Oncogene (2000) 19, 4151 ± 4158 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc The Bmx is activated by IL-3 and G-CSF in a PI-3K dependent manner

Niklas Ekman1,6, Elena Arighi1,2,6, Iiro Rajantie1, Pipsa Saharinen3, Ari RistimaÈ ki4, Olli Silvennoinen3,5 and Kari Alitalo*,1

1Molecular/Cancer Biology Laboratory and Ludwig Institute for Cancer Research, Haartman Institute, P.O.Box 21 (Haartmaninkatu 3), 00014 University of Helsinki, Finland; 2Department of Experimental Oncology, Istituto Nazionale Tumori, 20133 Milan, Italy; 3Department of Virology, Haartman Institute, P.O.Box 21 (Haartmaninkatu 3), 00014 University of Helsinki, Finland; 4Department of Obstetrics and Gynecology, Helsinki University Central Hospital, P.O. Box 140, 00029 Helsinki, Finland; 5Medical Biochemistry, University of Tampere, 33014 Tampere, Finland

Cytoplasmic tyrosine kinases play crucial roles in The Tec family of PTKs consists of ®ve members: the signaling via a variety of cell surface receptors. The Bmx founder member Tec, as well as Btk, Itk/Tsk/Emt, Txk tyrosine kinase, a member of the Tec family, is and Bmx tyrosine kinases. The Tec kinases are regulated expressed in hematopoietic cells of the granulocytic and by a variety of hematopoietic receptors. For example, the monocytic lineages. Here we show that Bmx is Bruton's tyrosine kinase, Btk, is activated through the catalytically activated by interleukin-3 (IL-3) and high anity IgE receptor in mast cells, by the antigen granulocyte-colony stimulating factor (G-CSF) recep- and CD72 receptors in B-cells, as well as by interleukin-3 tors. Activation of Bmx required phosphatidylinositol 3- (IL-3) and IL-5 receptors and the IL-6 cytokine family kinase (PI-3K) as demonstrated by the ability of PI-3K signal transducer, gp130 (Aoki et al., 1994; Deng et al., inhibitors to block the activation signal. A green 1998; Kawakami et al., 1994; Matsuda et al., 1995; Sato ¯uorescent protein (GFP) tagged Bmx was translocated et al., 1994; Venkataraman et al., 1998). Members of the to cellular membranes upon co-expression of a constitu- Tec family have also been shown to be activated by G tively active form of PI-3K, further indicating a role for , for example Gaq was shown to activate Btk PI-3K in signaling upstream of Bmx. The expression of both in vivo and in vitro (Bence et al., 1997), while Tec wild type Bmx in 32D myeloid progenitor cells resulted was shown to be involved in G protein-coupled receptor- in apoptosis in the presence of G-CSF, while cells and integrin-mediated signaling in human platelets expressing a kinase dead mutant of Bmx di€erentiated (Hamazaki et al., 1998). into mature granulocytes. However, Bmx did not The proteins of the Tec family share a characteristic modulate IL-3-dependent proliferation of the cells. These domain structure including an amino-terminal pleck- results demonstrate distinct e€ects of Bmx in cytokine strin homology (PH) domain, Tec homology (TH), src induced proliferation and di€erentiation of myeloid cells, homology 3 (SH3) and 2 (SH2) domains and a and suggest that the stage speci®c expression of Bmx is carboxy-terminal catalytic tyrosine kinase domain critical for the di€erentiation of myeloid cells. Oncogene (Mano, 1999; Okoh and Vihinen, 1999). Each kinase (2000) 19, 4151 ± 4158. has a speci®c expression pattern: expression of Btk is restricted to B-lymphoid and myeloid cell linages (de Keywords: Bmx; IL-3; G-CSF; PI-3K; di€erentiation; Weers et al., 1998), Itk is predominantly expressed in apoptosis cells of the T-lymphoid lineage (Gibson et al., 1993) and Txk is expressed in myeloid cells and T cells (Sommers et al., 1995). The prototype member of the Introduction family, the Tec tyrosine kinase, is most widely expressed, predominantly in the liver and in a wide Protein tyrosine kinases (PTKs) play an important role variety of hematopoietic cells (Mano et al., 1990, 1993). in transducing growth and di€erentiation signals from The Bmx was originally isolated while screening the plasma membrane to the cytoplasm. The majority for novel PTK expressed in the human bone of hematopoietic cytokine and antigen receptors rely marrow. Because the gene was mapped to