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

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P38 MAPK-Mediated Activation of NF-Kb by the Rhogef Domain of Bcr Oncogene (2002) 21, 4601 – 4612 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc 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).
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