P38 MAPK-Mediated Activation of NF-Kb by the Rhogef Domain of Bcr

Malgorzata Korus1,2, Gwendolyn M Mahon1,2, Li Cheng1 and Ian P Whitehead*,1

1Department of Microbiology and Molecular Genetics, UMDNJ – New Jersey Medical School, Newark, New Jersey, NJ 07103, USA

The oncogenic fusion protein p210 Bcr-Abl is causally et al., 1988; Fainstein et al., 1987; Konopka et al., associated with virtually all cases of chronic myelogenous 1984; Kurzrock et al., 1987; Walker et al., 1987). leukemia. The wild-type Bcr product has several Although animal models suggest that elevated levels of recognizable structural and functional motifs including Abl tyrosine kinase activity contribute to Bcr-Abl a domain that contains guanine nucleotide exchange mediated leukemogenesis (Lugo et al., 1990), the activity for Rho family GTPases (DH/PH domain). residues that distinguish the rearrangements associated Although this domain is retained within p210 Bcr-Abl, it with ALL (p185 Bcr-Abl) from the rearrangements has no known signaling activities in vivo. Here we report associated with CML (p210 Bcr-Abl) reside within the that a fragment of Bcr that encodes the isolated DH/PH Bcr protein. Thus, a complete understanding of the domain is a potent activator of the NF-kB transcription biological mechanisms that distinguish ALL from factor. Within the context of full length Bcr, this activity CML may depend upon the characterization of the is regulated by proximal flanking sequences that suppress signaling activities that reside within Bcr. the DH/PH domain encoded guanine nucleotide The major product of the bcr gene is a 160 kD exchange activity. NF-kB activation by Bcr is not protein that exists predominantly as a 600 kD oligomer mediated by nuclear translocation, but rather by p38 (McWhirter et al., 1993). Oligomerization is mediated mitogen-activated protein kinase (MAPK)-dependent by an amino terminal coiled coil domain (residues 1 – modification of the RelA/p65 transactivation domain. 40) that is required for the transforming activity of Although we were able to demonstrate that Bcr can Bcr-Abl chimerics. Four additional structural domains function as an exchange factor for Cdc42 in vivo, NF-kB have been identified within the Bcr protein. Contained activation appears to occur via a Cdc42-independent within the amino terminus of Bcr (residues 1 – 414) is a mechanism. These studies constitute direct evidence that serine/threonine kinase activity that when expressed the Bcr RhoGEF domain can function in vivo, and alone, or within the context of the full-length protein, identify a new signaling activity that may contribute to exhibits both autophosphorylation and transphosphory- the transforming potential of p210 Bcr-Abl. lation activity (Maru and Witte, 1991). The central Oncogene (2002) 21, 4601 – 4612. doi:10.1038/sj.onc. portion of Bcr (residues 501 – 870) consists of a tandem 1205678 DH/PH domain (Ron et al., 1991) that is shared by all members of the Rho specific guanine nucleotide Keywords: Bcr; RhoGEF; Dbl proteins; NF-kB; p38 exchange factors (RhoGEFs) (Whitehead et al., MAPK 1997). RhoGEFs have been shown to activate members of the Rho family of small GTPases by catalyzing the exchange of GDP for GTP. In in vitro assays, the isolated DH/PH domain of Bcr has been shown to Introduction catalyze exchange of GDP for GTP on the Rho family members RhoA, Cdc42 and Rac1 (Chuang et al., The breakpoint cluster region (bcr) gene is implicated 1995). The carboxyl terminus of Bcr (residues 1172 – in the pathogenesis of two human leukemias that are 1271) contains a GTPase activating protein (GAP) associated with the Philadelphia chromosome. Most activity that is also specific for members of the Rho chronic myelogenous leukemias (CML), and a subset family (Diekmann et al., 1991). GAPs stimulate the of the acute lymphocytic leukemias (ALL), can be intrinsic rate of hydrolysis of GTPases, thus converting attributed to the aberrant expression of fusion proteins them to the inactive GDP-bound form. Bcr-mediated that encode amino terminal sequences from Bcr and GAP activity has been demonstrated for Rac1, Rac2 carboxyl terminal sequences from the oncogenic non- and Cdc42 in vitro (Chuang et al., 1995; Diekmann et receptor tyrosine kinase Abl (Chan et al., 1987; Clark al., 1991), and Bcr-null mutant mice exhibit phenotypes that are consistent with elevated levels of activated Rac (Voncken et al., 1995). Also contained within the carboxyl terminus of Bcr (residues 911 – 1036) is a C2 *Correspondence: IP Whitehead; E-mail: [email protected] 2These authors contributed equally to this work domain. Such domains have been described in over 100 Received 26 February 2002; revised 15 May 2002; accepted 20 May mammalian proteins (including other GAPs) and have 2002 been shown to bind phospholipids in a calcium Activation of NF-kB by Bcr M Korus et al 4602 dependent manner (Rizo and Sudhof, 1998). The C2 whether the Bcr-encoded sequences have similar domain of Bcr has not been characterized and its properties. In the current study we have determined function is not currently known. that expression of the isolated DH/PH domain of Bcr The presence of the DH/PH module within Bcr is of is associated with the activation of Cdc42 (but not particular interest since many RhoGEFs have been Rac1 or RhoA) in vivo, and is associated with potent isolated based on their oncogenic activity, and in several stimulation of NF-kB response elements. Both