on receptor associated cytoplasmic PTKs for signaling. Xp22.2, it was called Bmx, Bone Marrow tyrosine kinase In many cases, cytokine binding to its cognate receptor gene in chromosome X (Tamagnone et al., 1994). Bmx is leads to activation of Jak (Janus) kinases and expressed mainly in di€erentiated cells of the granulo- subsequent activation of downstream signaling path- cytic and monocytic lineages (Kaukonen et al., 1996; ways including Stat (signal transducers and activators Weil et al., 1998), as well as in the heart endocardium and of transcription) transcription factors (Ihle et al., 1995). in the endothelium of large arteries (Ekman et al., 1997). However, also other kinases, for example members of Thus far, the signal transduction pathways involving the Src and Tec family, have been shown to be Bmx are poorly characterized. In prostate cancer cells, important for the signaling through cytokine receptors. IL-6 was shown to activate Bmx and in transfected cells, the a subunit of the G12 family of GTP-binding proteins activated Bmx, leading to stimulation of the serum response factor (Mao et al., 1998; Qui et al., 1998). Bmx is also capable of activating Stat transcription factors *Correspondence: K Alitalo 6The ®rst two authors contributed equally to this work and Bmx activation of Stat1 was inhibited by protein Received 8 May 2000; revised 21 June 2000; accepted 26 June 2000 kinase Cd (Saharinen et al., 1997; Wen et al., 1999). Bmx activation by IL-3 and G-CSF N Ekman et al 4152 The expression of Bmx in granulocytes led us to investigate the possibility that Bmx would be involved in signaling through IL-3 and G-CSF receptors (IL-3R and G-CSFR, respectively). Here we show that the kinase activity of Bmx is regulated by IL-3 and G-CSF via a PI-3K dependent pathway in IL-3 dependent 32D cells. Ectopic expression of Bmx did not a€ect the IL-3 induced proliferative responses of the 32D cells, but it caused apoptotic death of the cells induced to di€erentiate along the granulocytic lineage in response to G-CSF.

Results

The Bmx kinase activity is regulated by IL-3 and G-CSF in 32D cells Bmx has been shown to be expressed in hematopoie- tic progenitor cells, with increasing expression in di€erentiating and maturing cells of the granulocytic and monocytic lineages (Kaukonen et al., 1996; Weil et al., 1998). We therefore wanted to study the possible regulation of Bmx by IL-3 and G-CSF. For this purpose we chose to overexpress a HA tagged wild type Bmx (BmxWT) or a `kinase dead' BmxK445R (BmxKD) in the 32D cell line, an IL-3 dependent non-tumorigenic cell line derived from normal mouse myeloid progenitor cells. These cells can be induced by G-CSF to undergo granulocytic di€erentiation and thus they have been widely used for the study of hematopoietic cell di€erentiation (Valtieri et al., 1987). The 32D cells do not express endogenous Bmx (data not shown), which makes them suitable for studies of the e€ects of Bmx and its mutants on myeloid cell signaling and di€erentiation. Transfected cell clones expressing similar amounts of wild type and kinase dead Bmx were chosen for further studies. Figure 1 Bmx is activated by IL-3 and G-CSF in a PI-3K The transfected 32D cells were starved in IL-3- and dependent manner. (a) Following overnight starvation in IL-3- serum-free medium for 12 h, and then stimulated and serum-free medium, the Bmx expressing 32D cells were stimulated for the indicated times with IL-3 or G-CSF. with 50 ng/ml IL-3 or G-CSF for the indicated times. Immunoprecipitated Bmx was subjected to in vitro kinase assay As shown in Figure 1a, the in vitro autokinase and the activity was detected as autophosphorylation in SDS ± activity of immunoprecipitated Bmx increased after PAGE as described in the Materials and methods. A control both stimulations in a time-dependent manner for Western blotting using antibodies against the HA-epitope (a-HA) approximately 2 h. An enhanced level of kinase was performed to test equal amount of protein in each reaction (lower panel). (b) The 32D cells were stimulated for 30 min with activity persisted up to 20 h in the presence of IL- IL-3 or G-CSF in the presence or absence of 100 nM of the PI-3K 3, while the G-CSF induced activation started to inhibitor Wortmannin. The determination of autokinase activity decrease after 4 h, and was at background level after of immunoprecipitated Bmx (upper panel) and the control 8 h (data not shown). A similar kinetics of kinase blotting (lower panel) were performed as in a.