MIXED-LINEAGE KINASE CONTROL OF JNK AND p38 MAPK PATHWAYS

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1 MIXED-LINEAGE KINASE CONTROL OF JNK AND p38 MAPK PATHWAYS Kathleen A. Gallo* and Gary L. Johnson Mixed-lineage kinases (MLKs) are serine/threonine protein kinases that regulate signalling by the c-jun amino-terminal kinase (JNK) and p38 mitogen-activated-protein kinase (MAPK) pathways. MLKs are represented in the genomes of both Caenorhabditis elegans and Drosophila melanogaster. The Drosophila MLK Slipper regulates JNK to control dorsal closure during embryonic morphogenesis. In mammalian cells, MLKs are implicated in the control of apoptosis and are potential drug targets for many neurodegenerative diseases. SIGNALLING PHOSPHORELAYS Complex pathways in which phosphoryl groups are transferred through several signal-transduction proteins before reaching the target protein. CYCLOHEXIMIDE Antibiotic produced by some Streptomyces sp. that interferes with protein synthesis in eukaryotes by inhibiting peptidyltransferase activity of the 60S ribosomal subunit. *Departments of Physiology and of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824, USA. Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. s: doi: /nrm906 The mixed-lineage kinases (MLKs) are a family of serine/threonine protein kinases that function in a PHOS- PHORELAY module to control the activity of specific mitogen-activated-protein kinases (MAPKs) (BOX 1). MAPK cascades exist in all eukaryotes and orchestrate diverse cellular activities, including mitosis, programmed cell death, motility and metabolism. Substrates for MAPKs include transcription factors, phospholipases, other protein kinases, cytoskeleton-associated proteins and membrane receptors. All the known MLKs that have been produced by transfection in mammalian cell lines act as MAPK-kinase kinases (MKKKs) to activate c-jun amino-terminal kinase (JNK) pathways (FIG. 1). JNKs are MAPKs that were first characterized by their activation in response to CYCLOHEX- IMIDE-induced inhibition of protein synthesis 1. It was subsequently discovered that these kinases are activated in response to many different stimuli that stress cells, such as heat shock, inhibition of protein glycosylation, exposure to inflammatory cytokines and ultraviolet irradiation (reviewed in REF. 2). These stress-activated protein kinases were subsequently shown to bind to, phosphorylate and increase the transcriptional activity of c-jun 3. As a component of the ACTIVATOR-PROTEIN 1 (AP-1) TRAN- SCRIPTION-FACTOR COMPLEX, c-jun regulates the transcription of cytokine genes and many other genes 4. AP-1 is activated by environmental stress, radiation, cytokines and growth factors, stimuli that also activate JNKs 5,6. MLKs phosphorylate and activate MAPK kinases (MKKs), such as MKK4 and/or MKK7 (REFS 7 11), which, in turn, activate JNKs. However, it is not only the MLKs that can activate the JNK pathway; other MKKKs include the MEK kinases (MEKKs), apoptosis-inducing kinase 1 (ASK1) and transforming-growth factor β (TGFβ)-activated kinase 1 (TAK1) 12. When expressed in mammalian cells, some MLKs have been found to activate p38 (REFS 8,9,13 15). The p38 MAPK pathway is activated by multiple stimuli including many cytokines and cellular stresses that also activate JNKs 2. As with JNKs, other MKKKs can activate p38 including TAK1, ASK1 and MEKK4 (REF. 12). Of the 11 conserved subdomains of protein kinases, subdomains I VII of the MLKs resemble serine/threonine kinases, particularly the MEKKs and Raf (an MKKK in the MAPK/extracellular-signal-regulated kinase (ERK) 1/2 pathway), whereas subdomains VIII XI more closely resemble tyrosine kinases such as the fibroblast-growth-factor receptor and Src. Hence, when MLK genes were initially cloned, the hydroxyamino acid specificity of the corresponding gene products was unclear, which gave rise to the name mixed-lineage kinase 16.However,MLK3 was shown to autophosphorylate on serine and threonine residues 17, and the evidence so far indicates that all MLKs are bona fide serine/threonine kinases. Three subfamilies of MLKs Over the past several years, seven different mammalian MLKs have been identified. On the basis of domain NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 3 SEPTEMBER

2 Box 1 MAPK phosphorelay modules Raf, Mos, Tpl2 MEKKs, MLKs, TAK1, ASK1 MLK3, MEKK4, ASK1, TAK1 MEKK2/3 MKK1/2 MKK4/7 MKK3/6 MKK5 ERK/MAPK1/2 JNK p38 ERK5 The three defining kinases of all so-called mitogen-activated-protein kinase (MAPK) phosphorelay modules are conserved from yeast to humans. MAPKs are regulated by phosphorylation and are substrates for MAPK kinases (MKKs). MKKs are dualspecificity kinases that phosphorylate MAPKs on both a threonine and a tyrosine in the activation loop of the catalytic domain. This dual phosphorylation is absolutely required for activation of the MAPK and, in the case of the extracellular-signalregulated kinase (ERK), results in a 50,000-fold increase in specific activity 107.The MKKs must also be activated by phosphorylation within their activation loops. This is accomplished by a group of serine/threonine kinases known as the MAPK kinase kinases (MKKKs). In mammalian cells, the four best-characterized MAPK pathways are the ERK/MAPK1/2, the p38, the c-jun amino-terminal kinase (JNK) and the ERK5 pathways. Mixed-lineage kinases (MLKs) are MKKKs that regulate the JNK pathway. MLK3, dual-leucine-zipper-bearing kinase (DLK) and zipper sterile-α-motif kinase (ZAK) have also been shown to regulate the p38 pathway, and some MLKs might regulate the ERK5 pathway 14. As MKKKs, the MLKs phosphorylate and activate an MKK. Specific MKKs in each MAPK pathway phosphorylate and activate specific MAPKs. ASK, apoptosissignal-regulated kinase; Mos, Moloney sarcoma oncogene; TAK, transforming-growthfactor-β-activated kinase 1; Tpl, Triplolethal. DLKs. The second MLK subfamily, the DLKs, are typified by a kinase domain followed by two leucine-zipper motifs that are interrupted by a 31 amino acid spacer. DLK, like the MLKs, has a proline-rich carboxyl terminus but its regulatory function is undefined. The catalytic domains of the two human DLK family members, DLK (REF. 22) (also called zipper (leucine) protein kinase 23 and MAPK-upstream kinase 24 ) and leucine-zipper kinase (LZK) 25 are 87% identical. ZAKs. Recently, a third subgroup of MLKs has emerged that is represented by ZAK. ZAK is distinguished by the presence of a both a LEUCINE ZIPPER and a sterile-α motif (SAM). The SAM domain is an independently folding module of ~70 amino acids that is found (usually near the amino or carboxyl terminus) in many signalling molecules, including receptor tyrosine kinases, adaptor proteins and GTPase-activating proteins. Structural and biochemical studies indicate that SAM domains can mediate homo- or heterodimerization 26. In mammalian ZAK, the SAM domain of the ZAKα isoform is in the middle of the protein sequence 27. The ZAKβ splice-variant form (also known as MLK-like mitogen-activatedprotein triple kinase-β (MLTKβ) 14 and MLK-related kinase-β (MRKβ) 15 ) is identical to ZAKα from the amino terminus to the zipper domain but then diverges and terminates shortly thereafter, so it lacks a SAM domain (FIG. 2a). ACTIVATOR-PROTEIN 