activ- instances, this activity has been mapped to this tandem ities are suppressed by carboxyl- and amino-proximal structure (Whitehead et al., 1997). Although this domain flanking sequences when expressed in cis or in trans. is present in both p160 Bcr and p210 Bcr-Abl, no in vivo Activation of NF-kB by Bcr is not associated with functions have been attributed to it in either context. nuclear translocation, but rather with p38 MAPK- However, the recent observation that the DH/PH dependent transactivation of p65/RelA. This activity domain of p210 Bcr-Abl cannot be functionally replaced occurs independently of Cdc42 suggesting that Bcr may with homologous sequences from Cdc24 (a related utilize additional GTPase targets in vivo. These RhoGEF), or with irrelevant sequences of equivalent observations constitute direct evidence that the DH/ size (Kin et al., 2001), suggests that these sequences may PH domain of Bcr can exhibit independent signaling in fact contain transforming potential, which would be activity in vivo, and raises the possibility that such consistent with what has been observed for multiple activity may influence Philadelphia chromosome posi- members of the RhoGEF family (Whitehead et al., 1997). tive leukemias. It is generally thought that RhoGEFs regulate proliferative signaling pathways through the aberrant Results activation of their GTPase substrates, several of which have also been demonstrated to possess oncogenic Activation of NF-kB response elements by the isolated potential (Whitehead et al., 1997). However, the DH/PH domain of Bcr downstream signaling events that are triggered by chronic stimulation of Rho family GTPases, and which We have shown previously that many RhoGEFs share contribute to RhoGEF transformation, are still some- a common ability to stimulate signaling pathways that what obscure. Rho controlled signaling pathways have result in the activation of multiple transcription factors been shown to regulate multiple cellular behaviors (Westwick et al., 1998; Whitehead et al., 1999). In all including reorganization of the actin cytoskeleton cases that have been examined, this activity has been (Hall, 1994; Nobes and Hall, 1995; Ridley, 1995), mapped to the conserved DH/PH domain module. We regulation of the cell cycle (Olson et al., 1995) and the wished to determine whether such in vivo activity also activation of multiple transcription factors including resides within the DH/PH domain of Bcr. Towards this Elk-1, c-Jun, SRF and NF-kB (Coso et al., 1995; Hill end, a panel of Bcr truncation mutants was constructed et al., 1995; Minden et al., 1995; Perona et al., 1997). (Figure 1a), all of which contain the DH/PH domain This diversity of biological activity can be explained by (residues 491 – 876) along with varying amounts of the large, and equally diverse, collection of effector flanking sequence. All members of the panel were fused molecules that bind to activated Rho family GTPases to an HA epitope, and expression was confirmed in (Zohn et al., 1998). Recent reports have implicated COS-7 cells by Western blot with an anti-HA antibody several of these effectors in Rho and RhoGEF (F7, Santa Cruz Biotechnology, Santa Cruz, CA, USA; mediated transformation. A pharmacological inhibitor Figure 1b). In each case a cross-reactive band was that has been reported to be specific for the RhoA identified that corresponded in size to the predicted effector protein ROCK, can efficiently block the translation product of the various derivatives. transforming activity of RhoA, Dbl and Net1, in In order to determine whether expression of the Bcr NIH3T3 cells (Sahai et al., 1999). ROCK has been panel corresponded with increased activity on tran- implicated in Rho-mediated assembly of focal adhesion scriptional response elements, each panel member was complexes (Amano et al., 1997; Ishizaki et al., 1997; co-transfected with a luciferase-based reporter plasmid Leung et al., 1996; Watanabe et al., 1999). A genetic that was designed to measure either NF-kB-, Serum inhibitor that blocks activation of c-Jun by RhoA has response factor (SRF-), or c-Jun-mediated promoter also been shown to block RhoA-mediated transforma- activity. Although transient expression of the isolated tion. RhoA activation of c-Jun is thought to occur DH/PH domain of Bcr (Bcr(491 – 876)) was associated through a second effector protein, PKN (Marinissen et with activation of the NF-kB response element in COS- al., 2001). Finally, genetic inhibitors that block 7 cells (Figure 2a), and to a lesser extent in NIH3T3 transcription by NF-kB in NIH3T3 cells are potent cells (Figure 2b), very weak or no activation was inhibitors of Dbl and Dbs transformation in the same observed with all other panel members (complete data cell type (Whitehead et al., 1999). Although the effector for NIH3T3 cells is not shown). This suggests that the protein responsible for Rho-mediated activation of NF-kB signaling activity is regulated by sequences NF-kB has not yet been identified, NF-kB contributes located either carboxyl or amino proximal to the DH/ to the transforming activity of many oncogenes PH domain. All members of the panel, including including Bcr-Abl (Reuther et al., 1998). Bcr(491 – 876), failed to activate the c-Jun or SRF Since RhoGEF domains often exhibit potent signal- reporters (data not shown) suggesting that the ing and transforming activities, we wished to determine activation of NF-kB by Bcr is highly specific. Although

