activation of nonreceptor tyrosine kinase bmx/etk mediated by

Activation of Nonreceptor Tyrosine Kinase Bmx/Etk Mediatedby Phosphoinositide 3-Kinase, Epidermal Growth FactorReceptor, and ErbB3 in Prostate Cancer Cells*

Received for publication, April 24, 2007, and in revised form, August 28, 2007 Published, JBC Papers in Press, September 6, 2007, DOI 10.1074/jbc.M703412200

Xinnong Jiang, Robert A. Borgesi, Nicole C. McKnight, Ramneet Kaur, Christopher L. Carpenter, and Steven P. Balk1

From the Cancer Biology Program, Hematology-Oncology Division, Department of Medicine, Beth Israel Deaconess Medical Centerand Harvard Medical School, Boston, Massachusetts 02215

Pathways activated downstream of constitutively active phos-phatidylinositol (PI) 3-kinase in PTEN-deficient prostate can-cer (PCa) cells are possible therapeutic targets. We found thatthe nonreceptor Tec family tyrosine kinase Bmx/Etk was acti-vated by tyrosine phosphorylation downstream of Src and PI3-kinase in PTEN-deficient LNCaP and PC3 PCa cells and thatBmxdown-regulationby short interferingRNAmarkedly inhib-ited LNCaP cell growth. Bmx also associated with ErbB3 inLNCaP cells, and heregulin-�1 enhanced this interaction andfurther stimulated Bmx activity. Epidermal growth factor (EGF)similarly stimulated an interaction between Bmx and EGFreceptor and rapidly increased Bmx kinase activity. Bmx stimu-lation in response to heregulin-�1 and EGFwas Src-dependent,and heregulin-�1 stimulation of Bmx was also PI 3-kinase-de-pendent. In contrast, the rapid tyrosine phosphorylation andactivation of Bmx in response to EGFwasPI 3-kinase-independ-ent. Taken together, these results demonstrate that Bmx is acritical downstream target of the constitutively active PI 3-ki-nase in PTEN-deficient PCa cells and further show that Bmx isrecruited by the EGF receptor and ErbB3 and activated inresponse to their respective ligands. Therefore, Bmx may be avaluable therapeutic target in PCa and other epithelial malig-nancies in which PI 3-kinase or EGF receptor family pathwaysare activated.

The class I PI2 3-kinases produce phosphatidylinositol 3,4,5-triphosphate (PIP3), which recruits proteins containing PIP3-binding pleckstrin homology (PH) domains to the membrane(1). This PI 3-kinase pathway is negatively regulated by PTEN,which functions as a PIP3 phosphatase. PTEN loss and subse-

quent activation of PI 3-kinase signaling makes a major contri-bution to prostate cancer (PCa), with a large fraction of meta-static PCa and many high grade primary PCa being PTEN-deficient (2). Studies in mice have confirmed that lack of PTENpromotes the development of PCa, as mice completely lackingPTEN expression in the prostate develop high grade prostaticintraepithelial neoplasia (PIN) at early ages and progress to ade-nocarcinoma (3–5). The serine/threonine protein kinase Akt isthe best studied target of this PI 3-kinase pathway, with Aktfunctioning to inactivate a number of growth-suppressing orpro-apoptotic substrates (6). Prostate-specific expression of aconstitutively activated membrane-targeted Akt causes lesionsthat resemble PIN, but these lesions do not progress to adeno-carcinoma, indicating that additional proteins downstream ofPTEN loss and PI 3-kinase pathway activation contribute toPCa growth (7).One such candidate downstream target of PI 3-kinase is Bmx

(also termed Etk), a nonreceptor tyrosine kinasemember of theTec kinase family (8). Tec kinases are unique among tyrosinekinases in having a PH domain, whichmediates membrane tar-geting by binding PIP3. This exposes the kinase domain tomembrane-associated Src (or a Src family kinase), which phos-phorylates a regulatory tyrosine in the catalytic site (Tyr-566 inBmx) to activate the kinase (9). Tec kinases also contain SH2and SH3 domains, and the latter SH3 domain may mediate anintramolecular inhibitory interaction with the PH domain thatis also regulated by tyrosine phosphorylation (10–13). Bmxwasoriginally cloned as a novel tyrosine kinase expressed in mye-loid cells, but in contrast to other Tec kinases with limited tis-sue distribution, it is expressed in many cell types, includingarterial endothelium and epithelial cells (14–17). Bmx can beactivated by diverse stimuli, including cytokines and growthfactors, and it has been implicated in cellular functions, includ-ing differentiation, motility, and apoptosis (14, 18–25).Bmx in myeloid cells can be activated by multiple cytokines,

and this activation is PI 3-kinase-dependent (26). In contrast,Bmx in endothelial cells is activated by tumor necrosis factor(TNF) through an interaction with the type 2 TNF receptor(TNFR2), with subsequent Bmx-mediated stimulation of theVEGF receptor 2 (VEGFR2, KDR) and PI 3-kinase by unclearmechanisms (22, 27). Significantly, Bmx-deficient mice have adefect in ischemia-meditated angiogenesis that correlates withdecreased TNFR2 and VEGFR2 signaling in endothelium andbone marrow cells, indicating that Bmx functions physiologi-cally through these receptors in endothelial cells (28). In epithe-

* This work was supported by the DF/HCC Prostate SPORE and by the HersheyFamily Prostate Cancer Research Fund. The costs of publication of thisarticle were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

1 To whom correspondence should be addressed: Hematology-OncologyDivision, Beth Israel Deaconess Medical Center, HIM Bldg., Rm. 1050, 330Brookline Ave., Boston, MA 02215. Tel.: 617-667-0878; Fax: 617-667-5339;E-mail: [email protected].