(c) HUVE cells were starved overnight, followed by IL-3 stimulation for 30 min activation was obtained using poly(Glu, Tyr) as an in the presence or absence of Wortmannin. The kinase activity of exogenous substrate in the kinase reaction (data not endogenous Bmx was detected as previously described (upper shown). Only background levels of kinase activity panel). The lower panel shows blotting with endothelial speci®c a- were detected in corresponding immunoprecipitates of CD31 to con®rm equivalent amounts of protein in each reaction the 32DBmxKD cells. In addition to cells of hematopoietic origin, IL-3 has also been shown to induce endothelial cell PI-3K is involved in the activation of Bmx proliferation and new vessel formation (Dentelli et al., 1999). As Bmx is expressed in certain endothelial PI-3K has been shown to be a crucial mediator of both cells (Ekman et al., 1997), we wanted to test IL-3 and G-CSF signaling. PI-3K, activated by whether also endogenous Bmx was catalytically receptor associated Jak kinases, catalyzes the biosynth- regulated by IL-3. As in cells of hematopoietic esis of speci®c inositol phospholipids in the plasma origin, also endogenous Bmx from HUVE cells was membrane, enabling the anchorage and activation of activated by IL-3 (Figure 1c). Thus we conclude that several PH domain containing proteins (de Groot et IL-3 is a physiological regulator of Bmx kinase al., 1998; Hunter and Avalos, 1998; Migone et al., activity. 1998). We therefore tested whether the IL-3 and G-

Oncogene Bmx activation by IL-3 and G-CSF N Ekman et al 4153 CSF activation of Bmx is mediated by PI-3K. In the Bmx, we transfected the constructs into LEII endothe- presence of Wortmannin, an inhibitor of PI-3K, the lial cells, together with a control plasmid or with a increase in Bmx kinase activity following IL-3 or G- constitutively active catalytic subunit of PI-3K, p110- CSF stimulation of 32D cells was almost totally CAAX. As can be seen from Figure 3a, a homo- abolished (Figure 1b). A decrease of kinase activity geneous localization of Bmx-GFP ¯uorescence was was also observed for the endogenous Bmx in seen througout the cytoplasm in the absence of p110- Wortmannin treated HUVE cells (Figure 1c, lane 3). CAAX. In contrast, when p110-CAAX was coex- Another PI-3K inhibitor, LY294002 gave similar pressed with Bmx-GFP, much of the signal was results (data not shown). relocated to cellular membranes (Figure 3b). However, To further investigate the role of PI-3K in the Bmx was not con®ned to the plasma membrane but regulation of Bmx, we constructed expression plasmids showed a localization pattern similar to that previously encoding Bmx fused to green ¯uorescent protein (GFP) reported for Btk (Varnai et al., 1999). The same (Figure 2a). DKD-GFP lacks the Bmx kinase domain, pattern was also seen using the DKD-GFP construct while DPH-GFP lacks the pleckstrin homology do- (Figure 3c,d). In contrast, when the DPH-GFP main. The R29C mutation corresponds to the arginine construct was transfected together with p110-CAAX, to cysteine mutation (R28C) in Btk, which is no membrane localization of the signal could be responsible for the X-linked immunode®ciency (Xid) detected (Figure 3e,f). The R29C-GFP construct was syndrome in mice. The R28C mutation causes a localized similarly to DPH-GFP (Figure 3g,h), demon- dramatic reduction of the inositol binding activity of strating the critical function of the conserved arginine the PH domain, thereby inactivating the Btk protein in these mice (Fukuda et al., 1996). The E41K mutation of Btk, corresponding to the E42K mutation in Bmx, has been shown to activate Btk by causing constitutive membrane association (Li et al., 1995). Transfected Bmx-GFP constructs produced proteins of the expected sizes in 293T cells (Figure 2b). In order to examine the e€ects of the deletions and point mutations on the intracellular localization of