1 (AP-1) TRANSCRIPTION-FACTOR COMPLEX A transcription-factor complex that comprises a dimer of members of the Fos and Jun families of nuclear phosphoproteins. SRC-HOMOLOGY-3 (SH3) DOMAIN Protein sequence of ~50 amino acids that recognizes and binds sequences rich in proline. CRIB MOTIF A amino-acid sequence with eight conserved residues that is essential for the binding of signalling molecules to GTPbound forms of Rac and Cdc42. LEUCINE ZIPPER A leucine-rich domain within a protein that binds to other proteins with a similar domain. RHO-FAMILY GTPASES Ras-related GTPases involved in controlling the polymerization of actin. arrangements and sequence similarity within their catalytic domains, these MLKs cluster into three subgroups: the MLKs; the dual-leucine-zipper-bearing kinases (DLKs); and zipper sterile-α-motif kinase (ZAK). The simultaneous identification of the MLKs has resulted in multiple names for each kinase. TABLE 1 summarizes the nomenclature and synonyms of the MLK family members, and the domain arrangements of the human MLKs are outlined in FIG. 2a. MLKs. The first subgroup, which includes MLK1 MLK4, is characterized by an amino-terminal SRC-HOMOLOGY-3 (SH3) DOMAIN, followed sequentially by a kinase domain, a leucine-zipper region and a Cdc42/Rac-interactive binding (CRIB) MOTIF; as the name implies, the CRIB motif interacts with the RHO-FAMILY GTPASES Rac and Cdc42. MLK1 MLK4 share 75% sequence identity within their catalytic domains and ~65% sequence identity from their SH3 domains to their CRIB motif. The carboxyl termini of these proteins diverge, indicating that these regions might serve different regulatory functions, but all are proline-rich. The function of the proline-rich sequences is undefined. MLK3 has a Gly Pro-rich amino terminus that is absent from MLK1, MLK2 or MLK4. Of the MLK subfamily, only MLK2 (REF. 18) (also called MKN28-derived serine/threonine kinase 19 ), MLK3 (REF. 20) (also called SH3-domain-containing proline-rich kinase 17 ) and protein-tyrosine-kinase 1 (REF. 21) have been subjected to any biochemical characterization. Phylogenetic relationships. A dendrogram of the kinase domains of MLKs is shown in FIG. 2b.The Drosophila MLK, known as Slipper (Slpr), contains an amino-terminal SH3 domain, a kinase domain, a leucine zipper and a CRIB motif, in common with the MLK subfamily. Slpr is involved in regulating dorsal closure in the fly embryo 28. With the exception of Slpr, there are no genetic or biochemical data about MLKs in lower eukaryotes. MLKs are absent in yeast, but analysis of the NCBI (National Center for Biotechnology Information) protein database shows that, on the basis of sequence similarity within the catalytic and zipper domains, D. melanogaster and C. elegans each have a DLK homologue. In addition, a ZAK-like kinase, complete with a catalytic domain, zipper motif and SAM domain, is present in C. elegans. C. elegans also has the coding sequence for an SH3-domain-containing kinase, the catalytic domain of which is most related to the broad MLK family, but this protein kinase does not clearly fit into any one of the MLK subfamilies. Regulation of MLKs by dimerization In addition to their related catalytic domains, all seven MLKs contain some type of leucine zipper (FIG. 2a). Leucine zippers mediate protein dimerization or oligomerization by forming COILED COILS that are stabilized primarily by leucine or other non-aromatic hydrophobic residues that interact at the interfaces of opposing helices Although the zipper-regulated stoichiometries of MLKs have not been experimentally determined, we here use dimerization for simplicity. The leucine zippers of MLK1 MLK4 share ~70% 664 SEPTEMBER 2002 VOLUME 3

3 Figure 1 Mixed-lineage kinases. The seven mixed-lineage kinases (MLKs; MLK1 MLK4, DLK, LZK and ZAK) are members of a group of mitogen-activated-protein kinase (MAPK) kinase kinases that regulate the c-jun amino-terminal kinase (JNK) pathway through MAPK kinases (MKKs). All the MLK family members have been shown to activate the JNK pathway. MLK3 and dual-leucine-zipper-bearing kinase (DLK) have also been shown to activate the p38 MAPK pathway, but it is not known whether other MLKs can also activate p38. The different regulatory properties predicted for the different MLKs would allow integration of JNK and p38 activation with different cellular responses. Selected examples of the targets of these kinases are shown. ATF2, activating transcription factor 2; CHOP10, c/ebp-homologous protein 10; eef2k, eukaryotic elongation factor 2 kinase; Elk-1, ets-like-1; LZK, leucine-zipper kinase; MAPKAP-K, mitogenactivated protein kinase activated protein kinase; MEF2, myocyte enhancer factor 2; MNK, MAPK-interacting kinase; MSK, mitogen and stress-activated kinase; NFAT, nuclear factor of activated T cells; RNPK, ribonucleoprotein kinase; Shc, Src-homology-2-containing transforming protein; ZAK, zipper sterile-α-motif kinase. MLK1 MLK2 MLK3 MLK4 DLK LZK ZAK MKK4 MKK7 MKK3 MKK6 JNK Shc, p53, NFAT4, c-jun, ATF2, RNPK p38 MNK1/2, MEF2, CHOP10, Elk-1, MSK1/2, MAPKAP-K2/3, eef2k COILED-COIL A protein domain that forms a bundle of two or three α-helices. Short coiled-coil domains are involved in protein interactions but long coiled-coil domains, which form long rods, occur in structural or motor proteins. ACTIVATION LOOP A conserved structural motif in kinase domains that needs to be phosphorylated for full activation of most kinases. sequence identity but are only 35% identical to those of DLK and LZK, implying that the leucine-zipper domains of the different MLK subfamilies might regulate selectivity in protein protein interactions. Homodimerization. Dimerization is a common mechanism for the activation of protein kinases such as growth-factor-receptor tyrosine kinases 32 and c-raf-1 (REFS 33,34). The leucine zipper for DLK is required for DLK self-association, phosphorylation, activation and stimulation of the JNK pathway 35. A mutant of LZK in which the leucine-zipper domain was deleted indicated that this domain probably has the same function in this protein 36. A model in which the leucine zipper of DLK mediates homodimerization and transphosphorylation to activate the kinase is supported by the finding that induced dimerization of monomeric DLK promotes autophosphorylation and JNK activation 37. Consistent with the model that transphosphorylation leads to activation, the DLK leucine zipper, when overproduced, effectively inhibits the dimerization and activation of full-length DLK, presumably by forming inactive leucine-zipper DLK complexes. The dimerization of DLK requires neither kinase activity nor phosphorylation of DLK and is a function of the DLK leucine zipper 38. Like DLK, MLK3 dimerizes through its leucine zipper, and deletion of the entire leucine zipper results in an MLK3 variant that fails to autophosphorylate and to activate the JNK pathway 39. Studies with a form of MLK3 in which leucine-zipper-mediated dimerization has been disrupted by introducing a destabilizing proline residue support the finding that MLK3 dimerization is required for JNK activation 40. However, this mutant MLK3, like the wild-type version, could still be induced to autophosphorylate using active Cdc42, but failed to