2 The abbreviations used are: PI, phosphatidylinositol; PCa, prostate cancer;PIP3, phosphatidylinositol 3,4,5-triphosphate; PH, pleckstrin homology;PIN, prostatic intraepithelial neoplasia; SH, Src homology; siRNA, shortinterfering RNA; EGF, epidermal growth factor; EGFR, EGF receptor; VEGF,vascular endothelial growth factor; VEGFR, VEGF receptor; TBS, Tris-buff-ered saline; FBS, fetal bovine serum; HA, hemagglutinin; IL, interleukin;TNF, tumor necrosis factor; FAK, focal adhesion kinase; PP2,4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4d]pyrimidine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 45, pp. 32689 –32698, November 9, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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lial cells, Bmx colocalizes with focal adhesion kinase (FAK) inresponse to integrin signaling, and FAK-mediated phosphoryl-ation of tyrosine 40 in the Bmx PH domain may prime Bmx forsubsequent phosphorylation by Src (29). Bmx may then stimu-late migration by phosphorylation of p130Cas (30) and mayinteract with or activate additional proteins that regulatemotil-ity (RhoA, Rap1, and PAK1) (20, 31–33) or vesicular trafficking(caveolin and RUFY1) (34, 35). Bmx has also been reported tophosphorylate multiple Stats (Stat1, -3, and -5), and Bmx wasnecessary for Src-mediated activation of Stat3 and transforma-tion in epithelial cells (23, 24, 36).Bmxwas identified in PCa through screens to detect tyrosine

kinases expressed in PCa cell lines and clinical samples, and arecent immunohistochemical study indicates that its expres-sion is increased in PCa relative to normal prostate epithelium(16, 17, 37). Bmx in PCa cells can be activated by IL-6 or by aconstitutively activate p110 subunit of PI 3-kinase, and Bmxactivation by IL-6 is PI 3-kinase-dependent (19, 38). Neuropep-tides can also activate Bmx in PCa cells, and this activation isdependent on both FAK and Src but is independent of PI 3-ki-nase (21). Overexpression of Bmx can protect PCa cells fromradiation or drug-induced apoptosis, and this effect may bemediated through an interaction with the serine/threoninekinase Pim1 (38–40). Significantly, Tec kinase SH3 domainscan alsomediate an interactionwith p53, andBmx can suppressp53 transcriptional activity, indicating that Bmx may preventapoptosis through p53 (41). Finally, a recent study showed thattransgenic overexpression of Bmx in mouse prostate epithe-lium caused increased proliferation and the development oflesions resembling human PIN, further indicating that Bmxmay be a critical target of PI 3-kinase pathway activation in PCa(37).In this study we first confirmed that Bmx was activated

downstream of the constitutively active PI 3-kinase in PTEN-deficient PCa cells, and that down-regulation of Bmx couldmarkedly suppress PCa cell growth. We then used Bmx immu-noprecipitation and Tyr(P) immunoblotting to identify candi-date Bmxupstream activators or downstream substrates in PCacells, which revealed an interaction between Bmx and ErbB3.Significantly, heregulin-�1 treatment increased the Bmx inter-actionwith ErbB3 and enhancedBmx tyrosine phosphorylationand kinase activity. Moreover, EGF treatment similarlyenhanced Bmx activation and induced an interaction betweenBmxandEGFR. Bmx activation by heregulin-�1was dependenton both Src and PI 3-kinase, whereas Bmx activation by EGFwas Src-dependent but PI 3-kinase-independent. These studiesshow that Bmx is a downstream target of the activated PI 3-ki-nase pathway in PTEN-deficient PCa cells, and they provide thefirst direct link between activation of Bmx and receptor tyro-sine kinases. Moreover, they indicate that Bmx should be a val-uable therapeutic target in PCa and other cancers in which PI3-kinase and EGF family receptor pathways are activated.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents—Mouse anti-Bmx antibody wasfrom BD Biosciences. Mouse anti-phosphotyrosine antibody(4G10) and 4G10-conjugated agarose beads were fromUpstateBiotechnology, Inc. (Lake Placid, NY). Mouse anti-ErbB3 (2B5)

antibody was from Lab Vision Corp.(Fremont, CA). Rabbitpolyclonal antibodies raised against EGF receptor (EGFR),phospho-EGFR (Tyr-1068), ErbB2/Her2, Akt, phospho-Akt(Ser-473), Stat3, and phospho-Stat3 (Tyr-705) were from CellSignaling (Danvers, MA). Mouse anti-FLAG antibody (M2)-conjugated agarose beads, 3xFLAG peptide, IL-6, and PI 3-ki-nase inhibitor LY294002were fromSigma.Mouse anti-�-tubu-lin antibody was from Chemicon (Temecula, CA). Horseradishperoxidase-conjugated mouse IgG and rabbit IgG were fromPromega (Madison, WI). Recombinant human heregulin-�1and EGF were from R & D Systems (Minneapolis, MN). Srcinhibitor PP2was fromCalbiochem. Protease inhibitormixturetablets (EDTA-free) were from Roche Diagnostics. Bmx siRNAand control siRNAs were fromDharmacon RNA Technologies(Lafayette, CO). Bmx cDNA with 3xFLAG sequence at the 5�end was inserted into the pcDNA3 vector (Invitrogen) betweenNheI and NotI sites.Cell Culture and Transfection—The PTEN-deficient LNCaP

and PC3 human PCa cell lines, and the CWR22Rv1 PCa linewith intact PTEN, were from the ATCC and maintained inRPMI1640mediumsupplementedwith 10% fetal bovine serum(FBS). For transfectants, 10 �g of 3xFLAG-Bmx/pcDNA3DNA, HA-Bmx/pcDNA3, or PTEN-pcDNA DNA were trans-fected into LNCaP or PC3 cells in 10-cm plates at 50% conflu-ence using Lipofectamine, with empty vector for controls. Forstable transfectants, the media were replaced the next day, andcells were selected with G418 (900 �g/ml). Resistant cells weremaintained in medium containing 500 �g/ml G418, and theexpression of 3xFLAG-Bmx or HA-Bmx was analyzed byimmunoblotting. For Bmx knockdowns, siRNAs were trans-fected into LNCaP cells at 40 nM, andmedia were replaced 24 hlater. Cells were either maintained in RPMI 1640 medium sup-plemented with 10% FBS for 4 days or in serum-containingmedium for 2 days followed by serum starvation for 2 days.Cells were then trypsinized and counted.Immunoprecipitation and Immunoblotting—Cells were

treatedwith growth factors, inhibitors, or vehicle (0.1%Me2SO)as indicated. They were then washed twice with ice-cold Tris-buffered saline (TBS) and lysed with 1 ml of ice-cold RIPAbuffer (50 mM Tris-HCl, pH 8.0, containing 150 mM NaCl, 1%Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1mMEDTA, 1mMEGTA, 1 mM �-glycerophosphate, 1 mM pyrophosphate, 100mM sodium fluoride, 1 mM Na3VO4 and a mixture of proteaseinhibitors). Cell lysates were sonicated for 10 s and centrifugedat 13,000 rpm at 4 °C for 15 min to remove cell debris. Foranti-Tyr(P) immunoprecipitations, supernatants were trans-ferred to newmicrocentrifuge tubes, and equal amounts of pro-teins (1–5 mg) from each sample were mixed with 20–50 �l of4G10-conjugated agarose beads and incubated at 4 °C over-night with continuous agitation. Themixtures were then trans-ferred to MicroSpin columns (GE Healthcare). The beads werewashed with 600 �l of RIPA buffer six times followed by wash-ing with 600 �l of TBS twice. Subsequently, the columns wereplaced intomicrocentrifuge tubes and spun at 1200 rpm for 30 sto remove remnants of TBS. The bottoms of the columns wereplugged, and 10 �l of 2� Laemmli sample buffer without�-mercaptoethanol were added to each column followed byplacing the capped columns into microcentrifuge tubes. After