Oncogene Activation of NF-kB by Bcr M Korus et al 4603

Figure 2 Activation of an NF-kB responsive promoter element by the DH/PH domain module of Bcr. COS-7 cells (a)or NIH3T3 cells (b) were transiently transfected with 3 mgof pAX142 (vector) or pAX142 containing the indicated Bcr derivatives along with a luciferase reporter plasmid to determine stimulation of NF-kB-mediated transcriptional activity (NF-kB-luc), and pCMVnlac (0.25 mg) as an internal control for transfection Figure 1 Structure and expression of truncated derivatives of efficiency and/or growth inhibition. Luciferase and b-galactosi- human Bcr. (a) The domain structure of the full-length Bcr pro- dase activity was measured and expressed as fold activation rela- tein is illustrated in the upper line (O, oligomerization domain; ki- tive to the level of activation seen with the vector control. nase, serine/threonine kinase domain; DH, Dbl-homology Luciferase activity was then standardized relative to b-galactosi- domain; PH, pleckstrin homology domain; C2, calcium-binding dase activity. Data shown are representative of at least three inde- motif; GAP, GTPase activating protein domain) and the bars be- pendent assays performed on duplicate plates. The error bars low indicate the regions of the protein included in predicted trans- indicate standard deviations lation products of the various cDNA derivatives. cDNA designations indicate the amino acid residues from Bcr that are contained within the translation product. All derivatives are fused, at either the amino or carboxyl terminus (as indicated), two domains that we had identified as containing to an in-frame epitope from the hemagglutinin protein (HA) of inhibitory sequences failed to have any effect on influenza virus. (b) All cDNA derivatives were cloned into the endogenous NF-kB activity suggesting that they are pAX142 mammalian expression vector, transiently expressed in incapable of independently influencing the reporter COS-7 cells, and detected by Western blot using a mouse anti- HA monoclonal antibody (F-7, Santa Cruz Biotechnology, Santa element. In contrast, a genetic inhibitor that can Cruz, CA, USA) specifically block NF-kB nuclear translocation (IkBa(SS)), reduced the basal level of activity on this reporter element by 81%. Surprisingly, the amino terminal kinase domain also exhibited a modest yet activation of NF-kB by the DH/PH domain of Bcr is reproducible activation of the NF-kB reporter (three- consistent with what we have observed for several fold) suggesting that Bcr can regulate NF-kB other RhoGEFs (Whitehead et al., 1999), the strength activation via multiple, independent mechanisms. of this activation coupled with the failure to activate Next we wished to determine whether the two other response elements constitutes a novel signaling inhibitory domains could block NF-kB activation by profile within the family. Bcr when expressed in trans. Co-expression of either Bcr(415 – 490) or Bcr(876 – 1163) with the isolated DH/ PH domain module resulted in decreased activation of Inhibition of NF-kB activity by expression of flanking the NF-kB reporter (Figure 3d, 24 and 60% respec- sequences in trans tively). This suggests that each domain may be able to To confirm that the DH/PH domain of Bcr is flanked form independent inhibitory complexes with the by inhibitory sequences, additional epitope-tagged isolated GEF domain. The isolated kinase domain derivatives were constructed that encoded these did not exhibit any inhibition relative to vector putative inhibitory domains (Figure 3a; Bcr(415 – 490) controls thus confirming the specificity of the inhibi- and Bcr(876 – 1163)). As an additional control, a tion. construct was made that encoded the isolated kinase To further examine the specificity of the Bcr-encoded domain (Bcr(1 – 413)). Expression of all three inhibitory domains we also determined whether each of constructs was verified by transient transfection and the isolated domains could block activation of the NF- Western blot analysis in COS-7 cells (Figure 3b). kB reporter by a second RhoGEF. We have shown Initially we tested each derivative for their ability to previously that a derivative of Dbl that contains the regulate endogenous NF-kB activity (Figure 3c). The isolated DH/PH domain (Dbl-HA1) is a strong

Oncogene Activation of NF-kB by Bcr M Korus et al 4604

Figure 3 The DH/PH domain of Bcr is regulated by ﬂanking sequences. (a) The domain structure of the full-length Bcr protein is indicated in the upper line (see Figure 1 for explanation of abbreviations) and the bars below indicate the regions of the protein included in predicted translation products of the various cDNA derivatives. cDNA designations indicate the amino acid residues from Bcr that are contained within the translation products. All derivatives are fused to an in-frame epitope, at either the amino or carboxyl terminus (as indicated), from the hemagglutinin protein (HA) of inﬂuenza virus. (b) All derivatives were cloned into pAX142, transiently expressed in COS-7 cells, and detected by Western blot using a mouse anti-HA monoclonal antibody (F-7, Santa Cruz Biotechnology, Santa Cruz, CA, USA). (c – e) COS-7 cells were transiently transfected with 3 mg of pAX142, or pAX142 containing the indicated cDNA derivatives, along with NF-kB-luc and pCMVnlac. For c, relative luciferase units are expressed as a percentage relative to the number of units seen with vector controls. For d and e, data are calculated and presented as in Figure 2

activator of NF-kB response elements in NIH3T3 cells hypothesis in vivo. It has been shown previously that (Whitehead et al., 1999). A similar activation was the isolated DH/PH domain of Bcr contains GEF observed when we expressed this construct alone in activity for the Rho family members RhoA, Rac1 and COS-7 cells (Figure 3e). Co-expression with the amino Cdc42 in vitro (Chuang et al., 1995). To determine terminal inhibitory domain of Bcr (Brc(415 – 490)) whether this observation could be recapitulated in vivo, substantially reduced the activation of the reporter by we measured levels of RhoA-GTP, Cdc42-GTP and Dbl (79%) suggesting that this domain may be Rac1-GTP in COS-7 cells that were transiently promiscuous in its ability to form inhibitory complexes transfected with full-length Bcr (Bcr(1 – 1271)) and the with DH/PH domains. No significant inhibition was isolated DH/PH domain (Bcr(491 – 876)) (Figure 4a – observed when Dbl was co-expressed with the isolated c). For comparison we also transfected a transforming kinase domain, or with the carboxyl-terminal inhibi- derivative of Dbl (Dbl-HA1), which has in vitro tory domain. exchange activity for RhoA and Cdc42. Constitutively activated mutants of RhoA, Rac1 and Cdc42 that are locked in the GTP-bound state (RhoA(63L), The in vivo GEF activity of the Bcr DH/PH domain is Rac1(61L) and Cdc42(12V)) served as positive inhibited by flanking sequences controls. Although we were able to affinity purify the Our observation that the expression of the flanking constitutively activated GTPases out of COS-7 cell sequences of the Bcr DH/PH domain can inhibit Bcr- lysates, we were unable to detect any differences in mediated signaling in trans suggests that these endogenous Cdc42-GTP, RhoA-GTP or Rac1-GTP sequences may directly regulate the GEF activity levels between the vector and the Bcr lysates. Dbl-HA1 residing within Bcr. We wished to confirm this was able to activate endogenous RhoA in COS-7 cells,