phosphorylate residue T258 (where T represents threonine), one of the two activating phosphorylation sites in the ACTIVA- TION LOOP of the downstream target, MKK4 (REF. 40). This result indicates that Cdc42 regulation of MLK3 does not require stable leucine-zipper-mediated dimerization but that dimerization is required for proper substrate interaction and phosphorylation. Table 1 Nomenclature and synonyms of mixed-lineage kinases (MLKs) Subfamily Human Genome Synonyms Gene sequences Production in Drosophila Caenorhabditis Nomenclature mammals melanogaster elegans homologue Committee name homologue MLK MLK1 MAP3K9 Human GI: Epithelial cells Slipper GI: GI: MLK2 MAP3K10 MST Human GI: Brain, skeletal muscle, testes MLK3 MAP3K11 SPRK Human GI: Widely produced PTK1 Mouse GI: MLK4α Human α GI: MLK4β Human β GI: Unknown DLK DLK MAP3K12 ZPK Human GI: Brain (also keratinocytes GI: GI: MUK Mouse GI: and regenerating liver) Rat GI: LZK MAP3K13 Human GI: Widely produced ZAK ZAKα MRK Human α GI: Widely produced GI: ZAKβ MLTK Human β GI: MLK7 Mouse α GI: ZAK Mouse β GI: DLK, dual-leucine-zipper-bearing kinase; GI, gene identifier number in NCBI protein database; LZK, leucine zipper kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MLTK, MLK-like MAPK triple kinase; MRK, MLK-related kinase; MST, MKN28-derived serine/threonine kinase; MUK, MAPK upstream kinase; PTK1, protein tyrosine kinase 1; SPRK, Src-homology-3 (SH3) domain-containing proline-rich kinase; ZAK, zipper sterile-α-motif kinase; ZPK, zipper (leucine) protein kinase. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 3 SEPTEMBER

4 PRENYLATION The enzymatic addition of prenyl moieties (geranyl, farnesyl or geranylgeranyl groups) to a protein as a posttranslational modification. YEAST TWO-HYBRID SCREEN A technique used to test whether two proteins physically interact with each other. One protein is fused to the GAL4 activation domain and the other to the GAL4 DNA-binding domain, and both fusion proteins are introduced into yeast. Expression of a GAL4-regulated reporter gene indicates that the two proteins physically interact. GUANINE-NUCLEOTIDE- EXCHANGE FACTOR A protein that facilitates the exchange of GDP for GTP in the nucleotide-binding pocket of a GTP-binding protein. a b MLK DLK ZAK MLKs MLK1 MLK2 MLK3 MLK4α MLK4β DLK LZK ZAKα ZAKβ ZAKs DLKs Gly SH3 Kinase LZ CRIB SAM Figure 2 Comparison of the MLK family conserved domains and phylogenetic history. a Composite structure of human mixed-lineage kinases (MLKs), showing the relative positions of the Src-homology-3 (SH3), kinase, leucine-zipper (LZ) and Cdc42/Rac-interactive binding (CRIB) domains, and the sterile-α motif (SAM). See text for discussion. b Phylogenetic tree of the MLK subfamilies: MLKs, dualleucine-zipper-bearing kinases (DLKs) and zipper sterile-α-motif kinase (ZAK). Ce, Caenorhabditis elegans (nematode); Dm, Drosophila melanogaster (fruitfly); Hs, Homo sapiens (human); Mm, Mus musculus (mouse); Rn, Rattus norvegicus (rat) Hs DLK Rn DLK Mm DLK Hs LZK Dm DLK-like Ce DLK-like Hs ZAK Mm ZAK Ce ZAK-like Hs MLK3 Mm MLK3 Hs MLK4 Hs MLK1 Hs MLK2 Dm Slpr Ce SH3-containing MLK Heterodimerization. The DLK leucine zipper does not form efficient heterodimers with other MLK proteins, which indicates that this domain is unlikely to mediate heterodimerization of different MLKs. Heterodimerization of DLK and LZK has been observed, but this was localized to the DLK amino terminus and not to the leucine zipper 35. However, because no direct interaction between the amino termini of DLK and LZK could be shown, it was predicted that this interaction is a function of an intermediary protein such as JNK-interacting protein 1 (JIP1), which can itself oligomerize and which binds the amino termini of DLK and LZK (REF. 35). This has not been shown formally and there might be other proteins that could perform this function. SH3-mediated autoinhibition of MLK3 MLK1 MLK4 have amino-terminal SH3 domains that are absent from the other MLK subfamilies (FIG. 2a). SH3 domains are predicted to recruit MLK1 MLK4 to specific proteins that contain defined proline-rich motifs for the localization and regulation of signalling. The SH3 domain of MLK3 has been shown to autoinhibit its kinase activity, which is the first demonstration of an SH3 domain with this function in a serine/threonine kinase 41. Disruption of the SH3 domain of MLK3 by mutation of the conserved tyrosine residue at residue 52 to an alanine increased MLK3 kinase activity. The MLK3 SH3 domain seems to bind intramolecularly to a region between the leucine zipper and the CRIB motif, and mutation of the single proline here prevents SH3 binding and increases kinase activity. These results show that MLK3 is autoinhibited by binding of its SH3 domain to an autoregulatory sequence, which is similar to the autoinhibitory function of the SH3 domain in the Src family of tyrosine kinases The crucial proline in the SH3-binding region of MLK3 is conserved in MLK1, MLK2, MLK4 and Slpr, which indicates that this subfamily of MLKs use a common mechanism of SH3- mediated autoinhibition (FIG. 3). Regulation by Rho GTPases The Rho family of small GTPases, which includes several Rho and Rac isoforms, TC10 and Cdc42, regulates a wide array of cellular processes in eukaryotes, such as the control of normal and transformed growth, cellular transport, cell motility and the cytoskeleton (reviewed in REFS 45 47). Rho-family GTPases also modulate protein-kinase signalling pathways. Constitutively activated mutant forms of Rac and Cdc42 have been reported to activate the JNK and p38 protein-kinase cascades 48,49.The MLK subfamily members that have an SH3 domain (MLK1 MLK4) also contain a central CRIB motif. MLK3, which contains six of eight conserved residues in its CRIB motif, binds to activated forms of Cdc42 and Rac When MLK3 and activated Cdc42 are coexpressed, there is an increase in MLK3 activity and potentiation of MLK3-induced JNK activation, which is consistent with Rac and/or Cdc42 being upstream activators of MLK3 in the JNK pathway 51,52. The detailed mechanism by which Cdc42 and/or Rac activates MLKs is not completely understood. Coexpressing activated Cdc42 and MLK3 has been shown to promote MLK3 oligomerization 37,39. In addition, there are changes in the in vivo phosphorylation pattern of MLK3 if it is co-expressed with activated Cdc42 (REF. 52). Given the close proximity of the CRIB motif and the autoinhibitory SH3-binding sequence of MLK3 (FIG. 3), the interaction of Cdc42 with MLK3 might disrupt the SH3-mediated autoinhibitory interaction. Finally, Rhofamily GTPases associate with cellular membranes by virtue of post-translational PRENYLATION. Activated, GTPbound Cdc42 or Rac might well direct MLK3 to membrane compartments in the cell for localized activation of MAPK pathways. Although the experiments described above involved overexpression studies, the physiological relevance of Rac and/or Cdc42 in regulating the activity of SH3-containing MLKs is supported by the genetic evidence that links Drosophila Rac (drac) and the SH3-domain-containing Slpr in a JNK signalling pathway (see below). Furthermore, YEAST TWO-HYBRID SCREENS with the Rac-specific GUANINE-NUCLEOTIDE-EXCHANGE FACTOR (GEF) Tiam1 identified JIP2 (REF. 53) a MAPK scaffold that associates directly with MLK3 (REF. 54) and p38 (REFS 53,55) as 666 SEPTEMBER 2002 VOLUME 3

