Point mutations that cause JNK activation have not been identified (5
). For example, the replacement of the critical phosphorylation sites that cause JNK activation with acidic residues does not cause increased JNK activity. Nevertheless, activated JNK can be constructed using a fusion protein approach that was first reported by Cobb and colleagues (42
). This approach involves the fusion of a MAP kinase kinase with a MAP kinase. Thus, fusion of MEK1 to ERK2 causes constitutive ERK2 activity (42
). Gene disruption experiments demonstrate that two MAP kinase kinases (MKK4 and MKK7) cause JNK activation (51
). MKK4 can activate both JNK and p38 MAP kinase (8
). In contrast, MKK7 is a specific activator of JNK (20
). This specificity of MKK7 for JNK activation suggests that the fusion of MKK7 to JNK may cause constitutive JNK activation. We therefore prepared a series of proteins in which different JNK isoforms (JNK1α1, JNK2α2, and JNK3α2) were fused to one MKK7 isoform (MKK7β2). We also prepared two mutant MKK7-JNK fusion proteins with point mutations that catalytically inactivate MKK7 (active site Lys149
replaced with Ala) or JNK1 (tripeptide dual-phosphorylation motif Thr-Pro-Tyr replaced with Ala-Pro-Phe). In addition, we constructed a fusion protein with JNK1 and MKK7 in the reverse orientation. These fusion proteins are illustrated schematically in Fig. .
FIG. 1. Characterization of MKK7-JNK fusion proteins. (A) Plasmid expression vectors were constructed to express fusion proteins with an NH2-terminal epitope tag (Flag). The structure of the fusion proteins is schematically illustrated. The MKK7-JNK fusion proteins (more ...)
To test whether the MKK7-JNK fusion proteins exhibited constitutive JNK activity, we expressed the fusion proteins in CHO cells (Fig. ). Immunoblot analysis demonstrated that the amount of JNK, MKK7, and MKK7-JNK fusion proteins was similar. Immune complex protein kinase assays using the substrate c-Jun indicated that cotransfection of JNK1 together with MKK7 caused markedly increased JNK activity compared with assays with JNK1 alone. Increased kinase activity was also observed in assays using each of the fusion proteins, including MKK7-JNK1 and JNK1-MKK7 (Fig. ). These data indicate that the MKK7-JNK fusion proteins exhibit constitutive JNK activity. However, we found no evidence that these fusion proteins caused activation of endogenous JNK in transfection assays (data not shown). To confirm whether JNK in the fusion proteins was activated, we performed immunoblot analysis with an antibody that detects JNK phosphorylated on the tripeptide dual-phosphorylation motif Thr-Pro-Tyr. Each of the fusion proteins was found to contain activated JNK (Fig. ). In contrast, the mutated fusion protein MKK7-JNK1(APF) was found to lack JNK activity, while the mutated fusion protein MKK7(K>A)-JNK1 exhibited low basal JNK activity (Fig. ).
The JNK signaling pathway can regulate AP-1 transcription activity. We therefore tested the effect of the MKK7-JNK fusion proteins in cotransfection assays using an AP-1 reporter plasmid (Fig. ). Expression of JNK1 caused no significant change in AP-1 activity, as expected. In contrast, expression of MEKK1, a strong activator of the JNK pathway, caused markedly increased AP-1 transcription activity. Similarly, expression of the MKK7-JNK fusion proteins caused increased AP-1 activity. Increased activity was detected in experiments using JNK1, JNK2, and JNK3 fusion proteins and in experiments using both MKK7-JNK1 and JNK1-MKK7 fusion proteins. Interestingly, MEKK1 was unable to increase AP-1 activity in cells expressing the MKK7-JNK fusion proteins. Together, these data demonstrate that the constitutively activated JNK fusion proteins are functional in vivo and cause increased AP-1 activity (Fig. ). Studies of the transcription factor ATF2, which is phosphorylated and activated by JNK (18
), confirmed this conclusion (Fig. ).
FIG. 2. The MKK7-JNK fusion proteins increase AP-1 transcription activity. (A, C) AP-1 transcription activity was examined in a transfection assay using a reporter plasmid containing four AP-1 binding sites. CHO cells were cotransfected with the reporter plasmid (more ...)
