Oncogene (2007) 26, 3172–3184 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW Role of mitogen-activated protein kinase kinase 4 in cancer AJ Whitmarsh1 and RJ Davis2 1Faculty of Life Sciences, University of Manchester, Manchester, UK and 2Howard Hughes Medical Institute, Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA Mitogen-activated protein (MAP) kinase kinase 4 of the extracellular signal-regulated kinase (ERK), (MKK4) is a component of stress activated MAP kinase ERK5, c-Jun N-terminal kinase (JNK) and p38 (Chang signaling modules.It directly phosphorylates and activates and Karin, 2001). the c-Jun N-terminal kinase (JNK) and p38 families of The JNK and p38 MAP kinases are collectively MAP kinases in response to environmental stress, pro- referred to as stress-activated MAP kinases. They are inflammatory cytokines and developmental cues.MKK4 is activated in response to a variety of environmental ubiquitously expressed and the targeted deletion of the stresses and pro-inflammatory cytokines, and also play Mkk4 gene in mice results in early embryonic lethality. important roles in development (Davis, 2000; Kyriakis Further studies in mice have indicated a role for MKK4 in and Avruch, 2001). A number of MKKs can phosphor- liver formation, the immune system and cardiac hyper- ylate and activate JNK and p38. MKK3 and MKK6 trophy.In humans, it is reported that loss of function activate p38, MKK7 activates JNK, whereas MKK4 mutations in the MKK4 gene are found in approximately can activate both JNK and p38 (Davis, 2000; Kyriakis 5% of tumors from a variety of tissues, suggesting it may and Avruch, 2001) (Figure 1a). The JNK and p38 have a tumor suppression function.Furthermore, MKK4 pathways are implicated in tumor suppression (Kennedy has been identified as a suppressor of metastasis of and Davis, 2003; Bulavin and Fornace Jr, 2004) and this prostate and ovarian cancers.However, the role of MKK4 is supported by the presence of loss of function in cancer development appears complex as other studies mutations in the MKK4 gene in approximately 5% of support a pro-oncogenic role for MKK4 and JNK.Here human tumors from a variety of tissues (Teng et al., we review the biochemical and functional properties of 1997; Su et al., 1998). However, it is also reported that MKK4 and discuss the likely mechanisms by which it may MKK4 and JNK can participate in tumor formation regulate the steps leading to the formation of cancers. suggesting a more complex role for this pathway in Oncogene (2007) 26, 3172–3184. doi:10.1038/sj.onc.1210410 tumor development (Kennedy and Davis, 2003; Wang et al., 2004). Keywords: MAP kinase; MKK4; JNK; p38; tumor In this review, we describe the properties of MKK4 suppressor and examine how this protein kinase and its down- stream targets contribute to controlling the development of cancers. Introduction Biochemical properties of MKK4 The mitogen-activated protein (MAP) kinase signaling Cloning, structure and tissue distribution of MKK4 pathways are important mediators of cellular responses MKK4 was first identified in screens for novel MKK to extracellular signals that include growth factors, family members in Xenopus laevis and termed XMEK2 hormones, cytokines and environmental stresses (Chang (Yashar et al., 1993). Subsequently the Drosophila, and Karin, 2001). These pathways are evolutionarily mouse and human homologs were cloned and named conserved among eukaryotes and feature a triple kinase DMKK4, stress-activate protein kinase/extracellular- cascade comprised of the MAP kinase which is signal-regulated protein kinase kinase-1 and MKK4, phosphorylated and activated by a MAP kinase kinase respectively (Sanchez et al., 1994; De´ rijard et al., 1995; (MKK), which itself is phosphorylated and activated by Lin et al., 1995; Han et al., 1998). The human MKK4 a MAP kinase kinase kinase (MKKK) (Chang and gene is located on chromosome 17 and encodes a protein Karin, 2001). In mammals, four distinct MAP kinase of 399 amino acids (De´ rijard et al., 1995; Yoshida et al., pathways have been identified that lead to the activation 1999). Overall the mammalian MKK family share about 40% homology within their catalytic domains and Correspondence: Professor RJ Davis, Howard Hughes Medical MKK4 is most similar to MKK7 in this region (50% Institute, Program in Molecular Medicine, University of Massachu- setts Medical School, 373 Plantation Street, Worcester, MA 01605, identity; Tournier et al., 1997; Cuenda, 2000). The USA. catalytic domains of MKKs, like other Ser/Thr kinases, E-mail: [email protected] contain 11 subdomains (Hanks et al., 1988). The crystal MKK4 in cancer AJ Whitmarsh and RJ Davis 3173 a binding determinant is the domain for versatile docking Ras and Rho-family GTPases (DVD) located in the C-terminus of MKKs (Xia