From the
AP-1 is a sequence-specific transcriptional activator composed
of members of the Jun and Fos families (for review see Ref. 1). These
proteins, which belong to the bZIP group of DNA binding proteins (for
review see Ref. 2), associate to form a variety of homo- and
heterodimers that bind to a common site(1) . First identified by
its role in human metallothionein II
The reader should be aware that
although AP-1 DNA binding activity can be conveniently measured by
electrophoretic mobility shift or footprinting assays, changes in AP-1
DNA binding activity do not mirror the transcriptional activity of this
complex factor. Therefore, when dealing with AP-1, it is critical to
measure its ability to activate transcription of an AP-1-dependent
reporter gene. A useful promoter for such experiments is that of the
human collagenase gene (4). The reasons for this discrepancy are
several. First and foremost, several proteins can form complexes that
bind to AP-1 sites. These proteins, however, differ considerably in
their ability to activate transcription of target genes. For instance,
both c-Fos and Fra-1 form stable heterodimers with any of the Jun
proteins, and these heterodimers have similar DNA binding activities
and specificities, yet c-Fos has a potent transactivation domain that
is absent from the smaller Fra-1 protein(7) . Second,
phosphorylation at specific sites enhances the transactivating
potential of several AP-1 proteins, including c-Jun and c-Fos, without
having any effect on their DNA binding
activities(8, 9) .
Most of the genes that encode AP-1 components behave as
``immediate-early'' genes, i.e. genes whose
transcription is rapidly induced, independently of de novo protein synthesis, following cell stimulation. Among these, the
regulation of c-fos and c-jun transcription
is best understood. Several cis elements mediate c-fos induction in response to a diverse spectrum of extracellular
stimuli (reviewed in Ref. 10). A cAMP response element mediates
c-fos induction in response to neurotransmitters and
polypeptide hormones which, by using either cAMP or Ca
By
comparison with c-fos, the c-jun promoter is
somewhat simpler, and most of its inducers operate through one major cis element, the c-jun TRE (Fig. 1B).
This TRE differs from the consensus TRE sequence by 1-base pair
insertion(19) , and due to this subtle change it is more
efficiently recognized by c-Jun
The activities of both pre-existing and newly synthesized
AP-1 components are modulated through their phosphorylation. So far,
this form of posttranslational control was demonstrated for c-Jun,
c-Fos, and ATF2, but it is likely that other Jun and Fos proteins are
similarly regulated. In the case of c-Jun, phosphorylation at a cluster
of sites located next to its basic region inhibits DNA binding by c-Jun
homodimers but not by c-Jun
Interestingly, the
sequence surrounding the N-terminal phosphoacceptors of c-Jun is also
conserved in the C-terminal activation domain of c-Fos (33), suggesting
that phosphorylation at Thr-232, the homolog of Ser-73 of c-Jun,
potentiates c-Fos transcriptional activity. This prediction had turned
out to be correct. However, despite the similarity between the two
phosphoacceptor sites, Thr-232 of c-Fos is not phosphorylated by either
JNK1 or JNK2 but by a novel 88-kDa MAPK termed FRK(9) . Like the
ERKs and the JNKs, FRK is a proline-directed kinase, whose activity is
rapidly stimulated in response to Ha-Ras activation by growth factors.
Although the mechanism by which phosphorylation at Thr-232 stimulates
c-Fos transcriptional activity is not clear, in the context of a
c-Jun
A similar situation may apply for
c-Jun
As described above, three different types of MAPKs, the ERKs,
the JNKs, and FRKs, contribute to induction of AP-1 activity in
response to a diverse array of extracellular stimuli. It is of
considerable interest that each of these types of MAPKs is affecting
AP-1 activity through phosphorylation of a different substrate (Fig. 2). While the ERKs phosphorylate TCF/Elk-1 and thereby
induce c-Fos synthesis, they do not phosphorylate c-Jun or c-Fos on
sites that potentiate their transcriptional
activities(9, 17, 29) . In addition, the ERKs do
not appear to be involved in ATF2 phosphorylation (24). The JNKs, on
the other hand, phosphorylate the stimulatory sites of c-Jun and ATF2
but do not phosphorylate c-Fos(9, 24, 28) . The
JNKs are also capable of phosphorylation and activation of TCF/Elk-1,
suggesting they may be involved in c-fos induction under
certain circumstances.
