JNK2: A Negative Regulator of Cellular Proliferation

Abstract

The three different c-Jun N-terminal kinases (JNKs) are activated in multiple cell types by both apoptotic and mitogenic signals, and in turn regulate the activity of transcription factors such as c-Jun. Being highly homologous and ubiquitously expressed, the JNK1 and JNK2 proteins have been thought to perform redundant functions in many physiological process. However, our data from Jnk1-/- or Jnk2-/- cells and mice suggest that both JNK isozymes perform distinct functions in regulating cellular proliferation via differential regulation of c-Jun, which is a critical regulator of cell-cycle progression. Absence of JNK1, the positive regulator of c-Jun, leads to decreased fibroblast proliferation. In contrast, JNK2 deficiency leads to reduced c-Jun degradation, thereby augmenting c-Jun levels and cellular proliferation. Various cell types including fibroblasts, erythroblasts and hepatocytes from Jnk2-/- mice exhibit increased proliferation rates compared to their wild-type counterparts. These data therefore suggests that JNK2, in contrast to JNK1, is a negative regulator of cellular proliferation in multiple cell types.

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©2005 LANDES BIOSCIENCE. DO NOT DISTRIBUTE.
[Cell Cycle 3:12, 1520-1523; December 2004]; ©2004 Landes Bioscience
1520
Cell C
y
cle 2004; Vol. 3 Issue 12
Kanaga Sabapathy
1,
*
Erwin F. Wagner
2
1
National Cancer Centre; Singapore , Singapore
2
R
esearch Institute of Molecular Pathology; Vienna, Austria
*Correspondence to: Dr. Kanaga Sabapathy; National Cancer Centre; 11, Hospital
Drive; Singapore 169610 Singapore; Tel.: +65.6436.8349; Fax: + 65.6226.5694;
Email: cmrksb@nccs.com.sg
Received 10/15/04; Accepted 10/18/04
P
reviously published online as a
Cell
Cycle
E-publication:
http://www
.landesbioscience.com/journals/cc/abstract.php?id=1315
KEY WORDS
JNK, c-Jun, proliferation, cyclin D, erythropoiesis
ACKNOWLEDGEMENTS
We thank Wolfram Jochum for help with the
immunostaining.
This work was supported by the National
Medical Research Council of Singapore (K.S.) and
the Austrian Federal Ministry of Science, Transport
and the Arts (E.F.W.). The IMP is supported by
Boehringer Ingelheim.
Perspective
JNK2
A Negative Regulator of Cellular Proliferation
ABSTRACT
The three different c-Jun N-terminal kinases (JNKs) are activated in multiple cell types
by both apoptotic and mitogenic signals, and in turn regulate the activity of transcription
factors such as c-Jun. Being highly homologous and ubiquitously expressed, the JNK1
and JNK2 proteins have been thought to perform redundant functions in many physio-
logical process. However, our data from
Jnk1
-
/-
or Jnk2
-
/-
cells and mice suggest that both
JNK isozymes perform distinct functions in regulating cellular proliferation via differential
regulation of c-Jun, which is a critical regulator of cell-cycle progression. Absence of
JNK1, the positive regulator of c
-Jun, leads to decreased fibroblast proliferation. In
contrast, JNK2 deficiency leads to reduced c-Jun degradation, thereby augmenting c-Jun
levels and cellular proliferation. Various cell types including fibroblasts, erythroblasts and
hepatocytes from
Jnk2
-
/-
mice exhibit increased proliferation rates compared to their
wild-type counterparts. These data therefore suggests that JNK2, in contrast to JNK1, is
a negative regulator of cellular proliferation in multiple cell types.
JNK FUNCTIONS
Structural and functional similarities are frequently found among many proteins, and
such resemblance often suggests common modes of action or regulation. Many compo-
nents of cellular signaling modules, including kinases, are phylogenically grouped together
as families based on such homologies, the degree of which can sometimes be extremely
high. However, the question that remains unresolved is if such similar molecules are
evolutionarily conserved and carried through to perform exactly similar and redundant
functions, or if they have specific and different biological roles that have yet to be uncovered.
One such family of proteins is the c-Jun-N-terminal (JNK) kinases, that are present
ubiquitously in the organism. JNKs signal stress responses, thereby leading to apoptosis
induction, as well as pr
oinflammator
y and some form of mitogenic signals.
1-3
The JNK
subgr
oup consists of three members—JNK1, JNK2 and JNK3—which are highly homol-
ogous, and are present as multiple isoforms generated through alternate splicing at the
carboxy-terminus.