Figure 2 Bmx-GFP constructs and encoded polypeptides. (a)A Figure 3 E€ect of membrane-targeted PI-3K on the subcellular schematic illustration of Bmx-GFP fusion proteins encoded by the localization of Bmx-GFP proteins. LEII cells were transfected indicated cDNA constructs. (b) 293T cells were transfected with with indicated GFP constructs alone (left panels) or together with the indicated expression plasmids. Twenty-®ve mg total cell lysates a membrane targeted version of PI-3K, p110-CAAX (right were separated in a 7.5% SDS ± PAGE gel followed by anti-GFP panels). Figures show representative cells from several indepen- immunoblotting dent experiments

Oncogene Bmx activation by IL-3 and G-CSF N Ekman et al 4154 residue in anchoring the PH domain to phospholipids in the membrane. In contrast to the other constructs, the E42K-GFP mutation was localized to the mem- branes even in the absence of p110-CAAX (Figure 3i), and co-expression of p110-CAAX did not change its localization (Figure 3j). No membrane localization could be detected for GFP, expressed with or without p110-CAAX (data not shown). These data indicate that enhanced membrane localization of Bmx occurs upon an increase of PI-3K activity. The relocalization is independent of the kinase activity, but strictly dependent on speci®c residues in the PH domain. The results also suggest that the IL-3 and G-CSF receptors can activate Bmx via the activation of PI-3K, which catalyses phospholipid biosynthesis. The increased association of the Bmx PH domain with cellular membranes would enable Bmx kinase activation in this compartment.

Overexpression of Bmx induces apoptosis in G-CSF treated 32D cells While both IL-3 and G-CSF pathways control the proliferation and survival of 32D cells, only G-CSF is capable of inducing their terminal di€erentiation into mature granulocytes. In order to investigate the e€ects of Bmx during myeloid cell proliferation and di€er- Figure 4 32DBmxWT and 32DBmxKD cells show similar entiation, we next analysed the cytokine-induced growth curves in the presence of IL-3, but di€erences in survival proliferation and di€erentiation in 32D cells expressing in the presence of G-CSF. The columns display average cell numbers in cultures of four independent BmxWT and BmxKD wild type or kinase negative Bmx. In the presence of Bmx expressing clones. The standard deviations are marked with IL-3, the cells showed similar proliferation responses bars. (a) The cells were seeded on 96-well plates at 2000 cells/well (Figure 4a). This indicated that Bmx did not a€ect the in the presence of 2 ng/ml IL-3. The number of viable cells was IL-3-mediated growth or survival. The 32D cells were measured daily using the MTT assay. (b) The cells were seeded at next induced to undergo di€erentiation by removing all 4000 cells/well in 50 ng/ml G-CSF and viable cells were counted as above. & 32DBmxWT & 32DBmxKD IL-3 by extensive washing of the cells in IL-3 free medium followed by switching them into medium containing 50 ng/ml G-CSF. The 32DBmxKD cells continued to proliferate, although at a slower rate than in the IL-3 containing medium. After an initial growth period of 3 ± 4 days, they became growth arrested. In contrast, the 32DBmxWT cells did not survive in medium containing G-CSF (Figure 4b). After 2 weeks in G-CSF containing medium, the cultures were stained with May-GruÈ nwald Giemsa solution (MGG). At this point, most of the 32DBmxKD cells grown in G-CSF had a granulocytic phenotype, showing a clear nuclear segmentation and increased cytoplasm-to-nucleus ratio (Figure 5a), while no living 32DBmxWT cells could be detected (Figure 5b). For comparison, the 32DBmxKD or 32DBmxWT cells grown in IL-3 containing medium did not show signs of di€erentiation (Figure 5c,d). To con®rm these ®ndings, the 32D cells were stained simultaneously with ¯uorescent annexin V, which binds to cell surface phosphoserine in proapoptotic cells, and Figure 5 Only the 32DBmxKD cells di€erentiate in G-CSF with propidium iodide, which stains DNA in damaged containing medium. (a) MGG stained cytospin preparations of 32DBmxKD cells grown for 2 weeks in 50 ng/ml