5 H 2 N SH3 Inactive Kinase Zipper Active Kinase Cdc42 Figure 3 SH3-mediated autoinhibition of MLKs. A model in which the Src-homology-3 (SH3) domain in the amino terminus of mixed-lineage kinases (MLKs) binds to a proline (Pro)-containing sequence that is adjacent to the catalytic domain of the protein, which results in autoinhibition of kinase activity. Binding of GTP-bound Rac or Cdc42 through the Cdc42/Rac-interactive binding (CRIB) motif could compete with the autoinhibitory interaction, thereby inducing a conformational change and allowing the activation of MLK kinase activity. SCAFFOLDING PROTEIN A protein that has specific binding sites and is therefore important in the assembly, structure and function of larger molecular complexes. KINESIN Microtubule-based molecular motor, most often directed towards the plus end of microtubules. DORSAL ECTODERM The outer of the three embryonic germ layers; this gives rise to the entire central nervous system. SH3 NH 2 Pro Zipper CRIB Pro GDP Cdc42 GTP CRIB COOH COOH a binding partner. So, JIP2 might specifically co-ordinate Tiam1/Rac-induced MLK activation of p38. Several GEFs have been identified that can activate Rac and/or Cdc42 (reviewed in REFS 56,57), but their effects on MLK activity have not been tested. Discrete GTPase-dependent signalling complexes probably confer spatiotemporal control of MAPK signalling and other cellular functions, such as changes in the cytoskeletal architecture. Regulation of MLKs by phosphorylation Many protein kinases, including MKKKs, are regulated by phosphorylation. Within their catalytic domain, the activation loop often contains regulatory phosphorylation sites. The sequence TTXXS (residues ; where X represents any amino acid and S represents serine) is found in the activation loop of MLK3. Mutagenesis studies support the idea that T277 and S281 are positive regulatory (auto)phosphorylation sites (REF. 58). Because the sequence S/TXXXS is conserved in the activation loops of all mammalian MLKs, the analogous residues might serve similar functions in other MLKs. Mass spectrometry has been used to identify 11 in vivo phosphorylation sites of MLK3, most of which cluster at the carboxyl terminus 59. The finding that a proline residue immediately follows seven of the identified sites indicates that MLK3 is a target of prolinedirected kinases, which include the MAPKs and the cyclin-dependent kinases. One interesting possibility is that MLK3 activates the JNK (and/or p38) pathway, which phosphorylates MLK3 in a feedback loop. Indeed, although the exact sites of phosphorylation have not been identified, MLK2 can be phosphorylated in its carboxy-terminal region by JNK (REF. 60). JIPs and MLKs Consistent with the ability of MLKs to activate the JNK pathway, several MLKs, including MLK2 and MLK3 (REF. 54), DLK 37,54 and LZK 61 have been shown to interact with the JNK-pathway SCAFFOLDING PROTEIN JIP1/isletbrain 1 (IB1) (BOX 2). JIP1 binds MKK7 but not MKK4 (REF. 54), which indicates that at least in the context of JIP1-mediated signalling MKK7 is the relevant substrate for the MLK. Recent work indicates that JIP1 probably regulates DLK activation by preventing its oligomerization, and that the JIP complex is dynamic in nature and might act as a molecular switch 37. JIP2/IB2 binds JNK only very weakly 54 but has been recently shown to bind p38δ (REF. 55) and p38α in MLKmediated activation of the p38 MAPK pathway 53.Some activation of the p38 pathway by DLK (REF. 62), MLK3 (REF. 13), MLK2 (REFS 8,9) and ZAKα (REFS 14,15) has been observed in transfection experiments. These data, taken together with the recent findings that JIP2 interacts with both MLKs and p38 (REFS 53,55), indicate that MLKs can activate both the JNK and the p38 pathways. The structurally distinct scaffold JIP3 interacts with MLK3, MKK7, JNK1 and JNK3 (REF. 63). However, JNK/stress-activated-protein-kinase-associated protein 1 (JSAP1), a splice variant of JIP3, binds MEKK1, MKK4 and JNK1/2 (REF. 64). A genetic screen showed that disruption of the Drosophila homologue of JIP3, known as Sunday driver (Syd), leads to defects in KINESIN-dependent axonal transport 65. However, not only Syd/JIP3 but also JIP1 and JIP2 (REFS 66,67) interact with components of the kinesin motor complexes, which indicates that JIPs might be cargoes for kinesin-mediated transport and could have complex roles in subcellular localization and transport. Slpr: the Drosophila MLK In D. melanogaster, the JNK pathway regulates the process of dorsal closure during embryogenesis 68,69. Dorsal closure is a process of cell-sheet morphogenesis that involves the movement of the DORSAL ECTODERM from a lateral position to the dorsal midline and that encloses the embryo in a continuous protective epidermis. Study of the process of dorsal closure in the fly has identified a pathway that involves the GTPase drac1, an MKKKK called Misshapen (Msn), Slpr, the MKK7 homologue Hemipterous (Hep), the JNK homologue Basket (Bsk) and the AP-1 transcription factors djun and dfos, which is encoded by kayak (kay) (FIG. 4). Mutational analysis of this pathway shows the function of Slpr in epidermal-cell-sheet morphogenesis. Slpr is regulated by an upstream GTPase, drac1, and Msn. The slpr gene is functionally upstream of hep and bsk in the Drosophila JNK pathway 28. JNK has two regulatory functions during epithelial-cell-sheet morphogenesis. One is the control of AP-1-mediated gene expression. Decapentaplegic (Dpp) is the Drosophila homologue of bone morphogenic protein 4 (Bmp4), a member of the TGFβ family of cytokines 70,71.During dorsal closure, dpp is expressed at the leading edge of the epidermis and requires JNK signalling and AP-1 activation for its expression. Mutants in the slpr gene fail to express dpp at the leading edge of the epidermis during dorsal closure 28, which is consistent with a loss of function of JNK and AP-1 in slpr-mutant embryos. The NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 3 SEPTEMBER

6 Box 2 JNK-interacting proteins JIP/IB interacting proteins RhoGEF Tiam1 RasGRF1 ApoER2 APP Megalin LRP-1 Kinesin Stimulus Rac, Cdc42 Stimulus Rac, Cdc42 Rac, Cdc42 JIP1/IB1 DLK, LZK MLK2, MLK3 JIP2/IB2 DLK, MLK2 MLK3 JIP3 MLK3 MKK7 JNK Jun, ATF2 MKK7 JNK Jun, ATF2 MKK3 p38 Mef2C MKK7 JNK Jun, ATF2 c-jun amino-terminal kinase (JNK)-interacting proteins (JIPs) were cloned on the basis of their binding to JNKs. They function as putative scaffolding proteins for regulating JNK- and p38-mediated mitogen-activated-protein kinase (MAPK) signalling. JIPs also bind the MAPK kinases (MKKs) MKK3 or MKK7, and mixed-lineage kinases (MLKs). In addition to the MAPK signalling proteins, JIPs have been shown to bind several other proteins that have diverse biological functions, including the kinesin light chain. JIPs seem to be cargoes for the plus-end-directed microtubule-motor-protein kinesin-1. So, JIPs might be involved in localizing signalling complexes that involve not only the MLKs, JNK and p38 but also other proteins such as the small GTPase-regulatory proteins RhoGEF (REF. 108),Tiam-1 (REF. 53) and Ras-specific guanine-nucleotide-releasing