei werestained with propidium iodide (red). Since Btk–EGFP proteinwas detected as a green color, yellow spots indicated Btkproteins in the nuclei. As shown in Fig. 8(A), marked nuclearBtk staining, especially in the nuclear membrane area, wasobserved. These observations were consistence with theresults of Western blot analysis using subcellular fractionation(Fig. 7). When a control cytoplasmic protein, GAPDH (45),was expressed in Cos7 cells, GAPDH was not detected inthe nucleus (Fig. 8C). We also examined the relationshipbetween Btk localization and Btk activity. Transfectants of thekinase inactivated (K430R) Btk mutant appeared to show asimilar localization pattern in the nuclei to that of transfectantsof wild-type Btk (Fig. 8B). These results suggest that thelocalization of Btk in nuclei is independent of Btk activity. Wealso stained myc-BAM11 transfectants with anti-myc antibody.Results revealed that like human LTG19/ENL protein (44),most of the BAM11 existed within the nucleus (Fig. 8D).


Fig. 8. Subcellular localization of Btk–EGFP, Btk (K430R)–EGFP and myc-BAM11 fusion proteins in Cos7 cells. Cos7 cells expressing EGFPfusion constructs (green) (A) Btk–EGFP, (B) Btk (K430R)–EGFP and (C) GAPDH–EGFP were analyzed using CLSM. Cos7 cells expressingmyc-BAM11 (D) were stained with a mouse anti-myc antibody followed by FITC-conjugated anti-mouse IgG antibody (green). The nuclei werestained with propidium iodide (red).

Discussion

This study contains three major findings. (i) We identified amolecule, BAM11, which can bind to the PH domain of Btkboth in vitro and in vivo. A sequence homology searchrevealed that BAM11 has 89% homology to human LTG19/ENL at the amino acid level. (ii) Forced expression of atruncated BAM11 protein (amino acids 246–368) in Y16 cellsreduced the proliferative response induced by IL-5. The sametruncated BAM11 protein also inhibited Btk activity measuredby the in vitro kinase assay. (iii) Immunoblot analysis usinganti-Btk antibody and cell staining analysis using EGFP–Btkfusion protein demonstrated that a small proportion of Btkproteins exists within the nucleus.

The activity of Btk appears to be controlled by binding toother protein molecules. Full activation of Btk appears todepend on transphosphorylation of Tyr551 by a Src familykinase (46). Yao et al. (28) provided evidence that multipleisoforms of PKC interact with Btk and PKC-mediated phospho-

rylation down-regulates the enzymatic activity of Btk. The PHdomain of the cytoplasmic tyrosine kinases, including that ofBtk, is implicated as a protein interaction domain. Throughits physical association with the PH domain of Btk, we haveidentified BAM11 as a possible molecule which controlsBtk activity. Though BAM11 contains three possible tyrosinephosphorylation sites and can associate with Btk in Y16 cells,tyrosine phosphorylation of BAM11 upon IL-5 stimulation hasnot been observed (data not shown). This suggests thatBAM11 is not a major substrate of Btk in Y16 cells.

BAM11 has 89% homology to human LTG19/ENL at theamino acid level that was originally identified as one of thefusion partners involved in chromosomal translocations of11q23, MLL/ALL-1/HRX, in human leukemia cells (36,37). Thefunction of LTG19/ENL is not fully understood. Rubnitz et al.(47) reported that LTG19/ENL could activate transcription ofsynthetic reporter genes in both lymphoid and myeloid cells.In leukemia cells, it has been suggested that the t(11;19)


chromosomal translocation fuses a DNA binding domain,AT-hook, from the MLL/ALL-1/HRX protein with LTG19/ENLprotein, resulting in the formation of a new transcription factorthat may play an important role for leukemogenesis.

We have reported that the B cells of XID mice fail to respondto IL-5 (11,12) and Y16 exhibits not only IL-5-dependentproliferation but also enhanced Btk activity upon IL-5 stimula-tion (5). Li et al. (48) provided evidence that the expressionof a gain-of-function mutant of Btk (E41K) in Y16 inducedIL-5-independent growth. These observations indicate theimportance of the Btk signaling pathway in B cells to respondto IL-5 and we infer that the Btk signaling pathway is involvedin IL-5-dependent proliferation of Y16 cells. Here we haveshown the association of BAM11 with the PH domain of Btkand a truncated form of BAM11/aa240–368, that contains theBtk-binding region, reduced both IL-5-induced proliferationand IL-5-induced enhancement of Btk autophosphorylation inY16 cells. We also revealed that BAM11/aa240–368 partiallyinhibited Btk activity measured by the in vitro kinase assay.The extent of the suppression of IL-5-induced proliferation ofY16 transfectants depended on the amounts of expressedBAM11/aa240–368 protein, indicating that BAM11/aa240–368directly (or indirectly) affects the IL-5 signaling in Y16 cellsrather than the artifact of transfection experiments. However,it should be noted that full-length BAM11 affected neither IL-5 responsiveness nor Btk activity in this experimental system.BAM11 may have the potential to regulate Btk activity, butother factors may be required for the full expression of itsfunction. One possibility is that the binding of another moleculeto BAM11 may modulate the tertiary structure of BAM11 andthe modulated BAM11 exhibits functions shown for BAM11/aa240–368 in this study. This possibility is currently underinvestigation.

