From the Laboratory of Molecular Oncology, The Rockefeller
University, New York, New York 10021
Recently we have reported that the adaptor
protein Crk transmits signals to c-Jun kinase (JNK) through C3G, a
guanine-nucleotide exchange protein for the Ras family of small G
proteins. Transient expression of C3G in 293T cells induced JNK1
activation without a significant effect on extracellular signal-related
kinase 1 (ERK1), whereas mSos1 activated equally both JNK1 and ERK1.
Coexpression of the dominant negative form of Ras-N17 did not suppress
C3G-induced JNK1 activation but reduced the activity of JNK1 induced by
mSos1, suggesting that Ras is not required for JNK activation by C3G. Ras-independent activation of JNK was supported by the finding that
C3G-induced JNK activation was not inhibited by the dominant negative
forms of Rac or Pak, which are components of the signaling pathway from
Ras leading to JNK activation. In contrast, C3G-induced JNK1 activation
was strongly inhibited by coexpression of the kinase negative forms of
the mixed lineage kinase (MLK) family of proteins, MLK3 and dual
leucine zipper kinase (DLK). In addition, MLK3-induced JNK1 activation
was found to be suppressed by the kinase negative form of DLK, which
bound to MLK3. These results suggest that C3G activates JNK1 through a
pathway involving the MLK family of proteins.
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INTRODUCTION |
C3G was originally isolated as a binding partner for the SH3
domain of Crk (1). The sequence homology of the catalytic domain of C3G
suggested that C3G is a guanine-nucleotide exchange protein for the Ras
family of small G proteins (1). In fact, C3G complements the lack of
function of CDC25, a Ras guanine-nucleotide exchange protein
in yeast (1), and in vitro studies demonstrated that it
functions as an exchange factor for Ras family of proteins, Rap1 and
R-Ras (2, 3). Although C3G binds Crk in various tissues (4, 5), the
physiological relevance of the Crk-C3G complex is not clearly
understood. We recently analyzed the activity of the mitogen-activated
protein kinase (MAPK)1 family
of proteins that includes extracellular signal-related kinase 1 (ERK1)
and c-Jun N-terminal kinase 1 (JNK1) (6, 7) and found that C3G
transmits signals to JNK1 in fibroblasts (8). However, the responsible
small GTPase pathway that functions as a substrate for C3G and
transmits signals to JNK remains unidentified.
The small GTPase, Ras, has been reported to transmit a range of signals
in response to various stimuli, including a well established pathway to
ERK activation for cell cycle progression (9, 10). Sequential
activation of serine/threonine kinases such as c-Raf-1 and MAPK/ERK
kinase 1 (MEK1) downstream of Ras has been shown to be involved in ERK
activation (11). In addition, recent reports have shown that Ras also
activates JNK via the Rho family of small G proteins, Rac or
Cdc42 (12, 13) through sequential activation of serine/threonine
kinases, p21-activated kinase 1 (Pak1), MEK kinase 1 (MEKK1), and a
direct activator of JNK, Sek1/MKK4 (14-17). Because C3G has been shown
both to activate Ras in yeast (2) and to weakly increase the active
form of H-Ras when this was incubated with C3G in vitro (3),
in the present study we have examined the possibility that Ras may play
a critical role in C3G-mediated signal transduction in JNK
activation.
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MATERIALS AND METHODS |
Plasmids--
Mammalian expression plasmids, pCAGGS-C3G,
pCAGGS-C3G-F, pCAGGS-mSos1, and pCAGGS-mSos1-F were gifts from
Michiyuki Matsuda (NIH, Japan), pcDNA3-HA-JNK1, pCEF-GST-Pak-N,
pcDNA3-Ras-N17, pCEV29-RhoA-N19, pCEFL-MLK3, and pCEFL-MLK3-K114R
were from Silvio J. Gutkind (NIH). pCEP-EE-Rap-N17 was from Laurence
Quilliam, pEF-BOS-HA-Rac-N17 and pEF-BOS-HA-Cdc42-N17 were from Kouzo
Kaibuchi (NAIST, Japan), pcDNA3-Flag-DLK and
pcDNA3-Flag-DLK-K185A were from Lawrence B. Holzman
(University of Michigan), and pEBG-Sek1 and pEBG-Sek1-KR were from
Leonald I. Zon (Harvard University). Expression plasmids for
MEKK1
and
MEKK1-KR were constructed using pFC-MEKK (Stratagene). The
expression plasmid for TC21/R-Ras2-N17 was from Andrew M. Chang (Mount
Sinai Medical School, New York).