factor 1 (RasGRF1) 53, the low-density-lipoprotein-receptor-related proteins ApoER2 (REF. 109), Megalin 110 and low-density-lipoprotein-receptor-related protein 1 (LRP-1) 110, as well as amyloid precursor protein (APP) 111 and fibroblast-growth-factor-homologous factor 1 (FHF1) 55. ATF2, activating transcription factor 2; DLK, dual-leucine-zipper-bearing kinase; IB, islet brain; LZK, leucine-zipper kinase. MLKs (BOX 3). Characterization of MLK function in mammalian cells has primarily evolved around its role in regulating the activity of JNK, which, in turn, mediates the apoptosis of different cell types, particularly neurons. These studies are outlined below. MLK3 and transformation. JNK has been proposed to have a role in cellular transformation 80, which is consistent with the evidence that the AP-1 transcription complex contributes to transformation and tumorigenesis 80,81. Wild-type MLK3 overproduction has been shown to transform NIH3T3 fibroblasts, whereas catalytically inactive MLK3 is not transforming 82.The relevance of these observations is unclear and there is currently no evidence that any MLK family member functions as an oncogene in humans. Another recent report indicates that MLK3 inhibits Rac-mediated cellular transformation 83. The potential role, if any, of MLKs in cancer remains to be determined. The availability of inhibitors of MLKs should allow this question to be addressed in XENOGRAFT models of human tumours in athymic NUDE MICE. MLK3 and nuclear factor κb. Overexpression of MLK3 in JURKAT T-LYMPHOMA CELLS stimulates transcription of the cytokines tumour necrosis factor α (TNFα) 84 and interleukin-2 (IL-2) 85. Catalytically inactive MLK3 did not stimulate TNFα or IL-2 promoter activity and inhibited XENOGRAFT Tissue or organ graft between species. These grafts are usually rejected. NUDE MICE A mutation in mice that causes both hairlessness and defective formation of the thymus, which results in a lack of mature T cells. JURKAT T-LYMPHOMA CELLS Human leukaemic T-cell line used to study several aspects of T-cell biology and signalling, especially signal-transduction events initiated by the T-cell receptor. second defined function for JNK signalling during dorsal closure is the control of a stretched epithelial-cell morphology 76,77. The initiation of this function, however, is not inhibited in slpr-mutant embryos, although the cells cannot maintain the stretched morphology and the cells round up. The role of JNK in this process is poorly understood, but the fact that slpr-mutant embryos are defective in this process indicates a role for this MLK in controlling epithelial cell shape during dorsal closure. Interestingly, JNK functions not only in dorsal closure in the embryo but also in adult morphogenesis, epithelial planar polarity, innate immunity and apoptosis in the fly 78. Slpr does not seem to be required for JNK regulation of epithelial planar polarity or innate immunity. Regulation of the innate immune response in the fly involves dtak, and other MKKKs presumably control the JNK pathway during planar polarity 79. These studies clearly define the function of an MLK in the control of the JNK pathway in a specific regulatory process in the Drosophila embryo that involves the control of gene expression and cell shape. MLKs in mammalian cells MLKs clearly regulate the JNK pathway in mammalian cells. No targeted gene disruption of any MLK-family member has yet been reported but this is an area of active research by several laboratories. Consequently, most of the functional analysis of MLKs in mammalian cells has come from transfection experiments and from the use of CEP-1347, a small-molecule inhibitor of drac1 Epithelial cell shape Cell sheet movement Msn Slpr Hep Bsk djun Dpp MKKKK MLK MKK7 JNK c-jun/atf2 BMP4 Figure 4 Fly dorsal closure. Proposed c-jun amino-terminal kinase (JNK) pathway controlling the regulation of dorsal closure during Drosophila embryo morphogenesis. Slipper (Slpr) is the Drosophila MLK that controls the JNK pathway in dorsal closure. As with mammalian MLKs, Slpr has a Cdc42/Rac-interactive binding (CRIB) motif for binding the GTPase Rac. 668 SEPTEMBER 2002 VOLUME 3

7 Box 3 CEP-1347, a small-molecule inhibitor of MLKs CEP-1347 is a derivative of the natural product K252a, which was isolated from culture broths of Nocardiopsis. CEP-1347 has been found to have important neuroprotective properties 112, protecting primary neurons in culture from neurotrophic-factorwithdrawal-induced apoptosis 113. Furthermore, animal models of chemical-induced neural toxicity have shown that CEP-1347 is an effective neuroprotectant in vivo 114. CEP-1347 was found to block c-jun amino-terminal kinase (JNK), but not extracellularsignal-regulated-kinase 1/2 or p38 activation, in response to trophic-factor withdrawal. However, this is not due to the direct inhibition of JNK. Screening of upstream kinases in the JNK pathway showed that CEP-1347 inhibited MLKs, but not the mitogenactivated-protein kinase (MAPK) kinase kinases apoptosis-inducing kinase or MEKK1 (REF. 115). In addition to trophic-factor withdrawal, other stresses including ultravioletinduced apoptosis were blocked by CEP-1347 (REF. 113). The studies with CEP-1347 support a role for MLKs in stimulating JNK-regulated apoptosis. The obvious inference from the cumulative evidence for JNK involvement in neurodegenerative diseases including Huntington s, Alzheimer s and Parkinson s is that MLKs are logical therapeutic targets in the prevention of neuronal cell death. Indeed, CEP-1347 is currently in clinical trials for Parkinson s disease. DOMINANT-NEGATIVE A defective protein that retains interaction capabilities and so distorts or competes with normal proteins. Vav-stimulated IL-4 production 86. Nuclear factor κb (NF-κB) is also activated in Jurkat cells that overproduce MLK3 (REF. 87). Catalytically inactive MLK3 impaired NF-κB reporter-gene activity in response to T- cell stimulation but not in response to TNFα or IL-1. Endogenous MLK3 was phosphorylated in response to T-cell stimulation but no change in MLK3 activity was reported. The relevance of these findings in T-cell signalling is presently unclear but they indicate that MLK3 could be involved in T-cell activation in response to antigen challenge. Other MKKKs that regulate the JNK pathway (including MEKK1, MEKK2, MEKK3 and TAK1) have been shown to activate NF-κB (REFS 88 91). TAK1 seems to have a primary role in IL-1 activation of NF-κB. It is still unclear whether MEKKs or MLKs are relevant physiological regulators of NF-κB. Gene knockouts and the development and use of isoform-selective MLK inhibitors will be required to define the role of MLK3 and other MLKs in the regulation of NF-κB activity in T cells and other cell types. Neuronal apoptosis. Deprivation of growth factors leads to apoptosis of neurons in vivo and in vitro 92,93.Several studies have shown that c-jun is required for neuronal growth factor (NGF)-deprivation-induced apoptosis of neurons 94,95. Inhibition of c-jun function using DOMI- NANT-NEGATIVE c-jun or nuclear injection of anti-c-jun antibodies protects neurons from NGF-deprivationinduced death 94,95. In addition to regulation of c-jun, JNKs probably regulate the activity of