While a great deal remains to be learned about the mechanisms
that contribute to the regulation of AP-1 activity and most of the
important target genes whose expression is modulated by the different
forms of AP-1 are yet to be identified, quite a lot has been revealed
so far by focusing on this transcription factor and its response to
extracellular stimuli. Most importantly, the investigation of AP-1
regulation had revealed some of the general mechanisms by which protein
phosphorylation modulates transcription factor activity (reviewed in
Ref. 53) and the strategies used by cell surface receptors to
communicate with the nucleus (reviewed in Ref. 54). In addition to the
identification of important AP-1 target genes that will explain the
physiological functions of the different forms of this transcription
factor, a major challenge for the future is understanding the
mechanisms that confer biological specificity to the actions of protein
kinases and transcription factors. It is clear that even generic and
ubiquitous signaling proteins like the components of AP-1 and the MAPK
cascades can be involved in highly specific biological responses.
I thank P. Alford for preparation of the manuscript
and Drs. T. Kallunki and B. Su for preparation of the figures.
INTRODUCTION
Transcriptional Regulation of AP-1 Activity
Posttranslational Regulation of AP-1 Activity
Regulation and Specificity of MAPK Activity
Perspective
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
gene
regulation(3) , AP-1 was also found as a transcription factor
that mediates gene induction by the phorbol ester tumor promoter
12-O-tetradecanoylphorbol-13-acetate (TPA) and hence the name
TRE
(
)(TPA response element) for its recognition
site(4) . Following its discovery, AP-1 activity was found to be
induced by many other stimuli, including growth factors, cytokines, T
cell activators, neurotransmitters, and UV irradiation (1). Several
mechanisms are involved in induction of AP-1 activity and may be
classified as those that increase the abundance of AP-1 components and
those that stimulate their activity. A complete discussion of all the
mechanisms that regulate AP-1 activity, either positively or
negatively, is beyond the scope of this minireview, and the reader is
referred to an earlier, more comprehensive review(1) . The
present review focuses on the role of mitogen-activated protein kinases
(MAPKs) in regulation of AP-1 activity. The regulation and functions of
these important signal-transducing enzymes were recently
reviewed(5, 6) .
as second messengers, activated either protein kinase A or
calmodulin-dependent protein kinases, respectively(11) . A serum
response element (SRE) mediates c-fos induction by growth
factors, cytokines, and other stimuli that activate MAPKs(10) ,
and a Sis-inducible enhancer mediates induction by stimuli that
activate the JAK group of protein kinases (12). Given this complexity,
it is not surprising that c-fos transcription is rapidly
induced in response to almost any imaginable extracellular stimulus (Fig. 1A).
Figure 1:
Regulation of c-fos and c-jun transcription in response to
extracellular stimuli. The cis-acting elements in the c-fos and c-jun promoters that mediate their induction in
response to extracellular stimuli are illustrated. The protein kinases
that phosphorylate the transcription factors that interact with these
elements are indicated. PKA, protein kinase A; CaMK,
calmodulin-dependent protein kinase; SIE, Sis-inducible
enhancer; CRE, cAMP response
element.
The SRE is recognized by the serum
response factor, whose binding results in recruitment of the ternary
complex factor (TCF), which cannot bind to the SRE by
itself(10) . Following mitogenic stimulation, Elk-1, one of
several candidate TCFs(13) , is rapidly phosphorylated, most
likely by members of the ERK group of MAPKs(14, 15) .
Phosphorylation of Elk-1 was reported to facilitate formation of the
ternary complex composed of itself, the serum response factor, and the
SRE (14) and to stimulate its ability to activate transcription,
without affecting its DNA binding properties(15) . Since in
vivo the SRE appears to be constitutively occupied(10) ,
increased Elk-1 transcriptional activity is a more likely mechanism by
which ERK activation causes c-fos induction. The sites at
which Elk-1 is phosphorylated are clustered within its C-terminal
activation domain and are conserved in other candidate TCFs, such as
SAP-1(13) . Since the SRE also mediates c-fos induction
in response to stimuli such as UV irradiation(16) , which has
only a marginal effect on ERK activity(17) , it is possible that
Elk-1 or other TCFs are also phosphorylated by other MAPKs (see below).
Nevertheless, ERK activation leads to elevated AP-1 activity via
c-fos induction. This results in increased synthesis of c-Fos,
which upon translocation to the nucleus combines with pre-existing Jun
proteins to form AP-1 dimers that are more stable than those formed by
Jun proteins alone(18) . Increased stability results in higher
levels of AP-1 DNA binding activity because it shifts the equilibrium
toward dimer formation, which is essential for DNA binding.
ATF2 heterodimers than by
conventional AP-1 complexes(20) . Unlike c-Jun, ATF2 is a
constitutively expressed protein. However, despite its inducible
expression, most cell types contain some c-Jun protein prior to their
stimulation. Like the c-fos SRE, the c-jun TRE is
constitutively occupied in vivo(21) . Following
exposure to stimuli that activate members of the JNK group of
MAPKs(22) , both c-Jun (23) and ATF2 (24) are
rapidly phosphorylated. The constitutive occupancy of the c-jun TRE indicates that this phosphorylation occurs while the proteins
are bound to the c-jun promoter. Similarly, in the case of
c-fos, Elk-1 must be phosphorylated while bound to DNA.