Antibodies--
Anti-C3G (C-19), anti-Sos1/2 (D-21), anti-Rac1
(C-14), anti-Cdc42Hs (P1), anti-JNK1 (C-17), and anti-MLK3 (C18)
antibodies were purchased from Santa Cruz Inc. Anti-pan-Ras antibody
(Ab-3), anti-HA antibody, and anti-Flag antibody (M2) were obtained
from Calbiochem, Boehringer Mannheim, and Kodak, respectively.
Transient Transfection of Plasmid DNA--
293T cells were
cultured in Dulbecco's modified Eagle's medium with 10% calf serum
using a plastic dish 60 mm in diameter, and DNAs were introduced into
cells using a modified calcium-phosphate transfection system
(Stratagene). The procedures of cell lysis and immunoblotting were
described elsewhere (12).
In Vitro JNK and ERK Kinase Assays--
JNK and ERK kinase
activities were measured by in vitro kinase assay described
elsewhere using GST-c-Jun (amino acids 1-79) and myelin basic protein,
respectively, as substrates (12). Incorporated radio isotope activities
with each substrate were measured, and the results are shown as bar
graphs.
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RESULTS AND DISCUSSION |
C3G and Sos Have Different Specificities toward ERK1 and JNK in
293T Cells--
Because Ras in its GTP bound state can activate JNK by
a Rac, Cdc42, Pak, MEKK1, Sek1/MKK4 signaling cascade, we wanted to investigate whether C3G activated JNK via this pathway. To evaluate the
effects of C3G on Ras, we measured the potential of C3G to activate the
MAPKs, JNK1, and ERK1 and compared this with mSos1, which is an
authentic activator for Ras. cDNAs for C3G or mSos1 were
transiently coexpressed with either epitope tagged JNK1 or ERK1 in
human embryo kidney 293T cells. JNK1 or ERK1 activity was then measured
by an in vitro kinase assay using either GST-c-Jun or myelin
basic protein as substrates. As shown in Fig.
1A, C3G preferentially
activated JNK1 compared with ERK1 when compared with the positive
controls, which were UV irradiation or Ras-V12 expression. In contrast,
mSos1 activated both JNK1 and ERK1 to a similar extent. The protein
expression levels of each molecule in the JNK and ERK kinase assays
were examined by immunoblotting and are shown in Fig. 1 (B
and C, respectively). The results suggested that Ras may not
be involved in the C3G-induced activation of JNK1.

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Fig. 1.
In vitro kinase assays for JNK1 and
ERK1 in 293T cells expressing C3G or mSos1. A, 4 µg of
mammalian expression plasmids pCAGGS for C3G, C3G-F, mSos1, or mSos1-F
were cotransfected with either 2 µg of expression plasmid for HA-JNK1
or GST-ERK1 into 293T cells for the JNK1 or ERK1 assays, respectively.
UV irradiation and c-Ha-Ras-V12 were employed as positive controls in
the JNK1 and ERK1 assays, respectively. The averages of radioisotope
counts from three independent experiments are shown as a bar graph. The open bars show the results of the JNK1 assay, and the
closed bars show those of the ERK1 assays. B, a
typical result of the JNK1 assay employing GST-c-Jun (1-79) is shown
at the top. Protein expression levels of HA-JNK1, C3G, and
mSos1 were confirmed by immunoblotting (IB) with anti-HA,
C3G, and Sos antibodies using total cell lysates containing 20 µg of
protein/lane. Protein expression in each lane is as follows: vector
control (lane 1), HA-JNK1 (lane 2), C3G and
HA-JNK1 (lane 3), C3G-F and HA-JNK1 (lane 4),
mSos1 and HA-JNK1 (lane 5), mSos1-F and HA-JNK1 (lane
6), and HA-JNK1 and UV irradiation (lane 7).
C, a typical result of the ERK1 assay using myelin basic
protein (MBP) is displayed at the top. Protein expression levels of GST-ERK1, C3G, mSos1, and H-Ras were confirmed by
immunoblotting (IB) with anti-HA, C3G, Sos, and pan-Ras
antibodies. Protein expression in each lane is as follows: control
(lane 1), GST-ERK1 (lane 2), C3G and GST-ERK1
(lane 3), C3G-F and GST-ERK1 (lane 4), mSos1 and
GST-ERK1 (lane 5), mSos1-F and GST-ERK1 (lane 6),
and GST-ERK1and H-Ras (lane 7).