It has been reported that the activation of Btk dependson both transphosphorylation by a Src family kinase andmembrane localization (49). The membrane localization ofBtk is controlled by the binding of phospholipid moieties(1,35). In this study, we showed by means of biochemicalanalysis using parental Y16 cells that Btk was detected in thenucleus at a similar extent as in the cytoplasm on a perprotein basis (Fig. 7). Btk was detected in the cytoplasm butwas hardly detectable in the nucleus in per cell number basisthat may be due to a low sensitivity to detect a tiny amountof Btk in the nucleus. Results of confocal microscopic analysisusing Cos7 transfectants revealed a significant localization ofBtk in the nucleus. Overexpression of a particular proteinmight lead to inappropriate localization of the protein.However,this does not exclude the possibility that Btk may localize innucleus. These results may reflect the fact that the Btksequence contains the bipartite type of nuclear localizationsignal, KRSQQKKKTSPLNFKKR at amino acid residue 12,which is composed of two basic residues, 10 residues as aspacer and another basic region consisting of at least threebasic residues out of five residues (50). To clarify the quantityof Btk in the nucleus, further experiments using a morephysiological setting would be necessary in future. Althoughthe current role of Btk in the nucleus has not sufficiently beenclarified at present, Btk might regulate transcription factoractivity of LTG19/ENL (BAM11). The possibility that Btk isinvolved in leukemogenesis will also be considered. By analyz-

ing the role of Btk from the viewpoint of the crisis mechanismof leukemia, we may be able to clarify the function of Btk inthe nucleus.

Analysis of BAM11-deficient mice would be considered asanother approach to clarify the functional relationship betweenBAM11 and Btk. The tissue distribution of BAM11 is substan-tially broader than that of Btk, which is restricted to hematopoi-etic cells. BAM11 may also associate with other cellularproteins in B-lineage cells than Btk, although BAM11 is ableto associate with the PH domain of Btk in preference to thatof Tec kinase. Further analysis of BAM11 and the role of Btkin the nucleus should bring new insight regarding molecularmechanisms of Btk-mediated B cell development and prolif-eration.

Acknowledgments

We are grateful to Drs Tatsuo Kinashi and Satoshi Takaki for theirvaluable suggestions. We are also grateful to Drs Yoshihiro Takemotoand Yasuhiro Hashimoto for their helpful advice on the screening ofBAM11. We thank Drs T. Yamamoto, T. Kurosaki, K. Horikawa, and T.Kouro for providing pME18S-myc vector, pApuro vector, purified Btkexpressed by insect cells and anti-Btk antibody respectively. Wethank Dr Paul W. Kincade and Gavin Maxwell for critical reading ofthe manuscript. This study was supported in part by a Grant-in-Aidfor Scientific Research from the Ministry of Education, Science, Sportsand Culture of Japan, and by Kato Memorial Bioscience Foundation.

Abbreviations

Btk Bruton’s tyrosine kinaseCLSM confocal laser scanning microscopyGST glutathione S-transferasePIP3 phosphatidylinositol[3,4,5]triphosphatePH pleckstrin homologyPKC protein kinase CTH Tec homologyXid X-linked immune deficiencyXLA X-linked agammaglobulinemia

Note added in proof

The nucleotide sequence of mouse BAM11 has been deposited inthe GenBank database (accession No. AF298887).

References

1 Kurosaki, T. 1999. Genetic analysis of B cell antigen receptorsignaling. Annu. Rev. Immunol. 17:555.

2 Satterthwaite, A. B., Li, Z. and Witte, O. N. 1998. Btk function inB cell development and response. Semin. Immunol. 10:309.

3 Kawakami, Y., Yao, L., Miura, T., Tsukada, S., Witte, O. N. andKawakami, T. 1994. Tyrosine phosphorylation and activation ofBruton tyrosine kinase upon Fc epsilon RI cross-linking. Mol. Cell.Biol. 14:5108.