Oncogene Activation of NF-kB by Bcr M Korus et al 4605

Figure 4 The in vivo GEF activity of Bcr is regulated by flanking sequences. COS-7 cells were transiently transfected with 3 mg pAX142 (vector) or pAX142 that contained the indicated cDNAs. Lysates were collected at 48 h and aliquots were examined by Western blot for expression of RhoA (a: Total-RhoA), Cdc42 (b: Total-Cdc42), Rac1 (c: Total-Rac1) and the HA-tagged Dbl and Bcr derivatives (d: anti-HA). The remainder of the lysates were then split into three parts, each of which was normalized for expression of either RhoA, Rac1 or Cdc42. Each lysate was then subjected to affinity purification using immobilized GST- Pak (Rac1 and Cdc42) or GST-C21 (RhoA). GTP-bound Cdc42 (b: GTP-Cdc42) and Rac1 (c: GTP-Rac1) that were precipitated with GST-PAK, or GTP-bound RhoA (a: GTP-RhoA) that was precipitated with GST-C21, were visualized by Western blot. Ad- ditional lysates were prepared from cells that were transfected with constitutively activated versions of the relevant GTPases (either RhoA(63L), Rac1(61L) or Cdc42(12V)) as indicated. These lysates were also subjected to affinity purification with the relevant GST fusion protein and were included as positive controls. (e, f) COS-7 cells were transiently co-transfected with the indicated plasmids along with a plasmid that encodes wild-type Cdc42. Lysates were collected at 48 h and affinity purifications were performed as described. GTP-Cdc42 and Total-Cdc42 levels were determined by phosphoimage analysis. Each value was first normalized to the vector control, and then GTP-Cdc42 levels were further normalized relative to Total-Cdc42 levels. All experiments were performed a minimum of three times and data shown constitutes a representative data set but not Rac1 or Cdc42. The failure of Bcr to activate Bcr. Thus, although the DH/PH domain of Bcr RhoA, Rac1 or Cdc42 could not be attributed to poor appears competent to catalyze exchange when expression of Bcr in the lysates (Figure 4d). expressed in COS-7 cells, it does not appear to regulate If we increased the endogenous pool of GTPases by endogenous steady state levels of RhoA, Rac1 or co-transfecting Bcr with cDNAs that encode wild-type Cdc42. versions of RhoA, Rac1 or Cdc42, we were able to To determine whether the inhibitory flanking detect elevated Cdc42-GTP in the Bcr-transfected cells sequences of the Bcr DH/PH domain could diminish (Figure 4e), but not elevated RhoA or Rac1 (not the Cdc42 specific GEF activity, we measured Cdc42- shown). The level of activity was equivalent to what we GTP loading in COS-7 cells that were transiently co- observed with the transforming derivative of Dbl and transfected with Bcr(415 – 876) or Bcr(491 – 1162) and was only observed with the isolated DH/PH domain of wild-type Cdc42 (Figure 4f). Although both of these

Oncogene Activation of NF-kB by Bcr M Korus et al 4606 derivatives were expressed at comparable levels to the isolated DH/PH domain neither was able to induce an increase in Cdc42-GTP loading. Thus, the GEF activity that resides within Bcr appears to be under autoinhibitory control that is mediated by residues located within ﬂanking domains.

Bcr activates NF-kB through a Cdc42-independent mechanism Since activated derivatives of Cdc42 have been shown to stimulate transcription from NF-kB responsive reporters in COS-7 cells (Montaner et al., 1998; Perona et al., 1997), and the isolated DH/PH domain of Bcr can activate both Cdc42 and NF-kB response element in this same cell type, we wondered whether Bcr activates NF-kB in a Cdc42-dependent manner. It is possible that endogenous Cdc42 is cycling more rapidly in the Bcr transfected cells, and that this increased cycling is not reflected in discernible increases in steady-state levels of Cdc42-GTP. To explore this possibility further, we wished to determine if Bcr stimulation of transcription from the NF-kB reporter is Figure 5 Bcr activates NF-kB through a Cdc42-independent impaired by co-expression with specific inhibitors of mechanism. COS-7 cells were transiently transfected with 3 mg Cdc42 function. pAX142-cdc42(17N) encodes a of pAX142 and/or pAX142 containing the indicated cDNAs, along with NF-kB-luc and pCMVnlac. Data are calculated and GTPase defective dominant-inhibitory mutant of presented as in Figure 2 Cdc42 that has been used extensively to analyse Cdc42-mediated signaling activities. pyDF30-WASP- GBD encodes the GBD of WASP (an effector protein specific for Cdc42) and has been shown to specifically tion and reduce the nuclear pool of NF-kB. IkBa(SS) is inhibit Cdc42 mediated functions. Co-expression of Bcr a derivative of IkBa that has been mutated on serines with Cdc42(17N) failed to block activation of the NF- 32 and 36. This mutant cannot be phosphorylated by kB reporter while co-expression with wild-type Cdc42 the IKK complex and forms irreversible complexes produced levels of activation that are more consistent with NF-kB in the cytoplasm. To determine whether with additivity than with synergy (Figure 5a). activation of the NF-kB response element by Bcr is Similarly, WASP-GBD failed to block reporter activa- mediated by the nuclear pool of NF-kB, we co- tion by Bcr but did block activation of the same transfected Bcr(491 – 876) with an expression plasmid reporter by an activated derivative of Cdc42(12V); that contains IkBa(SS) (Figure 6a). In the presence of Figure 5b). Collectively, these observations would the NF-kB inhibitor, activation of the reporter by Bcr suggest that activation of NF-kB reporters by Bcr was dramatically reduced (88%). Importantly, this cannot be accounted for by activation of endogenous reagent was also effective in blocking endogenous Cdc42 in COS-7 cells. activity at the NF-kB reporter (Figure 3c) suggesting that a nuclear pool of transcriptionally active NF-kBis present within COS-7 cells in the absence of Bcr Activation of the NF-kB response element by Bcr is expression. mediated by nuclear NF-kB Despite our inability to identify the in vivo substrate Activation of the NF-kB response element by Bcr is not for Bcr in COS-7 cells, we wished to further explore the associated with increased nuclear translocation mechanism through which Bcr was stimulating NF-kB activation. The canonical mechanism of NF-kB Our observation that Bcr activation of NF-kB response activation is through its dissociation from the elements can be blocked by IkBa(SS) suggests that Bcr regulatory IkB molecules (Karin, 1999). In response may regulate the nuclear accumulation of NF-kB. to extracellular stimuli such as IL-1 or TNFa,IkB Upon release from IkB, NF-kB is free to translocate to becomes phosphorylated and targeted for degradation the nucleus where it can bind to NF-kB responsive by ubiquitin-mediated proteolysis. IkB phosphoryla- elements. This event that can be detected using tion is mediated by the IkB kinase (IKK) complex, a electrophoretic mobility shift assays (EMSA). Using heterodimeric kinase whose catalytic core is comprised such an assay we were unable to detect increased of IKKa and IKKb. Upon release from IkB, NF-kBis nuclear translocation of NF-kB in COS-7 cells free to translocate to the nucleus where it can bind to transfected with either the isolated DH/PH domain, NF-kB responsive elements. Several genetic inhibitors or full-length Bcr (Figure 6b). In contrast, treatment of have been described that can block IkB phosphoryla- the cells with TNFa for 30 min prior to harvesting the