4-6
Among the three members of the JNK family, JNK3 is expressed pre-
dominantly in the brain whereas both JNK1 and JNK2 are expressed ubiquitously.
6
Combinatorial use of the various JNKs and their upstream kinases are thought to lead to
differ
ential regulation of substrate proteins in response to multiple stimuli, thereby estab-
lishing signal-specificity. Nevertheless, the specific roles of the different JNK proteins have
not been fully addressed, although the different JNK isozymes may have evolved for specific
biological functions, pr
obably depending on the activ
ating stimuli and r
esponding cell
type.
Upon stimulation, the activated JNKs phosphorylate transcription factors like c-Jun
and ATF-2, which participate in the activation and formation of the AP-1 complex.
7
H
ither
to, most in vitro studies have used c-Jun as the model substrate to investigate the
natur
e of JNK
-
substrate binding activity and specificity
. All thr
ee JNKs hav
e been sho
wn
to bind to the delta domain of c-Jun and phosphorylate it on serines 63 and 73, leading
to its activation.
5
Of the two, JNK2 has been shown to have a twenty-five fold higher
binding affinity for c-Jun than JNK1.
5
Moreover, although the various isoforms of both
JNK1 and JNK2 w
er
e sho
wn to bind and phosphor
ylate the substrates to various extents
in in vitro experiments, the JNK1 isoforms appear to be slightly more efficient in phospho-
r
ylating c
-Jun.
8
Besides being involved in phosphorylation and subsequent activation of substrates
upon signal stimulation, the JNKs hav
e been suggested to cause degradation of the same

substrates under nonstressed conditions.
9
The inactive JNKs have
been shown to cause degradation of transcription factors such as
c
-Jun, ATF2 and p53—a function that was thought to be dependent
on binding of JNKs to the substrates, and occurs in the absence of
phosphor
ylation of the substrates.
9
D
egradation of the substrates is
inhibited in the presence of activating stimuli, which now signal
through the activated JNKs and stabilize the substrates by phoshory-
lating them.
10
Thus, the dual activity of the JNKs in targeting c-Jun
and other substrates for ubiquitin-mediated degradation or in
protecting them from entering the degradation pathway via phospho-
rylation points to an intricate role of JNK in regulating c-Jun stability.
Nonetheless, the specific role of the different JNK proteins in regu-
lating substrate metabolism was hitherto unknown.
JNKS AND PROLIFERATION CONTROL
The biological effects of the JNK proteins on cellular proliferation
have been alluded to indirectly in many previous studies. Oncogenic
Ha-ras has been shown to activate the JNK pathway.
11
In addition,
the transforming small GTP binding proteins Rac1 and Cdc42 have
been shown to activate the JNKs.
12,13
Moreover, it was recently
shown that the JNK pathway is activated in some tumor cell types,
indicating that the JNK pathway might be essential for tumorigene-
sis.
14
In support of these indirect studies, we and others have
demonstrated a causal role for the JNKs in regulating cellular prolif-
eration using fibroblasts lacking both JNK1 and JNK2.
15,16
The
Jnk1
-/-
Jnk2
-/-
fibroblasts were found to exhibit a proliferation defect
and were also defective in c-Jun phosphorylation, which has been
shown to be essential for cellular proliferation.
17,18
In vivo studies
using cells from knockout and transgenic mice have indicated that
c-Jun is essential for efficient transition of the G
1
-S phase of the cell
cycle, and cells lacking c-Jun have a severe proliferation defect.
17
In
addition, it was demonstrated that c-Jun N-terminal phosphorylation
is important for efficient cellular proliferation.
19
Fibroblasts from
mice carrying a mutant c-jun allele having the JNK phosphoacceptor
serines 63 and 73 changed to alanines (junAA) proliferate slower
than wild-type cells, though this defect was not as severe as found
with c-
jun
-/-
fibroblasts.
19
SPECIFIC ROLES OF JNK1 AND JNK2 IN REGULATING
C-JUN-DEPENDENT PROLIFERATION
In our recent findings published in Molecular Cell, we demon-
strated that JNK1 and JNK2 perform distinct functions in regulating
cellular pr
oliferation.