specific Bcl-2- family proteins that control mitochondrial integrity and cytochrome c release 96,97. Studies in neuronal systems indicate that MLK2, MLK3 and DLK might be important MKKKs that mediate trophic-factor-deprivation-induced neuronal apoptosis Overexpression of MLK2, MLK3 or DLK in PC12 cells induces apoptosis 99. NGF-withdrawalinduced apoptosis of superior cervical ganglion (SCG) sympathetic neurons requires the functional activity of Cdc42 and activation of JNK 100. When SCG neurons are deprived of NGF, there is an increase in MLK3 kinase activity and activation of JNK, but it is unclear whether NGF withdrawal affects the levels of MLK3 protein. Overexpression of MLK3 in SCG neurons activates JNK and induces apoptosis. By contrast, a catalytically inactive MLK3 mutant protein blocked apoptosis of SCG neurons in response to NGF deprivation. It seems that MLK3 is activated upon NGF withdrawal and that MLK3 or a related MKKK is involved in JNK activation in response to NGF withdrawal. Jnk3-knockout mice show a decreased susceptibility to kainate-induced epileptic seizures and neuronal apoptosis 102, which is similar to the phenotype of mice that lack the kainate receptor glutamate-receptor 6 (GluR6) subunit 100. The resistance to neuronal excitotoxicity of GluR6-deficient and Jnk3 / mice indicated a potential link between GluR6 and Jnk3 activation. Overexpression of either MLK2 or MLK3 in the rat hippocampal cell line HN33 induces apoptosis, whereas a catalytically inactive mutant of MLK2 or MLK3 suppresses GluR6-induced apoptosis of these cells 103. The postsynaptic-density protein PSD-95 couples MLK2 and/or MLK3 to the GluR6 receptor complex in HN33 cells. MLK2 and MLK3 can be co-immunoprecipitated with PSD-95 from HN33 cells and rat brain lysates, which indicates that they exist in a complex in cells. PSD-95 through its SH3 domain binds MLK2 and MLK3, and deletion of the PSD-95 SH3 domain abolishes PSD-95 association with MLK2 and MLK3. Finally, deletion of the PSD-95-binding site of GluR6 inhibited GluR6-induced JNK activation and apoptosis of HN33 cells. These findings indicate that PSD-95 couples the JNK pathway to GluR6 by binding MLK2 and/or MLK3. How MLK2 or MLK3 is activated in this complex is not yet defined. MLK2 and MLK3 regulation of JNK activation in neurons almost certainly has functions that are independent of the induction of apoptosis. JNK signalling probably has many functions that are involved in the control of neuronal homeostasis. In the event of NGF withdrawal, the apoptotic response probably dominates, in part because of the loss of pro-survival signals involving the activation of Akt, a pro-survival factor. Polyglutamine-expanded huntingtin. Huntington s disease is a neurodegenerative disorder that is characterized by mental impairment, choreiform movement and cognitive deficits. The gene involved in Huntington s disease is ubiquitously expressed and encodes a 350-kDa cytoplasmic protein called huntingtin 104,105. The huntingtin protein is found in neuronal cell bodies, dendrites and nerve terminals associated with synaptic vesicles and microtubules. Changes in the HUNTINGTIN gene that are associated with disease involve the expansion of a polyglutamine stretch near the amino terminus of huntingtin, and the length of the polyglutamine expansion correlates with the severity of disease. The huntingtin protein has been shown to immunoprecipitate with MLK2 in co-transfected 293T cells and in NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 3 SEPTEMBER

8 a Huntingtin MLK2 (inactive) JNK activation Neuronal survival Aggregation JNK activation PolyQ MLK2 (increased activity) Figure 5 Possible MLK2 huntingtin interaction. a Mixedlineage kinase 2 (MLK2) and huntingtin are proposed to be in a complex under normal circumstances. b In Huntington s disease, polyglutamine-expanded (polyq) huntingtin is aggregated and does not seem to interact with MLK2. Aggregation of polyglutamine-expanded huntingtin might lead to a stress response that involves increased activity of c-jun amino-terminal kinase (JNK). The loss of the MLK2 huntingtin interaction and the consequent alteration of MLK2 activity could also contribute to the JNK response and neuronal apoptosis. HN33 cells 98. The SH3 domain of MLK2 binds to the proline-rich amino terminus of huntingtin. The polyglutamine-expanded version of huntingtin, however, fails to bind MLK2 and activates JNK and induces apoptosis. However, the catalytically inactive form of MLK2, MKK4 or MKK7 decreased JNK activity and apoptosis induced by this variant of huntingtin. It has been proposed 98 that, in normal neuronal cells, MLK2 is sequestered in an inactive form by binding of its SH3 domain to the amino terminus of huntingtin and that, in Huntington s disease, the failure of polyglutamine-expanded huntingtin to interact with MLK2 allows the free MLK2 to be activated and to induce JNK-mediated apoptosis (FIG. 5). In support of this model, expression of the amino terminus of normal huntingtin protected HN33 cells from toxicity induced by MLK2 and a mutated form of huntingtin containing 48 polyglutamine repeats. These findings are consistent with huntingtin s being a negative regulator of MLK2. However, the failure of MLK2 to interact with polyglutamine-expanded huntingtin might simply be due to the aggregation of the disease-associated form of huntingtin. Polyglutamine-expanded huntingtin probably has other dominant effects in the pathology of Huntington s disease. Further studies are obviously required to define the importance of polyglutamine expansions of huntingtin and the interaction of huntingtin with MLK2 in Huntington s disease. Future directions The MLKs have emerged as an important family of MKKKs. MLKs regulate the JNK pathway, and some MLKs, such as DLK and MLK3, regulate the p38 pathway. b Neuronal apoptosis Because their altered regulation might contribute to neurogenerative diseases, the MLKs have surfaced as attractive therapeutic targets for the treatment of diseases such as Huntington s. Several questions, however, still remain unresolved. For example, why should there be seven members of the MLK family, as well as the MEKKs, ASK1 and TAK1, regulating the JNK and p38 pathways? Some MLKs are restricted in their cell and tissue production, whereas several others are widely produced. The diverse regulatory domains that are present in the MLKs indicate that these proteins are probably regulated differently by different upstream stimuli and participate in selective interactions with other proteins that target MLKs to different subcellular locations. The physiological roles of MLKs have not been defined and most studies of MLKs have relied on the transfection of cultured cells. The exception is the studies in Drosophila, in which Slpr is important in the expression of dpp and in embryonic morphogenesis. MLKs probably have similar roles in mammalian cells in the control of gene expression, as occurs in response to cytokines in different cell types. What is needed is an understanding of the subcellular location and identification of extracellular and upstream stimuli that selectively regulate different MLKs. MLK1 MLK4 are regulated by Cdc42/Rac and their activation is probably coordinated