Oncogene Activation of NF-kB by Bcr M Korus et al 4607

Figure 6 Bcr-mediated activation of the NF-kB response element is not associated with increased nuclear translocation. (a) COS-7 cells were transiently transfected with 3 mg of pAX142 or pAX142 containing the indicated cDNAs, along with NF-kB-luc and pCMVnlac. Data are calculated and presented as in Figure 2. (b) EMSA performed with nuclear extracts collected from COS-7 cells that were either treated with 10 ng/ml TNFa for 30 min prior to lysate collection (lanes 1 and 2), or transiently transfected with 3 mg pAX142 (lane 3), pAX142-bcr(491 – 876) (lane 4) or pAX142-bcr(1 – 1271) (lane 5). All lysates except lane 1 were incubated with a radiolabeled oligonucleotide probe containing an NF-kB binding site, and resolved on 5% polyacrylamide gels. NF-kB complexes were identified through competition and supershift assays (not shown) and are indicated with arrows. (c) COS-7 cells were transfected with 3 mg pAX142 or pAX142-bcr(491 – 876), or were treated with 10 ng/ml TNFa for the indicated time period prior to harvesting lysates. Media was changed and fresh TNFa added every 3 h as required. NF-kB-luc and pCMVnlac were included in each transfection. Data are calculated and presented as in Figure 2 lysates was associated with a measurable increase in the mechanism to activate NF-kB, we co-transfected nuclear pool of the transcriptionally active p50/p65 COS-7 (Figure 7a) and NIH3T3 (Figure 7b) cells with heterodimer. The identities of the NF-kB complexes Bcr(491 – 876), and with a luciferase reporter construct were confirmed using competition and supershift assays fused to a promoter containing five Gal4 DNA-binding (data not shown). To explore the possibility that TNFa sites (Gal-luc) along with expression constructs encod- may be a more robust activator of NF-kB than Bcr, ing the Gal4 DNA-binding domain fused to and that our failure to detect nuclear translocation in transactivation domain 1 (TA1) of RelA/p65. This response to Bcr expression reflects a lack of sensitivity reporter system was designed to detect modifications at of the EMSA, we compared TNFa and Bcr in their the transactivation domain of RelA/p65. Transient ability to activate the NF-kB reporter. Regardless of expression of Bcr(491 – 876), but not full-length Bcr, the duration of time during which we exposed COS-7 activates the transcriptional function of Gal4-p65 in cells to TNFa prior to harvesting the lysates, we were both NIH3T3 and COS-7 cells. Interestingly, TNFa, unable to detect a level of activation equivalent to that which is a potent stimulator of the NF-kB-luc reporter seen with Bcr (Figure 6c). Thus, our analysis would (Figures 6c and 7a), failed to activate the Gal4-p65 suggest that the activation of NF-kB by Bcr is reporter in COS-7 cells. This further suggests that TNFa mechanistically distinct from that seen with TNFa, and Bcr activate NF-kB by distinct mechanisms and and not associated with any readily discernible increase supports a model in which Bcr activates NF-kBby in nuclear translocation. modification of the transactivation domain of p65/ RelA. Bcr activates the transactivation domain of RelA/p65 Bcr activates NF-kB through a p38 MAPK-dependent Despite the apparent lack of nuclear accumulation of mechanism NF-kB in Bcr transfected cells, activation of NF-kB response elements is observed. One possibility is that Previous studies have shown that p38 MAPK can Bcr is exerting its influence on a preexisting nuclear stimulate the transcriptional activity of NF-kB through pool of NF-kB. Thus, expressing IkBa(SS) would clear a mechanism that does not require nuclear accumula- the nucleus of this pool and reduce the number of tion (Bergmann et al., 1998; Madrid et al., 2001; targets for Bcr activation. It has been shown previously Wesselborg et al., 1997). Additionally, we have shown that a second mechanism of NF-kB activation can previously that many RhoGEFs share a common occur involving phosphorylation of the RelA/p65 ability to activate p38 MAPK in COS-7 cells transactivation domain. Such a mechanism has been (Westwick et al., 1998). To determine whether Bcr observed for activation of NF-kB by Ras, Bcr-Abl and activates NF-kB through a p38 MAPK-dependent Akt. To determine whether Bcr utilizes a similar mechanism, we measured Bcr activation of the NF-