Bmx/Etk Activation in Prostate Cancer Cells

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incubating at 65 °C for 15 min (to elute bound proteins), thecolumnswere unplugged, transferred into newmicrocentrifugetubes, and spun at 2000 rpm for 30 s to collect samples. 1 �l of�-mercaptoethanol was added to each sample, and the sampleswere boiled for 5 min. Samples were then resolved by 4–12%NuPAGE gel (Invitrogen) followed by membrane transfer. Themembranes were blocked with 5%milk in TBS containing 0.1%Tween 20 (TBS/T) at room temperature for 2 h and incubatedwith primary antibodies overnight at 4 °C. After washing withTBS/T, the membranes were incubated with appropriatehorseradish peroxidase-conjugated secondary antibodies atroom temperature for 1 h, washed, and developed withenhanced chemiluminescence (ECL) reagents (PerkinElmerLife Sciences).For Bmx immunoprecipitations by M2-conjugated agarose

beads, cells were lysed with TBS containing 1%Triton X-100, 1mM Na3VO4, and a mixture of protease inhibitors. Equalamounts of proteins (1–5 mg) from each sample were thenmixed with 20–50 �l of M2-conjugated agarose beads andincubated at 4 °C for 2 h with continuous agitation. The beadswere then transferred to MicroSpin columns, washed, andincubated with 10 �l of 3xFLAG peptide (100 �g/ml) at 4 °Covernight with continuous agitation to elute 3xFLAG-Bmx andassociated proteins. The columns were spun at 2000 rpm for30 s to collect samples. 3 �l of 6� Laemmli sample buffer and 1�l of �-mercaptoethanol were added to each sample, and thesamples were boiled for 5 min. Electrophoresis and immuno-blotting were then performed as described above.For immunoprecipitation of endogenous Bmx, cells were

lysed with TBS containing 1%Triton X-100, 1mMNa3VO4, anda mixture of protease inhibitors. 5 mg of proteins from eachsample were mixed with 5 �g of mouse anti-Bmx antibody (BDBiosciences) and 5 �g of rabbit anti-Bmx antibody (Cell Signal-ing) and then incubated overnight at 4 °C with continuous agi-tation. Equal amounts of mouse IgG and rabbit IgG were usedas negative controls. 20 �l of protein A/G beads (Pierce) werethen added to each sample and incubated at 4 °C for 2 h withcontinuous agitation. Elution of the immunocomplexes andelectrophoresis and immunoblotting were performed asdescribed above.In Vitro Kinase Assay—3xFLAG-Bmx was immunoprecipi-

tated from LNCaP cells, treated with or without growth factorsor LY294002, and elutedwith 3xFLAGpeptide. ElutedBmxwasthen mixed with kinase buffer (final 20 mM HEPES, pH 7.5, 10mMMgCl2, 20mM �-glycerophosphate, 1mM dithiothreitol, 20�MATP, 5mMNa3VO4) and 1�Ci of [�-32P]ATP (PerkinElmerLife Sciences). The kinase assay was performed at 30 °C for 30min and stopped with 10 mM EDTA and Laemmli samplebuffer. The samples were resolved by 4–12% NuPAGE gel, andBmx autophosphorylation was visualized by autoradiography.

RESULTS

Bmx Is Constitutively Activated Downstream of PI 3-Kinaseand Src in PTEN-deficient PCa Cells—PTEN-deficient LNCaPPCa cells have high levels of constitutive PI 3-kinase pathwayactivation evenwhen cultured in serum-freemedium, and inhi-bition of PI 3-kinase with LY492002 or wortmannin markedlysuppresses LNCaP cell growth. To determinewhether Bmxwas

constitutively activated by endogenous PI 3-kinase in PTEN-deficient cells, we generated LNCaP cells thatwere stably trans-fected with an epitope-tagged (3xFLAG) Bmx, which could beefficiently immunoprecipitated using anti-FLAG antibodies.As noted above, LNCaP cells grown in serum-free mediummaintained high levels of PI 3-kinase pathway activation, asassessed by immunoblotting for pAkt (Fig. 1A). Immunopre-cipitation of Bmx from these cells grown in serum-freemediumfollowed by anti-Tyr(P) immunoblotting indicated that Bmxwas partially activated, and it could be further activated whenthe cells were grown in serum-containing medium (10% FBS)(Fig. 1A, lanes 1 and 3). Consistent with previous data, IL-6treatment rapidly increased Bmx phosphorylation and Stat3activation (Fig. 1A, lane 2). Significantly, treatment with a PI3-kinase inhibitor (LY294002) abrogated Akt activation anddecreased the levels of Bmx tyrosine phosphorylation, indicat-ing that PI 3-kinase contributes to Bmx activation under theseconditions (Fig. 1A, lanes 4 versus 1). The basal level of Bmxtyrosine phosphorylation was also reduced by an Src inhibitor(Fig. 1A, PP2, lane 5), and the combination of LY294002 andPP2 resulted in nearly complete loss of basal Bmx phosphoryl-ation (lane 6).When cells were grown in serum-containing medium (10%

FBS), we also found that Bmx tyrosine phosphorylation couldbe markedly decreased by treatment with LY294002 and PP2(Fig. 1B). Similar results were obtained in other independentstable LNCaP clones expressing FLAG-Bmx or expressing anHA-tagged Bmx (Fig. 1C and data not shown). We also exam-ined another PTEN-deficient PCa cell line, PC3, which was sta-bly transfected with the 3xFLAG-Bmx. In these cells PP2 alonewas more effective than LY294002, but the combination com-pletely blocked Bmx phosphorylation (Fig. 1D).We next used anti-Tyr(P) immunoprecipitation followed by