To examine whether the effect of the MKK7-JNK fusion proteins on transcription activity depends upon increased JNK activity, we examined the effect of catalytically inactive fusion proteins on AP-1 activity in luciferase reporter assays (Fig. ) and ATF2 luciferase reporter assays (Fig. ). These data demonstrated that no increased transcription activity was detected in experiments using inactivated JNK (replacement of the tripeptide dual-phosphorylation motif Thr-Pro-Tyr with Ala-Pro-Phe) in the fusion protein MKK7-JNK1(APF). Similarly, no increased transcription activity was detected in experiments using inactive MKK7 (replacement of active site Lys149 with Ala) in the fusion protein MKK7(K>A)-JNK1. Thus, catalytically active MKK7 and JNK are required for the fusion proteins to increase AP-1 activity.
A small increase in AP-1 and ATF2 transcription activity was observed when the two inactive fusion proteins were coexpressed [MKK7-JNK1(APF) plus MKK7(K>A)-JNK1]. This may be caused by functional complementation between the two mutated fusion proteins. To test this hypothesis, we examined JNK activation by immunoblot analysis using an antibody that binds dually phosphorylated JNK. As expected, the MKK7-JNK1(APF) fusion proteins did not contain activated JNK (Fig. ), but a low level of activated JNK was detected in experiments using the MKK7(K>A)-JNK1 fusion protein. In contrast, activated JNK was detected when the two inactive fusion proteins were coexpressed. These data indicate partial complementation between the defective functions of these inactive fusion proteins. The simplest hypothesis is that MKK7 in the MKK7-JNK1(APF) fusion protein can phosphorylate and activate JNK in the MKK7(K>A)-JNK1 fusion protein when these two proteins are coexpressed.
Activated JNK causes cell death.
The JNK signal transduction pathway can contribute to stress-induced apoptosis (5
). However, it is unclear whether activated JNK is sufficient to induce an apoptotic response. We therefore tested the effect of activated JNK on the survival of cultured cells (Fig. ). In initial experiments, we cotransfected GFP together with the inactive fusion protein MKK7-JNK1(APF). Fluorescence microscopy demonstrated numerous transfected cells expressing GFP. In contrast, a marked reduction in GFP-positive cells was detected in cultures transfected with the activated fusion protein MKK7-JNK1 (Fig. ). As a positive control, we demonstrated that a strong inducer of apoptosis, truncated Bid (tBid), also caused a marked reduction in the number of GFP-positive cells. These data indicate that activated JNK can induce cell death. To confirm this observation, we measured viable cells by crystal violet staining of the adherent population (Fig. ). A marked reduction in cell viability was detected in cultures expressing activated JNK1, JNK2, or JNK3 fusion proteins, but not when these cultures were transfected with wild-type JNK protein kinases (Fig. ). Similar results were obtained in experiments using an alternative assay of cell viability that employs a luciferase reporter plasmid (Fig. ). Control studies demonstrated that the inactive fusion proteins MKK7-JNK1(APF) and MKK7(K>A)-JNK1 did not cause marked cell death (Fig. ). However, coexpression of these inactive fusion proteins did cause some cell death (Fig. ), consistent with the observed partial functional complementation between these inactive fusion proteins (Fig. ).
FIG. 3. Activated JNK causes cell death. (A) CHO cells were cotransfected with a GFP expression vector together with expression vectors for the MKK7-JNK1 fusion protein (wild type and phosphorylation defective) or an active fragment of the BH3-only protein Bid (more ...)
The cell death caused by activated JNK could be the result of nonspecific toxicity. To exclude this possibility, we examined whether the cell death caused by activated JNK could be rescued by increased survival signaling. The Akt signaling pathway is established as an important mediator of cell survival (4
). We therefore examined the effect of overexpression of Akt on activated JNK-induced cell death. Expression of wild-type Akt caused no significant reduction in JNK-induced death (Fig. ). In contrast, activated Akt (modified by NH2
-terminal myristoylation) caused a marked reduction in activated JNK-induced cell death (Fig. ). These data suggest that the cell death caused by activated JNK is not the result of nonspecific toxicity but is the result of increased signal transduction by JNK.
FIG. 4. JNK-stimulated apoptosis is inhibited by Akt. (A) CHO cells were transfected with vector control, Akt, or activated Akt (myristoylated Akt) together with the indicated plasmid expression vectors. Decreased cell survival caused by activated JNK (more ...)