et al., 1998; Takekawa et al., 2005; Figure 1b). The interac- tions between MKKs and the upstream and downstream kinases of the cascade appear to be critical for efficient MEKK MLK TAK1 ASK1 TPL2 signal transfer through MAP kinase pathways (Xia et al., 1998; Ho et al., 2003; Takekawa et al., 2005). MKK4 mRNA is ubiquitously expressed in adult MKK7MKK4 MKK3 MKK6 mouse and human tissue with the highest levels of expression in brain, in particular in the cerebral cortex, hypothalamus, hippocampus and cerebellum (Sanchez et al., 1994; De´ rijard et al., 1995; Carboni et al., 1997; JNK p38 Lee et al., 1999). In early embryogenesis in mice (up to embryonic day 10 (E10)), Mkk4 transcripts are confined to the central nervous system. Later, starting at E12, Cyclin D1 MAPKAPK2 Mkk4 becomes highly expressed in the developing liver c-Jun Bax p53 p53 Bcl2 coincident with a period of active differentiation and an Bax increase in liver size (Lee et al., 1999). Subcellular 14-3-3 Bim AR CDC25 RXRα Bmf localization studies demonstrate that MKK4 protein is RARα Bcl-XL mainly found in the cytoplasm, although some nuclear AR Mcl-1 localization has been detected (Tournier et al., 1999; Coffey et al., 2000). 257 261 b S-I-A-K-T 1 94 399 D KD DVD MKK4 activation of JNK and p38 MAP kinases 39 45 364 387 MKK4 is unique among the mammalian MKK family Figure 1 (a) Mammalian stress-activated MAP kinase pathways. in its ability to phosphorylate and activate two MAP JNK is activated by phosphorylation by MKK4 and MKK7 kinase groups: JNK and p38 (De´ rijard et al., 1995; Lin whereas p38 is activated by MKK4, MKK3 and MKK6. These et al., 1995). MKK3 and MKK6 are specific for p38, MKKs can be activated by many different MKKKs depending on whereas MKK7 is a specific JNK activator (Davis, 2000; the stimulus. JNK and p38 phosphorylate many known regulators of tumorigenesis. (b) Domain structure of MKK4. The kinase Kyriakis and Avruch, 2001). MKK4 activates all the domain (KD) of MKK4 contains eleven subdomains. MKK4 is mammalian JNK isoforms (JNK1, JNK2 and JNK3) activated by phosphorylation of the Ser and Thr residues (in bold) and a subset of p38 isoforms (p38a, p38b) (De´ rijard within the S-I-A-K-T motif located between subdomains VII and et al., 1995; Lin et al., 1995; Jiang et al., 1996). VIII. At the N-terminus there is a D-domain (D) motif for binding to JNK and p38, and at the C-terminus a DVD domain that All MAP kinases are activated by phosphorylation of mediates interactions with various MKKKs. The numbers refer to the Thr and Tyr residues of a Thr-X-Tyr motif located amino acids in human MKK4. within kinase subdomain VIII (Chang and Karin, 2001). Both residues need to be phosphorylated for full activation of the MAP kinase (Chang and Karin, 2001). However, it was observed in vitro that MKK4 structures of MEK1 and MEK2, the MKKs in the ERK preferentially phosphorylated the Tyr residue on JNK pathway, demonstrate that MKKs fold into a small b- (Sanchez et al., 1994; Lin et al., 1995), whereas the stranded N-terminal lobe and a larger helical C-terminal second JNK activator, MKK7, preferentially targeted lobe (Ohren et al., 2004). The adenosine triphosphate the Thr residue (Lawler et al., 1998). These observations binding site is located in the cleft formed between the led to the hypothesis that JNK isoforms are activated two lobes and is surrounded by residues that are synergistically by MKK4 and MKK7 (Lawler et al., conserved between the MKK family members (Cuenda, 1998; Fleming et al., 2000; Lisnock et al., 2000). Some 2000; Ohren et al., 2004). in vivo evidence to support this model has come from MKK4, similar to the other MKKs, also contains studies using mouse embryonic stem (ES) cells and docking sites for both upstream and downstream mouse embryonic fibroblasts (MEFs) that feature components of the JNK and p38 signaling cascades targeted deletions of the Mkk4 and Mkk7 genes (Xia et al., 1998; Ho et al., 2003; Takekawa et al., 2005; (Tournier et al., 2001; Wada et al., 2001; Kishimoto Figure 1b). At the N-terminus there is a D-domain type et al., 2003). These studies also demonstrated that docking site that binds to JNK and p38 (Ho et al., distinct stimuli might differentially utilize MKK4 and 2003). In addition to MKKs, D-domain docking sites MKK7. For example, the activation of JNK in response that bind to MAP kinases are found in many proteins to the pro-inflammatory cytokines tumor necrosis involved in MAP kinase signaling including MAP factor-a (TNFa) and interleukin-1 (IL-1) was almost kinase phosphatases, substrates, and scaffold or adaptor completely abolished in Mkk7À/À MEFs, but reduced proteins (Sharrocks et al., 2000). The region encom- to around 50% activation in Mkk4À/À cells (Tournier passing the D-domain may also participate in the et al., 2001).