Oncogene Activation of NF-kB by Bcr M Korus et al 4608

Figure 7 Bcr activates the transactivation domain of RelA/p65. COS-7 cells (a) or NIH3T3 cells (b) were either treated with TNFa (10 ng/ml for 24 h prior to lysate collection), or were transfected with plasmids encoding the indicated Bcr derivatives (3 mg), along with either NF-kB-luc or expression vectors for the Gal4 DNA binding domain fused to transactivation domain 1 of p65 (0.25 mg Gal-p65) and a Gal4 luciferase reporter (2.5 mg Gal4-luc). For each condition, pCMVnlac was also included in the transfection. Data are calculated and presented as in Figure 2

kB-luc and Gal4-p65 reporters in the presence or Whitehead et al., 1999). These include activation of absence of a highly specific pharmacological inhibitor MEK MAPKK and the NF-kB transcription factor. of p38 MAPK (SB203580). Our initial examination of Although Bcr encodes a canonical DH/PH domain cell lysates revealed elevated levels of activated p38 motif, with demonstrated GEF activity in vitro MAPK (Figure 8a) in the Bcr-transfected cells, which (Chuang et al., 1995), little is known about the fate was not observed in control cells, or in the presence of of this activity within the context of the full-length the inhibitor. This suggests that Bcr can activate protein, or within the context of an in vivo system. endogenous p38 MAPK, and that the inhibitor can Such an analysis would be prerequisite to under- block this activation. Next we determined the effects of standing the contribution of these sequences to the the inhibitor on NF-kB activation. In the presence of transforming activity associated with the human the inhibitor, activation of both the NF-kB-luc and oncogene p210 Bcr-Abl. Our observation that this Gal4-p65 reporters by Bcr was substantially reduced domain can trigger p38 MAPK-dependent NF-kB (82 and 69% respectively) relative to controls (Figure transcriptional activity in vivo, is in accordance with 8b). Since the concentration of inhibitor that we used is what has been observed for other members of the extremely low (400 nM) the effect that we observed is RhoGEF family (Whitehead et al., 1999), and suggests not likely to be due to non-specific activity. Non- that this domain can establish productive interactions specific activity has only been observed with this with GTPase substrates in vivo. Consistent with this, inhibitor at concentrations in the 3 – 5 mM range (Lali we were able to observe elevated levels of Cdc42-GTP et al., 2000). These results suggest that Bcr regulates in Bcr-transfected cells. NF-kB through its ability to stimulate endogenous p38 Our finding that the signaling activity associated MAPK. with the Bcr DH/PH domain is subject to autoinhibitory control is also consistent with findings from other family members. Autoinhibition as a mechanism of Discussion GEF regulation has been invoked for many RhoGEFs including Dbl, Vav, Ost, Ect2 and Tiam1 (Aghazadeh The RhoGEF family consists of a large collection of et al., 2000; Habets et al., 1994; Horii et al., 1994; Miki structurally diverse proteins that share a common DH/ et al., 1993; Ron et al., 1991). RhoGEFs generally exist PH domain tandem motif (Whitehead et al., 1997). in a quiescent state and require activation to exhibit Numerous studies have now established that the DH signaling and transforming activity. The mechanisms of domain mediates Rho protein binding and GEF activation are diverse and include interaction with catalytic activity while the PH domain is required for upstream signals such as phosphoinositides, Ga appropriate subcellular localization. Both domains subunits of heterotrimeric G proteins, and protein contribute to the potent transforming activity that is kinases. Often such signals appear to relieve intramo- associated with activated derivatives of many lecular constraints upon GEF activity converting RhoGEFs (Whitehead et al., 1997). When expressed RhoGEFs to an active state. For example, the amino in their activated forms, RhoGEFs will stimulate terminal sequences of the Vav protein bind to the DH multiple signaling pathways, some of which contribute domain active site blocking access to GTPase to cellular transformation (Westwick et al., 1998; substrates (Aghazadeh et al., 2000). Phosphorylation

Oncogene Activation of NF-kB by Bcr M Korus et al 4609 such as Dbl and Bcr are regulated remains to be determined. Our observation that the two autoinhibitory domains that we have identified within the Bcr protein can block signaling when expressed in trans, leads us to speculate that residues contained within these domains can form direct contacts with the DH/PH domain. This is supported by our observation that these domains can individually block Bcr-mediated activity in vivo. The amino-terminal inhibitory domain (residues 415 – 490) has no obvious similarities with other proteins and contains no known motifs. Structural data on the folding of this domain relative to the GEF core may be required to gain further insights into the molecular basis of this inhibition. Interestingly, the carboxyl terminal inhibitory domain (residues 876 – 1163) contains a C2 domain (residues 911 – 1036). Such domains have been described in numerous signaling molecules and are thought to regulate phospholipid binding in a calcium-dependent manner (Rizo and Sudhof, 1998). Although the function of the C2 domain in Bcr is not known, this domain appears to be necessary for Bcr-mediated GAP activity in vivo (IP Whitehead, unpublished observations) and may function as membrane localization signal that facilitates interactions between Bcr and its in vivo target Rac1. If so, the C2 domain may compete with the PH domain for cellular localization of Bcr, thus preventing Bcr from associating with other GTPase substrates such as Cdc42. However, our observation that expression of this domain can block signaling by Bcr when expressed in trans suggests that this domain may also contain residues that form direct contacts with the DH/PH domain module. Although experiments using a bacterially purified DH/PH domain module of Bcr have revealed a Figure 8 Bcr activates NF-kB through a p38 MAPK-dependent mechanism. COS-7 cells were transiently transfected with 3 mg relatively promiscuous GEF activity in vitro (RhoA, pAX142 or pAX142-bcr(491 – 876), along with either NF-kB-luc Rac1 and Cdc42) (Chuang et al., 1995), we have been (2.5 mg), or Gal-p65 (0.25 mg) and Gal4-luc (2.5 mg). For each unable to completely recapitulate these observations in condition pCMVnlac was included in the transfection. Three live cells. We have detected increased levels of GTP- hours prior to lysate collection, SB203580 (400 nM) or water (control) was added to the media. Lysates were collected at 48 h and Cdc42 in cells transfected with Bcr, but we have been were analysed by Western blot to determine endogenous levels of unable to observe any change in levels of GTP-RhoA total (C-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or GTP-Rac1. We have observed similar incongruities and phosphorylated (9211-CST, New England Biolabs, Beverly, when comparing the in vivo and in vitro GEF MA, USA) p38MAPK (a), or for luciferase activity (b). Data specificities of the Dbs protein in NIH3T3 cells (IPW, are calculated and presented as in Figure 2 unpublished data). This suggests that cellular context may be the critical determinant of GEF specificity and that in vitro data may not be a reliable predictor of in of Vav on tyr-174 (which is contained within these vivo function. Our observation that genetic inhibitors inhibitory sequences) by Lck, or removal of these of Cdc42 function fail to block Bcr-mediated signaling amino-terminal sequences by truncation, exposes the activity further suggests that all the GTPase substrates oncogenic potential of the protein (Han et al., 1997). for Bcr have not yet been identified. Similarly, autoinhibitory sequences have been identified Bcr joins a growing list of signaling molecules that within the amino terminus of the Dbl protein that bind have been shown to activate NF-kB through a directly to the PH domain blocking access to GTPase mechanism that does not require increased nuclear substrates and masking the intracellular targeting accumulation or DNA binding. Recent studies on cell function of the PH domain (Bi et al., 2001). Removal lines derived from T2K and GSK-3 knockout mice of this flanking sequence by truncation is necessary to have suggested that this is a physiologically relevant expose the Dbl encoded exchange and transforming mechanism of NF-kB activation (Bonnard et al., 2000; activity. The mechanisms through which the catalytic Hoeflich et al., 2000). These cell lines are competent in activities of full-length natively expressed RhoGEFs the nuclear translocation of NF-kB but are completely

