REVIEWS
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.
e-mails: [email protected],
[email protected]
doi:10.1038/nrm906
The mixed-lineage kinases (MLKs) are a family of serine/threonine protein kinases that function in a PHOSPHORELAY 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 CYCLOHEX1
IMIDE-induced inhibition of protein synthesis . 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-Jun3.
As a component of the ACTIVATOR-PROTEIN 1 (AP-1) TRANSCRIPTION-FACTOR COMPLEX, c-Jun regulates the transcription
of cytokine genes and many other genes4. AP-1 is activated by environmental stress, radiation, cytokines and
growth factors, stimuli that also activate JNKs5,6. MLKs
phosphorylate and activate MAPK kinases (MKKs), such
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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 JNKs2. 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 residues17, 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
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REVIEWS
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 activity107. 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
pathway14. 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.
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 14–16-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.
664
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 kinase19), MLK3 (REF. 20) (also called
SH3-domain-containing proline-rich kinase17) and protein-tyrosine-kinase 1 (REF. 21) have been subjected to
any biochemical characterization.
| SEPTEMBER 2002 | VOLUME 3
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
kinase23 and MAPK-upstream kinase24) 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 heterodimerization26. In mammalian
ZAK, the SAM domain of the ZAKα isoform is in the
middle of the protein sequence27. 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).
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
embryo28. 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 helices29–31. 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%
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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.
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 kinases32 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 pathway35. A mutant of LZK in
which the leucine-zipper domain was deleted indicated that this domain probably has the same function
in this protein36. 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 activation37.
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
MLK1 MLK2 MLK3 MLK4
DLK
LZK
ZAK
MKK4 MKK7
MKK3 MKK6
JNK
p38
Shc, p53, NFAT4,
c-Jun, ATF2, RNPK
MNK1/2, MEF2, CHOP10, Elk-1,
MSK1/2, MAPKAP-K2/3, eEF2K
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 zipper38.
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 pathway39. 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 activation40.
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 ACTIVATION 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
Nomenclature
Committee name
Synonyms
Gene sequences
Production in
mammals
Drosophila
melanogaster
homologue
Caenorhabditis
elegans homologue
Human GI:12005724
Epithelial cells
Slipper
GI:15554294
GI:17562526
Brain, skeletal
muscle, testes
Widely produced
GI:7293761
GI:17507127
MLK
MLK1
MAP3K9
MLK2
MAP3K10
MST
Human GI:4505263
MLK3
MAP3K11
SPRK
PTK1
Human GI:4505195
Mouse GI:11528494
Human α GI:17736729
Human β GI:17736731
MLK4α
MLK4β
DLK
DLK
MAP3K12
LZK
MAP3K13
ZAK
ZAKα
ZAKβ
ZPK
MUK
Human GI:21735552
Mouse GI:1083502
Rat GI:7446400
Human GI:4758696
Human α GI:7706601
Human β GI:19526767
Mouse α GI:10798808
Mouse β GI:10798810
MRK
MLTK
MLK7
ZAK
Unknown
Brain (also keratinocytes
and regenerating liver)
Widely produced
Widely produced
GI: 17554622
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.
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a
SH3
Kinase
LZ
CRIB
MLK1
1066
MLK2
954
Gly
MLK
MLK3
847
MLK4α
570
MLK4β
1036
DLK
DLK
859
LZK
SAM
966
ZAKα
ZAK
800
ZAKβ
455
b
DLKs
ZAKs
MLKs
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
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).
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-NUCLEOTIDEEXCHANGE FACTOR
A protein that facilitates the
exchange of GDP for GTP in the
nucleotide-binding pocket of a
GTP-binding protein.
666
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 zipper35. 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
| SEPTEMBER 2002 | VOLUME 3
SH3 domain with this function in a serine/threonine
kinase41. 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 kinases42–44. 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 SH3mediated 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 cascades48,49. 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
Rac50–52. 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 pathway51,52.