Phosphorylation of c-Jun and ATF2 stimulates their ability to activate
transcription, thereby leading to c-jun induction. Thus, part
of the increase in AP-1 activity in response to JNK-activating stimuli
(such as tumor necrosis factor
, UV irradiation) is due to
increased c-Jun synthesis and possibly c-Fos synthesis (as the JNKs may
also phosphorylate and activate Elk-1; see below). Another part of the
increase in AP-1 activity is due to c-Jun phosphorylation.
c-Fos heterodimers(25) . On the
other hand, phosphorylation of c-Jun at Ser-73 and Ser-63, located
within its transactivation domain, potentiates its ability to activate
transcription as either a homodimer (26, 27) or a
heterodimer with c-Fos(9) . These residues, which do not affect
DNA binding activities, are phosphorylated by the newly discovered
members of the MAPK family, the Jun kinases or
JNKs(22, 28) . So far, the JNKs are the only protein
kinases found to efficiently phosphorylate the N-terminal sites of
c-Jun. Interestingly, neither ERK1 nor ERK2 phosphorylates the
N-terminal stimulatory sites of c-Jun and instead phosphorylate one of
the inhibitory sites located next to the C-terminal DNA binding domain
(17, 29). Using an altered specificity mutant of c-Jun that is
phosphorylated by protein kinase A instead of JNK, phosphorylation of
Ser-73 (and Ser-63 to a lesser extent) was demonstrated to be directly
responsible for potentiating the transactivation function(30) .
Phosphorylation may potentiate c-Jun transcriptional activity through
recruitment of CREB binding protein (CBP), a protein that was
originally identified by virtue of its binding to phospho-CREB, another
bZIP transcription factor that is activated by protein kinase
A(31, 32) . Following phosphorylation of its N-terminal
sites, but not the C-terminal sites, c-Jun can bind CBP and CBP can
potentiate its ability to activate transcription(31) . CBP is
postulated to connect the phosphorylated activation domains of CREB or
c-Jun to the basal transcriptional machinery.
c-Fos heterodimer, phosphorylation of each protein makes a
similar contribution to stimulation of transcriptional activity,
suggesting that both activation domains interact with the
transcriptional machinery.
ATF2 heterodimers, as ATF2 phosphorylation at Thr-63 and
Thr-71 within its N-terminal activation domain was recently shown to
stimulate its transcriptional activity (22). Like c-Jun, ATF2 is also
phosphorylated by the JNKs(24) . Transactivation by ATF2 is also
potentiated upon binding of Rb or E1A, probably through recruitment of
additional activation domains to the DNA-bound ATF2 dimer(34) .
Both E1A and Rb act in concert with phosphorylation of
ATF2(24) . Although E1A can induce c-jun transcription(20) , it represses AP-1 activity(35) .
This repression could be mediated through competition for CBP, which is
very similar to the p300 E1A binding protein(36) . Indeed, it
was recently shown that p300 and CBP are functionally interchangeable
and that E1A can inhibit the coactivation function of both
factors(37, 38) .
(
)FRK, so far, is only
known to affect c-Fos activity(9) .
Figure 2:
Three
distinct MAPKs contribute to induction of AP-1 activity.
Phosphorylation of TCF/Elk-1 bound to the c-fos promoter by
the ERKs stimulates its transcriptional activity, thus leading to
c-fos induction. JNK-mediated phosphorylation of ATF2 and
c-Jun bound to the c-jun promoter stimulates their
transcriptional activities leading to c-jun induction. The
newly synthesized c-Fos and c-Jun proteins combine to form stable AP-1
heterodimers. A further increase in AP-1 activity is brought about by
the JNKs and FRK, which phosphorylate c-Jun and c-Fos, respectively, on
sites that augment their transcriptional activities. SRF,
serum response factor.
These results clearly
indicate that MAPKs are highly specific in their choice of substrates
and do not phosphorylate just any Ser or Thr residue that is followed
by a Pro, as previously assumed. The molecular mechanisms underlying
this high degree of substrate specificity are being explored with the
Jun-JNK interaction as a paradigm. Efficient phosphorylation by the
JNKs requires a docking site located between residues 30 and 60 of
c-Jun(28, 39) . In vitro, this site mediates
binding of c-Jun to the JNKs, and although c-JunJNK complexes
have not yet been detected in living cells, the integrity of the
docking site is essential for phosphorylation and stimulation of c-Jun
activity(28) . The docking site is not the only feature of c-Jun
that ensures efficient phosphorylation by the JNKs, because in
vitro JunB also binds to the JNKs through a similar region but is
not phosphorylated by them.