G-CSF. Note cells. Flow cytometric analysis of the apoptotic changes the granulocytic phenotype with clearly segmented nucleus. (b) was then carried out. Already after 24 h in the G-CSF 32DBmxWT cells grown similarly. No viable cells can be containing culture medium, 32% of the 32DBmxWT detected. (c) 32DBmxKD cells grown in 2 ng/ml IL-3. (d) cells stained positive for both markers of apoptosis, 32DBmxWT cells grown similarly while only 9% of the 32DBmxKD cells showed such signs of apoptosis (data not shown). As shown in Figure 6, after 48 h, 12% of the 32DBmxKD cells were of the myeloperoxidase (MPO) mRNA in di€erent 32D apoptotic, as compared to 87% of the 32DBmxWT cell clones. The upregulation of the MPO gene has cells. been shown to be a neutrophil-speci®c di€erentiation We next examined the involvement of Bmx in G- marker (Morishita et al., 1987). As shown in Figure 7, CSF-induced di€erentiation, by studying the expression only 32D cells expressing kinase dead Bmx upregulated

Oncogene Bmx activation by IL-3 and G-CSF N Ekman et al 4155 dependent on the activity of PI-3K, which by producing phosphorylated inositol residues, enables the Tec family proteins to bind to the plasma membrane through their PH domains (Corvera and Czech, 1998). For example, a Btk-GFP fusion protein was shown to be membrane localized in a PI-3K dependent manner following -, G-protein-coupled receptor-, or immunoreceptor acti- vation (Nore et al., 2000). Several point mutations in Btk a€ecting the ability of its PH domain to bind inositol phospholipids have been shown to cause human X-linked agammaglobulinemia (XLA) and in mice, a point mutation in the PH domain, R28C, is responsible for the analogous Xid phenotype (Mo- hamed et al., 1999). Further evidence for the coupling between PI-3K and Btk was recently provided by the Figure 6 32DBmxWT cells undergo apoptosis when grown in ®ndings that p85a knock-out mice show a nearly G-CSF containing medium. 1.56106 cells were incubated on ice identical phenotype to the Xid mice, including in ¯uorescent annexin V and propidium iodide containing binding impaired B cell development, reduced numbers and bu€er for 10 min and then analysed with FACScan. Cells negative for both propidium iodide and annexin V staining were proliferative responses of mature B cells and no T cell- considered as live cells, propidium iodide-negative and annexin V- independent antibody production (Fruman et al., 1999; positive staining as early apoptotic cells, while propidium iodide- Suzuki et al., 1999). and annexin-positive cells were primarily in late stage of apoptosis Because we earlier found Bmx in myeloid cells, we have here studied its coupling to hematopoietic cytokine signals regulating myeloid cell di€erentiation. Compared to other cytoplasmic kinases activated by IL-3 or G-CSF, Bmx showed a slower activation pattern. For example, stimulation of exogenously expressed gp130 in BAF-B03 cells results in Btk and Tec phosphorylation within 5 min (Matsuda et al., 1995), while in the case of Bmx, approximately a 30 min stimulation was needed for a detectable increase in the kinase activity, and the peak occurred after 2 h of stimulation. One di€erence between these studies is that in our experiments Bmx was activated through endogenous IL-3R and G-CSFR, that may induce a less ecient signal than the exogenously overexpressed cytokine receptors. However, we could also demonstrate a PI-3K sensitive, slow activation of endogenous Bmx by IL-3 in endothelial cells, indicat- ing a physiological role for Bmx signaling from the IL- Figure 7 Analysis of the mRNAs encoding the MPO di€erentia- 3R complex. tion marker and G-CSFR. The cells were washed with factor free The binding properties of the Bmx PH domain may medium followed by stimulation with 50 ng/ml G-CSF for the provide one explanation for the slow kinetics of indicated times. The cells were lysed, the mRNA was isolated and activation of its tyrosine kinase. It has been previously analysed by Northern blotting and hybridization using the reported that, when compared to the PH domains of indicated 32P-labeled cDNA probes Btk and Tec, the Bmx PH domain shows much less anity against inositol