Bmx immunoblotting to assess the activation of endogenousBmx in LNCaP cells. There was no detectable activation ofendogenous Bmx (above the basal level) by IL-6 in serum-starved cells, although the IL-6 still markedly activated Stat3(Fig. 1E, lane 2). Similarly to the transfected Bmx, tyrosinephosphorylation of endogenous Bmx was increased by growthin serum-containing medium. Importantly, inhibition of PI3-kinase or Src with LY294002 or PP2, respectively, markedlydecreased the phosphorylation of endogenous Bmx in serum-containing medium. Moreover, the combination of LY294002andPP2 decreasedBmxphosphorylation to below its basal levelin serum-free medium.In contrast to these results in PTEN-deficient LNCaP cells,

tyrosine phosphorylation of Bmx could not be detected underthe same conditions in a PCa cell line with intact PTEN(CWR2Rv1), although endogenous Bmx could be readilydetected in whole cell lysates from these cells (Fig. 1F). Finally,transfection of wild-type PTEN into the 3xFLAG-Bmx express-ing LNCaP cells markedly reduced both PI 3-kinase pathwayactivation, as assessed by pAkt immunoblotting, and tyro-sine phosphorylation of Bmx (Fig. 1G). Taken together, theseresults indicated that Bmx was constitutively active inLNCaP PCa cells grown in serum-free medium, that it wasfurther activated in medium with 10% FBS, and that themajor pathway mediating Bmx activation was PI 3-kinase-




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dependent membrane recruitment followed by Src-depend-ent tyrosine phosphorylation.Down-regulation of Bmx Suppresses PCa Cell Growth—To

address directly whether endogenous Bmx may be playing acritical role in LNCaP cell growth downstream of PI 3-kinase,we next assessed the effects of Bmx siRNA on cell growth inmediumwith 10% FBS and in serum-freemedium. LNCaP cellswere transfected with Bmx or control siRNA and then culturedfor 4 days in medium with 10% FBS or for 2 days in 10% FBSmedium followed by 2 days in serum-freemedium. Cell growthwas onlymodestly decreased in serum-freemedium, consistentwith the constitutive activity of the PI 3-kinase signaling path-way (Fig. 2, A and B). Cell recovery was moderately decreasedby the control siRNA but was markedly decreased by the BmxsiRNA under both conditions, indicating that Bmx plays a sig-nificant role in regulating cell growth downstream of PI 3-ki-nase. Immunoblotting confirmed that Bmx expression wasdown-regulated by the Bmx siRNA (Fig. 2C).

Identification of Bmx-associatedTyrosine-phosphorylated Proteins—In addition to membrane recruit-ment mediated by the Bmx PHdomain, the SH2 domain presum-ably mediates Bmx binding to tyro-sine-phosphorylated proteins thatmay be upstream activators ordownstream targets of Bmx. Toidentify candidate proteins interact-ing with the Bmx SH2 domain, weused anti-FLAG antibody-conju-gated beads to immunoprecipitate3xFLAG epitope-tagged Bmx fromstably transfected LNCaP cells. Thebeads were eluted sequentially with3xFLAG peptide and then SDS, as avariable amount of the 3xFLAG Bmxremains associated with the beadsafter peptide elution. The eluted pro-teins were then immunoblotted withanti-Tyr(P). As a further control forproteins binding nonspecifically tothebeads,wealsocarriedout theanti-FLAG immunoprecipitations onLNCaP cells stably transfected withan empty expression vector.Fig. 3A is a Tyr(P) immunoblot of

whole cell lysates from Bmx (B) andvector control (V) stably transfectedLNCaP cells, before and after theanti-FLAG immunoprecipitation. Alarge number of tyrosine-phospho-rylated proteins were observed inboth cells, with several bandsappearing tobe increased in theBmx-expressing cells. Strikingly, a singlemajor tyrosine-phosphorylated pro-tein of �180 kDa was detected in thepeptide eluted material from the

3xFLAG-BmxexpressingLNCaPcells versus the control cells (Fig.3B). Further elutionwith SDS revealed additional specific bands at�110 and �80 kDa. Similar bands were obtained by peptideand/or SDS elution in independent experiments, with the upperband at �180 kDa being most prominent (Fig. 3C). The tyrosine-phosphorylated band migrating at �80 kDa was consistent withthe epitope-tagged Bmx,whichmigrates at this position. It shouldagain be noted that although the 3xFLAG peptide elution is veryspecific, it only removes a variable fraction of the 3xFLAG-Bmx,and the tyrosine-phosphorylated fractionmay elute less readily.We then carried out further immunoblotting of candidate

proteins to identify the major tyrosine-phosphorylated pro-tein(s) at�180 kDa. Significantly, ErbB3 (�185 kDa)was foundby immunoblotting to be highly enriched in the specific peptideand SDS-eluted material (Fig. 3D). ErbB3 can be phosphoryla-ted by ErbB2 and EGFR in response to growth factor stimula-tion and can form heterodimers with these receptor tyrosinekinases. However, we did not detect a consistent interaction

FIGURE 1. Bmx is constitutively activated downstream of PI 3-kinase and Src in PTEN-deficient PCa cells.A and B, tyrosine phosphorylation (pTyr) of Bmx in LNCaP cells stably transfected with 3xFLAG-tagged Bmx(LNCaP-FLAG-Bmx). Cells were either serum-starved for 2 days (A) or maintained in medium supplementedwith 10% FBS (B and lane 3 in A). Cells were treated with IL-6 (10 ng/ml, 15 min), LY294002 (LY; 20 �M, 8 h), PP2(10 �M, 8 h), or LY�PP2 (8 h). Unless otherwise stated, cells were treated similarly with IL-6, LY294002, or PP2 inother experiments. Cell lysates were immunoblotted as indicated, or 1 mg was immunoprecipitated (IP) withanti-FLAG, peptide-eluted, and then immunoblotted with Tyr(P) antibody 4G10. C, tyrosine phosphorylation ofBmx in LNCaP cells stably transfected with HA-tagged Bmx (LNCaP-HA-Bmx). Serum-starved cells (2 days, lanes1 and 2) were stimulated with IL-6 (lane 2). Cells maintained in 10% FBS containing medium were treated withLY, PP2, or LY�PP2 (lanes 3– 6), and cell lysates were immunoblotted as indicated, or 1 mg was immunoprecipi-tated with 4G10-conjugated agarose beads and then immunoblotted with Bmx antibody. D, tyrosine phos-phorylation of Bmx in PC3 cells stably transfected with 3xFLAG-tagged Bmx (PC3-FLAG-Bmx). The experimentswere performed as described in C. E and F, tyrosine phosphorylation of endogenous Bmx in LNCaP andCWR22Rv1 cells. The experiments were performed on untransfected cells as described in C, vehicle control was0.1% Me2SO (DMSO). G, LNCaP-FLAG-Bmx cells in 10% FBS medium were transiently transfected for 24 h withPTEN or control pcDNA vector (under conditions that yield 80 –90% transfection efficiency), cultured for anadditional 24 h, and then examined for Bmx phosphorylation. Bar graphs show the ratios of anti-Tyr(P) oranti-Bmx versus anti-tubulin band intensities, and data are representative of at least three experiments. SFM,serum-free medium.