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 oligomerization37,39. 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
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(REF. 54), DLK37,54 and LZK61 have been shown to interact
GDP
Inactive
Cdc42
Kinase
SH3
Zipp
NH2
er
Pro
COOH
CRIB
GTP
Cdc42
Active
H2N
SH3
Kinase
Zipper
Pro
CRIB
COOH
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.
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.
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 switch37.
JIP2/IB2 binds JNK only very weakly54 but has been
recently shown to bind p38δ (REF. 55) and p38α in MLKmediated activation of the p38 MAPK pathway53. 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
65
KINESIN-dependent axonal transport . 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
Regulation of MLKs by phosphorylation
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.
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 277–281;
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 terminus59. 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).
DORSAL ECTODERM
The outer of the three
embryonic germ layers; this gives
rise to the entire central nervous
system.
JIPs and MLKs
Consistent with the ability of MLKs to activate the JNK
pathway, several MLKs, including MLK2 and MLK3
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In D. melanogaster, the JNK pathway regulates the
process of dorsal closure during embryogenesis68,69.
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 pathway28. 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 cytokines70,71. During
dorsal closure, dpp is expressed at the leading edge of the
epidermis72–75 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 closure28, which is consistent with a loss of
function of JNK and AP-1 in slpr-mutant embryos. The
VOLUME 3 | SEPTEMBER 2002 | 6 6 7
REVIEWS
Box 2 | JNK-interacting proteins
JIP1/IB1
JIP/IB
interacting proteins
RhoGEF
Tiam1
RasGRF1
ApoER2
APP
Megalin
LRP-1
Kinesin
Stimulus
Rac,
Cdc42
DLK,
LZK
MLK2,
MLK3
MKK7
JNK
Jun, ATF2
JIP2/IB2
Stimulus
Rac,
Cdc42
DLK,
MLK2
MKK7
JNK
Jun, ATF2
MLK3
MKK3
p38
Mef2C
MKK7
JNK
Jun, ATF2
JIP3
Rac,
Cdc42
MLK3
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),
Megalin110 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.
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.
668
second defined function for JNK signalling during dorsal
closure is the control of a stretched epithelial-cell morphology76,77. 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
fly78. 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 polarity79. 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
| SEPTEMBER 2002 | VOLUME 3
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 transformation80, which is
consistent with the evidence that the AP-1 transcription complex contributes to transformation and
tumorigenesis80,81. Wild-type MLK3 overproduction has
been shown to transform NIH3T3 fibroblasts, whereas
catalytically inactive MLK3 is not transforming82. 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 transformation83. 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
dRac1
Epithelial cell shape
Msn
MKKKK
Slpr
MLK
Hep
MKK7
Bsk
JNK
dJun
c-Jun/ATF2
Dpp
BMP4
Cell sheet movement
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.
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REVIEWS
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
properties112, protecting primary neurons in culture from neurotrophic-factorwithdrawal-induced apoptosis113. Furthermore, animal models of chemical-induced
neural toxicity have shown that CEP-1347 is an effective neuroprotectant in vivo114.
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.
Vav-stimulated IL-4 production86. 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 Tcell 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.
DOMINANT-NEGATIVE
A defective protein that retains
interaction capabilities and so
distorts or competes with
normal proteins.
Neuronal apoptosis. Deprivation of growth factors leads
to apoptosis of neurons in vivo and in vitro92,93. Several
studies have shown that c-Jun is required for neuronal
growth factor (NGF)-deprivation-induced apoptosis of
neurons94,95. Inhibition of c-Jun function using DOMINANT-NEGATIVE c-Jun or nuclear injection of anti-c-Jun
antibodies protects neurons from NGF-deprivationinduced death94,95. In addition to regulation of c-Jun,
JNKs probably regulate the activity of specific Bcl-2family proteins that control mitochondrial integrity and
cytochrome c release96,97.