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We also tested the effects of membrane targeted forms of C3G or mSos1
using a farnesylation acceptable sequence on JNK1 or ERK1 activation in
a transient protein expression system, because it is known that stably
expressed Sos-F strongly activates ERK1 in murine fibroblasts compared
with wild type mSos1 (Ref. 18 and data not shown). Unlike the results
obtained in the stable protein expression system, we found that the
presence of a farnesylation targetting sequence on either C3G or mSos1
did not result in a significant increase in ERK1 or JNK1 activity
compared with the cytoplasmic derivatives (Fig. 1A). It is
conceivable that high levels of C3G or mSos1 expression in the
transient expression system may permit easy contact with their
substrates localized in the vicinity of the membrane, and thus
farnesylation does not result in further increases in kinase
activation. Therefore we used wild type C3G and mSos1 for further
analysis of the downstream signaling pathway of C3G-induced JNK
activation.
Expression of the Dominant Negative Form of Ras Suppressed
mSos1-induced, but Not C3G-induced, Activation of JNK1 in 293T
Cells--
A reciprocal experiment to test whether
C3G-dependent activation of JNK is regulated by Ras
involves dominant negative Ras-N17 protein. Although coexpression of
Ras-N17 significantly reduced mSos1-induced JNK activation,
cotransfection of Ras-N17 did not have any effect on C3G-induced JNK1
activation even on the mild 2.5-fold activation resulting from
transfection of reduced amounts of C3G (Fig.
2A). In agreement with Fig. 1,
these data suggest that C3G activates JNK1 in a Ras-independent manner.
C3G has been reported to activate Rap1 in an in vitro
guanine-nucleotide exchange assay (2). Therefore, we tested whether a
dominant negative form of Rap1, Rap-N17, can suppress C3G-induced JNK1
activation. As shown in Fig. 2B, the coexpression of
Rap1-N17 did not suppress either C3G or mSos1-induced JNK1 activation,
suggesting that Rap1 is also not involved in JNK activation. In
addition, N17 TC21/R-Ras2, a dominant negative form of another member
of the Ras family of proteins, did not inhibit C3G-induced JNK1
activation.2 Recently, it has
been shown that JNK can be activated by Rac, a member of the Rho family
of GTPase. Although Rac1-N17 strongly suppressed mSos1-induced JNK1
activation (Fig. 2B), such a mutant again failed to have an
observable effect on C3G-mediated JNK activation. Similar results were
obtained by RhoA-N19 or Cdc42-N17 (data not shown), suggesting that
none of the Rho family GTPase binds C3G to JNK activation. It should be
noted that Rac1-N17 caused a more profound suppression of mSos1-induced
JNK1 activation than Ras-N17 with 90 and 60% reductions, respectively,
being observed (Fig. 2B). This discrepancy can be explained
if we assume that Rac1 can be directly activated by mSos1 through
the Dbl homology domain of mSos1.

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Fig. 2.
Dominant negative form of Ras-N17 does not
suppress C3G-induced JNK1 activation in 293T cells. A,
comparison of the effect of Ras-N17 on C3G- or mSos1-induced JNK1
activation. 4 µg of the plasmid for Ras-N17 expression was
cotransfected with the indicated amount of plasmids for C3G or mSos1
into 293T cells, and JNK1 activity measured. 2 µg of plasmid for
HA-JNK1 was cotransfected in all samples. B, effects of
dominant negative forms of Ras, Rap1, and Rac in C3G- or mSos1-induced
JNK1 activation in 293T cells. 4 µg of plasmids for Ras-N17,
EE-Rap1-N17, or HA-Rac-N17 expression were cotransfected with plasmid
for C3G or mSos1 and measured for JNK1 activity. 2 µg of plasmid for
HA-JNK1 expression was cotransfected in all samples. Representative
results of the JNK1 assays were shown below the bar graph
(GST-c-Jun (1-79)). Protein expression levels of HA-JNK1
measured by immunoblotting using anti-HA antibody are shown in the
bottom panel (IB).
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Suppression of C3G-induced JNK1 Activation by the Kinase Negative
Form of Mixed Lineage Kinase MLK3--
Because one of the homologues
of the yeast Ste20, serine/threonine kinase Pak1 that bound
to Rac and Cdc42 has been reported to activate JNK (14), we examined
the involvement of Pak1 in C3G-induced JNK1 activation. In 293T cells,
the N terminus region of Pak1 (Pak-N) lacking the kinase domain that
has been reported to have a dominant negative function against Rac- or
Cdc42-induced JNK activation (19) was found not to suppress C3G-induced
JNK1 activation, whereas Pak-N did block Cdc42-induced JNK1 activation in our system as has been previously reported (19) (Fig.