phospholipids (Kojima et al., 1997). Provided that membrane localization is neces- MPO mRNA, while no expression could be detected in sary for the activation of Bmx, the low anity towards cells expressing wild type Bmx. The failure of the PI-3K lipid products could explain the long stimulation BmxWT expressing cells to respond to G-CSF was not times needed for Bmx activation. It is interesting to due to lack of G-CSF receptors, however. The note that also the reported stimulation time required Northern blot in Figure 7 shows that both for IL-6 activation of Bmx is approximately 30 min 32DBmxWT and KD cells express G-CSFR mRNA, (Qui et al., 1998), and thus comparable with the although the amount of the mRNA in the 32DBmxWT present results. cells was slightly less than in the 32DBmxKD cells Our ®ndings that the R29C and DPH mutations (when compared with the b-actin signals). Both clones prevented PI-3K-induced membrane localization of also upregulated the G-CSFR mRNA after stimulation Bmx are consistent with the view that a lipid anchoring with G-CSF. mechanism is indeed involved in the membrane binding and activation of the tyrosine kinase (Okoh and Vihinen, 1999). The E41K mutation in Btk has been Discussion reported to result in increased membrane localization, phosphorylation and kinase activity of the Btk protein The activation signal from the receptor to the Tec (Li et al., 1995). We also found that the corresponding kinases has in many cases been demonstrated to be mutation in Bmx relocalizes the kinase to the cell

Oncogene Bmx activation by IL-3 and G-CSF N Ekman et al 4156 membranes. However, in contrast to the results with di€erentiation process is lethal for the cells, thus a Btk, the E42K mutation has been reported to di€erentiation stage-dependent regulation of Bmx inactivate Bmx in prostate cancer cells (Qui et al., would be critical for cell survival. Gene targeting and 1998), which would indicate that the activation biochemical characterization of other Tec family mechanism of Bmx di€ers from that of Btk. members have demonstrated critical roles for Btk, Itk The di€erentiation of hematopoietic progenitor cells and Txk kinases in intracellular signaling of B cells and is a tightly regulated process involving modulation of T cells (Mano, 1999; Schae€er et al., 1999). In both cell proliferation and apoptosis. Many cytokines accordance with these studies, our present results not only stimulate cell growth, but are also necessary suggest that Bmx may be involved in functional for cell survival. In the absence of appropriate modulation of di€erentiated myeloid cells rather than cytokines, the progenitor cells undergo programmed regulation of proliferation and di€erentiation of the cell death (Miyajima et al., 1999). We found that Bmx precursor cells. is involved in maintaining this crucial balance between cell proliferation and di€erentiation in cytokine- dependent myeloid progenitor cells. The wild type Bmx in 32D cells induced an apoptotic phenotype in Materials and methods response to G-CSF stimulation, while the di€erentia- Antibodies, growth factors and inhibitors tion process was not a€ected by expression of the kinase dead Bmx. Yet IL-3, which is a strong survival The monoclonal anti-hemagglutinin (HA)-epitope antibody factor for myeloid cells, was able to suppress such (ab) and CD31 ab were from Berkeley Antibody Company (Richmond, CA, USA), while the polyclonal anti-GFP ab di€erences in the survival of the transfected cells. was from Clontech (San Diego, CA, USA). The polyclonal Elevated Bmx kinase activity may thus predispose anti-Bmx ab has been previously described (Tamagnone et myeloid cells to apoptosis upon G-CSF-stimulation, al., 1994). Human G-CSF (hG-CSF) was a gift from Drs but not during their proliferation as immature cells Riitta Alitalo and Petri Salven, mouse IL-3 (mIL-3) was when IL-3 is present. Enhanced apoptosis of the Bmx obtained from Calbiochem Novabiochem (La Jolla, CA, overexpressing cells is also compatible with the fact USA) and human IL-3 (hIL-3) from PeproTech (London, that during normal myeloid di€erentiation the highest UK). For inhibition of PI-3K activity, 100 nM of Wortman- Bmx