Oncogene (2003) 22, 5946–5957 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc CrkII induces serum response factor activation and cellular transformation through its function in Rho activation Toshinori Iwahara1, Tsuyoshi Akagi1, Tomoyuki Shishido1 and Hidesaburo Hanafusa*,1 1Laboratory of Molecular Oncology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan CrkII belongs to the adaptor protein family that plays a interesting biological activities despite the lack of an crucial role in signal transduction. In order to better enzymatic domain. For example, v-Crk induces onco- understand the biological functions of CrkII, we focused genic transformation of chicken embryo fibroblasts on the regulation of gene expression by CrkII. Various (CEF) and the elevation of tyrosine phosphorylation transcriptional control elements were examined for their levels of several proteins (Birge et al., 1996; Feller, activation by CrkII-expression, and we found that CrkII 2001). CrkL, which has a similar structure to CrkII, has selectively activates the serum response element (SRE), a also been reported to induce the anchorage independent transcriptional control element of immediate-early genes. growth of Rat1 cells. Even though cellular CrkII has not This SRE activation induced by CrkII-overexpression was been reported to have oncogenic potential, it has been mediated by the serum response factor (SRF) via Rho. shown to regulate several biological activities, including Indeed, we confirmed that the amount of activated Rho mitogenesis and reorganization of the cytoskeleton was increased in the CrkII-expressing cells. Moreover, we (Klemke et al., 1998; Nakashima et al., 1999; Cho and showed that when overexpressed, CrkII induces the Klemke, 2000; Gumienny et al., 2001). To date, v-Crk cellular transformation of NIH 3T3 cells and that a studies have reported its activation of several signaling dominant negative mutant of Rho suppresses this trans- pathways, such as the JNK, PI3K/AKT, and Rho small formation, strongly suggesting that activation of Rho is GTPase pathways (Tanaka et al., 1997; Altun-Gultekin essential for the transforming activity by CrkII. Further- et al., 1998; Akagi et al., 2000), however, little is known more, we also found that CrkII and Ga12, a member of about the transcriptional factors which are affected by the heterotrimeric G proteins, synergistically activates Crk downstream in its signaling pathways. In this study, Rho as well as the SRF, and that an SH3 mutant of CrkII therefore, we initially asked the question as to whether can inhibit the Ga12-induced activation of SRF. These or not CrkII is involved in the regulation of gene results strongly suggest that CrkII is involved in the expression, and analyzed the molecular events affected activation of Rho and SRF by Ga12. Our study provides or controlled by the expression of cellular CrkII. strong evidence that Rho activation plays a crucial role in Various oncogenic signals and mitogen-stimulated CrkII-mediated signals to induce gene expression and signals result in the transcriptional activation of cellular transformation. immediate-early genes (IEGs), such as c-fos and Egr-1. Oncogene (2003) 22, 5946–5957. doi:10.1038/sj.onc.1206633 IEGs play a critical role in mitogenesis and contribute to cellular transformation (Greenberg and Ziff, 1984; Lau Keywords: CrkII; Rho; transformation and Nathans, 1985; Herschman, 1991; Meijne et al., 1997). Several IEGs contain a common cis-acting transcriptional control element, termed the serum Introduction response element (SRE) (Herschman, 1991; Treisman, 1995). The SRE is essential for the transcriptional The Crk family of adaptor proteins was originally activation of IEGs by a number of growth stimulating identified as the oncogene product of the avian sarcoma factors acting on tyrosine kinases and G-protein- virus CT10, v-Crk (Mayer et al., 1988). The mammalian coupled receptors (GPCRs) (Treisman, 1995). In addi- cellular homologues of v-Crk, including CrkI, CrkII, tion, the SRE can be activated in the absence of growth and closely related CrkL, have subsequently been factor stimulation by oncogene products, such as v-Ras, identified (Matsuda et al., 1992; Reichman et al., 1992; v-Src, and v-Raf (Treisman, 1995). Two c-fos SRE- ten Hoeve et al., 1993). Crk family proteins are binding proteins, the serum response factor (SRF) and composed primarily of the Src homology (SH) 2 and the ternary complex factor (TCF) of the Ets family have SH3 domains, and the widely expressed CrkII has an N- been shown to be important for SRE-mediated gene terminal SH2 domain and two SH3 domains (Matsuda expression. TCFs are phosphorylated and transacti- et al., 1992; Reichman et al., 1992). The most interesting vated in response to activation of the mitogen-activated feature of the Crk family is its ability to induce many protein kinases (MAPKs) signaling pathway (Gille et al., 1992; Treisman, 1996). In contrast, SRF activity is *Correspondence: H Hanafusa; E-mail: [email protected] regulated by a signaling pathway in which the