From the
The closely related MAP kinases,
Because no other MEK substrates have
been identified, MEKs are viewed as dedicated kinases that
phosphorylate only the MAP kinases. Kinases related to ERK1 and ERK2,
in spite of retaining a similar arrangement of activating
phosphorylation sites (Fig. 1), are poor in vitro substrates of MEK1(27) . Thus, the marked specificity of
MEKs contributes to the selective activation of their downstream
targets.
Phosphorylation by MEK on two sites is required for MAP
kinase activation. The two activating phosphorylation sites, a tyrosine
and a threonine (Tyr-185 and Thr-183 of ERK2, Fig. 2and 3), lie
1 residue apart on the MAP kinases (49) in the phosphorylation
lip. In vivo and in vitro, phosphorylation of
tyrosine precedes phosphorylation of threonine(50, 51) ,
although phosphorylation of either residue can occur in the absence of
the other(52, 53) . Because both tyrosine and threonine
phosphorylations are required to activate the MAP kinases, phosphatases
that remove phosphate from either site will inactivate them. Certain
dual specificity phosphatases selectively inactivate MAP kinases by
dephosphorylating both sites (reviewed in Ref. 54).
In cAPK, a phosphothreonine residue located in the phosphorylation
lip interacts with basic residues, one of which is located in the
N-terminal domain, to stabilize the closed domain structure. Similar
interactions are likely to stabilize the closed state of ERK2. A domain
rotation within ERK2 would bring homologous basic residues, including
Arg-65 in the N-terminal domain (Fig. 3), into position to bind
the phosphate group on Thr-183.
The phosphorylation lip, which
contains the Thr-183 and Tyr-185 phosphorylation sites, blocks access
of substrates to the active site. The side chain of Tyr-185 lies buried
near the active site, and its main chain occupies the substrate binding
site. A local conformational change occurs upon phosphorylation,
displacing Tyr-185 and creating a lip structure compatible with high
catalytic activity.
The
findings of these structural studies have important implications for
the regulation of ERK2 and related kinases. The disorder observed in
the mutants indicates that the phosphorylation lip is not a stable
structure and suggests that modest amounts of binding energy are
sufficient to induce conformational changes in this region(57) .
The phosphorylation lip must acquire a different conformation to be
phosphorylated by MEK and, after phosphorylation, another conformation
that is compatible with high catalytic activity. Tyr-185 is buried in
the low activity conformation of ERK2, yet in the activation process it
is phosphorylated first. The binding energy provided by interaction of
ERK2 with MEK may be sufficient to dislodge Tyr-185 from its buried
position allowing it access to the active site of MEK.
The three recessive FUS3 mutations are buried in the N-terminal
domain (Fig. 3). These are the least likely to affect
interactions with other molecules. In ERK2 these residues are in close
proximity and are involved in packing the
The dominant mutations lie
on the surface and could involve interactions with other molecules.
Here we have analyzed other possible effects of these dominant
mutations. One FUS3 mutation (His-230 of ERK2) results in a loss of
charge on the substrate binding face near the putative phosphotyrosine
binding site and most likely affects interactions with substrates or
regulators. A second FUS3 mutant, Glu-58 of ERK2, lies in a part of the
structure unique to ERKs that replaces the B helix of cAPK. This
region, near the putative phosphothreonine binding site, may be
important for interactions in the activated structure(57) . A
Val to Leu mutation (V171L in ERK2) in FUS3 lies at the beginning of
the phosphorylation lip. The mutation may release steric constraint
associated with a
The
gain-of-function mutations in FUS3 (59) and the rolled gene
product (58) are listed with the corresponding residue numbers in ERK2
and are grouped as dominant or recessive.
We would like to thank Elliott Ross, Jessie Hepler,
Meg Phillips, and Megan Robinson (UT Southwestern) for critical reading
of the manuscript and suggestions about figures, members of the
Goldsmith and the Cobb laboratories for their efforts and insights, and
Jo Hicks for preparation of the manuscript.