4 Kawakami, Y., Kitaura, J., Hata, D., Yao, L. and Kawakami, T.1999. Functions of Bruton’s tyrosine kinase in mast and B cells.J. Leuk. Biol. 65:286.

5 Sato, S., Katagiri, T., Takaki, S., Kikuchi, Y., Hitoshi, Y., Yonehara,S., Tsukada, S., Kitamura, D., Watanabe, T., Witte, O. N. andTakatsu, K. 1994. IL-5 receptor-mediated tyrosine phosphorylationof SH2/SH3-containing proteins and activation of Bruton’s tyrosineand Janus 2 kinases. J. Exp. Med. 180:2101.

6 Rawlings, D. J., Saffran, D. C., Tsukada, S., Largaespada, D. A.,Grimaldi, J. C., Cohen, L., Mohr, R. N., Bazan, J. F., Howard, M.,Copeland, N. G., Jenkins, N. A. and Witte, O. N. 1993. Mutation


of unique region of Bruton’s tyrosine kinase in immunodeficientXID mice. Science 261:358.

7 Thomas, J. D., Sideras, P., Smith, C. I., Vorechovsky, I., Chapman,V. and Paul, W. E. 1993. Colocalization of X-linkedagammaglobulinemia and X-linked immunodeficiency genes.Science 261:355.

8 Tsukada, S., Saffran, D. C., Rawlings, D. J., Parolini, O., Allen, R.C., Klisak, I., Sparkes, R. S., Kubagawa, H., Mohandas, T., Quan,S., Belmont, J. W., Cooper, M. D., Conley, M. E. and Witte, O. N.1993. Deficient expression of a B cell cytoplasmic tyrosine kinasein human X- linked agammaglobulinemia. Cell 72:279.

9 Vetrie, D., Vorechovsky, I., Sideras, P., Holland, J., Davies, A.,Flinter, F., Hammarstrom, L., Kinnon, C., Levinsky, R., Bobrow,M., Smith, C. I. E. and Bentley, D. R. 1993. The gene involved inX-linked agammaglobulinaemia is a member of the src family ofprotein-tyrosine kinases [published erratum appears in Nature1993; 364(6435):362]. Nature 361:226.

10 Smith, C. I., Islam, K. B., Vorechovsky, I., Olerup, O., Wallin, E.,Rabbani, H., Baskin, B. and Hammarstrom, L. 1994. X-linkedagammaglobulinemia and other immunoglobulin deficiencies.Immunol. Rev. 138:159.

11 Hitoshi, Y., Sonoda, E., Kikuchi, Y., Yonehara, S., Nakauchi, H.and Takatsu, K. 1993. IL-5 receptor positive B cells, but noteosinophils, are functionally and numerically influenced in micecarrying the X-linked immune defect. Int. Immunol. 5:1183.

12 Koike, M., Kikuchi, Y., Tominaga, A., Takaki, S., Akagi, K., Miyazaki,J., Yamamura, K. and Takatsu, K. 1995. Defective IL-5-receptor-mediated signaling in B cells of X-linked immunodeficient mice.Int. Immunol. 7:21.

13 Go, N. F., Castle, B. E., Barrett, R., Kastelein, R., Dang, W.,Mosmann, T. R., Moore, K. W. and Howard, M. 1990. Interleukin10, a novel B cell stimulatory factor: unresponsiveness of Xchromosome-linked immunodeficiency B cells. J. Exp. Med.172:1625.

14 Hata, D., Kawakami, Y., Inagaki, N., Lantz, C. S., Kitamura, T.,Khan, W. N., Maeda-Yamamoto, M., Miura, T., Han, W., Hartman,S. E., Yao, L., Nagai, H., Goldfeld, A. E., Alt, F. W., Galli, S. J.,Witte, O. N. and Kawakami, T. 1998. Involvement of Bruton’styrosine kinase in FcepsilonRI-dependent mast cell degranulationand cytokine production. J. Exp. Med. 187:1235.

15 Kikuchi, Y., Yasue, T., Miyake, K., Kimoto, M. and Takatsu, K.1995. CD38 ligation induces tyrosine phosphorylation of Brutontyrosine kinase and enhanced expression of interleukin 5-receptoralpha chain: synergistic effects with interleukin 5. Proc. Natl Acad.Sci. USA 92:11814.

16 Yamashita, Y., Miyake, K., Kikuchi, Y., Takatsu, K., Noda, S.,Kosugi, A. and Kimoto, M. 1995. A monoclonal antibody againsta murine CD38 homologue delivers a signal to B cells forprolongation of survival and protection against apoptosis in vitro:unresponsiveness of X-linked immunodeficient B cells.Immunology 85:248.