Rho Received 13 January 2003; revised 1 April 2003; accepted 7 April 2003 family of small GTPases are involved (Hill et al., 1995). Crk-mediated SRF activation and transformation via Rho T Iwahara et al 5947 This pathway can stimulate SRF-mediated transcription The SRE-mediated gene expression by CrkII was through a TCF-independent mechanism (Hill and almost completely blocked by the mutation in the SRE Treisman, 1995; Hill et al., 1995). For example, some that prevents SRF binding (Figure 1b). This implies that GPCR-mediated stimuli, like lysophosphatidic acid, CrkII activates the SRE through an SRF-dependent selectively induce Rho-dependent SRF activation with- mechanism. To further confirm the SRF as the target of out significant TCF activation (Hill et al., 1995). CrkII in signaling to the SRE, we used another reporter Moreover, the constitutively active forms of the a plasmid containing an altered c-fos SRE, SRE.L, which subunits of the Gq and G12 class of heterotrimeric G binds only to the SRF, and not to TCF, and has been proteins have been reported to activate SRF via Rho shown to be regulated by SRF independently of TCF activation (Fromm et al., 1997; Mao et al., 1998b,c; (Hill et al., 1995). As shown in Figure 1c, we found that Fukuhara et al., 1999), indicating that Rho activation is expression of CrkII gave a 10-fold increase in the essential for transmission of these kinds of signals to the SRE.L-luciferase activity. As a positive control, we nucleus via SRF activation (Hill et al., 1995). consistently confirmed that constitutively activated By using reporter gene assays for various transcrip- RhoA (RhoV14) could also induce the SRE.L-luciferase tional control elements, here we show that CrkII- activity under the same experimental condition expression results in selective transcriptional activation (Figure 1c). Next, to examine the contributions of each mediated by the SRE. This SRE activation by CrkII SH2 and SH3 domain of CrkII in its transcriptional occurs through an SRF-dependent pathway via Rho activity, an SH2-deficient mutant (R38 K), and two activation. Moreover, we found that CrkII-expression SH3-deficient mutants (W169K and W275 K) were results in cellular transformation and that Rho activa- transiently transfected into NIH3T3 cells (Figure 1d). tion is required for this transforming activity by CrkII. We found that all of the three SH domains were In addition, we also provide evidence that CrkII is likely required for complete SRF activation by CrkII to be involved in the Rho activation pathway induced by (Figure 1e). Taken together, these results indicate the heterotrimeric G protein subunit Ga12. Therefore, that CrkII can activate SRE-mediated gene expression we have here characterized a novel CrkII-mediated through an SRF-dependent pathway in NIH 3T3 signal that potently activates Rho, which consequently cells. regulates transcriptional activation as well as cellular transformation. CrkII activation of SRF is mediated by Rho Since it has been reported that the Rho family of small Results G proteins, Rho, Cdc42, and Rac induce SRF activation (Hill et al., 1995), we investigated the Rho family- CrkII activates SRE-mediated gene expression through mediated signaling pathways in CrkII-overexpressing SRF activation cells. We examined the effect of the simultaneous expression of CrkII and the Clostridium botulinum C3 To assess whether expression of CrkII activates intracel- transferase, which specifically ADP-ribosylates and lular signaling pathways leading to transcriptional deactivates Rho proteins but not Cdc42 nor Rac (Hill activation, we used reporter plasmids carrying the et al., 1995). As shown in Figure 2a, the coexpression of luciferase gene under the control of various well-defined C3 almost completely abolished the SRF-mediated gene transcriptional control elements, which are known to be transcription induced by CrkII. Consistent with pre- regulated by many signaling pathways. The reporter vious studies, C3 prevented the induction of SRF- plasmids pSRE-luc, pMyc-luc, pAP-1-luc, and pNF-kB- mediated gene transcription by constitutively activated luc contain the CArG box of the c-fos SRE (Akagi et al., RhoA (RhoV14), but neither by activated Cdc42 1997), the Myc–Max heterodimer binding sequence (Cdc42V12), activated Rac1 (RacV12) nor by v-Src, (CACGTG) (Ayer et al., 1996), the AP-1 binding TRE which has been demonstrated to activate SRF both elements, and the NF-kB site from the murine Ig k gene through Cdc42 and Rac1 (Mao et al., 1998a), suggesting (Akagi et al., 1997), respectively. NIH 3T3 cells were that C3 specifically prevents the Rho-mediated activa- transiently cotransfected with the CrkII expression vector tion pathway (Figure 2a). In addition, coexpression of a and each of these luciferase reporter constructs. These dominant inhibitory mutant of Rho (Rho N19) (Khos- luciferase reporter assays showed that SRE-mediated ravi-Far et al., 1995) also suppressed SRF activation by gene transcription was most prominently activated by CrkII, but not by activated RacV12 (Figure 2b).