INTRODUCTION
Control of the MAP Kinase Cascade
Parallel MAP Kinase Pathways
Activation and Inactivation of the MAP Kinases
Three-dimensional Structure of ERK2
MAP Kinase Mutants and Structural Implications
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)extracellular signal-regulated protein kinases 1 and
2 (ERK1 and ERK2), are ubiquitous components of signal transduction
pathways. ERK1 and ERK2 are activated by diverse extracellular stimuli
and by protooncogene products that induce proliferation or enhance
differentiation (reviewed in Refs. 1 and 2). MAP kinase
phosphorylations have an impact on processes in the cytoplasm, the
nucleus, the cytoskeleton, and the membrane. The variety of signals
that conscript the MAP kinase pathway demonstrates that this cascade
serves a myriad of purposes, and the consequences of its activation
will depend on cellular context. Because of the pleiotropic potential
of these kinases, their activation needs to be tightly controlled. This
review discusses the complexity of upstream regulation of the MAP
kinase pathway, parallel cascades, and concepts that are likely to
apply to many MAP kinase family members developed from analysis of the
crystal structure of ERK2.
Receptor Tyrosine Kinases
The best understood
means of activating the MAP kinase pathway (reviewed in Refs. 1 and 3)
is that used by receptor tyrosine kinases. Ligands cause receptors to
autophosphorylate on tyrosine residues; the phosphotyrosine residues of
autophosphorylated receptors then bind the SH2 domains of adapters,
such as Grb2 (growth factor receptor-bound protein 2). The adapters
recruit guanine nucleotide exchange factors with proline-rich SH3
domain-binding sites to the membrane in proximity to the isoprenylated
small G proteins they activate. Exchange factors promote the
association of Ras with GTP. The GTP-bound form of Ras binds the
protein kinases Raf-1 and B-Raf, thereby targeting one or both Raf
isoforms to the membrane where Raf protein kinase activity is
increased. MAP kinase kinases 1 and 2 (MKK), also called MAP/ERK
kinases (MEK)(4, 5, 6) , are phosphorylated and
activated by Raf-1 and B-Raf and are the upstream activators of the MAP
kinases. Receptor tyrosine kinases have also been reported to activate
the cascade in rat fibroblasts via a Ca-dependent but
protein kinase C (PKC)- and Ras-independent pathway(7) .
Receptors that do not contain intrinsic tyrosine kinase activity but
that harbor sites for tyrosine phosphorylation may also activate the
cascade via association of phosphotyrosine residues on the receptors or
the activated tyrosine kinases with adapters(8) .
G Protein-coupled Receptors
The MAP kinase cascade
can also be activated by certain heterotrimeric G
proteins(9, 10) . Most require Ras and are believed to
exploit the steps described for tyrosine kinases, but Ras-independent
activation has been reported (9-12).
PKC
PKC is used by many receptors types to
regulate the MAP kinase pathway, alone or with other
mechanisms(13, 14) , and may act at several steps in the
cascade. The effects of phorbol esters are Ras-dependent in PC12 cells (11) and Jurkat cells (15) but Ras-independent in
fibroblasts(16) , consistent with multiple sites of action of
PKC. PKC may directly activate Raf-1(17) , but mutation of the
site phosphorylated by PKC does not interfere with activation of Raf by
many stimuli including phorbol ester(18) . Other sites of action
of PKC are likely to be either farther upstream or at the level of MAP
kinase inactivation.
Regulation and Specificity of MEKs
All known
signaling pathways are believed to use the two dual specificity protein
kinases MEK1 and MEK2 to phosphorylate and activate MAP
kinase(6) . MEK1 and -2 are activated not only by Raf-1 and
B-Raf (19, 20) but also by the Mos protooncogene
product(21, 22) , MEK kinase 1
(MEKK1)(
)(23) , and other probably
distinct, growth factor-stimulated activities(24, 25) .
The mechanisms controlling MEKK1 are unknown, although Ras may be
required(26) . In oocytes, Mos is believed to be controlled by
its synthesis and degradation.
