Although several elements in the apoptotic signaling pathway that lead to JNK and c-Jun activation have been defined, the molecules that couple Rac1 and Cdc42 to activation of MKK4 and MKK7 in this pathway are less well understood. In this light, the drug CEP-1347 provided both a clue and a challenge. That is, since CEP-1347 blocks JNK activation (42
) and appears to inhibit neither JNKs, MKKs, Rac1, nor Cdc42 directly (42a
), there is another implied constituent that must be in the pathway and that is sensitive to the drug. The data provided here implicate MLK family members in this role.
Multiple forms of evidence indicate a role for the MLK family in the neuronal apoptotic JNK/c-Jun pathway. First, transcripts for all members of the MLK family are expressed in both naive and neuronally differentiated PC12 cells and in sympathetic neurons. Moreover, at least two members of the family (MLK3 and DLK) were found here to be present at the protein level in PC12 cells. Multiple members of the MLK family are also present in brain (12
) (data not shown). Second, overexpression of different members of the MLK family, including MLK1, MLK2, MLK3, and DLK, induced significant cell death in both neuronally differentiated and naive PC12 cells. In addition, the two family members tested (DLK and MLK3) were also potent inducers of apoptosis in cultured sympathetic neurons. This process was mediated by activation of the JNK pathway, as indicated by its blockade by coexpression of dominant-negative MKK4, dominant-negative MKK7, or dominant-negative c-Jun. These observations are consistent with past findings in nonneuronal cells demonstrating that MLK2, MLK3, and DLK activate the JNK pathway though the activation of MKK7 and/or SEK1 (MKK4) and that expression of inactive SEK1 inhibits basal and MLK3-activated JNK activity (4
). Third, dominant-negative forms of MLK family members effectively protected neuronally differentiated PC12 cells and sympathetic neurons from apoptosis induced by NGF deprivation. Fourth, apoptotic stimuli led to both elevated levels and apparent activation of endogenous DLK in neuronal PC12 cells. Lastly, CEP-1347, which protects sympathetic neurons and neuronally differentiated PC12 cells from death evoked by NGF deprivation as well as a number of other insults (42
), also protected the cells from death elicited by overexpression of MLK family members. In contrast, the compound did not protect against death caused by overexpression of MKK4 or ΔN ASK1. This is consistent with recent findings identifying MLK family members as specific targets of CEP-1347 (42a
Our use of various JNK/c-Jun pathway constructs as well as of CEP-1347 permitted us to verify the order of the constituents of this pathway. Rac1 and Cdc42 have been implicated as acting upstream of MKK4 and -7, JNKs, and c-Jun (1
). Consistent with prior studies, our observations that dominant-negative MLKs and CEP-1347 suppress death evoked by constitutively activated forms of Rac1 and Cdc42 also place these GTPases upstream of the MLKs. Furthermore, as anticipated, blockade of MLK-induced death by dominant-negative MKK4 and -7 as well as by dominant-negative c-Jun positions MLKs upstream of MKK4 and/or MKK7. A scheme summarizing this pathway is shown in Fig. .
FIG. 13 Scheme for the MLK-dependent pathway for apoptotic neuronal death. NGF deprivation of neuronal cells induces the activation of Cdc42 and Rac1, which in turn activate the MLK family members, very likely with the mediation of a scaffold protein(s). Activated (more ...)
Cytoplasmic cytochrome c
is known to cause neuronal death (11
). It was recently reported that JNK null cells exhibit a defect in the mitochondrial death signaling mechanism, including the failure to release cytochrome c
in response to apoptotic stimuli (64
). This indicated that JNK activity is upstream of mitochondrial events in the death pathway. Our observation that MLK3 or DLK overexpression leads to loss of cytochrome c
staining is consistent with this interpretation. The finding that cell death induced by overexpression of MLK family members can be prevented by general caspase inhibitors further suggests that MLKs act upstream of caspase activation.
JNK/c-Jun pathway activation has been found to elevate transcription of FAS ligand in some systems, and this has been suggested to contribute, in turn, to apoptotic death (40
). We observed in neuronal PC12 cells that a dominant-negative form of FADD, which should interfere with FAS signaling, provided only a very limited protection from death elicited by MLK3 or DLK overexpression (Z. Xu, unpublished results). This suggests that regulated pathways other than those involving FAS and FADD are likely to play more major roles in death evoked by MLK or JNK activation. Alternatively, it is possible that FAS ligand may not depend completely on FADD to kill cells in our system.
What are the possible mechanisms by which MLK family members might be activated and by which they, in turn, phosphorylate their targets? In addition, what does the capacity, observed here, of various MLK dominant-negative forms to block death caused by other MLK family members tell us about these mechanisms? Overexpressed MLK3 and DLK can form homodimers through leucine zipper interactions, leading to autophosphorylation and activation, and such dimerization is a prerequisite for subsequent activation of JNKs (41
). Vacratsis and Gallo (65
) recently reported that constitutively activated Cdc42 fully activates a monomeric MLK3 leucine zipper mutant in terms of both autophosphorylation and histone phosphorylation activity and induces the same in vivo phosphorylation pattern as that of wild-type MLK3. However, this catalytically active monomeric MLK3 mutant is unable to stimulate JNK activation because it fails to phosphorylate one of the two activating phosphorylation sites, Thr258, of MKK4. These studies indicate that zipper-mediated MLK3 oligomerization is not required for MLK3 activation by Cdc42 but instead is critical for proper interaction and phosphorylation of a downstream target(s). This further suggests that one mechanism by which MLK and DLK dominant-negative forms act is by interfering with homodimerization required for substrate interaction.