16
Consistent with the notion that c
-
J
un phos
-
phor
ylation is r
equir
ed for efficient pr
oliferation, w
e sho
w
ed that
Jnk1
-/-
fibr
oblasts, in which c
-
J
un phosphor
ylation is reduced, exhib-
ited a pr
oliferation defect. H
o
w
ever,
Jnk2
-/-
fibr
oblasts w
er
e found to
hav
e incr
eased proliferation rates, and a concomitant increase in the
levels of phosphorylated c-Jun. Moreover, JNK2 deficiency also led
to increased proliferation of erthyroblasts suggesting that the negative
effect of JNK2 on cellular pr
oliferation was not r
estricted to fibr
ob
-
lasts. O
ur r
esults indicate that despite their biochemical and str
uc
tural
similarities and earlier expectations, JNK1 and JNK2 ex
er
t differ
ent
effects on c-Jun turnover and activity, which correlates with their
different effects on cell proliferation. JNK1 appears to be a positive
regulator of cellular proliferation, being required for phosphorylation
and activation of c-Jun. On the contrary, JNK2 appears to primarily
function as a negative regulator of c-Jun stability, correlating with its
negative effect on cell pr
oliferation.
At a molecular level, our findings indicate that JNK1 contributes
to c-Jun phosphorylation more than JNK2, especially after cellular
stimulation. Very little JNK1 was associated with c-Jun in unstimu-
lated cells, but the interaction between the two is increased dramat-
ically upon cell stimulation.
16
As JNK1 is also the major c-Jun
kinase present in the cells, this resulted in increased c-Jun N-terminal
phosphorylation and elevated AP-1 activity. By contrast, c-Jun was
mostly bound to JNK2 in unstimulated cells, although this interac-
tion did not result in c-Jun phosphorylation. Instead, JNK2 appeared
to be responsible for targeting c-Jun to degradation, correlating with
previous reports that inactive JNKs can target c-Jun and other sub-
strates for degradation.
9
These findings made under physiological
conditions, correlate with findings based on in vitro experiments
showing that inactive JNK2 has 25x more affinity for c-Jun binding
than JNK1.
5
In addition to decreasing the amount of JNK2 bound
c-Jun and increasing the binding of JNK1 to c-Jun, cellular stimula-
tion also resulted in JNK1-mediated c-Jun phosphorylation, activa-
tion and stabilization. Previously, transient transfection experiments
demonstrated that both JNK1 and JNK2 could phosphorylate c-Jun
and other targets,
8
giving rise to the expectation that there are no
major differences between the kinase activities of JNK1 and JNK2.
We also obtained similar results in transient transfections,
16
but
under physiological conditions, there were clear differences regarding
the relative roles of JNK1 and JNK2 in c-Jun regulation.
Other recent reports have also focused on c-Jun turnover. Nateri
et al. demonstrated that c-Jun is degraded by the E3 ligase
Fbw7-containing Skp/Cullin/F-box protein complex (SCF
Fbw7
),
only when it is phosphor
ylated on the JNK phosphor
ylation sites
(i.e., serines 63 and 73).
20
SCF
Fbw7
-dependent c-Jun degradation
was shown to occur in neuronal cells, where SCF
Fb
w7
is highly
expressed, suggesting that this could be a cell-type dependent pathway
(B
ehrens A, personal communication). Moreover, Goa et al. have
recently showed that the E3 ligase Itch, controlled c-Jun turnover in
T cells.
21
I
tch was sho
wn to be efficiently phosphor
ylated b
y JNK1,
rather than JNK2, that led to activ
ation of its E3 ligase activity
.
Itch-mediated c-Jun degradation was found to be independent of
JNK-mediated c-Jun phosphorylation.
21
Furthermore, human
De-Etiolated-1 gene product, whose expression was highest in the
ovary and some lymphoid organs was shown to regulate c-Jun stability
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Cell C
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JNK2
Figure 1. Increased hepatocyte proliferation after partial hepatactomy in
JNK2 deficient livers. H&E staining of Jnk2
+/+
and Jnk2
-
/-
livers 30 h after
partial hepatectomy (top panel). BrdU-immunostaining of wild-type and
JNK2 deficient livers 30 h after hepatectomy are shown in the lower panel.
Arrows point to some BrdU-positive hepatocytes. n = 3; Magnification: 40x.

JNK2 NEGATIVELY REGULATES PROLIFERATION
OF HEPATOCYTES AND KERATINOCYTES
Similar differences in the control of cellular
pr
oliferation in various other cell types have also
been noted. S
ince it was shown that c-Jun was
r
equired for proper hepatocyte proliferation after
partial hepatectomy (PH),
23
we have also evaluated
the effect of JNK2 loss during PH by determining
the number of S-phase cells by BrdU incorporation.
We noticed that liver regeneration after partial
hepatectomy occurred much faster in
Jnk2
-/-
mice
compared to the wild-type counterparts (Sabapathy
an Wagner, unpublished data). Compared with
controls, the rate of BrdU incorporation by hepato-
cytes was distinctly increased in
Jnk2
-/-
mice (Fig. 1),
suggesting that the absence of JNK2 leads to accel
-
erated hepatocyte proliferation. Moreover, recent
data from Weston et al. suggested that keratinocyte
proliferation was also differentially altered by JNK1
and JNK2.