Figure 1:
Alignment of phosphorylation
lip sequences of ERK/MAP kinase family members. ERK1, ERK2, ERK3, HOG1,
and JNK1 are mammalian enzymes. MPK1, KSS1, and FUS3 are from budding
yeast and SPK1 is from fission yeast. The lip sequence of the Drosophila rolled gene product is identical to ERK1. Dots indicate identities; dashes indicate deletions. The
phosphorylation sites are denoted by an asterisk. The 17
residues disordered in the ERK2 Tyr-185 mutants extend from the Asp
preceding the FUS3 insertion to the conserved Arg preceding the
sequence WYRAPE.
The ERK Protein Kinase Subfamily
ERK1 and ERK2
were the first of the ERK/MAP kinase subfamily to be
cloned(28, 29, 30) . Other related mammalian
enzymes have been detected including: two ERK3
isoforms(29, 31, 32) , ERK4(33) , Jun
N-terminal kinases/stress-activated protein kinases
(JNK/SAPKs)(34, 35) , p38/HOG1(36, 37) ,
and p57 MAP kinases (38). The presence of at least six MAP kinases in
yeast suggests that there are more in mammals. Sequence signatures of
the ERK family are most apparent in subdomains V, VII, IX, and XI (39) and include a long insert between subdomains X and XI. The
sequences of the regulatory phosphorylation lip (surface loop between
subdomains VII and VIII, see below) are also related, with conserved
dual phosphorylation sites (Fig. 1).
The MEK Protein Kinase Subfamily
Several
laboratories have uncovered additional MEKs, for which some substrates
have been defined. A mammalian homolog of a MEK first identified in Xenopus (40) is called MAP kinase kinase 4 (MKK4), SAPK/ERK
kinase (SEK), or JNK kinase (JNKK), because in vitro it
activates JNK/SAPK and p38/HOG1 (27, 41, 42) but
not ERK1 or ERK2(27) . Yet another newly cloned MEK, MKK3,
selectively activates p38/HOG1 in transfected cells (42).
MAP Kinase Modules Mediate Distinct Signaling
Events
The consistent appearance of 3-kinase cascades, first
recognized in yeast, has engendered the concept of distinct MAP kinase modules(43) (Fig. 2). The
modules convey information to target effectors and coordinate incoming
information from parallel signaling pathways. A canonical MAP kinase
module consists of three protein kinases that act sequentially within
one pathway: a MEKK (a MEK activator), a MEK (a MAP kinase activator),
and a MAP kinase (any ERK homolog). Raf-1 (or B-Raf), MEK1 (or MEK2),
and ERK2 (or ERK1) constitute the best known mammalian MAP kinase
module. The second mammalian MAP kinase module to be defined apparently
consists of MEKK1, MKK4 (the MEK), and SAPK/JNK (the
ERK)(44, 45) . MKK3 and p38/HOG1 appear to define yet a
third cascade. MEKK1 can activate MKK4, MKK3, MEK1, and
MEK2(27, 46, 47) , suggesting that MEKKs have a
broader substrate specificity than MEKs. Thus, enzymatic specificity of
the MEK, not the MEKK, may limit cross-cascade noise. Additional
contributions to specificity may be provided through subcellular
targeting of the enzymes(48) .
Figure 2:
Mammalian MAP kinase modules. There are
multiple MAP kinase modules in mammalian cells. Three that can be
distinguished at present are the MAP kinase pathway, the JNK/SAPK
pathway, and the HOG/p38 pathway. A MAP kinase module is a 3-kinase
cascade consisting of an ERK or MAP kinase, which is activated by a MEK
or MAP kinase kinase that in turn is activated by a MAP kinase kinase
kinase or MEKK.
General Features
The three-dimensional structure
of the unphosphorylated form of ERK2 provides a picture of its low
activity state(55) . It consists of a smaller N-terminal domain
and a larger C-terminal domain connected by a linker or crossover
region (Fig. 3), similar to other protein kinases. ATP binds at a
site deep in the catalytic cleft, formed at the interface between the
two domains, whereas protein substrates bind on the surface.
