Gene Ther Mol Biol Vol 4,
83-98. December 1999.
Mammalian c-Jun
N-terminal kinase pathway and STE20-related kinases
Review Article
Yi-Rong Chen and
Tse-Hua Tan*
Department of Immunology, Baylor
College of Medicine, Houston, Texas 77030, USA
______________________________________________________________________________________
*Correspondence: Department of Immunology, M929, One Baylor Plaza,
Houston, TX 77030; Tel: (713) 798-4665; Fax: (713) 798-3033; E-mail: ttan@bcm.tmc.edu
Key
words: c-Jun, N-terminal Kinase,
STE20-related Kinases, JNK signaling, MEKK1, MAPK
Received: 6 August, 1999; accepted 11 October,
1999
Summary
The
c-Jun N-terminal kinases (JNKs) belong to a subgroup of mitogen-activated
protein kinases (MAPKs) that are activated by environmental stress,
proinflammatory cytokines, and mitogenic stimuli in mammalian cells. Studies on
the JNK pathway in mammalian cells demonstrate that JNK regulates the
transcriptional activities of many transcription factors, and that JNK is
required for the regulation of cell proliferation and apoptosis. Studies on jnk-deficient mice reveal that JNK is involved in the
response to immunological stimuli and in embryonic morphogenesis. JNK, as other
MAPKs, is regulated by a kinase cascade. JNK activation is mediated by dual
phosphorylation on the motif, Thr-Pro-Tyr. To date, this phosphorylation is
known to be mediated by the MAPK kinases (MAP2Ks), MKK4 and MKK7. MKK4 and MKK7
are activated by MEKK1 and other MAPK kinase kinases (MAP3Ks). The MAPK kinase
kinase kinases (MAP4Ks) including HPK1, GCK, and homologous kinases, which have
a kinase domain related to yeast STE20, can activate the JNK signaling cascade.
These mammalian STE20-related MAP4Ks may be involved in integrating various
stimuli to the JNK cascade. The signaling specificity of mammalian JNK pathway
may be controlled by scaffold proteins that interact with kinases at different
levels in the pathway.
I.
Introduction
Mitogen-activated
protein kinases (MAPKs) are important mediators for intracellular signaling in
cells (Schaeffer and Weber, 1999). Mammalian MAPKs consist of three major groups including extracellular
signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs, also known as
stress-activated protein kinases, SAPKs), and p38-MAPKs (Schaeffer
and Weber, 1999). All of MAPKs share a common character: they are activated by
phosphorylation at a Thr-X-Tyr motif (X is Glu in ERKs, Pro in JNKs, and Gly in
p38-MAPKs) in kinase subdomain VIII (Schaeffer
and Weber, 1999). The major targets for MAPK kinases are transcription factors that
regulate gene expression. MAPKs are involved in signaling induced by various
extracellular or intracellular stimuli. Currently, the JNK pathway is one of
the known cellular signaling pathways that respond to the most diverse stimuli (Ip
and Davis, 1998; Schaeffer and Weber, 1999). JNK is activated by mitogenic signals such as epidermal growth factor (Minden
et al., 1994b), lymphocyte activation signals (Su
et al., 1994; Sakata et al., 1995; Berberich et al., 1996; Chen et al., 1996a; Chen et al., 1996b), and oncogenic Ras (Derijard
et al., 1994). JNK is also activated by pro-inflammatory cytokines (TNF-a and IL-1) (Kyriakis
et al., 1994; Sluss et al., 1994), lipopolysaccharide (Hambleton
et al., 1996), G protein-coupled receptors (Collins
et al., 1996; Coso et al., 1996), shear stress (Li et al., 1996), osmotic shock (Galcheva-Gargova et al., 1994), protein synthesis inhibitors (Kyriakis
et al., 1994), and apoptotic stimuli such as growth factor withdrawal (Xia
et al., 1995), heat shock (Kyriakis et al., 1994; Zanke et al.,
1996), ceramides (Westwick et al., 1995), DNA-damaging chemicals (Saleem
et al., 1995; Zanke et al., 1996), UV radiation (Derijard et al., 1994; Chen et al.,
1996b; Zanke et al., 1996), and g radiation (Kharbanda
et al., 1995; Chen et al., 1996a; Chen et al., 1996b). The diversity of JNK-activating stimuli imply that mammalian cells may
be equipped with multiple upstream regulators that link various cellular
signals to the JNK pathway, and the accumulated experimental evidence proves
that is the case. To date, the JNK pathway consists of JNKs and various MAP2Ks,
MAP3Ks, and MAP4Ks (Figure 1). The subtle regulation of the JNK
pathway by its regulators in conjunction with other signaling pathways may
allow JNK to regulate a variety of cellular functions. In this review, we will
discuss the known components in the JNK pathway and how the emerging mammalian
scaffold proteins may control signaling diversity and specificity in this
signaling pathway.