Oncogene Activation of NF-kB by Bcr M Korus et al 4610 defective in any transactivation function. A series of of the amino-terminal inhibitory domain that we have recent studies has begun to assemble the components identified within Bcr, or may reflect the absence of of a signaling pathway that may contribute to RelA/ specific co-factors in the 32D cell line that are required p65 phosphorylation and transactivation (Finco et al., for activation of the Bcr encoded GEF activity. 1997; Madrid et al., 2001; Sizemore et al., 1999). IL-1b, Nonetheless, our observation that both the DH/PH Ras and Akt have been shown to stimulate the domain and the amino-terminal serine/threonine kinase transactivation function of RelA/p65, with activation domain of Bcr can activate a signaling pathway that by IL-1b occurring in an Akt-dependent manner has demonstrated roles in tumorigenesis suggests that (Sizemore et al., 1999). In turn, Akt has been shown these activities need to be examined in greater detail in to stimulate the transactivation function of RelA/p65 the context of the Bcr-Abl oncogenes. in an IkBb and p38 MAPK-dependent manner (Madrid et al., 2001). Although the mechanism(s) Materials and methods through which either IKKb or p38 MAPK cause phosphorylation of p65 remains unclear, our observa- Molecular constructs tion that Bcr activation of NF-kB is dependent on p38 The pAX142 mammalian expression vector has been MAPK function, suggests that Bcr may be stimulating described previously (Whitehead et al., 1995). pAX142- NF-kB in a manner that is either similar to or bcr(1 – 1271), pAX142-bcr(1 – 876), pAX142-bcr(415 – 876), dependent upon Akt. We are currently exploring this pAX142-bcr(491 – 876), pAX142-bcr(491 – 1163), pAX142- possibility. bcr(491 – 1271), pAX142-bcr(1 – 413), pAX142-bcr(415 – 490), The presence of the Bcr DH/PH domain within the and pAX142-bcr(876 – 1163) contain cDNAs that encode the p210 Bcr-Abl chimeric protein raises the possibility indicated amino acids of the human Bcr protein fused to an that it may contribute to Bcr-Abl-mediated oncogen- HA epitope tag. All PCR products that were used in the esis. However, there is conflicting evidence concerning synthesis of these derivatives were sequenced in their entirety. the role of the Bcr encoded DH/PH domain in Detailed descriptions of the cloning strategies used to transformation. The transforming potential of p185 generate each derivative are available upon request. Construction of pAX142-IkBa(SS), pAX142-cdc42(17N), Bcr-Abl, which lacks the DH/PH domain, is stronger pAX142-cdc42(12V), pAX142-cdc42(WT), pAX142-rac1(61L), than that of p210 Bcr-Abl suggesting that this domain pAX142-rhoA(63L), and pAX142-dbl(HA1) have been may inhibit transformation. However, there have been described previously (Whitehead et al., 1998, 1999). several recent studies that suggest that this may not be pyDF30-WASP-GBD encodes the GTPase binding domain the case. First, replacement of the DH domain of Bcr (GBD) of WASP (the product of the Wiskott-Aldrich with a homologous domain from Cdc24, or with syndrome locus) and was kindly provided by M Symons irrelevant sequences, diminishes transforming activity (Qiu et al., 1997). in Rat1 cells (Kin et al., 2001). This suggests that p185 The reporter constructs utilized in the luciferase-coupled Bcr-Abl and p210 Bcr-Abl may possess distinct transcriptional assays have been described previously: NF- transforming properties, and that the DH/PH domain kB-luc contains the luciferase gene under control of a minimal promoter that contains tandem copies of the HIV may perform a specific function within the context of NF-kB binding sequences (Galang et al., 1996); Gal-luc p210. Second, this domain has been reported to contains the luciferase gene under the control of a promoter activate Rac1 in vitro (Chuang et al., 1995; Diekmann that contains five Gal4 DNA-binding sites (Su et al., 1994); et al., 1991), and dominant negative Rac1 blocks Bcr- Gal4-p65 contains the Gal4 DNA-binding domain fused to Abl transformation (Skorski et al., 1998). Finally, transactivation domain 1 (TA1) of RelA/p65 (Schmitz and xeroderma pigmentosum group B protein (XPB), which Baeuerle, 1991). pCMVlac encodes the sequences for the b- is a helicase of the basal class II transcription factor H galactosidase gene under the control of the CMV promoter (TFIIH), binds to the DH domain in the context of (provided by J Samulski). Bcr-Abl (Maru et al., 1999). This interaction reduces the helicase and ATPase activities of XPB and reduces Cell culture and reagents its repair activity in rodent models. Since progression COS-7 and NIH3T3 cells were maintained in Dulbecco’s of CML from the chronic to acute phases is associated modified Eagle medium (DMEM; high glucose) supplemen- with genetic instability, it has been proposed that Bcr- ted with 10% fetal bovine serum (Sigma, St. Louis, MI, Abl may confer a mutator phenotype via its inactiva- USA) or 10% bovine calf serum (JRH, Lenexa, KS, USA) tion of XPB (Maru, 2001). respectively. The specific p38 MAPK pharmacological We have shown previously that NF-kB activation inhibitor SB203580 (Calbiochem, San Diego, CA, USA) contributes to the transforming potential of several was used at a working concentration of 400 nM unless RhoGEFs (Whitehead et al., 1999), and others have otherwise indicated. shown that NF-kB is required for Bcr-Abl-mediated transforming activity (Reuther et al., 1998). However, Transient expression reporter gene assays activation of NF-kB by Bcr-Abl in 32D cells has been For transient expression reporter assays, cells were trans- attributed to the Abl encoded tyrosine kinase activity fected by calcium phosphate co-precipitation (NIH3T3) or by and a p210 Bcr-Abl derivative that lacks tyrosine DEAE-dextran (COS-7) as described previously (Clark et al., kinase activity is not able to activate NF-kB-dependent 1995; Whitehead et al., 1995). Cells were allowed to recover response elements (Reuther et al., 1998). The failure of for 30 h, and were then starved in DMEM that was this mutant to activate NF-kB may reflect the presence supplemented with 0.5% serum for 14 h before lysate