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betweenBmx andErbB2 or EGFR in unstimulated cells (see Fig.6). These results indicated that ErbB3 was associated with Bmxin LNCaP cells, although there may clearly be additional asso-ciated proteins at �180 kDa or other molecular masses.Bmx Is Activated by Heregulin-�1—The interaction between

Bmx and ErbB3 suggested that ErbB2, the major kinase medi-ating ErbB3 phosphorylation, may be a direct or indirectupstream activator of Bmx. To test this hypothesis, we assessedtyrosine phosphorylation of the 3xFLAG-Bmx in serum-starved LNCaP cells in response to heregulin-�1, which stimu-lates ErbB2/ErbB3 dimerization and ErbB2 kinase activity. Asexpected, heregulin-�1 treatment stimulated the tyrosinephosphorylation of ErbB2 andErbB3, as assessed by anti-Tyr(P)immunoprecipitation and immunoblotting, with the increasedErbB3 tyrosine phosphorylation persisting throughout the timecourse (Fig. 4A). Significantly, this correlated with a rapidincrease in Bmx tyrosine phosphorylation, which peakedwithin 15 min and then declined to below base-line levels over8 h. This decline was associated with a decrease in total Bmx,and declines in total and phosphorylated ErbB2, but not ErbB3.

To confirm that the anti-Tyr(P)immunoprecipitation of Bmx wasbecause of tyrosine phosphorylationof Bmx, versus Bmx association withother tyrosine-phosphorylated pro-teins, we directly immunoprecipi-tated Bmx with anti-FLAG beads.Immunoblotting with Tyr(P) thenconfirmed that Bmx tyrosine phos-phorylation was rapidly and stronglyincreased in response toheregulin-�1(Fig. 4B). Interestingly, we alsodetected an increased associationbetween Bmx and a tyrosine-phos-phorylated �185-kDa protein inresponse to heregulin-�1, which wasconsistent with ErbB3 (see Fig. 6).

Finally, we determined whether endogenous Bmx was simi-larly activated by heregulin-�1. Nontransfected LNCaP cellswere serum-starved and then stimulated with heregulin-�1 for15 min to 8 h, and lysates were immunoprecipitated with anti-Tyr(P). Immunoblotting confirmed the expected activation ofErbB2, with a marked increase in ErbB3 phosphorylation (Fig.4C). Interestingly, phosphorylation of ErbB2 was more persis-tent, whereas ErbB3wasmore transient in these cells versus theBmx overexpressing LNCaP cells, although we do not know ifthis is a direct effect of increased Bmx. In any case, consistentwith the above data, there was also a marked increase in thephosphorylation of endogenous Bmx in response to heregulin-�1. Taken together, these data indicated that ErbB2/ErbB3maymediate the recruitment and activation of Bmx.Bmx Is Activated by EGF—Although we did not detect an

interaction between Bmx and EGFR in LNCaP cells in 10%serum, a previous study in breast cancer cells found that trans-fected Bmx could be activated by EGF treatment (42). There-fore, similar experimentswere carried out to examineBmx acti-vation in response to EGF. Treatment of LNCaP cells with EGFresulted in a rapid (within 5min) increase in tyrosine phospho-rylation of EGFR and subsequent receptor down-regulation,with a similar increase in ErbB2 phosphorylation that presum-ably reflects EGFR/ErbB2 heterodimerization (Fig. 5A). Phos-phorylation of ErbB3 was only transiently increased, versus thepersistent increase observed above with heregulin-�1. Signifi-cantly, Bmx tyrosine phosphorylationwas also increasedwithin5min by EGF, whichwas confirmed by first immunoprecipitat-ing Bmx with anti-FLAG and then immunoblotting for Tyr(P)(Fig. 5B). In contrast to stimulation with heregulin-�1, Bmxphosphorylation in response to EGFwasmore transient anddidnot result in a decrease in total Bmx. A tyrosine-phosphoryla-ted band consistent with EGFR or ErbB3 was also associatedwith Bmx after EGF stimulation (see Fig. 6).We next examined tyrosine phosphorylation of endogenous

Bmx in response to EGF stimulation. Nontransfected LNCaPcells were serum-starved and then stimulated with EGF for5–120 min, and lysates were then immunoprecipitated withanti-Tyr(P) and immunoblotted. EGF again stimulatedmarkedincreases in the phosphorylation of EGFR, ErbB2, and ErbB3 inthese nontransfected LNCaP cells (Fig. 5C). Significantly, EGF

FIGURE 2. Down-regulation of Bmx suppresses LNCaP cell growth. Bmx and control siRNAs (40 nM) weretransfected into LNCaP cells, and media were replaced 24 h later. Cells were either maintained in mediumsupplemented with 10% FBS for 4 days or in serum-containing medium for 2 days followed by serum starvationfor 2 days. Cells were then trypsinized, and cell numbers were counted in triplicate using a hemocytometer.A, images of LNCaP cells 4 days post-transfection (�20 magnification). B, effects of Bmx siRNA on cell growth.C, equal amounts of protein from Bmx or control siRNAs transfected LNCaP cells immunoblotted for Bmx. Dataare representative of three experiments. SFM, serum-free medium.

FIGURE 3. Identification of Bmx-associated tyrosine-phosphorylatedproteins. LNCaP cells stably transfected with 3xFLAG-tagged Bmx (B) orempty vector (V) control in medium with 10% FBS were lysed and immuno-precipitated (IP) with M2-conjugated agarose beads (4 °C, 2 h), and theneluted with FLAG peptide (100 �g/ml, 1 h at 4 °C) and SDS sequentially. A, 20�g of whole cell lysates (pre- and post-immunoprecipitation) immuno-blotted with 4G10. B, immune complexes eluted from M2 beads analyzed byimmunoblotting with 4G10 (B) or ErbB3 (D) antibodies. C, results of an inde-pendent experiment (immunoprecipitation with M2 beads, peptide elution,and immunoblotting with 4G10 antibody).