In addition to blocking death caused by overexpression of their own wild-type counterparts, dominant-negative MLK3 and dominant-negative DLK also suppressed death evoked by other family members. One potential explanation is that various family members act in a cascade. For instance, on the basis that dominant-negative DLK suppresses MLK3-induced JNK1 activation, it was suggested that DLK functions downstream of MLK3 (60
). However, in apparent disagreement with this possibility, we observed that dominant-negative MLK3 also effectively suppresses death evoked by DLK. A second model is that MLK3 and DLK form heterodimers whose function can be blocked by dominant-negative forms of either partner. Consistent with this, the two proteins can be coimmunoprecipitated (60
). However, Nihalani et al. (50
) recently provided evidence that MLK3 and DLK do not form heterodimers.
A third model, which is also consistent with coimmunoprecipitation, is that the MLK3 and DLK compete for binding to a common intermediary protein. For instance, it is possible that MLK3 and DLK (and their dominant-negative forms) compete for interaction with upstream activators such as Cdc42 and Rac1 and/or with the downstream kinases MKK4 and MKK7. For example, there is evidence that MLK2 and MLK3 associate with the activated (GTP-bound) forms of Rac1 and Cdc42 (48
). Moreover, coexpression of activated Cdc42 with MLK3 leads to a substantial increase in MLK3 dimerization as well as altered MLK3 serine/threonine phosphorylation (4
). However, unlike the case of the PAK family of protein kinases, the activation of MLK3 by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins (4
). Such studies thus suggest that an additional component is required for MLK3 activation by Cdc42. Good candidates for this role would be scaffold proteins, such as JIP1 or POSH. The multidomain protein POSH binds to the constitutively active form of Rac1, which is known to regulate the activity of MLKs, while JIP1 binds to MLKs and additional components of the JNK pathway and appears to be capable of activating MLKs (50
). Thus, it is attractive to consider the possibility that dominant-negative MLKs may act, at least in part, by competing for binding to a common scaffold protein that is required for activation of the JNK death pathway.
One question that is not currently resolved by our experiments is whether all or only a subset of MLK family members participate in the JNK apoptotic pathway. All appear to be expressed in the neuronal cell systems studied here, and all tested members of the family elicited death upon overexpression. This would suggest that multiple family members may indeed contribute to JNK activation and thereby to the death process.
Another kinase that has been raised as a potential upstream component of the JNK apoptotic pathway in neurons is ASK1. Overexpression of ΔN ASK1 evokes death of neuronal PC12 cells and sympathetic neurons, apparently by activation of the JNK/c-Jun pathway, and dominant-negative ASK1 partially protects such cells from death caused by NGF withdrawal (32
). We observed that death stimulated by active ASK1, in contrast to death evoked by NGF deprivation and other apoptotic stimuli, was not blocked by CEP-1347 and that dominant-negative ASK1 partially suppressed death caused by MLK3 and DLK overexpression. Two main interpretations of these findings are currently possible. One is that ASK1 lies downstream of MLKs in the JNK pathway. However, our observation that dominant-negative MLK3 and dominant-negative DLK reciprocally inhibit death evoked by constitutively active ASK1 is inconsistent with this possibility. The other interpretation is that ASK1 is not a major player in the JNK death pathway activated by NGF withdrawal and that the protective effects of dominant-negative ASK1 overexpression reflect nonspecific competition with MLKs for interaction with Rac1, Cdc42, scaffold proteins, or downstream targets.
We noted that death promoted by MLK family members and other elements of the JNK pathway was suppressed by coexpression of a constitutively active form of AKT (Fig. and data not shown). AKT plays an important role in mediating antiapoptotic activities of growth factors and appears to do so in part by phosphorylating and affecting the activities of a number of proapoptotic proteins, including caspase 9, BAD, IκB kinase, GSK3β, and FKHR (16
; reviewed in references 33
). Although AKT was recently reported to inhibit the Rac1 signal transduction pathway (37
), an additional mechanism must be invoked in the present studies, since it also provided protection from elements that are downstream of Rac1 in the JNK death pathway. This indicates that AKT must suppress the JNK-dependent death mechanism not only upstream or at the level of Rac1 activation but also at some point downstream of MLKs (which appear to lack consensus sites for AKT phosphorylation). Although the identity of the downstream AKT-sensitive element(s) is unknown, it is of interest that many known substrates seem unlikely. Rat (the species used in our studies) caspase 9 lacks the consensus site for phosphorylation by AKT, and there is no current evidence that BAD is involved in death evoked by NGF deprivation. Also, GSK3β and FKHR do not appear to be likely downstream targets of the JNK pathway. This raises the possibility that downstream of the JNK pathway there lies an additional novel AKT substrate that regulates survival and death.