with regulation of the cytoskeleton; for the DLK and ZAK subfamily members, this is less clear. Some MLK members are probably involved in embryonic development, which is similar to the function of Slpr in Drosophila, whereas others probably function primarily in differentiated cell types. Genetic studies involving targeted gene disruptions will be required for the unequivocal definition of function for each MLK. The association of MLK proteins, but not other MKKKs, with JIPs is a very important discovery. JIPs interact with proteins such as kinesin motor complexes, receptors and regulators of Rho GTPases, which indicates that MLKs might be involved in regulating JNK and p38 signalling during vesicular transport, endocytosis and cytoskeletal assembly. MLK2 has also been shown to interact with clathrin and to influence clathrin-coated-vesicle transport 106. An important question is whether MLKs function to regulate MAPK pathways only when complexed with or in the vicinity of JIPs, or whether they also signal independently of JIPs. Small-molecule inhibitors of JNKs, p38 and MLKs are now entering clinical trials for different diseases: JNKs and p38 primarily for cancer and inflammation, and MLKs for neurogenerative disorders. The outcomes of these trials will help to establish the relevance of these MAPK pathways and the MLKs in human disease and their value as therapeutic targets. 1. Kyriakis, J. M. & Avruch, J. pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-llysine. J. Biol. Chem. 265, (1990). 2. Kyriakis, J. M. & Avruch, J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81, (2001). 3. Derijard, B. et al. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-jun activation domain. Cell 76, (1994). 4. Macian, F., Lopez-Rodriguez, C. & Rao, A. Partners in transcription: NFAT and AP-1. Oncogene 20, (2001). 5. Wagner, E. F. AP-1 introductory remarks. Oncogene 20, (2001). 6. Toone, W. M., Morgan, B. A. & Jones, N. Redox control of AP-1-like factors in yeast and beyond. Oncogene 20, (2001). 7. Rana, A. et al. The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1. J. Biol. Chem. 271, (1996). 8. Hirai, S. et al. MST/MLK2, a member of the mixed lineage kinase family, directly phosphorylates and activates SEK1, 670 SEPTEMBER 2002 VOLUME 3

9 an activator of c-jun N-terminal kinase/stress-activated protein kinase. J. Biol. Chem. 272, (1997). 9. Cuenda, A. & Dorow, D. S. Differential activation of stressactivated protein kinase kinases SKK4/MKK7 and SKK1/MKK4 by the mixed-lineage kinase-2 and mitogenactivated protein kinase kinase (MKK) kinase-1. Biochem. J. 333, (1998). 10. Hirai, S. et al. Differential activation of two JNK activators, MKK7 and SEK1, by MKN28-derived nonreceptor serine/threonine kinase/mixed lineage kinase 2. J. Biol. Chem. 273, (1998). 11. Merritt, S. E. et al. The mixed lineage kinase DLK utilizes MKK7 and not MKK4 as substrate. J. Biol. Chem. 274, (1999). 12. Fanger, G. R. et al. MEKKs, GCKs, MLKs, PAKs, TAKs, and tpls: upstream regulators of the c-jun amino-terminal kinases? Curr. Opin. Genet. Dev. 7, (1997). 13. Tibbles, L. A. et al. MLK-3 activates the SAPK/JNK and p38/rk pathways via SEK1 and MKK3/6. EMBO J. 15, (1996). 14. Gotoh, I., Adachi, M. & Nishida, E. Identification and characterization of a novel MAP kinase kinase kinase, MLTK. J. Biol. Chem. 276, (2001). 15. Gross, E. A. et al. MRK, a mixed lineage kinase related molecule that plays a role in γ-radiation-induced cell cycle arrest. J. Biol. Chem. 277, (2002). 16. Dorow, D. S., Devereux, L., Dietzsch, E. & de Kretser, T. Identification of a new family of human epithelial protein kinases containing two leucine/isoleucine-zipper domains. Eur. J. Biochem. 213, (1993). 17. Gallo, K. A. et al. Identification and characterization of SPRK, a novel Src-homology 3 domain-containing prolinerich kinase with serine/threonine kinase activity. J. Biol. Chem. 269, (1994). This paper first showed that a mixed lineage kinase (MLK3) is a serine/threonine kinase. 18. Dorow, D. S. et al. Complete nucleotide sequence, expression, and chromosomal localisation of human mixedlineage kinase 2. Eur. J. Biochem. 234, (1995). 19. Katoh, M., Hirai, M., Sugimura, T. & Terada, M. Cloning and characterization of MST, a novel (putative) serine/threonine kinase with SH3 domain. Oncogene 10, (1995). 20. Ing, Y. L. et al. MLK-3: identification of a widely-expressed protein kinase bearing an SH3 domain and a leucine zipperbasic region domain. Oncogene 9, (1994). 21. Ezoe, K., Lee, S. T., Strunk, K. M. & Spritz, R. A. PTK1, a novel protein kinase required for proliferation of human melanocytes. Oncogene 9, (1994). 22. Holzman, L. B., Merritt, S. E. & Fan, G. Identification, molecular cloning, and characterization of dual leucine zipper bearing kinase. A novel serine/threonine protein kinase that defines a second subfamily of mixed lineage kinases. J. Biol. Chem. 269, (1994). 23. Reddy, U. R. & Pleasure, D. Cloning of a novel putative protein kinase having a leucine zipper domain from human brain. Biochem. Biophys. Res. Commun. 202, (1994). 24. Hirai, S. et al. Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK. Oncogene 12, (1996). 25. Sakuma, H. et al. Molecular cloning and functional expression of a cdna encoding a new member of mixed lineage protein kinase from human brain. J. Biol. Chem. 272, (1997). 26. Stapleton, D., Balan, I., Pawson, T. & Sicheri, F. The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization. Nature Struct. Biol. 6, (1999). 27. Liu, T. C. et al. Cloning and expression of ZAK, a mixed lineage kinase-like protein containing a leucine-zipper and a sterile-α motif. Biochem. Biophys. Res. Commun. 274, (2000). 28. Stronach, B. & Perrimon, N. Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, Slipper. Genes Dev. 16, (2002). This paper provides evidence that Slipper is the Drosophila MLK that is downstream of the GTPase drac and controls the JNK pathway during epithelial migration in the developing fly embryo. 29. Hodges, R. S., Zhou, N. E., Kay, C. M. & Semchuk, P. D. Synthetic model proteins: contribution of hydrophobic residues and disulfide bonds to protein stability. Peptide Res. 3, (1990). 30. Hu, J. C., O Shea, E. K., Kim, P. S. & Sauer, R. T. Sequence requirements for coiled-coils: analysis with λ repressor GCN4 leucine zipper fusions. Science 250, (1990). 31. O Shea, E. K., Klemm, J. D., Kim, P. S. & Alber, T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254, (1991). 32. Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 103, (2000). 33. Luo, Z. et al. Oligomerization activates c-raf-1 through a Ras-dependent mechanism. Nature 383, (1996). 34. Farrar, M. A., Alberol, I. & Perlmutter, R. M. Activation of the Raf-1 kinase cascade by coumermycin-induced dimerization. Nature 383, (1996). 35. Nihalani, D., Merritt, S. & Holzman, L. B. Identification of structural and functional domains in mixed lineage kinase dual leucine zipper-bearing kinase required for complex formation and stress-activated protein kinase activation. J. Biol. Chem. 275, (2000). This paper shows that the leucine zipper of DLK mediates homodimerization but that the DLK zipper does not mediate heterodimerization with other MLKs, including LZK. 36. Ikeda, A. et al. Identification and characterization of functional domains in a mixed lineage kinase LZK. FEBS Lett. 488, (2001). 