Studies in neuronal systems indicate that MLK2,
MLK3 and DLK might be important MKKKs that
mediate trophic-factor-deprivation-induced neuronal
apoptosis98–101. Overexpression of MLK2, MLK3 or DLK
in PC12 cells induces apoptosis99. NGF-withdrawalinduced apoptosis of superior cervical ganglion (SCG)
sympathetic neurons requires the functional activity of
Cdc42 and activation of JNK100. When SCG neurons are
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© 2002 Nature Publishing Group
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) subunit100. 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 cells103. 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 huntingtin104,105. 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
VOLUME 3 | SEPTEMBER 2002 | 6 6 9
REVIEWS
a
b
PolyQ
Huntingtin
Aggregation
MLK2
(inactive)
MLK2
(increased activity)
JNK activation
JNK activation
Neuronal survival
Neuronal apoptosis
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 cells98. 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 proposed98 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.
1.
3.
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, 17355–17363 (1990).
Kyriakis, J. M. & Avruch, J. Mammalian mitogen-activated
protein kinase signal transduction pathways activated by
stress and inflammation. Physiol. Rev. 81, 807–869 (2001).
Derijard, B. et al. JNK1: a protein kinase stimulated by UV
670
| SEPTEMBER 2002 | VOLUME 3
2.
4.
5.
6.
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 transport106. 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.
light and Ha-Ras that binds and phosphorylates the c-Jun
activation domain. Cell 76, 1025–1037 (1994).
Macian, F., Lopez-Rodriguez, C. & Rao, A. Partners in
transcription: NFAT and AP-1. Oncogene 20, 2476–2489
(2001).
Wagner, E. F. AP-1 — introductory remarks. Oncogene 20,
2334–2335 (2001).
Toone, W. M., Morgan, B. A. & Jones, N. Redox control of
7.
8.
AP-1-like factors in yeast and beyond. Oncogene 20,
2336–2346 (2001).
Rana, A. et al. The mixed lineage kinase SPRK
phosphorylates and activates the stress-activated protein
kinase activator, SEK-1. J. Biol. Chem. 271, 19025–19028
(1996).
Hirai, S. et al. MST/MLK2, a member of the mixed lineage
kinase family, directly phosphorylates and activates SEK1,
www.nature.com/reviews/molcellbio
© 2002 Nature Publishing Group
REVIEWS
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32. Schlessinger, J. Cell signaling by receptor tyrosine kinases.
Cell 103, 211–225 (2000).
33. Luo, Z. et al. Oligomerization activates c-Raf-1 through a
Ras-dependent mechanism. Nature 383, 181–185 (1996).
34. Farrar, M. A., Alberol, I. & Perlmutter, R. M. Activation of the
Raf-1 kinase cascade by coumermycin-induced
dimerization. Nature 383, 178–181 (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, 7273–7279 (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, 190–195 (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, 3447–3458 (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, 16888–16896 (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, 32408–32415 (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, 27893–27900
(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, 45598–45603 (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, 602–609 (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, 757–775
(1997).
44. Xu, W., Harrison, S. C. & Eck, M. J. Three-dimensional
structure of the tyrosine kinase c-Src. Nature 385, 595–602
(1997).
45. Bar-Sagi, D. & Hall, A. Ras and Rho GTPases: a family
reunion. Cell 103, 227–238 (2000).
46. Ridley, A. J. Rho family proteins: coordinating cell
responses. Trends Cell Biol. 11, 471–477 (2001).
47. Takai, Y., Sasaki, T. & Matozaki, T. Small GTP-binding
proteins. Physiol. Rev. 81, 153–208 (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, 1137–1146 (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, 1147–1157 (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,
29071–29074 (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, 27225–27228 (1996).
an activator of c-Jun N-terminal kinase/stress-activated
protein kinase. J. Biol. Chem. 272, 15167–15173 (1997).
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, 11–15 (1998).
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, 7406–7412 (1998).