The observation that activated Akt, but not wild-type Akt, blocked cell death mediated by activated JNK is interesting because we had not anticipated that overexpressed wild-type Akt would not suppress JNK-induced cell death. One possible explanation is that activated myristoylated Akt has different signaling properties than wild-type Akt (35
). Indeed, we found that activated JNK caused decreased amounts of activated wild-type Akt, but not membrane-bound myristoylated and activated Akt (Fig. ). This decrease in the amount of activated Akt was mediated, in part, by decreased expression of Akt (Fig. ). This observation prompted us to examine other signaling pathways in cells expressing activated JNK. These studies demonstrated that the expression of activated JNK did not cause changes in the activity of endogenous JNK (data not shown). However, activated JNK did markedly inhibit activation of the ERK1/2 MAP kinases caused by phorbol ester (data not shown) and MEK1 (Fig. ). In contrast, activated JNK caused no change in the p38 MAP kinase signaling pathway (Fig. ). The selective effect of activated JNK to suppress ERK1/2 activation could be mediated by increased expression of a MAP kinase phosphatase (2
). Together, these data demonstrate that activated JNK alters the regulation of Akt and the ERK-1/2 MAP kinases. Since these signaling pathways have been implicated in survival signaling by previous studies (4
), it is possible that these changes may contribute to cell death caused by activated JNK.
FIG. 5. Activated JNK causes decreased ERK activity. (A) CHO cells were transfected with an empty expression vector (control) or with an expression vector for activated MEK1 (HA-ΔN3-MEK1 [S218E, S222D]) together with the indicated expression vectors. (more ...) Activated JNK causes apoptosis.
The cell death caused by activated JNK could be mediated by several different mechanisms. To test if the cell death was caused by apoptosis, we examined cells expressing activated JNK for biochemical characteristics of the apoptotic response. The activation of effector caspases, including caspase-3, is a key step in the apoptotic program. Expression of activated JNK caused a reduction in the amount of 32-kDa pro-caspase-3 and a corresponding increase in the amount of processed and activated caspase-3 (Fig. ). The activation of caspase-3 was confirmed by probing immunoblots with an antibody that binds selectively to a 20-kDa processed fragment of caspase-3 (Fig. ). These data indicate that JNK causes caspase activation. One caspase-dependent process in apoptotic cells is nucleosomal fragmentation of genomic DNA. Indeed, increased nucleosomal DNA fragmentation was caused by activated JNK1, JNK2, and JNK3 (Fig. ). The effects of activated JNK on DNA fragmentation were blocked by incubation of the cells with the caspase inhibitor zVAD-fmk (data not shown).
FIG. 6. Activated JNK causes apoptosis. (A) Cell lysates prepared at 40 h posttransfection were examined by immunoblot analysis using antibodies to the Flag epitope tag (lower panel), activated caspase-3 (middle panel), and caspase-3 (upper panel). Equal amounts (more ...) JNK causes release of mitochondrial cytochrome c by a caspase-independent mechanism.
JNK could activate apoptosis by stimulating death receptor signal transduction or by inducing the intrinsic cell death pathway involving mitochondria. To test the involvement of the intrinsic pathway, we examined cytochrome c release in cells expressing activated JNK. The active fusion protein MKK7-JNK1 was sufficient to induce cytochrome c release and was not inhibited by the caspase inhibitor zVAD-fmk (Fig. ). In contrast, the inactive fusion protein MKK7-JNK1 (APF) failed to release cytochrome c. These data indicate that JNK causes cytochrome c release by a caspase-independent mechanism.
JNK causes a rapid apoptotic response.
The observation that activated JNK causes apoptosis in transfection assays does not provide reliable information concerning the time course of this effect of JNK. It is possible that JNK causes apoptosis by an indirect mechanism that requires long periods of JNK activation. In contrast, activated JNK may lead to rapid apoptosis. To distinguish between these possibilities, we performed microinjection experiments. The wild-type fusion protein MKK7-JNK1 caused cytochrome c
release (Fig. ). Time course analysis demonstrated that 33% of the microinjected cells were dead within 3 h postinjection and that 84% of the microinjected cells were dead within 4 h (Fig. ). No marked changes in the viability of cells microinjected with the inactive fusion protein MKK7-JNK1(APF) were detected during this time period (Fig. ). Control studies using a tBid expression vector demonstrated that this protein caused apoptosis (39% of cells within 1 h and 88% of cells within 2 h postinjection) that was more rapid than that caused by activated JNK (data not shown). The kinetics of tBid-stimulated cytochrome c
release are consistent with the known function of tBid to bind, induce conformational changes in, and cause mitochondrial redistribution of Bax and Bak (9
). The slightly slower kinetics of JNK-stimulated apoptosis may either reflect a lower expression threshold for the function of tBid or indicate that several biochemical steps separate JNK activation from the mechanism of cytochrome c
release. Nevertheless, these data demonstrate that activated JNK does cause rapid apoptosis and that the observed cell death is not a long-term consequence of JNK activity.