(
)JunB is not
phosphorylated by the JNKs because its homologs of Ser-63 and Ser-73 of
c-Jun are not followed by prolines. Once prolines are inserted after
these serines in JunB, the resulting variant becomes
JNK-responsive.
(
)In addition to the docking site
and Pro at the P+1 position, efficient phosphorylation of Jun
proteins by the JNKs requires specific residues flanking the
phosphoacceptor site. These residues, however, are not a part of the
docking site and do not affect JNK binding.
When the
binding of the two human JNKs to c-Jun was compared, JNK2 was found to
bind much better than JNK1(40) . Consequently, the K
of JNK2 toward c-Jun is lower than the K
of JNK1 toward c-Jun and its V
is higher(40) . The catalytic
properties of JNK2 measured with other substrates are not considerably
different from those of JNK1, and JNK1 may be the more effective kinase
for other substrates. The basis for the higher affinity of JNK2 toward
c-Jun was traced to a small region of approximately 20 residues located
near its catalytic pocket(40) . This region, which is variable
among all MAPKs, is not a part of the catalytic pocket itself. Most
likely, it is the element of JNK2 that interacts with the docking sites
on c-Jun, as illustrated in Fig. 3.
Figure 3:
Mechanism of c-Jun phosphorylation by
JNK2. The first step in this reaction involves the binding of JNK2
through its docking element (rectangularindentation)
to the docking site in the N-terminal activation domain of c-Jun (rectangularprotrusion). Next JNK2 dissociates from
the docking site on c-Jun, but, due to the high local concentration of
its substrate (c-Jun), it binds through its catalytic pocket (triangularindentation) to a peptide loop that
contains the phosphoacceptor sites of c-Jun (triangularprotrusion). This results in c-Jun phosphorylation and
dissociation of JNK2, allowing the phosphorylation of another substrate
molecule.
In addition to the
differences in substrate specificities, the three types of MAPKs that
affect AP-1 activity differ in their responses to extracellular
stimuli. The ERKs are most efficiently stimulated by growth factors and
phorbol esters(6, 17, 41) , whereas FRK responds
to growth factors but not to phorbol esters(9) . Neither FRK nor
ERK activities are considerably stimulated by exposure to UV
irradiation or tumor necrosis factor, stimuli that are effective for
JNK activation (9, 17, 41). Compared with the FRKs and the ERKs, JNK
activity is modestly stimulated by growth
factors(17, 38, 41) . The largest increases in
JNK activity are observed after UV irradiation (22, 28) or costimulatory activation of T
cells(40) . Although all three types of kinases are stimulated
in response to Ras
activation(19, 42, 43, 44) , the JNKs
also respond to Ras-independent signals(42) . However, even Ras
activation affects ERK and JNK through different kinase cascades (Fig. 4). The major pathway leading from Ras to ERK is based on
the Ras-mediated recruitment of Raf-1 to the plasma
membrane(45) . This results in activation of Raf-1, a Ser/Thr
kinase that phosphorylates and activates the dual specificity kinases
MEK1 and MEK2 (46). The latter are responsible for phosphorylation and
activation of the ERKs(47) . Ras also activates MEKK1, a Ser/Thr
kinase unrelated in its primary structure to
Raf(48, 49) . So far, no direct interaction between Ras
and MEKK1 has been observed, and it is not clear how it is activated by
Ras. Although in vitro MEKK1 is an efficient MEK
activator(48, 49) , in vivo it mostly activates
JNKK1 (also called SEK1 or MKK4), a dual specificity kinase that
phosphorylates and activates the
JNKs(42, 50, 51, 52) . In the future it
would be of interest to identify the factors that contribute to
limiting the specificity of MEKK1 action and confine it to the JNK
activation cascade. Raf-1, on the other hand, has only a marginal
effect on JNKK1 activity, and none of the MEKs can activate the JNKs
(42, 52). Likewise, JNKK1 does not activate the
ERKs(51, 52) . Thus, the kinase cascades responsible for
ERK and JNK activation are distinct and exhibit very little cross-talk (Fig. 4).
Figure 4:
Two
distinct Ras-dependent protein kinase cascades lead to ERK and JNK
activation. The relationships between the different signaling proteins
involved in the Ras-dependent activation of ERK and JNK in response to
growth factors are illustrated. The mechanism by which Ras activates
MEKK1 is not known, as indicated by the questionmark. In vitro and upon overexpression in
vivo, MEKK1 can lead to MEK activation, as indicated by the brokenarrow. In vivo, however, this does
not result in ERK activation. The major function of MEKK1 is therefore
JNKK1 activation and consequently JNK
activation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.