Merritt, S. E. et al. The mixed lineage kinase DLK utilizes
MKK7 and not MKK4 as substrate. J. Biol. Chem. 274,
10195–10202 (1999).
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, 67–74 (1997).
Tibbles, L. A. et al. MLK-3 activates the SAPK/JNK and
p38/RK pathways via SEK1 and MKK3/6. EMBO J. 15,
7026–7035 (1996).
Gotoh, I., Adachi, M. & Nishida, E. Identification and
characterization of a novel MAP kinase kinase kinase,
MLTK. J. Biol. Chem. 276, 4276–4286 (2001).
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, 13873–13882 (2002).
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, 701–710 (1993).
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, 15092–15100 (1994).
This paper first showed that a mixed lineage kinase
(MLK3) is a serine/threonine kinase.
Dorow, D. S. et al. Complete nucleotide sequence,
expression, and chromosomal localisation of human mixedlineage kinase 2. Eur. J. Biochem. 234, 492–500 (1995).
Katoh, M., Hirai, M., Sugimura, T. & Terada, M. Cloning and
characterization of MST, a novel (putative) serine/threonine
kinase with SH3 domain. Oncogene 10, 1447–1451 (1995).
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, 1745–1750 (1994).
Ezoe, K., Lee, S. T., Strunk, K. M. & Spritz, R. A. PTK1, a
novel protein kinase required for proliferation of human
melanocytes. Oncogene 9, 935–938 (1994).
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, 30808–30817 (1994).
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, 613–620
(1994).
Hirai, S. et al. Activation of the JNK pathway by distantly
related protein kinases, MEKK and MUK. Oncogene 12,
641–650 (1996).
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, 28622–28629 (1997).
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,
44–49 (1999).
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,
811–816 (2000).
Stronach, B. & Perrimon, N. Activation of the JNK pathway
during dorsal closure in Drosophila requires the mixed
lineage kinase, Slipper. Genes Dev. 16, 377–387 (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.
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, 123–137 (1990).
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,
1400–1403 (1990).
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, 539–544 (1991).
NATURE REVIEWS | MOLECUL AR CELL BIOLOGY
© 2002 Nature Publishing Group
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
This paper was the first to show a role for Rac and/or
Cdc42 in the activation of MLK3 and in MLK3mediated activation of the JNK pathway in cells.
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, 14231–14241 (2000).
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, 4073–4085 (2002).
This work shows that MLK3 can activate the p38
signalling pathway in a Tiam1/JIP2-dependent
fashion.
Yasuda, J. et al. The JIP group of mitogen-activated protein
kinase scaffold proteins. Mol. Cell. Biol. 19, 7245–7254
(1999).
This paper describes the association of MLKs with
the JIP scaffold proteins for the regulation of the JNK
pathway.
Schoorlemmer, J. & Goldfarb, M. Fibroblast growth factor
homologous factors are intracellular signaling proteins. Curr.
Biol. 11, 793–797 (2001).
Hoffman, G. R. & Cerione, R. A. Signaling to the Rho
GTPases: networking with the DH domain. FEBS Lett. 513,
85–91 (2002).
Van Aelst, L. & D’Souza-Schorey, C. Rho GTPases and
signaling networks. Genes Dev. 11, 2295–2322 (1997).
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, 1961–1967 (2001).
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, 5613–5624 (2002).
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,
10801–10810 (2001).
Ikeda, A. et al. Mixed lineage kinase LZK forms a functional
signaling complex with JIP-1, a scaffold protein of the c-Jun
NH2-terminal kinase pathway. J. Biochem. 130, 773–781
(2001).
Fan, G. et al. Dual leucine zipper-bearing kinase (DLK)
activates p46SAPK and p38MAPK but not ERK2. J. Biol. Chem.
271, 24788–24793 (1996).
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, 1030–1043
(2000).
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, 7539–7548
(1999).
Bowman, A. B. et al. Kinesin-dependent axonal transport is
mediated by the Sunday driver (SYD) protein. Cell 103,
583–594 (2000).