levels are obtained in mature cells (over ®vefold nin (Sigma, St Louis, MO, USA) or 100 mM of LY294002 increase, see Weil et al., 1998), which in the case of (Sigma) was used to treat cells for 30 min prior to growth neutrophils are known to have the shortest life span factor stimulations. among leukocytes. These cells spend only 6 ± 10 h in circulation before marginating and entering tissues Cell cultures and growth factor stimulations where they survive for 1 ± 2 days, followed by apoptotic WEHI-3D and 293T cells were grown in Dulbecco's Modi®ed death (Boggs, 1967). Eagle's Medium (DMEM), while LEII endothelial cells were The reported roles of the Tec proteins in hemato- grown in MEM. 32D cells were maintained in RPMI1640 poietic cell survival suggest rather complex functions medium, containing 5% medium conditioned by the WEHI- for these proteins. For example, Btk promotes 3D cells as a source of IL-3. The HUVE cells were isolated apoptosis in murine mast cells (Kawakami et al., according to the standard protocol (Ja€e et al., 1973) and 1997) and is necessary for radiation-induced apoptosis cultured on gelatin-coated dishes in Medium 199 (Bio of DT-40 lymphoma B cells (Uckun et al., 1996). On Whittaker, Walkersville, MD, USA). All media were supplemented with 10% fetal calf serum (FCS), glutamine the other hand, Btk was recently shown to prevent the and antibiotics, the Medium 199 in addition with Endothelial Fas induced death pathway by binding to the Fas Cell Growth Supplement (50 mg/ml; Upstate Biotechnology receptor and thereby blocking FADD and FLICE Inc., Lake Placid, NY, USA), heparin (5 u/ml, Sigma). activation (Vassilev et al., 1999). Btk has also been Where indicated, the HUVE cells were treated with 20 ng/ shown to be crucial for B cell survival, and in the ml hIL-3 for 30 min and the 32D cells with either 50 ng/ml absence of wild-type Btk, B cells undergo apoptotic cell hG-CSF or with 50 ng/ml mIL-3 for the indicated periods of death in response to anti-IgM stimulation (Anderson et time. All stimulation using G-CSF were carried out in the al., 1996). Also Bmx has been implicated in both cell abcense of IL-3. The cell cultures were assayed for the total survival and apoptosis. In prostate cancer cells, Bmx number of viable cells by the MTT assay (Mosmann, 1983) was shown to be needed for IL-6 induced neuroendo- and May-GruÈ nwald Giemsa (MGG) stained cytospin pre- parations were used to evaluate granulocytic di€erentiation. crine di€erentiation (Qui et al., 1998) and for protection against therapy-induced apoptosis (Xue et al., 1999). On the other hand, Bmx was shown to be DNA constructs able to reconstitute apoptosis in Btk de®cient chicken The expression vector for human full-length Bmx cDNA B cells (Tomlinson et al., 1999). Recently, Bmx was containing the HA epitope in the carboxy terminus has been also shown to link Src to Stat3 activation in Src- described before (Saharinen et al., 1997). The Bmx mutants, mediated cell transformation (Tsai et al., 2000). Such R29C, E42K and K445R were generated by site speci®c dual e€ects of the Tec kinases suggest that the proteins mutagenesis, using the GeneEditor (Promega, Madison, WI, may have di€erent functions in di€erent types of cells USA) and the oligonucleotides 5'-AAT AAT TAC AAA or even in the same cell lineage during di€erent stages GAA TGT CTT TTT GTT TTG ACC-3',5'-TGT TGC TGT TAG GAT GAT CAA GG-3' and 5'-TGT TGC TGT of development and di€erentiation. TAG GAT GAT CAA GG-3', respectively, as primers. The In normal myeloid cells the expression of Bmx is BmxHA expression vector was used as template. For increased during di€erentiation and maturation of intracellular localization studies, full-length Bmx cDNA was granulocytes and macrophages (Weil et al., 1998). PCR ampli®ed and inserted in front of SuperGFP using the Our results demonstrate that the expression of Bmx in GFP Fusion TOPO Cloning Kit (Invitrogen, San Diego, CA, myeloid precursor cells during the initiation of the USA). The forward primers used for the ampli®cation were