II. c-Jun N-terminal kinases
The
human JNKs are encoded by three genes jnk1, jnk2, and jnk3 (Derijard
et al., 1994; Kallunki et al., 1994; Sluss et al., 1994; Gupta et al., 1996). The corresponding genes have also been identified in rats (Kyriakis
et al., 1994). JNK3 is preferentially expressed in neuronal tissues, while JNK1 and
JNK2 are widely expressed in many tissues. Ten isoforms of JNK, generated by
alternative splicing of the transcripts from the three genes, have been
identified (Gupta et al., 1996). The protein products of the JNK isoforms have molecular weights of 46
kDa or 55 kDa. The 55 kDa JNK isoforms contain a C-terminal extension, a result
of alternative splicing, which distinguishes them from the 46 kDa isoforms (Gupta
et al., 1996). No apparent functional differences exist among the 46 kDa and 55 kDa
isoforms encoded by the same JNK gene (Gupta
et al., 1996). An additional alternative splicing exists in the kinase domains of
JNK1 and JNK2, but not in JNK3 (Gupta
et al., 1996). The alternative splicing in the kinase domains of JNK1 and JNK2
changes the specificity of interaction between JNKs and their substrates (Gupta
et al., 1996), suggesting that JNK isoforms may target different substrates in
vivo. The JNK binding sites are
different from the sites of phosphorylation on the substrates (Kallunki
et al., 1996). Deletion of the binding site prevents phosphorylation of the substrate
by JNK (Kallunki et al., 1996). However, a substrate lacking a JNK-binding site can also be
phosphorylated through association with a protein containing the JNK-binding
region (Kallunki et al., 1996).
The
known substrates for JNK family members include the transcription factors c-Jun
(Hibi et al., 1993; Derijard et al.,
1994; Kyriakis et al., 1994;
Gupta et al., 1996), JunD (Gupta et al., 1996), ATF-2 (Gupta et al., 1995; van Dam et al.,
1995; Whitmarsh et al., 1995), ATFa (Bocco et al., 1996), Elk-1 (Cavigelli et al., 1995; Whitmarsh et al., 1995; Zinck et al., 1995), Sap-1a (Janknecht and Hunter, 1997), GABPa, GABPb (Hoffmeyer
et al., 1998), and the tumor suppressor p53 (Milne
et al., 1995; Alder et al., 1997). Generally, phosphorylation of these factors by JNK increases their
transcriptional activity.
The
physiological functions of JNK have been examined by genetic analysis. The jnk1-/-, jnk2-/-, and jnk3-/- single mutant mice have no global
abnormality (Yang et al., 1997b; Dong et al.,
1998; Yang et al., 1998). The T cells in jnk1-/- and jnk2-/-
mice preferentially differentiate into Th2 rather than Th1 cells (Dong
et al., 1998; Yang et al., 1998). The jnk1-/- T
cells also hyper-proliferate and exhibit decreased activation-induced apoptosis
(Dong et al., 1998). Excitotoxicity-induced apoptosis in the hippocampus is absent in jnk3-/- mice in comparison to normal mice (Yang
et al., 1997b). The jnk1/jnk3 and
jnk2/jnk3 deficient mice also
develop normally (Kuan et al., 1999); however, jnk1/jnk2
deficient mice are embryonically lethal and have severe dysregulation of
apoptosis in the brain (Kuan et al., 1999). These results indicate that JNK1 and JNK2 may have overlapping
functions, and are important in regulation of immune response and embryonic
development. JNK3 may have its unique functions in the neuronal tissues. These
studies also provide animal models which support the accumulated evidence on
the role of JNK in apoptotic signaling in mammalian cells (Ip
and Davis, 1998).
III.
MAP2Ks in the JNK pathway
The
activation of JNK is dependent on the phosphorylation on Thr-183 and Tyr-185.
MKK4 (also known as SEK1 or JNKK1) is a physiological activator of JNK (Sanchez
et al., 1994; Derijard et al., 1995; Lin et al., 1995). MKK4 phosphorylates and activates JNK in vitro and in vivo (Sanchez
et al., 1994; Derijard et al., 1995; Lin et al., 1995). However, recombinant wild-type JNK proteins are phosphorylated at Tyr,
Ser and Thr residues in the presence of recombinant MKK4, whereas a
kinase-inactive JNK is phosphorylated predominantly on Tyr (Sanchez
et al., 1994). This suggests that recombinant MKK4 does not have apparent dual
specificity toward JNK. It is possible that the phosphorylation on Thr-183 is
caused by the proline-directed kinase activity of JNK itself, occurring after
MKK4-mediated Tyr-phosphorylation. Another possibility is that MKK4 obtains
dual-specific kinase activity only after activation by upstream kinases. Two
isoforms of MKK4 have been reported through the differential usage of
translation initiation sites (Derijard
et al., 1995; Lin et al., 1995).