Verhey, K. J. et al. Cargo of kinesin identified as JIP
scaffolding proteins and associated signaling molecules. J.
Cell. Biol. 152, 959–970 (2001).
Whitmarsh, A. J. et al. Requirement of the JIP1 scaffold
protein for stress-induced JNK activation. Genes Dev. 15,
2421–2432 (2001).
Knust, E. Drosophila morphogenesis: movements behind
the edge. Curr. Biol. 7, R558–R561 (1997).
Noselli, S. JNK signaling and morphogenesis in Drosophila.
Trends Genet. 14, 33–38 (1998).
Spencer, F. A., Hoffmann, F. M. & Gelbart, W. M.
Decapentaplegic: a gene complex affecting morphogenesis
in Drosophila melanogaster. Cell 28, 451–461 (1982).
Affolter, M., Marty, T., Vigano, M. A. & Jazwinska, A. Nuclear
interpretation of Dpp signaling in Drosophila. EMBO J. 20,
3298–3305 (2001).
Glise, B. & Noselli, S. Coupling of Jun amino-terminal kinase
and Decapentaplegic signaling pathways in Drosophila
morphogenesis. Genes Dev. 11, 1738–1747 (1997).
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, 1728–1737 (1997).
Kockel, L. et al. Jun in Drosophila development: redundant
and nonredundant functions and regulation by two MAPK
signal transduction pathways. Genes Dev. 11, 1748–1758
(1997).
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
VOLUME 3 | SEPTEMBER 2002 | 6 7 1
REVIEWS
during dorsal closure. Genes Dev. 11, 1717–1727 (1997).
76. Glise, B., Bourbon, H. & Noselli, S. hemipterous encodes a
novel Drosophila MAP kinase kinase, required for epithelial
cell sheet movement. Cell 83, 451–461 (1995).
77. Riesgo-Escovar, J. R., Jenni, M., Fritz, A. & Hafen, E. The
Drosophila Jun-N-terminal kinase is required for cell
morphogenesis but not for DJun-dependent cell fate
specification in the eye. Genes Dev. 10, 2759–2768 (1996).
78. Stronach, B. E. & Perrimon, N. Stress signaling in
Drosophila. Oncogene 18, 6172–6182 (1999).
79. Vidal, S. et al. Mutations in the Drosophila dTAK1 gene
reveal a conserved function for MAPKKKs in the control of
Rel/NF-κB-dependent innate immune responses. Genes
Dev. 15, 1900–1912 (2001).
80. Behrens, A., Jochum, W., Sibilia, M. & Wagner, E. F.
Oncogenic transformation by Ras and Fos is mediated by cJun N-terminal phosphorylation. Oncogene 19, 2657–2663
(2000).
81. Vogt, P. K. Jun, the oncoprotein. Oncogene 20, 2365–2377
(2001).
82. Hartkamp, J., Troppmair, J. & Rapp, U. R. The JNK/SAPK
activator mixed lineage kinase 3 (MLK3) transforms NIH 3T3
cells in a MEK-dependent fashion. Cancer Res. 59,
2195–2202 (1999).
83. Lambert, J. M. et al. Role of MLK3-mediated activation of
p70 S6 kinase in Rac1 transformation. J. Biol. Chem. 277,
4770–4777 (2002).
84. Hoffmeyer, A. et al. Different mitogen-activated protein
kinase signaling pathways cooperate to regulate tumor
necrosis factor α gene expression in T lymphocytes. J. Biol.
Chem. 274, 4319–4327 (1999).
85. Hoffmeyer, A. et al. The GABP-responsive element of the
interleukin-2 enhancer is regulated by JNK/SAPK-activating
pathways in T lymphocytes. J. Biol. Chem. 273,
10112–10119 (1998).
86. Hehner, S. P. et al. Vav synergizes with protein kinase Cθ to
mediate IL-4 gene expression in response to CD28 costimulation in T cells. J. Immunol. 164, 3829–3836 (2000).