Oncogene Bmx activation by IL-3 and G-CSF N Ekman et al 4157 5'-GAG ACG GAT GAT AAT ATG GAT A-3' and 5'-AAT sodium vanadate. The Bmx protein was immunoprecipitated ATG GGC TCC CAG CCA CCA TCT-3' for the full length from the lysates by incubation with either polyclonal anti- Bmx and for Bmx deleted of the PH domain (DPH) Bmx ab or monoclonal anti-HA antibodies for 1 h, followed respectively, while the reverse primers 5'-GCT TGT CTT by binding to protein-A-Sepharose (Amersham Pharmacia TTT CCC GAA GT-3' and 5'-GGG ACA CAG AGT CGG Biotech AB) or protein-G-Sepharose (Amersham Pharmacia GGA-3' were used for ampli®cation of full-length and DKD Biotech AB) for 1 h with gentle agitation. The immunopre- forms, respectively. The PCR reaction conditions were: cipitates were washed twice with the lysis bu€er, once with denaturation at 948C for 45 s, annealing at 508C for 30 s wash bu€er (150 mM NaCl, 20 mM HEPES pH 7.4) and and extension at 728C for 150 s for 20 cycles. The PCR twice with kinase assay bu€er (10 mM HEPES pH 7.4, 5 mM ampli®cations were carried out using Dynazyme (Finnzyme, MnCl2, sodium vanadate), and resuspended in 5 mlof Finland) polymerase. The constitutive active PI-3K catalytic reaction bu€er (kinase assay bu€er, 10 mM unlabeled ATP, subunit, p110-CAAX cloned into pcDNA3 (Invitrogen) 5 mCi gamma-ATP). The kinase reactions were carried out at mammalian expression vector, was a kind gift from Dr 308C for 10 min and stopped by adding 50 ml of stop bu€er Julian Downward. The cDNAs encoding human myeloper- (4 mM unlabeled ATP, 40 mM EDTA, 20 mM HEPES oxidase (MPO), human G-CSFR and b-actin were obtained pH 7.4, 100 mg BSA). The sepharose bound Bmx was eluted from American Type Culture Collection (Manassas, VA, in Laemmli sample bu€er, and separated in 7.5% SDS ± USA), Dr Shigekazu Nagata and from Invitrogen, respec- PAGE. The gel was dried and Bmx autokinase activity was tively. detected by exposure on ®lm.

Transfections and generation of stable 32D cell clones Flow cytometric analysis of apoptosis expressing Bmx The cells were stained by ¯uorescein isothiocyanate-conju- Transient transfections of LEII and 293T cells were done gated annexin V and by the ¯uorescent dye propidium iodide using the Calcium Phosphate Transfection kit (GIBCO, Life (PI). Cells negative for both PI and annexin V staining were Technologies, Gaithersburg, MD, USA) according to considered as live cells, PI-negative, annexin V-positive cells manufacturer's instructions. Stable BmxHA and BmxHA as early apoptotic cells, PI-positive and annexin V-positive K445R expressing 32D cells were generated by electropora- cells as primarily cells in late stage of apoptosis. About tion (240 V, 960 mF) followed by selection in 500 mg/ml 1.56106 cells were harvested and incubated on ice in annexin neomycin. V and PI containing binding bu€er for 10 min and analysed with FACScan (Becton Dickinson, Franklin Lakes, NJ USA) Isolation and analysis of RNA Poly(A)+ mRNA fraction was isolated by lysing cells in lysis bu€er (10 mM Tris-HCl pH 8, 100 mM NaCl, 2 mM EDTA, 0.4 mg/ml Protease K) and binding of mRNA to oligo(dT) Acknowledgments cellulose (Amersham Pharmacia Biotech AB, Uppsala, We are grateful to Drs Riitta Alitalo, Julian Downward, Sweden). For Northern blots, 10 mg of mRNA was Shigekazu Nagata and Petri Salven for kindly providing electrophoresed through 0.8% formaldehyde-agarose gels, the reagents speci®ed in the Materials and methods and to transferred to Hybond-N ®lters (Amersham, Aylesbury, UK) Mari HelanteraÈ and Tapio Tainola for the excellent in 106SSC and hybridized at 428C with 32P-labeled cDNA technical assistance. This work was supported by the probes. Finnish Cancer Organization, the Finnish Academy, the Sigrid Juselius Foundation, the University of Helsinki and the European Union BioThechnology program (BIO4- In vitro Bmx kinase assay CT98-0142). In addition, N Ekman was supported by For the kinase assays, the cells were lysed in TKB lysis bu€er grants from Helsinki University Biomedical Graduate (1% NP-40, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM School, Medicinska UnderstoÈ dsfoÈ reningen Liv och HaÈ lsa EDTA) supplemented with aprotinin, leupeptin, PMSF and and from the Blood Disease Research Foundation.

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Oncogene