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also stimulated a rapid increase in the tyrosine phosphorylationof endogenous Bmx. Taken together, these results show thatEGFR can mediate Bmx recruitment and activation, althoughthe activation of both EGFR and ErbB2 in response to EGFindicates that this Bmx activation might be ErbB2-mediated.Finally, we carried out in vitro kinase assays to confirm that

the increased tyrosine phosphorylation of Bmx correlated withincreased kinase activity. Serum-starved LNCaP cells werestimulatedwith EGFor heregulin-�1 and then immunoprecipi-tated with anti-FLAG. Bmx was peptide eluted, and autophos-

phorylation activity was assessed byincubation in kinase buffer with[32P]ATP. Consistent with theTyr(P) immunoblotting, basalkinase activity was precipitated byanti-FLAG from the 3xFLAG-Bmxexpressing LNCaP cells, and thiscould be rapidly increased by EGFor heregulin-�1 (Fig. 5D).Growth Factors Enhance Bmx

Associationwith ErbB3 andEGFR—To determine whether heregu-lin-�1 and EGF were enhancingdirect interactions between ErbB3and EGFR, respectively, we exam-ined Bmx-associated proteins aftergrowth factor stimulations. Serum-starved LNCaP cells expressing3xFLAG-Bmx were stimulated withheregulin-�1, immunoprecipitatedwith anti-FLAG beads, and thenimmunoblotted for ErbB3, as well asEGFR and ErbB2. As shown in Fig.6A, heregulin-�1 stimulated thetyrosine phosphorylation of Bmxand increased the level of ErbB3 thatwas associated with Bmx. In con-trast, there was no detectable asso-ciation between the FLAG-taggedBmx and EGFR or ErbB2 before orafter stimulation with heregulin-�1.To assess interactions with endoge-nous Bmx, nontransfected LNCaPcells were heregulin-�1-stimulated,and lysates were immunoprecipi-tated with anti-Bmx or negativecontrol Abs. The interactionbetween ErbB3 and endogenousBmx was similarly enhanced byheregulin-�1, despite a markeddecline in total ErbB3 levels,whereas there was no interactionwith EGFR or ErbB2, and no inter-actions in the control Ab precipi-tates (Fig. 6B and data not shown).

Serum-starved LNCaP cells wereanalyzed similarly after stimulationwith EGF. As shown in Fig. 6C using

3xFLAG-tagged Bmx, EGF increased Bmx tyrosine phospho-rylation and stimulated an interaction between the FLAG-Bmxand EGFR that was not detectable in the unstimulated cells andwas substantial relative to the decreased levels of EGFR afterEGF stimulation. In contrast, EGF did not stimulate interac-tions with ErbB2 or ErbB3 (although the basal Bmx-ErbB3interaction was not clearly detected in this experiment). Signif-icantly, EGF also stimulated a specific association betweenendogenous Bmx and EGFR in untransfected LNCaP cells (Fig.6D). These results indicated that both ErbB2/ErbB3 and EGFR

β

ββ

FIGURE 4. Bmx is activated by heregulin-�1. A and B, Bmx activation in LNCaP-FLAG-Bmx cells. Cells wereserum-starved for 2 days and then stimulated with heregulin-�1 (40 ng/ml) for 0.25, 0.5, 1, 2, 4, or 8 h. A, cellswere lysed with RIPA buffer containing a mixture of protease inhibitors and 1 mM Na3VO4. 5 mg of cell lysateswere incubated with 50 �l of 4G10-conjugated agarose beads overnight at 4 °C, and SDS eluted material wasimmunoblotted with Bmx, ErbB2, or ErbB3 antibodies. Total protein levels of Bmx, ErbB2, and ErbB3 were alsoanalyzed by immunoblotting. B, reciprocal immunoprecipitation of Bmx. Cell lysates were immunoprecipi-tated (IP) with anti-FLAG, peptide-eluted, and immunoblotted with 4G10 antibody. Molecular standards areindicated at the left margin. C, endogenous Bmx activation in untransfected LNCaP cells. Growth factor stimu-lation, immunoprecipitations, and subsequent immunoblotting were performed the same as described in A.Bar graphs show the ratios of anti-Tyr(P) or anti-Bmx versus anti-tubulin band intensities, and data are repre-sentative of at least three experiments.

FIGURE 5. Bmx is activated by EGF. A and B, Bmx activation in LNCaP-FLAG-Bmx cells. Cells were serum-starved for 2 days and then stimulated with EGF (20 ng/ml) for 5, 10, 15, 30, 60, or 120 min. A, cells were lysed,immunoprecipitated (IP) with anti-Tyr(P) (A) or anti-FLAG (B), and immunoblotted as in Fig. 4. C, endogenousBmx activation in LNCaP cells. EGF stimulation, anti-Tyr(P) immunoprecipitations, and subsequent immuno-blotting were performed the same as described in A and Fig. 4. Bar graphs show the ratios of anti-Tyr(P) oranti-Bmx versus anti-tubulin band intensities, and data are representative of at least three experiments. D, invitro kinase assay. LNCaP-FLAG-Bmx cells or nontransfected LNCaP cells were serum-starved for 2 days andthen stimulated with EGF (20 ng/ml, 5 min) or heregulin-�1 (40 ng/ml, 15 min). Bmx was immunoprecipitatedfrom 2 mg of cell lysates by using 30 �l of M2-conjugated agarose beads. Peptide eluted Bmx was then mixedwith kinase buffer and 1 �Ci of [�-32P]ATP at for 30 min at 30 °C, and the reaction was stopped with 10 mM EDTAand Laemmli sample buffer. The samples were resolved by 4 –12% NuPAGE gel, and Bmx autophosphorylationwas visualized by autoradiography. Bar graph shows the relative intensities of the labeled bands.




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could mediate Bmx recruitment in response to stimulation bytheir respective ligands.Bmx Phosphorylation in Response to Heregulin-�1 Is Src- and

PI 3-Kinase-dependent—Although these results showed thatEGFR and ErbB2 could mediate Bmx recruitment, it was notclear whether they also mediated Bmx phosphorylation andactivation independently of PI 3-kinase or Src. Therefore, wenext determined whether LY294002 or PP2 could block Bmxphosphorylation in response to heregulin-�1. Brief pretreat-ment of serum-starved 3xFLAGBmx-LNCaP cells withLY294002 (0.5–2 h) markedly decreased PI 3-kinase activity, asassessed by Akt activation, although heregulin-�1 still caused aslight increase in Akt activation (Fig. 7A). LY294002 treatmentfor 2 h modestly decreased basal activation of Bmx, as assessed

by anti-FLAG immunoprecipitationand anti-Tyr(P) immunoblotting,and more substantially decreasedBmx phosphorylation in responseto heregulin-�1 (Fig. 7A). Signifi-cantly, LY294002 also abrogated thephosphorylation of endogenousBmx in response to heregulin-�1, asassessed by anti-Tyr(P) immuno-precipitation and Bmx immuno-blotting (Fig. 7B). Interestingly,although endogenous Bmx phos-phorylation was blocked, LY294002again did not completely blockheregulin-�1-mediated activationof Akt.Treatment with PP2 more mark-

edly suppressed basal phosphoryla-tion of 3xFLAG and endogenousBmx and strongly inhibited Bmxphosphorylation in response toheregulin-�1 (Fig. 7, A and B). Incontrast, heregulin-�1-mediatedactivation of Akt was not blocked byPP2 (Fig. 7, A and B). Takentogether, these data indicate thatBmx activation by heregulin-�1 isdependent on both PI 3-kinase andSrc (or an Src family kinase).Bmx Phosphorylation in Response

to EGF Is Src-dependent and PI3-Kinase-independent—Similarly toits effect on heregulin-�1 stimula-tion, PP2 abrogated the phosphoryl-ation of 3xFLAG and endogenousBmx in response to EGF (Fig. 8, Aand B). In contrast, LY294002 didnot block EGF-stimulated phospho-rylation of transfected nor endoge-nous Bmx (Fig. 8, A and B). Signifi-cantly, this did not appear to bebecause of residual PI 3-kinase acti-vation, as basal and EGF-stimulatedAkt phosphorylation were almost

completely suppressed by the LY294002 treatment.Finally, to further assess whether Bmx activation by EGFwas