87. Hehner, S. P. et al. Mixed-lineage kinase 3 delivers
CD3/CD28-derived signals into the IκB kinase complex.
Mol. Cell. Biol. 20, 2556–2568 (2000).
88. Lee, F. S., Hagler, J., Chen, Z. J. & Maniatis, T. Activation of
the IκBα kinase complex by MEKK1, a kinase of the JNK
pathway. Cell 88, 213–222 (1997).
89. Lee, F. S., Peters, R. T., Dang, L. C. & Maniatis, T. MEKK1
activates both IκB kinase α and IκB kinase β. Proc. Natl
Acad. Sci. USA 95, 9319–9324 (1998).
90. Zhao, Q. & Lee, F. S. Mitogen-activated protein kinase/ERK
kinase kinases 2 and 3 activate nuclear factor-κB through
IκB kinase-α and IκB kinase-β. J. Biol. Chem. 274,
8355–8358 (1999).
91. Ninomiya-Tsuji, J. et al. The kinase TAK1 can activate the
NIK-I κB as well as the MAP kinase cascade in the IL-1
signalling pathway. Nature 398, 252–256 (1999).
672
92. Putcha, G. V., Deshmukh, M. & Johnson, E. M., Jr.
BAX translocation is a critical event in neuronal apoptosis:
regulation by neuroprotectants, Bcl-2, and caspases.
J. Neurosci. 19, 7476–7485 (1999).
93. Deckwerth, T. L. & Johnson, E. M., Jr. Temporal analysis of
events associated with programmed cell death (apoptosis)
of sympathetic neurons deprived of nerve growth factor.
J. Cell. Biol. 123, 1207–1222 (1993).
94. Estus, S. et al. Altered gene expression in neurons
during programmed cell death: identification of c-Jun as
necessary for neuronal apoptosis. J. Cell. Biol. 127,
1717–1727 (1994).
95. Ham, J. et al. A c-Jun dominant negative mutant protects
sympathetic neurons against programmed cell death.
Neuron 14, 927–939 (1995).
96. Tournier, C. et al. Requirement of JNK for stress-induced
activation of the cytochrome c-mediated death pathway.
Science 288, 870–874 (2000).
97. Eilers, A. et al. Role of the Jun kinase pathway in the
regulation of c-Jun expression and apoptosis in sympathetic
neurons. J. Neurosci. 18, 1713–1724 (1998).
98. Liu, Y. F., Dorow, D. & Marshall, J. Activation of MLK2mediated signaling cascades by polyglutamine-expanded
huntingtin. J. Biol. Chem. 275, 19035–19040 (2000).
99. Xu, Z. et al. The MLK family mediates c-Jun N-terminal
kinase activation in neuronal apoptosis. Mol. Cell. Biol. 21,
4713–4724 (2001).
100. Mota, M., Reeder, M., Chernoff, J. & Bazenet, C. E.
Evidence for a role of mixed lineage kinases in neuronal
apoptosis. J. Neurosci. 21, 4949–4957 (2001).
101. Harris, C. A. et al. Inhibition of the c-Jun N-terminal kinase
signaling pathway by the mixed lineage kinase inhibitor
CEP-1347 (KT7515) preserves metabolism and growth of
trophic factor-deprived neurons. J. Neurosci. 22, 103–113
(2002).
102. Yang, D. D. et al. Absence of excitotoxicity-induced
apoptosis in the hippocampus of mice lacking the Jnk3
gene. Nature 389, 865–870 (1997).
103. Savinainen, A. et al. Kainate receptor activation induces
mixed lineage kinase-mediated cellular signaling cascades
via postsynaptic density protein 95. J. Biol. Chem. 276,
11382–11386 (2001).
104. Mulle, C. et al. Altered synaptic physiology and reduced
susceptibility to kainate-induced seizures in GluR6-deficient
mice. Nature 392, 601–605 (1998).