3A).

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Fig. 3.
Suppression of C3G-induced JNK1 activation by
the kinase negative form of MLK3-K114R in 293T cells. A, a
kinase deletion form of Pak1 (Pak-N) did not suppress C3G-induced JNK1
activation. 4 µg of plasmid for GST-Pak-N was cotransfected with 4 µg of plasmids for C3G or Cdc42-V12 as indicated by + or below the graph. B, a kinase negative form of MLK3
suppressed C3G-induced JNK1 activation. 4 µg of plasmid for
MLK3-K144R was cotransfected with plasmids for C3G, Cdc42-V12, or
MEKK1 as indicated by + or below the graph. In A
and B, 2 µg of HA-JNK1 was cotransfected in all samples. Representative results of the JNK assay are shown at the top
(GST-c-Jun (1-79)). Protein expression levels of HA-JNK1 as
detected by immunoblotting are also shown (IB).
C, verification of JNK1 activation by MLK3 and DLK. 4 µg
of expression plasmid for MLK3 or DLK is cotransfected with HA-JNK1,
and measured JNK activity is shown in the top panel. Protein
expression in each lane is as follows: vector control (lanes
1 and 2), MLK3 (lane 3), MLK3-K114R
(lane 4), Flag-DLK (lane 5), and Flag-DLK-K185A
(lane 6). HA-JNK1 is expressed in lanes 2-6.
Expression levels of MLK3, DLK, and HA-JNK1 as detected by
immunoblotting are shown (IB).
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In addition to the Ste20 homologues, a newly emerging class
of kinases, the mixed lineage kinase (MLK) family of proteins including
MLK1 (20), MLK2/MST (21, 22), MLK3/SPRK/PTK1 (23-25), and dual leucine
zipper kinase (DLK) (26) have been reported to regulate JNK activation.
Specifically, MLK3, which has a SH3 domain and a CRIB (Cdc42 and Rac
interactive binding) domain (27), has been shown to play a Pak1-like
role such as MAPK kinase kinase and transmit signals to JNK (28). We
therefore examined the role of MLK3 in C3G-dependent
signaling to JNK1. Interestingly, co-expression of the kinase negative
form of MLK3-K144R suppressed C3G-induced JNK1 activation (Fig.
3B). In these experiments, we used Cdc42-V12 and a truncated
form of
MEKK1 that has been shown to activate JNK1 as positive and
negative controls, respectively, for the estimation of the dominant
negative effect of MLK3 (Fig. 3B), because MLK3 has been
shown to function between Cdc42 and MEKK1 (28). In control experiments
(Fig. 3C) JNK activation could only be observed with native
MLK3 but not kinase negative MLK3. The results (Fig. 3, A
and B) suggest the possible involvement of MLK3 rather than
Pak1 in C3G-induced JNK1 activation.
Suppression of C3G-induced JNK1 Activation by Kinase Negative
Form of DLK--
Because MLK3 has been shown to transmit signals to
MEKK1 (28), we examined a role for MEKK1 in C3G-dependent
JNK1 activation. As shown in Fig.
4A, expression of the
kinase negative form of
MEKK1-KR attenuated by approximately 50%
the C3G-induced JNK1 activation. Because the suppression of C3G-induced
JNK1 activation by
MEKK1-KR was partial, it is possible that another
molecule functions as MAPK kinase kinase for JNK1 instead of MEKK1.
Because one of the MLK family of proteins, DLK, has been shown to
function as a MEKK1-like protein (29), we examined a possible role for DLK and found that coexpression of kinase negative form of DLK-K185A clearly suppressed the C3G-dependent JNK1 activation in
293T cells (Fig. 4B). Moreover, it can be demonstrated that
wild type DLK can activate JNK1 in 293T cells (Fig. 3C,
lane 5).

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Fig. 4.