17 Setoguchi, R., Kinashi, T., Sagara, H., Hirosawa, K. and Takatsu,K. 1998. Defective degranulation and calcium mobilization ofbone-marrow derived mast cells from Xid and Btk-deficient mice.Immunol. Lett. 64:109.

18 Tsukada, S., Rawlings, D. J. and Witte, O. N. 1994. Role of Bruton’styrosine kinase in immunodeficiency. Curr. Opin. Immunol. 6:623.

19 Cheng, G., Ye, Z. S. and Baltimore, D. 1994. Binding of Bruton’styrosine kinase to Fyn, Lyn, or Hck through a Src homology 3domain-mediated interaction. Proc. Natl Acad. Sci. USA 91:8152.

20 Park, H., Wahl, M. I., Afar, D. E., Turck, C. W., Rawlings, D. J.,Tam, C., Scharenberg, A. M., Kinet, J. P. and Witte, O. N. 1996.Regulation of Btk function by a major autophosphorylation sitewithin the SH3 domain. Immunity 4:515.

21 Kurosaki, T. and Kurosaki, M. 1997. Transphosphorylation ofBruton’s tyrosine kinase on tyrosine 551 is critical for B cellantigen receptor function. J. Biol. Chem. 272:15595.

22 Yasue, T., Nishizumi, H., Aizawa, S., Yamamoto, T., Miyake, K.,Mizoguchi, C., Uehara, S., Kikuchi, Y. and Takatsu, K. 1997. Acritical role of Lyn and Fyn for B cell responses to CD38 ligationand interleukin 5. Proc. Natl Acad. Sci. USA 94:10307.

23 Matsushita, M., Yamadori, T., Kato, S., Takemoto, Y., Inazawa, J.,Baba, Y., Hashimoto, S., Sekine, S., Arai, S., Kunikata, T., Kurimoto,

M., Kishimoto, T. and Tsukada, S. 1998. Identification andcharacterization of a novel SH3-domain binding protein, Sab,which preferentially associates with Bruton’s tyrosine kinase (BtK).Biochem. Biophys. Res. Commun. 245:337.

24 Yamadori, T., Baba, Y., Matsushita, M., Hashimoto, S., Kurosaki,M., Kurosaki, T., Kishimoto, T. and Tsukada, S. 1999. Bruton’styrosine kinase activity is negatively regulated by Sab, the Btk-SH3 domain-binding protein. Proc. Natl Acad. Sci. USA 96:6341.

25 Musacchio, A., Gibson, T., Rice, P., Thompson, J. and Saraste,M. 1993. The PH domain: a common piece in the structuralpatchwork of signalling proteins. Trends Biochem. Sci. 18:343.

26 Lemmon, M. A., Ferguson, K. M. and Schlessinger, J. 1996. PHdomains: diverse sequences with a common fold recruit signalingmolecules to the cell surface. Cell 85:621.

27 Tsukada, S., Simon, M. I., Witte, O. N. and Katz, A. 1994. Bindingof beta gamma subunits of heterotrimeric G proteins to thePH domain of Bruton tyrosine kinase. Proc. Natl Acad. Sci.USA 91:11256.

28 Yao, L., Kawakami, Y. and Kawakami, T. 1994. The pleckstrinhomology domain of Bruton tyrosine kinase interacts with proteinkinase C. Proc. Natl Acad. Sci. USA 91:9175.

29 Li, Z., Wahl, M. I., Eguinoa, A., Stephens, L. R., Hawkins, P. T.and Witte, O. N. 1997. Phosphatidylinositol 3-kinase-gammaactivates Bruton’s tyrosine kinase in concert with Src familykinases. Proc. Natl Acad. Sci. USA 94:13820.

30 Fluckiger, A. C., Li, Z., Kato, R. M., Wahl, M. I., Ochs, H. D.,Longnecker, R., Kinet, J. P., Witte, O. N., Scharenberg, A. M.and Rawlings, D. J. 1998. Btk/Tec kinases regulate sustainedincreases in intracellular Ca2� following B-cell receptor activation.EMBO J. 17:1973.

31 Yang, W. and Desiderio, S. 1997. BAP-135, a target for Bruton’styrosine kinase in response to B cell receptor engagement. Proc.Natl Acad. Sci. USA 94:604.

32 Novina, C. D., Kumar, S., Bajpai, U., Cheriyath, V., Zhang, K.,Pillai, S., Wortis, H. H. and Roy, A. L. 1999. Regulation of nuclearlocalization and transcriptional activity of TFII- I by Bruton’styrosine kinase. Mol. Cell. Biol. 19:5014.