MKK4
has been found to be mutated or deleted in some tumor cells, suggesting that it
may be a tumor suppressor gene (Teng
et al., 1997; Su et al., 1998). Homologous deletion in mkk4 genes is embryonically lethal in mice, indicating that MKK4 is essential
for embryonic development (Nishina et al., 1997; Yang et al.,
1997a). Studies in mkk4-/-/rag-/- chimaeric mice reveal that MKK4 protects thymocytes
from apoptosis mediated by CD95 and CD3 (Nishina
et al., 1997), and is required for maintenance of a normal peripheral lymphoid
compartment but not for lymphocyte development (Swat
et al., 1998). Mkk4-/- T
cells derived from mkk4-/-/rag-/- chimaeric mice are defective in heat shock and
anisomycin-induced JNK activation, but normal in osmotic shock-induced JNK
activation (Nishina et al., 1997). These results indicate that MKK4 is one but not the only activator of
JNK in mammalian cells.
Recently,
a novel kinase MKK7 (also named as JNKK2) has been cloned and found to
specifically activate JNK, but not p38-MAPK or ERK (Moriguchi
et al., 1997; Tournier et al., 1997; Wu et al., 1997; Yao et al., 1997). MKK7 is related to MKK4 and belongs to the mammalian MAPK kinase
superfamily (Tournier et al., 1997; Yao et al., 1997). MKK7 is also closely related to the Drosophila protein kinase hemipterous (HEP) (Tournier
et al., 1997; Yao et al., 1997), which is the activator of Drosophila JNK (DJNK).
Both
MKK4 and MKK7 are widely expressed in human and murine tissue, whereas the
relative abundance of each MKK differs among tissues (Tournier
et al., 1997; Yao et al., 1997). Both MKK4 and MKK7 mediate signals from the same panel of
extracellular stimuli (Wu et al., 1997); however, studies show that they are preferentially activated by
different MAP3Ks (Hirai et al., 1998; Merritt et al.,
1999; Tournier et al., 1999). Furthermore, the MKK7 gene can encode six isoforms of protein products
through alternative splicing of the mRNA transcripts (Tournier
et al., 1999). These MKK7 isoforms respond differently to extracellular stimuli and
upstream kinases (Tournier et al., 1999). The differential regulation of MKK4 and MKK7 isoforms by their
upstream activators needs to be further examined.
IV. MAP3Ks in the JNK pathway
Multiple
upstream MAPK/ERK kinase kinases or MAP kinase
kinase kinases (MEKK or MAP3Ks) have been reported to activate
the JNK pathway via MKK4 and/or MKK7 (Figure 1). These MEKK-like kinases include MEKK1-4,
ASK1/MAPKKK5, MAPKKK6, TAK1, Tpl-2/Cot, MLK2/MST, MLK3/SPRK/PTK1, MUK/DLK/ZPK,
and LZK.
A. MEKKs
MEKK1
is the first identified MAP3K that activates JNK (Minden
et al., 1994a; Yan et al., 1994). MEKK1 was cloned on the basis of its homology with the yeast STE11 and
Byr2 kinases (Lange-Carter et al., 1993; Xu et al., 1996). To date, four kinases have been cloned and named MEKK1-4 (Lange-Carter
et al., 1993; Blank et al., 1996; Gajewski and Thompson, 1996; Xu et al., 1996; Ellinger-Ziegelbauer et al., 1997; Gerwins et al., 1997; Takekawa et al., 1997). The four MEKKs (ranging from 69.5-195 kDa in size) have homologous
kinase domains in the C-termini of the proteins; however, their N-terminal
domains have little homology. MEKK1 and MEKK4 can interact with GTP-binding
proteins Cdc42 and Rac (Fanger et al., 1997; Gerwins et al., 1997). MEKK1 also binds to Ras in a GTP-dependent manner (Russell
et al., 1995). All four MEKKs (MEKK1-4) activate the JNK pathway (Lange-Carter
et al., 1993; Blank et al., 1996; Gajewski and Thompson, 1996; Xu et al., 1996; Ellinger-Ziegelbauer et al., 1997; Gerwins et al., 1997; Takekawa et al., 1997). Besides the JNK pathway, MEKKs also regulate other cellular signaling
pathways. MEKK1, MEKK2, and MEKK3 activate the ERK pathway (Lange-Carter
et al., 1993; Blank et al., 1996; Ellinger-Ziegelbauer et al., 1997), and also activate the NF-kB through the IkB
kinases (IKKs) (Lee et al., 1997; Zhao and Lee, 1999). MEKK3 and MEKK4 have been shown to activate the p38-MAPK pathway
through MKK6 (Takekawa et al., 1997; Deacon and Blank, 1999).
B.
TAK1
TGF-b activated kinase 1 (TAK1) was
identified by its ability to rescue STE11 mutants in S cerevisiae (Yamaguchi
et al., 1995). TAK1 is a 579 amino acid protein with the kinase domain in its
N-terminus (Yamaguchi et al., 1995). The C-terminal region has no distinct domain structures but interacts
with TAK1 binding protein (TAB) 1 and 2 (Yamaguchi
et al., 1995; Shibuya et al., 1996). Association of TAK1 and TAB1 enhances the kinase activity of TAK1