Suppression of C3G-induced JNK1 activation by
the kinase negative form of DLK-K185A in 293T cells. A, a
kinase negative form of MEKK1 partially suppressed C3G-induced JNK1
activation. 4 µg of expression plasmid for the kinase negative form
of MEKK1-KR was cotransfected with plasmids for C3G or Sek1/MKK4 as
indicated by + or below the bar graph, and
cotransfected JNK1 activity was measured. B, a kinase
negative form of DLK suppressed C3G-induced JNK1 activation. 4 µg of
expression plasmid for the kinase negative form of DLK-K185A was
cotransfected with plasmids for C3G, Cdc42-V12, or Sek1/MKK4 as
indicated by + or below the bar graph, and JNK1 activity was measured. C, a kinase negative form of DLK
suppressed MLK3-induced JNK1 activation. 4 µg of expression plasmid
for the kinase negative form of DLK-K185A was cotransfected with
plasmids for MLK3 as indicated by + or below the bar
graph. In A, B, and C, 2 µg of
HA-JNK1 was cotransfected in all samples. A representative result of
the JNK assay is shown at the top (GST-c-Jun
(1-79)). Protein expression levels of HA-JNK1 as detected by
immunoblotting are shown (IB). D, interaction of
MLK3 and DLK-K185A in 293T cells. Expressed proteins in each lane are
as follows: vector control (lane 1), MLK3 (lane
2), Flag-DLK-K185A (lane 3), and MLK3 and Flag-DLK-K185A (lane 4). Protein expression levels of MLK3
were confirmed by anti-MLK3 antibody using total cell lysates
containing 20 µg of protein (top panel). Interaction of
MLK3 and Flag-DLK-K185A was examined by immunoprecipitation using
anti-Flag antibody following immunoblotting with anti-MLK3 antibody
(middle panel). The same filter was reprobed with anti-Flag
antibody to confirm the amount of precipitated Flag-DLK-K185A
(bottom panel).
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Because C3G-induced JNK1 activation was suppressed by dominant negative
forms of both MLK3 and DLK, we examined whether MLK3 activates JNK
through DLK in 293T cells. Because MLK3 has been reported to function
as a Pak1-like protein (28) and DLK as a MEKK1-like protein (29), it is
likely that MLK3 lies upstream to DLK. We found that the kinase
negative form of DLK-K185A strongly suppressed the MLK3-induced JNK1
activation (Fig. 4C). We could also demonstrate the
interactions of overexpressed MLK3 and DLK-K185A in 293T cells (Fig.
4D). Although the mechanisms of the interactions of MLK3 and
DLK are unknown, this association may well contribute to the
suppression of MLK-induced JNK1 activation by DLK-K185A. The data
suggest that both MLK3 and DLK may be involved in the signaling pathway
in C3G-induced JNK1 activation in 293T cells (Fig.
5).

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Fig. 5.
Signaling pathway from C3G for JNK activation
by Ras-independent mechanism. Two of the MLK family of proteins,
MLK3 and DLK, possibly transmit signals from C3G leading to JNK
activation. The well described pathway from Ras and Rac/Cdc42 to JNK
through sequential activation of serine/threonine kinase Pak1 and MEKK1 is also shown.
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Our present studies suggest that C3G-mediated signaling pathway to JNK1
occurs in a Ras-independent mechanism. Consistent with this idea, Rac,
which is a major signal transducer for Ras to JNK1, does not have any
role in C3G-induced JNK1 activation. Furthermore, we have shown that
another member of the Ras family of proteins Rap1 does not induce JNK1
activation downstream of C3G. Downstream signaling of Rap1 may involve
the activation of ERK1 through B-Raf under cAMP stimulation (30).
Recently, R-Ras has been shown to be activated by both C3G and
Ras-GRF/Cdc25Mm in in vitro assays (3), and it
has been reported that R-Ras could transmit signals to Akt through
phosphatidylinositol 3-kinase but not to ERK and JNK (31). We are
currently investigating the responsible small GTPase that may serve as
a substrate of C3G and at the same time induce JNK1 activation. In this
study, we have examined the involvement of serine/threonine kinases in C3G-induced JNK activation and clearly showed that kinase negative forms of the MLK family of proteins, MLK3 and DLK, suppress C3G-induced JNK1 activation in 293T cells. Although one should be cautious about
the possibility that a dominant negative form of kinases transiently
overexpressed in cells could block the function of related family of
proteins, our observations strongly suggest a possible role for the MLK
family of proteins in C3G signaling to JNK1.
We thank Michiyuki Matsuda (NIH, Japan),
Silvio J. Gutkind (NIH), Laurence Quilliam (Indiana University), Kouzo
Kaibuchi (NAIST, Japan), Lawrence B. Holzman (University of
Michigan), Leonald I. Zon (Harvard), and Andrew M. Chang
(Mount Sinai Medical School, New York) for plasmid DNAs. We also thank
Lawrence B. Holzman for useful discussion and Raymond Birge, Alvaro
Monteiro and William W. Hall for critical reading of the manuscript,
and Hiroaki Hiraga for preparation of digital figures.