105. Kornau, H. C., Schenker, L. T., Kennedy, M. B. & Seeburg,
P. H. Domain interaction between NMDA receptor subunits
and the postsynaptic density protein PSD-95. Science 269,
1737–1740 (1995).
106. Akbarzadeh, S. et al. Mixed lineage kinase 2 interacts with
clathrin and influences clathrin-coated vesicle trafficking. J.
Biol. Chem. (in the press).
107. Prowse, C. N. & Lew, J. Mechanism of activation of ERK2
by dual phosphorylation. J. Biol. Chem. 276, 99–103 (2001).
| SEPTEMBER 2002 | VOLUME 3
108. Meyer, D., Liu, A. & Margolis, B. Interaction of c-Jun aminoterminal kinase interacting protein-1 with p190 rhoGEF and
its localization in differentiated neurons. J. Biol. Chem. 274,
35113–35118 (1999).
109. Stockinger, W. et al. The reelin receptor ApoER2 recruits
JNK-interacting proteins-1 and -2. J. Biol. Chem. 275,
25625–25632 (2000).
110. Gotthardt, M. et al. Interactions of the low density lipoprotein
receptor gene family with cytosolic adaptor and scaffold
proteins suggest diverse biological functions in cellular
communication and signal transduction. J. Biol. Chem. 275,
25616–25624 (2000).
111. Matsuda, S. et al. c-Jun N-terminal kinase (JNK)-interacting
protein-1b/islet-brain-1 scaffolds Alzheimer’s amyloid
precursor protein with JNK. J. Neurosci. 21, 6597–6607
(2001).
112. Kaneko, M. et al. Neurotrophic 3,9-bis[(alkylthio)methyl]and-bis(alkoxymethyl)-K-252a derivatives. J. Med. Chem.
40, 1863–1869 (1997).
113. Maroney, A. C. et al. Motoneuron apoptosis is blocked by
CEP-1347 (KT 7515), a novel inhibitor of the JNK signaling
pathway. J. Neurosci. 18, 104–111 (1998).
114. Saporito, M. S., Brown, E. M., Miller, M. S. & Carswell, S.
CEP-1347/KT-7515, an inhibitor of c-Jun N-terminal kinase
activation, attenuates the 1-methyl-4-phenyl
tetrahydropyridine-mediated loss of nigrostriatal
dopaminergic neurons in vivo. J. Pharmacol. Exp. Ther. 288,
421–427 (1999).
115. Maroney, A. C. et al. CEP-1347 (KT7515), a semisynthetic
inhibitor of the mixed lineage kinase family. J. Biol. Chem.
276, 25302–25308 (2001).
This paper describes CEP-1347, which inhibits the
mixed-lineage kinases (MLKs). CEP-1437 might be
useful in the treatment of neurodegenerative diseases
in humans.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
ApoER2 | APP | ASK1 | ATF2 | Bcl-2 | Bmp4 | Cdc42 | DLK |
eEF2K | ERK5 | FHF1 | GluR6 | huntingtin | IB1 | IB2 | IL-2 | IL-4 |
JIP1 | JIP2 | JIP3 | JNK1 | JNK3 | Jun | LRP-1 | LZK | MEF2 |
Megalin | MEKK1 | MEKK4 | MKK4 | MKK7 | MLK1 | MLK2 |
MLK3 | Mos | MSK | p38α | p38δ | Raf-1 | RasGRF1 | RhoGEF |
TAK1 | TC10 | Tiam1 | TNFα | Tpl | Vav | ZAK
Flybase: http://flybase.bio.indiana.edu/
Bsk | dJun | Dpp | dRac | dTAK | Hep | kayak | Msn | Slipper |
Sunday driver
ProSite: http://ca.expasy.org/prosite/
CRIB | SAM | SH3
Access to this interactive links box is free online.
www.nature.com/reviews/molcellbio
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