Oncogene Activation of NF-kB by Bcr M Korus et al 4611 preparation. Analysis of luciferase expression was as of nuclear extract was combined with 4 ml56 binding buffer described previously with enhanced chemiluminescent (250 mM NaCl, 50 mM Tris, pH 7.7, 50% glycerol, 5 mM reagents and a Monolight 3010 luminometer (Analytical DTT, 0.5 M EDTA), 1 ml(2mg/ml) POLY (dI-dC)-POLY(dI- Luminescence, San Diego, CA, USA) (Hauser et al., 1995). b- dC) (Amersham Pharmacia, Piscataway, NJ, USA), galactosidase activity was determined using Lumi-Gal 15 000 c.p.m. radiolabeled oligonucleotide, and water to substrate (Lumigen, Southfield, MI, USA) according to the 20 ml. Reactions were spun for 5 s, incubated at room manufacturer’s instructions. All assays were performed in temperature for 15 min, and then resolved on a 5% triplicate. polyacrylamide gel. Gels were dried and exposed to Kodak X-Omat Blue Imaging Film. Supershift assays were performed on anti-p50 and anti-p65 polyclonal antibodies Protein expression (C-20 and NLS respectively, Santa Cruz Biotechnology, Protein expression in transiently transfected COS-7 cells was Santa Cruz, CA, USA). determined by Western blot analysis as described previously (Whitehead et al., 1995). Protein was visualized with Cdc42, Rac1 and RhoA activation assays enhanced chemiluminescence reagents (Amersham Pharma- cia, Piscataway, NJ, USA). The p21 binding domains of Pak3 (Rac1/Cdc42; GST-PBD) (Glaven et al., 1999) or Rhotekin (RhoA; GST-C21) (Reid et al., 1996) were expressed as GST fusions in BL21(DE3) cells Electrophoretic mobility shift assays and immobilized by binding to glutathione-coupled Sephar- Nuclear extracts from COS-7 cells were prepared as follows. ose 4B beads (Amersham Pharmacia, Piscataway, NJ, USA). Cells were collected, washed three times in cold phosphate- The immobilized Rho binding domains were then used to buffered saline, and then gently resuspended in 10 mM precipitate activated GTP-bound Rac1, Cdc42 (GST-PBD) or HEPES, pH 7.6, 60 mM KCl, 1 mM EDTA, 0.275% NP-40, RhoA (GST-C21) from COS-7 cell lysates. Cells were washed 1mM DTT, 2.5 mg/ml aprotinin, 5 mg/ml leupeptin, 2.5 mg/ml in cold phosphate-buffered saline and then lysed in 50 mM pepstatin, 1 mM PMSF (CE Buffer), and then incubated on Tris-HCl, pH 7.4, 2 mM MgCl2, 100 mM NaCl, 10% glycerol, ice for 3 – 4 min. Nuclei were then pelleted at 15 000 r.p.m. 1% NP-40, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mg/ml for 4 min at 48C, washed with 100 ml of CE buffer lacking aprotinin, and 1 mg/ml PMSF. Cell lysates were then cleared NP-40, and then spun again at 15 000 r.p.m. for 4 min at by centrifugation at 10 000 g for 10 min at 48C. The 48C. The supernatant was then removed and replaced with expression of proteins was confirmed by Western blotting two pellet volumes of 20 mM Tris, pH 8.0, 420 mM NaCl, prior to affinity purification. Lysates used for affinity 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 2.5 mg/ml purification were normalized for endogenous Rho levels. aprotinin, 5 mg/ml leupeptin, 2.5 mg/ml pepstatin, 0.5 mM Affinity purifications were carried out at 48C for 1 h, washed PMSF. Salt concentration was adjusted to 400 mM with three times in an excess of lysis buffer, and then analysed by 5 M NaCl and then incubated on ice for 10 min with Western blot. Cdc42, RhoA and Rac1 were detected by the vortexing. Nuclear lysates were clarified by centrifugation at B-8 and 26C4 monoclonal, and C-14 polyclonal antibodies 14 000 r.p.m. for 10 min and then glycerol was added to a respectively (Santa Cruz Biotechnology, Santa Cruz, CA, final concentration of 20%. USA). g-32P labeled NF-kB consensus oligonucleotides were prepared using reagents from the Gel Shift Assay System (Promega, Madison, WI, USA) using protocols provided by the manufacturer. Unincorporated nucleotide was removed Acknowledgements and per cent incorporation determined using G-25 Sephadex This work was supported by Public Health Service grant Columns (Boehringer Mannheim, Indianapolis, IN, USA). CA-77493 (IP Whitehead) from the National Cancer DNA binding reactions were performed as follows. Three ml Institute.

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Oncogene