FIG. 7. JNK causes rapid release of cytochrome c and apoptosis. (A) Activated JNK causes cytochrome c release from the mitochondria. CHO cells were microinjected with the MKK7-JNK1 expression plasmid together with dog IgG. The injected cells were visualized by (more ...) Activated JNK may regulate the function of Bcl2 family proteins.
It has been established in previous studies that JNK can phosphorylate the antiapoptotic proteins Bcl2 and Bcl-XL
in vitro (6
). This phosphorylation has been reported to inhibit the prosurvival function of Bcl2 and Bcl-XL
), although this conclusion is controversial since some studies indicate that phosphorylation may enhance the antiapoptotic actions of Bcl2 (6
) and may stabilize Bcl2 (and thus increase prosurvival signaling) by preventing ubiquitin-mediated degradation of Bcl2 (3
). Taken together, these data indicate that Bcl2 and Bcl-XL
represent potential sites of regulation by JNK, although the exact role of JNK-regulated phosphorylation of these proteins is unclear.
We examined Bcl2 phosphorylation in cells expressing the activated JNK fusion proteins. Immunoblot analysis demonstrated that the MKK7-JNK1 fusion protein caused a marked decrease in the electrophoretic mobility of Bcl2, consistent with phosphorylation (Fig. ). However, only a fraction of the Bcl2 was found to become phosphorylated in cells overexpressing MKK7-JNK1 (Fig. ). This partial phosphorylation of Bcl2 was unexpected because previous studies have suggested that Bcl2 is a JNK substrate and we had expected that the overexpression of activated JNK would lead to stoichiometric phosphorylation of Bcl2. To examine further the extent of Bcl2 phosphorylation caused by activated JNK, we investigated the effect of changes in the expression of Bcl2. These studies demonstrated that Bcl2 phosphorylation was only observed when Bcl2 was expressed at a low level (Fig. ). Interestingly, expression of Bcl2 partially inhibited apoptosis caused by the MKK7-JNK1 fusion protein (Fig. ).
FIG. 8. Regulation of Bcl2 by activated JNK. (A) Activated JNK causes Bcl2 phosphorylation in vivo. CHO cells were transfected without and with a plasmid expression vector for human Bcl2 (20 ng). The cells were cotransfected with the empty expression vector or (more ...)
The observation that only a fraction of the total population of Bcl2 molecules can be phosphorylated in cells with activated JNK is interesting. Whether this partial phosphorylation of Bcl2 is sufficient to account for the ability of activated JNK to cause apoptosis is unclear. If this phosphorylation does cause reduced antiapoptotic signaling (13
), it is possible that the partial phosphorylation decreases Bcl2 function below a threshold that is required to maintain survival. In contrast, if Bcl2 phosphorylation causes increased antiapoptotic signaling (3
), it is not obvious how the partial Bcl2 phosphorylation might contribute to JNK-stimulated apoptosis.
In addition to the antiapoptotic protein Bcl2, previous studies have also implicated the proapoptotic BH3-only protein Bid in JNK-stimulated apoptosis (52
). It was observed that UV radiation caused Bid cleavage by a caspase-independent mechanism and that this processing of Bid was absent in Jnk
-null cells (52
). These data suggested that Bid might contribute to JNK-stimulated apoptosis. To test this hypothesis, we examined the effect of activated JNK on wild-type and Bid−/−
fibroblasts (Fig. ). The MKK7-JNK1 fusion protein efficiently killed the Bid−/−
cells (Fig. ). Control studies demonstrated that Jnk
-null cells were also efficiently killed by the MKK7-JNK1 fusion protein (Fig. ). In contrast, the kinase-inactive mutant fusion protein MKK7-JNK1(APF) did not cause apoptosis of wild-type, Jnk
-null, or Bid−/−
cells. These data demonstrate that Bid does not play an essential role in JNK-dependent apoptosis.
FIG. 9. The proapoptotic Bcl2 family protein Bid is not required for JNK-dependent apoptosis. (A) Bid is not required for JNK-dependent apoptosis. Wild-type and Bid−/− mouse fibroblasts were transfected with empty vector (control) or expression (more ...)