Figure 3:
The positions of gain-of-function
mutations of MAP kinase mapped onto the three-dimensional structure of
ERK2. Red denotes oxygen atoms; all other atoms (C, N, S, H)
except as noted below are shown in purple. Yellow indicates Thr-183 and Tyr-185, the phosphorylated side chains; darkblue denotes basic residues likely to be
involved in binding the phosphorylated side chains. Brightgreen and turquoise indicate dominant and
recessive mutations, respectively. The residue numbers of ERK2
corresponding to the mutations are indicated. A, standard
kinase view (profile); B, a second view rotated 80°
looking into the phosphorylation lip.
Conformational Changes
Phosphorylation probably
activates ERK2 by causing both global and local conformational changes.
The two domains of ERK2 are rotated 17° farther apart than
these domains in the structure of cAMP-dependent protein kinase (cAPK)
(56). Therefore, a rotation of the N- and C-terminal domains must occur
to cause closure of the active site and align the catalytic residues.
A Possible Binding Site for Phosphate on
Tyr-185
Arg-189 and -192, residues not highly conserved among
the protein kinases, create an anion binding site (Fig. 3) on the
surface of ERK2 near the phosphorylation lip(57) . In the
refined, low activity structure this site was filled with a sulfate ion
acquired during crystallization. Interaction of the phosphate group of
Tyr-185 with these residues may help to stabilize the conformation of
the lip in the active structure.
The Phosphorylation Lip Controls the Activity of the
MAP Kinases
Biochemical and structural analyses of mutations of
the activating phosphorylation sites suggest how phosphorylation
increases ERK2 activity. The structure of the ERK2 mutant T183E and its
basal activity are similar to wild type, but it is activated
100-fold following a single phosphorylation on
Tyr-185(53) , suggesting that glutamate in part mimics the
negative charge of threonine phosphate. The crystal structures of three
ERK2 mutants at Tyr-185 (57) suggest changes in local
conformation upon ERK2 activation. In these mutants, 15 residues of the
phosphorylation lip from Asp-173 to Ala-187 (Fig. 1) are
disordered(57) . Because any change to Tyr-185 introduces
disorder into the low activity structure, Tyr-185 likely has an
essential role in creating the low activity conformation.
Locations of Mutations Identified in Genetic Selections
for Activated MAP Kinases
Thus far, no mutations have been
identified that greatly increase MAP kinase activity in vitro;
however, gain-of-function mutations have been found in two MAP kinases,
the product of the Drosophila rolled gene (58) and
FUS3(59) , a component of the pheromone response pathway in
budding yeast. The mutations and the corresponding residues in ERK2 are
listed in and displayed on the ERK2 structure in Fig. 3. The mutations are characterized as dominant or recessive.
-ribbon (residues
6-18) that replaces the A helix found in cAPK. This ribbon
contributes to the positioning and rigidity of the core
-sheet of
the N-terminal domain and may influence the open conformation of the
two domains in the inactive enzyme. These mutations may increase
flexibility of this part of the molecule.
-branched residue, influencing refolding of the
lip. The mutation identified in the rolled gene product
(Asp-319 in ERK2) is just C-terminal to the conserved protein kinase
core near the crossover region between the N- and C-terminal domains.
Asp-319 forms a network of ionic interactions with residues conserved
among MAP kinases to create a hinge bridging the N- and C-terminal
domains. Thus, this mutation may affect the domain structure or
orientation.
Conclusion
Thus far, no constitutively active MAP
kinases are known, despite attempts at their genetic selection and
site-directed mutagenesis. Such failure suggests that cells cannot
tolerate the continuous activity of MAP kinase. Constitutively active
mutants of MEK transform cells and generate tumors in nude
mice(60) . However, effects of activated MEKs could be
compensated for in a regulatable fashion by increasing phosphatase
activity to inactivate MAP kinases. Perhaps the catastrophe that a cell
might encounter if MAP kinases were constitutively active accounts for
the diabolically complex mechanisms to activate these protein kinases
and the multiplicity of mechanisms to inactivate them.
Table: Gain-of-function mutations in MAP kinases
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.