PI 3-kinase-independent, we carried out in vitro kinase assays.Serum-starved 3xFLAGBmx-LNCaP cells were EGF-stimu-lated, and Bmx was isolated by anti-FLAG immunoprecipita-tion and 3xFLAG peptide elution. Bmx kinase activity, asassessed by autophosphorylation in in vitro kinase assays, wasrapidly stimulated by EGF (Fig. 8C). Treatment with LY294002decreased basal Bmx kinase activity and completely sup-pressed Akt phosphorylation, but it did not inhibit Bmx acti-vation in response to EGF. These data are consistent with theabove Bmx tyrosine phosphorylation results and confirmthat the rapid activation of Bmx in response to EGF is PI3-kinase-independent.

β β

FIGURE 6. Bmx association with ErbB3 and EGFR is enhanced by growth factor stimulation. LNCaP-FLAG-Bmx cells (A and C) or nontransfected LNCaP (B and D) cells were serum-starved for 2 days and then stimulatedwith heregulin-�1 (40 ng/ml; 0.25, 0.5, or 1 h; A and B) or EGF (20 ng/ml; 5, 15, 30 min; C and D). A and C, 3xFLAGBmx was immunoprecipitated (IP) from 3 mg of cell lysates using 30 �l of M2-conjugated agarose beads.Peptide eluted protein migrating at �80 kDa was then immunoblotted with 4G10 (upper panels), and elutedproteins migrating between �120 –220 kDa were blotted for EGFR, ErbB2, or ErbB3 as indicated. Total proteinlevels of Bmx, EGFR, ErbB2, ErbB3, and �-tubulin in the whole cell lysates (1% of input) were also analyzed byimmunoblotting. B and D, endogenous Bmx was immunoprecipitated from LNCaP cell lysates using anti-Bmxor negative control antibodies and blotted for EGFR, ErbB2, and ErbB3, as indicated (no bands were detected inthe negative control antibody immunoprecipitates; data not shown). Total EGFR, ErbB2, ErbB3, and �-tubulinin 1% of the cell lysates used for immunoprecipitation are also shown. Data are representative of two (B and D)or three (A and C) experiments.

β β

FIGURE 7. Bmx phosphorylation in response to heregulin-�1 is Src- and PI 3-kinase-dependent. A, LNCaP-FLAG-Bmx cells were serum-starved for 2 days. Heregulin-�1 (40 ng/ml) stimulation of the cells was performed15 or 30 min before the end of a 2-h incubation with LY294002 (LY, 20 �M) or PP2 (10 �M). Bmx was immuno-precipitated (IP) from 1 mg of cell lysates by using 20 �l of M2-conjugated agarose beads. Peptide eluted Bmxwas analyzed by immunoblotting with 4G10 antibody. B, nontransfected LNCaP cells were serum-starved for 2days and treated with LY294002, PP2, or heregulin-�1 as described in A. Tyrosine-phosphorylated proteinswere immunoprecipitated from 5 mg of cell lysates by 30 �l of 4G10-conjugated agarose beads, and SDSeluted proteins were immunoblotted with Bmx antibody. Immunoblotting with pAkt was performed to assessthe activity of PI 3-kinase. Bar graphs show the ratios of anti-Tyr(P) or anti-Bmx versus anti-tubulin band inten-sities, and data are representative of at least three experiments.




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DISCUSSION

PTEN loss and PI 3-kinase activation make a major contri-bution to PCa, and pathways activated downstream of PI 3-ki-nase are candidate therapeutic targets. This study shows thatBmx is activated downstream of the constitutively active PI3-kinase pathway in PTEN-deficient LNCaP and PC3 PCa cells,and that Bmx down-regulation by RNA interference markedlyinhibits cell growth, indicating that Bmx is a critical down-stream effector of PI 3-kinase in these PCa cells. Moreover,Bmx was further rapidly stimulated in response to activation ofErbB2/ErbB3 and EGFR in PCa cells. Treatment of LNCaP cellswith an ErbB2/ErbB3 ligand, heregulin-�1, enhanced an inter-action betweenBmx andErbB3 and stimulated the activation ofBmx. EGF treatment similarly stimulated an interactionbetween EGFR and Bmx and rapid Bmx activation. Althoughheregulin-�1 stimulated a Bmx-ErbB3 interaction, Bmx activa-tion by heregulin-�1 was still PI 3-kinase-dependent, as it wasblocked by LY294002. In contrast, Bmx activation by EGF wasPI 3-kinase independent. Finally, Bmx activation in response toboth heregulin-�1 and EGF was blocked by inhibition of Src,indicating that Src mediates Bmx phosphorylation subsequentto its membrane recruitment by PI 3-kinase, ErbB3, or EGFR.Taken together, these results show that Bmx is a critical down-stream target of the constitutively active PI 3-kinase in PTEN-deficient PCa cells and demonstrate the first direct linkbetween activation of Bmx and receptor tyrosine kinases.Moreover, they indicate that Bmxmaybe a valuable therapeutictarget in PCa and other epithelial malignancies in which PI3-kinase or EGF receptor family pathways are activated.The activation of Bmx and other Tec kinases is dependent on

phosphorylation of a regulatory tyrosine in the catalytic site,which is mediated by Src or Src family kinases (9). Moreover,activation of Bmx and other Tec kinases by many stimuli is PI3-kinase-dependent, although previous studies indicate thatthis activation in response to some stimuli may be independentof PI 3-kinase (21, 29, 43). In contrast to Src family kinases thatare inhibited by an interaction between their SH2 domain and aC-terminal phosphotyrosine, Tec kinasesmay be autoinhibitedby an SH3 domain-mediated intramolecular interaction (10–13). This inhibitory interaction in Bmx may be disrupted byPIP3 binding to the PH domain or by FAK binding to the PHdomain and subsequent phosphorylation of Tyr-40 in the PHdomain (29). Therefore, the PI 3-kinase dependence of heregu-lin-�1-mediated Bmx activation may reflect a requirement forPIP3 binding to expose the Bmx kinase domain for phosphoryl-ation by Src. Alternatively, PIP3may be required to stabilize theinteraction between Bmx and ErbB3 or to enhance Bmx target-ing to an Src-enriched membrane domain.A final alternative is that Bmx is activated in response to

heregulin-�1 independently of its recruitment by ErbB3, withErbB3 serving as a scaffold or possibly a substrate for activatedBmx. In this regard, it may be significant that ErbB3 tyrosinephosphorylation in response to heregulin-�1 was persistent inthe Bmx overexpressing LNCaP cells versus more transient inthe control cells. In any case, as ErbB3 is a potent activator of PI3-kinase, these data indicate that Bmx may be particularlyresponsive to PI 3-kinase activation through ErbB2/ErbB3 and