33 Yao, L., Janmey, P., Frigeri, L. G., Han, W., Fujita, J., Kawakami,Y., Apgar, J. R. and Kawakami, T. 1999. Pleckstrin homologydomains interact with filamentous actin. J. Biol. Chem. 274:19752.

34 Li, T., Rawlings, D. J., Park, H., Kato, R. M., Witte, O. N. andSatterthwaite, A. B. 1997. Constitutive membrane associationpotentiates activation of Bruton tyrosine kinase. Oncogene15:1375.

35 Takata, M. and Kurosaki, T. 1996. A role for Bruton’s tyrosine kinasein B cell antigen receptor-mediated activation of phospholipase C-gamma 2. J. Exp. Med. 184:31.

36 Tkachuk, D. C., Kohler, S. and Cleary, M. L. 1992. Involvementof a homolog of Drosophila trithorax by 11q23 chromosomaltranslocations in acute leukemias. Cell 71:691.

37 Yamamoto, K., Seto, M., Komatsu, H., Iida, S., Akao, Y., Kojima,S., Kodera, Y., Nakazawa, S., Ariyoshi, Y., Takahashi, T. and Ueda,R. 1993. Two distinct portions of LTG19/ENL at 19p13 are involvedin t(11;19) leukemia. Oncogene 8:2617.

38 Takaki, S., Tominaga, A., Hitoshi, Y., Mita, S., Sonoda, E.,Yamaguchi, N. and Takatsu, K. 1990. Molecular cloning andexpression of the murine interleukin-5 receptor. EMBO J. 9:4367.

39 Takemoto, Y., Furuta, M., Li, X. K., Strong-Sparks, W. J. andHashimoto, Y. 1995. LckBP1, a proline-rich protein expressed inhaematopoietic lineage cells, directly associates with the SH3domain of protein tyrosine kinase p56lck. EMBO J. 14:3403.

40 Takaki, S., Murata, Y., Kitamura, T., Miyajima, A., Tominaga, A.and Takatsu, K. 1993. Reconstitution of the functional receptorsfor murine and human interleukin 5. J. Exp. Med. 177:1523.

41 Chen, R. H., Sarnecki, C. and Blenis, J. 1992. Nuclear localizationand regulation of erk- and rsk-encoded protein kinases. Mol. Cell.Biol. 12:915.

42 Storrie, B. and Madden, E. A. 1990. Isolation of subcellularorganelles. Methods Enzymol. 182:203.

43 Bence, K., Ma, W., Kozasa, T. and Huang, X. Y. 1997. Directstimulation of Bruton’s tyrosine kinase by G(q)-protein alpha-subunit. Nature 389:296.


44 Joh, T., Kagami, Y., Yamamoto, K., Segawa, T., Takizawa, J.,Takahashi, T., Ueda, R. and Seto, M. 1996. Identification of MLLand chimeric MLL gene products involved in 11q23 translocationand possible mechanisms of leukemogenesis by MLL truncation.Oncogene 13:1945.

45 Shashidharan, P., Chalmers-Redman, R. M., Carlile, G. W., Rodic,V., Gurvich, N., Yuen, T., Tatton, W. G. and Sealfon, S. C.1999. Nuclear translocation of GAPDH–GFP fusion protein duringapoptosis. Neuroreport 10:1149.

46 Rawlings, D. J., Scharenberg, A. M., Park, H., Wahl, M. I., Lin, S.,Kato, R. M., Fluckiger, A. C., Witte, O. N. and Kinet, J. P. 1996.Activation of BTK by a phosphorylation mechanism initiated bySRC family kinases. Science 271:822.

47 Rubnitz, J. E., Morrissey, J., Savage, P. A. and Cleary, M. L. 1994.ENL, the gene fused with HRX in t(11;19) leukemias, encodes anuclear protein with transcriptional activation potential in lymphoidand myeloid cells. Blood 84:1747.

48 Li, T., Tsukada, S., Satterthwaite, A., Havlik, M. H., Park, H.,Takatsu, K. and Witte, O. N. 1995. Activation of Bruton’s tyrosinekinase (Btk) by a point mutation in its pleckstrin homology (PH)domain. Immunity 2:451

49 Mahajan, S., Fargnoli, J., Burkhardt, A. L., Kut, S. A., Saouaf,S. J. and Bolen, J. B. 1995. Src family protein tyrosine kinasesinduce autoactivation of Bruton’s tyrosine kinase. Mol. Cell.Biol. 15:5304.

50 Dingwall, C. and Laskey, R. A. 1991. Nuclear targetingsequences—a consensus? Trends Biochem. Sci. 16:478.