24
Absence of JNK1 led to thinner epi-
dermis compared to wild-type mice, due to a
marked reduction of the proliferating cells in the
stratum basale. In contrast, skin from
Jnk2
-/-
mice
revealed keratinocyte hyperplasia, resulting in
increased number of epithelial cell layers, which was
attributed to enhanced proliferation. In addition to
increased proliferation due to lack of JNK2, a
recent report also suggested that specific inhibition
of JNK2 resulted in aneuploidy in human cells.
25
This was proposed to be due to defects in chromo-
some segregation and central spindle formation
during anaphase, which eventually resulted in poly-
ploidy. Together, the emerging trend points to
increased proliferation in multiple cell types in the
absence of JNK2 (Table 1).
OUTLOOK
Hitherto, there are no reports identifying JNK2
mutations in human cancers, though the c-Jun/JNK
pathway has been sho
wn to be activated in many
cancers.
14
Thus, the possibility exists that JNK2’
s
degradation inducing activity is reduced in cancer
cells by specific activation of a JNK2-specific
inhibitor(s), or vice-versa. Further investigations are
r
equired to explore these possibilities. Nevertheless,
the genetic data suggest that JNK2 has specifically
evolved to keep a check on total JNK activity within
the cell, which if left uncontrolled, could lead to
pathological conditions.
Ther
efore, one could envis-
age a model, as depicted in F
igur
e 2, wher
eby both
JNK1 and JNK2 regulates substrate turnover and
function in a r
ecipr
ocal fashion, thereby creating a
regulatory circuit that controls JNK activity. The
data also imply that JNK2-specific pharmacological
activ
ators that specifically incr
ease its degradation
activity or JNK1-specific inhibitors that reduce JNK1 kinase activity
would be useful clinically in human disease, where JNK activity is
elev
ated, such as cancers and neur
onal disor
ders.
Figure 2. Proposed model of JNK1 and JNK2 action. The model shows that when wild-type
cells are in a quiescent state, c-Jun is predominantly bound to inactive JNK2. This probably
results in c-Jun being targeted for degradation (dashed arrows), thereby maintaining low basal
levels of c-Jun. JNK1 bound to c-Jun phosphorylates c-Jun at a reduced rate (solid arrows). After
cell stimulation, JNK2 dissociates from c-Jun, giving access for activated JNK1 to interact with
c-Jun. This results in increased JNK1-mediated c-Jun phosphorylation. The degradative signal
by JNK2 is also inhibited, as less JNK2 is c-
Jun bound, while activated JNK2 makes a minor
contribution to c-Jun phosphorylation. The net effect is increased c-Jun stability and activity. In
Jnk1
-/-
cells, absence of JNK1 leads to loss of basal c-Jun phosphorylation and increased
targeting by JNK2 of c-Jun to degradation, resulting in reduced basal c-Jun levels and activity.
Upon stimulation, c-Jun is only weakly phosphorylated and partially stabilized. By contrast,
absence of JNK2 results in c
-
Jun being less degraded in the absence of stimulating signals,
thereby resulting in higher basal levels. Upon stimulation, the stable c-Jun is further phospho-
rylated by JNK1, resulting in increased activity and stability. → Represents phosphorylation
sig
nals;
dashed line represents degradation signal.
T
able 1
ABSENCE
OF
JNK2 RESUL
TS
IN
INCREASED PROLIFERA
TION
IN VARIOUS CELL-TYPES
Cell-type Jnk1
-/-
Jnk2
-/-
R
eference
Fibroblasts Reduced proliferation Increased proliferation 16
Erythroblasts No difference Increased proliferation 16
Hepatocytes
Not tested
Increased proliferation
This repor
t
Keratinocytes Reduced proliferation Increased proliferation 24
1522 Cell Cycle 2004; Vol. 3 Issue 12
by assembling a CUL4A ubiquitin ligase.
22
T
ogether with our findings,
it is evident that c-Jun stability is controlled by several distinct mech-
anisms that appear to be cell-type specific.
JNK2

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Cell C
ycle 1523
Altogether, it is evident that structurally and functionally similar
pr
oteins can be evolutionarily selected to perform distinct and oppo-
site functions, which can be unrav
eled by genetic studies. The JNK
family of pr
oteins would thus serve as a paradigm for other functional
studies aiming to elucidate the specific functions of highly similar
proteins.
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