The observation that JNK activation does regulate Bcl2 phosphorylation (Fig. ) and Bid processing (52
) suggests that Bcl2 family proteins may contribute to JNK-stimulated apoptosis. However, Bid is not required for JNK-dependent apoptosis and the importance of Bcl2 phosphorylation is unclear (Fig. and ). These considerations suggest that other members of the Bcl2 family may contribute to JNK-dependent apoptosis. Other members of the Bcl2 family that are potentially relevant include the proapoptotic proteins Bax and Bak.
Bax and Bak are required for JNK-dependent apoptosis.
Members of the proapoptotic Bax subfamily of Bcl2-like proteins represent potential targets of the JNK-dependent apoptosis signaling pathway (36
). Recent studies indicate redundant functions of Bax and the related protein Bak (30
). Fibroblasts with compound mutations in both the Bax
genes are remarkably resistant to stress-induced cytochrome c
release and apoptosis (59
). This phenotype is similar to JNK-deficient primary fibroblasts (52
). Thus, Bax and Bak may be required for JNK-dependent apoptosis. To test this hypothesis, we examined fibroblasts derived from wild-type and Bax−/− Bak−/−
mice (Fig. ). Expression of activated JNK caused apoptosis of wild-type fibroblasts. In contrast, the Bax−/− Bak−/−
fibroblasts were resistant to the apoptotic effects of activated JNK (Fig. ). Together, these data demonstrate that the proapoptotic proteins Bax and Bak are essential for JNK-dependent apoptosis.
FIG. 10. The proapoptotic Bcl2 family proteins Bax and Bak are required for JNK-dependent apoptosis. (A) Bax and Bak are required for JNK-dependent apoptosis. Wild-type and Bax−/− Bak−/− mouse fibroblasts were transfected with empty (more ...)
The defect in JNK-stimulated apoptosis observed in Bax−/− Bak−/− fibroblasts (Fig. ) suggests that Bax and Bak may act as downstream components of a JNK-stimulated apoptotic signaling pathway. If this mechanism is correct, it would be predicted that Jnk-null cells would not be resistant to the proapoptotic effects of Bax and Bak. Control studies demonstrated that the BH3-only protein tBid caused apoptosis of wild-type and Jnk-null cells (Fig. ). Similarly, the expression of Bax or Bak also caused apoptosis of wild-type and Jnk-null cells (Fig. ). These data are consistent with the hypothesis that Bax and Bak function as downstream components of a JNK-stimulated apoptosis signaling pathway.
JNK is required for the stress-induced activation of Bax.
The requirement of Bax and Bak for JNK-dependent apoptosis (Fig. ) suggests that JNK deficiency may cause a defect in the normal function of Bax or Bak. To test this hypothesis, we examined the effect of UV radiation on Bax activation using a conformation-specific monoclonal antibody that binds activated Bax (21
). Immunofluorescence microscopy demonstrated that UV radiation caused Bax activation in wild-type fibroblasts but not in JNK-deficient (Jnk1−/− Jnk2−/−
) fibroblasts (Fig. ). Control studies using immunoblot analysis demonstrated that the wild-type and JNK-deficient fibroblasts expressed similar amounts of Bax (52
). Together, these data demonstrate that Bax activation is JNK dependent in cells exposed to stress.
The defective Bax activation in JNK-deficient cells exposed to stress (Fig. ) might reflect a specific role of JNK in the response of cells to particular stimuli, or it might reflect a general requirement of JNK for Bax function. To distinguish between these possibilities, we examined the effect of the BH3-only protein tBid, which is known to induce Bax and Bak oligomerization and insertion into the mitochondrial outer membrane (9
). The effects of tBid on cytochrome c
release and apoptosis require Bax and Bak (58
). Microinjection experiments demonstrated that tBid caused efficient cytochrome c
release and apoptosis in both wild-type and JNK-deficient (Jnk1−/− Jnk2−/−
) fibroblasts (data not shown). Furthermore, transfection assays confirmed that tBid can kill Jnk
-null cells (Fig. ). Thus, JNK is required for Bax and Bak activation caused by exposure of cells to stress (Fig. ) but is not required for cell death initiated by BH3-only proteins (Fig. ). Together, these data indicate that BH3-only members of the Bcl2 family may function downstream of JNK and upstream of Bax and Bak in a stress-induced apoptotic signaling pathway.