37. Nihalani, D., Meyer, D., Pajni, S. & Holzman, L. B. Mixed lineage kinase-dependent JNK activation is governed by interactions of scaffold protein JIP with MAPK module components. EMBO J. 20, (2001). This paper investigates the mechanism by which JIP1 regulates MLK activation and signalling, demonstrates that JIP1 can prevent oligomerization of DLK and provides evidence for the dynamic nature of the JIP1 complex. 38. Mata, M. et al. Characterization of dual leucine zipperbearing kinase, a mixed lineage kinase present in synaptic terminals whose phosphorylation state is regulated by membrane depolarization via calcineurin. J. Biol. Chem. 271, (1996). 39. Leung, I. W. & Lassam, N. Dimerization via tandem leucine zippers is essential for the activation of the mitogenactivated protein kinase kinase kinase, MLK-3. J. Biol. Chem. 273, (1998). This was the first demonstration of homodimerization of an MLK and of the requirement for dimerization in activating JNK. 40. Vacratsis, P. O. & Gallo, K. A. Zipper-mediated oligomerization of the mixed lineage kinase SPRK/MLK-3 is not required for its activation by the GTPase Cdc 42 but is necessary for its activation of the JNK pathway. Monomeric SPRK L410P does not catalyze the activating phosphorylation of Thr258 of murine mitogen-activated protein kinase kinase 4. J. Biol. Chem. 275, (2000). This paper indicates that dimerization of MLK3 is required for proper interaction and phosphorylation of a downstream MKK leading to JNK activation. 41. Zhang, H. & Gallo, K. A. Autoinhibition of mixed lineage kinase 3 through its Src homology 3 domain. J. Biol. Chem. 276, (2001). This paper shows that MLK3 is autoinhibited through an interaction between the SH3 domain and a sequence located between the zipper and CRIB motifs, and that MLK1 MLK4 are probably autoregulated in an analogous fashion. 42. Sicheri, F., Moarefi, I. & Kuriyan, J. Crystal structure of the Src family tyrosine kinase Hck. Nature 385, (1997). 43. Williams, J. C. et al. The 2.35 Å crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions. J. Mol. Biol. 274, (1997). 44. Xu, W., Harrison, S. C. & Eck, M. J. Three-dimensional structure of the tyrosine kinase c-src. Nature 385, (1997). 45. Bar-Sagi, D. & Hall, A. Ras and Rho GTPases: a family reunion. Cell 103, (2000). 46. Ridley, A. J. Rho family proteins: coordinating cell responses. Trends Cell Biol. 11, (2001). 47. Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 81, (2001). 48. Coso, O. A. et al. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell 81, (1995). 49. Minden, A. et al. Selective activation of the JNK signaling cascade and c-jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell 81, (1995). 50. Burbelo, P. D., Drechsel, D. & Hall, A. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J. Biol. Chem. 270, (1995). 51. Teramoto, H. et al. Signaling from the small GTP-binding proteins Rac1 and Cdc42 to the c-jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family. J. Biol. Chem. 271, (1996). This paper was the first to show a role for Rac and/or Cdc42 in the activation of MLK3 and in MLK3- mediated activation of the JNK pathway in cells. 52. Bock, B. C., Vacratsis, P. O., Qamirani, E. & Gallo, K. A. Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo. Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation. J. Biol. Chem. 275, (2000). 53. Buchsbaum, R. J., Connolly, B. A. & Feig, L. A. Interaction of Rac exchange factors Tiam1 and Ras-GRF1 with a scaffold for the p38 mitogen-activated protein kinase cascade. Mol. Cell. Biol. 22, (2002). This work shows that MLK3 can activate the p38 signalling pathway in a Tiam1/JIP2-dependent fashion. 54. Yasuda, J. et al. The JIP group of mitogen-activated protein kinase scaffold proteins. Mol. Cell. Biol. 19, (1999). This paper describes the association of MLKs with the JIP scaffold proteins for the regulation of the JNK pathway. 55. Schoorlemmer, J. & Goldfarb, M. Fibroblast growth factor homologous factors are intracellular signaling proteins. Curr. Biol. 11, (2001). 56. Hoffman, G. R. & Cerione, R. A. Signaling to the Rho GTPases: networking with the DH domain. FEBS Lett. 513, (2002). 57. Van Aelst, L. & D Souza-Schorey, C. Rho GTPases and signaling networks. Genes Dev. 11, (1997). 58. Leung, I. W. & Lassam, N. The kinase activation loop is the key to mixed lineage kinase-3 activation via both autophosphorylation and hematopoietic progenitor kinase 1 phosphorylation. J. Biol. Chem. 276, (2001). 59. Vacratsis, P. O., Phinney, B. S., Gage, D. A. & Gallo, K. A. Identification of in vivo phosphorylation sites of MLK3 by mass spectrometry and phosphopeptide mapping. Biochemistry 41, (2002). 60. Phelan, D. R., Price, G., Liu, Y. F. & Dorow, D. S. Activated JNK phosphorylates the C-terminal domain of MLK2 that is required for MLK2-induced apoptosis. J. Biol. Chem. 276, (2001). 61. Ikeda, A. et al. Mixed lineage kinase LZK forms a functional signaling complex with JIP-1, a scaffold protein of the c-jun NH 2 -terminal kinase pathway. J. Biochem. 130, (2001). 62. Fan, G. et al. Dual leucine zipper-bearing kinase (DLK) activates p46 SAPK and p38 MAPK but not ERK2. J. Biol. Chem. 271, (1996). 63. Kelkar, N., Gupta, S., Dickens, M. & Davis, R. J. Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3. Mol. Cell. Biol. 20, (2000). 64. Ito, M. et al. JSAP1, a novel Jun N-terminal protein kinase (JNK)-binding protein that functions as a scaffold factor in the JNK signaling pathway. Mol. Cell. Biol. 19, (1999). 65. Bowman, A. B. et al. Kinesin-dependent axonal transport is mediated by the Sunday driver (SYD) protein. Cell 103, (2000). 66. Verhey, K. J. et al. Cargo of kinesin identified as JIP scaffolding proteins and associated signaling molecules. J. Cell. Biol. 152, (2001). 67. Whitmarsh, A. J. et al. Requirement of the JIP1 scaffold protein for stress-induced JNK activation. Genes Dev. 15, (2001). 68. Knust, E. Drosophila morphogenesis: movements behind the edge. Curr. Biol. 7, R558 R561 (1997). 69. Noselli, S. JNK signaling and morphogenesis in Drosophila. Trends Genet. 14, (1998). 70. Spencer, F. A., Hoffmann, F. M. & Gelbart, W. M. Decapentaplegic: a gene complex affecting morphogenesis in Drosophila melanogaster. Cell 28, (1982). 71. Affolter, M., Marty, T., Vigano, M. A. & Jazwinska, A. Nuclear interpretation of Dpp signaling in Drosophila. EMBO J. 20, (2001). 72. Glise, B. & Noselli, S. Coupling of Jun amino-terminal kinase and Decapentaplegic signaling pathways in Drosophila morphogenesis. Genes Dev. 11, (1997). 73. Hou, X. S., Goldstein, E. S. & Perrimon, N. Drosophila Jun relays the Jun amino-terminal kinase signal transduction pathway to the Decapentaplegic signal transduction pathway in regulating epithelial cell sheet movement. Genes Dev. 11, (1997). 74. Kockel, L. et al. Jun in Drosophila development: redundant and nonredundant functions and regulation by two MAPK signal transduction pathways. Genes Dev. 11, (1997). 75. Riesgo-Escovar, J. R. & Hafen, E. Drosophila Jun kinase regulates expression of decapentaplegic via the ETSdomain protein Aop and the AP-1 transcription factor DJun NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 3 SEPTEMBER