FIGURE 8. Bmx phosphorylation in response to EGF is Src-dependent andPI 3-kinase-independent. A, LNCaP-FLAG-Bmx cells were serum-starved for2 days. EGF (20 ng/ml) stimulation of the cells was performed 5 or 30 minbefore the end of a 2-h incubation with LY294002 (20 �M) or PP2 (10 �M). Bmxwas immunoprecipitated (IP) from 1 mg of cell lysates by using 20 �l ofM2-conjugated agarose beads, eluted from the M2 beads by FLAG peptide,and analyzed by immunoblotting with 4G10 antibody. B, nontransfectedLNCaP cells were serum-starved for 2 days and treated with LY294002, PP2, orEGF the same as described in A. Cell lysates (5 mg) were immunoprecipi-tated with 30 �l of 4G10-conjugated agarose beads, SDS eluted, andimmunoblotted with Bmx antibody. Bar graphs show the ratios of anti-Tyr(P) or anti-Bmx versus anti-tubulin band intensities, and data are rep-resentative of at least three experiments. C, in vitro kinase assay. LNCaP-FLAG-Bmx cells were treated and immunoprecipitated as described in A.Peptide eluted Bmx was mixed with kinase buffer, and the kinase assaywas performed at 30 °C for 30 min. The samples were resolved by 4 –12%NuPAGE gel, and Bmx autophosphorylation was visualized by autoradiog-raphy. Bar graph shows the relative intensities of the labeled bands. Wholecell lysates were immunoblotted with Bmx and anti-pAkt antibody to con-firm inhibition of PI 3-kinase.




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may function physiologically to integrate signaling through thePI 3-kinase and ErbB2/ErbB3 pathways. In contrast to Bmxactivation by heregulin-�1, EGF-mediated activation of Bmxwas not dependent on PI 3-kinase. Thismay reflect Bmx target-ing to a distinct membrane microdomain by EGFR or confor-mational changesmediated by EGFR (possibly because of phos-phorylation at Tyr-40 or other sites) that expose the Bmxkinasedomain for phosphorylation by Src, independently of PIP3binding.Previous studies have indicated that Bmxmay be activated by

receptor tyrosine kinases. Studies in endothelial cells showedthat Bmx could be activated by VEGF or by transfected overex-pressed Tie-2 and VEGF receptor 1 (14, 25). However, it is notknown whether Bmx interacts directly with these receptors,and Bmx activation by the endogenous receptors has not beenestablished. Another study inMDA-MB-468 breast cancer cellsshowed that EGF could stimulate tyrosine phosphorylation ofadenovirus transduced Bmx, which could be partially blockedby Src and PI 3-kinase inhibitors (42). This study also used aphospho-specific antibody to show EGF stimulated phospho-rylation of Tyr-40 in the Bmx PH domain, but it is not clearwhether this is mediated by EGFR or another kinase, includingFAK. Interestingly, EGF stimulates Stat1 activation and apo-ptosis in these MDA-MB-468 cells, and both could be blockedby a kinase domain-deleted Bmx (42). Further studies areneeded to determine whether these observations inMDA-MB-468 cells reflect a direct interaction between EGFR and Bmxand to determine the basis for the apoptotic response.Bmx activation in PCa cells can also be stimulated by neu-

ropeptides (bombesin and neurotensin) and IL-6, and Bmx isrequired for neuroendocrine differentiation induced by theseagents in LNCaP cells (19, 21). Neuropeptide activation of Bmxis mediated by FAK and Src and is PI 3-kinase-independent. Incontrast, IL-6-mediated Bmx activation is PI 3-kinase-depend-ent. We observed IL-6 activation of transfected Bmx, but notendogenous Bmx, despite robust Stat3 activation in response toIL-6. The basis for this difference is not clear. A previous studyalso indicated that Bmx is a mediator of Src-induced Stat3 acti-vation, although these studies were done in rat hepatoma cells(36). In any case, given the marked effects of Bmx down-regu-lation on PCa cell growth, it will clearly be important to identifyits critical downstream substrates in PCa cells. Previous studieshave identified p130Cas as a possible Bmx substrate activateddownstream of integrin signaling in epithelial cells (1), but fur-ther Bmx substrates remain to be identified.In summary, this study demonstrates that Bmx is activated

downstream of endogenous constitutively active PI 3-kinase inPTEN-deficient PCa cells and that it plays a critical role in sup-porting PCa cell growth. Moreover, we identify an additionalmechanism of Bmx activation through recruitment by ErbB2/ErbB3 and EGFR in response to their respective growth factors.Taken together, these results show that ErbB2/ErbB3 andEGFR are upstream activators of Bmx and provide the firstdirect link between Bmx and receptor tyrosine kinases. Signif-icantly, PCa progression has been associated with increasedErbB2 expression, aswell as PTEN loss and PI 3-kinase pathwayactivation, indicating that Bmx may be a critical point of con-vergence for these signaling pathways in advanced PCa (44, 45).

Finally, these studies indicate that Bmx may be activated and atherapeutic target in other epithelial cancers with increasedErbB2 or EGF receptor activity.

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Carpenter and Steven P. BalkXinnong Jiang, Robert A. Borgesi, Nicole C. McKnight, Ramneet Kaur, Christopher L.

Prostate Cancer CellsPhosphoinositide 3-Kinase, Epidermal Growth Factor Receptor, and ErbB3 in

Activation of Nonreceptor Tyrosine Kinase Bmx/Etk Mediated by

doi: 10.1074/jbc.M703412200 originally published online September 6, 20072007, 282:32689-32698.J. Biol. Chem.

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http://www.jbc.org/lookup/doi/10.1074/jbc.M703412200

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http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=282/45/32689&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/282/45/32689

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/282/45/32689.full.html#ref-list-1

http://www.jbc.org/

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