Abstract
Neuronal apoptotic death induced by nerve growth factor (NGF) deprivation is reported to be in part mediated through a pathway that includes Rac1 and Cdc42, mitogen-activated protein kinase kinases 4 and 7 (MKK4 and -7), c-Jun N-terminal kinases (JNKs), and c-Jun. However, additional components of the pathway remain to be defined. We show here that members of the mixed-lineage kinase (MLK) family (including MLK1, MLK2, MLK3, and dual leucine zipper kinase [DLK]) are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling. Overexpression of MLKs effectively induces apoptotic death of cultured neuronal PC12 cells and sympathetic neurons, while expression of dominant-negative forms of MLKs suppresses death evoked by NGF deprivation or expression of activated forms of Rac1 and Cdc42. CEP-1347 (KT7515), which blocks neuronal death caused by NGF deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members. In addition, NGF deprivation or UV irradiation leads to an increase in both level and phosphorylation of endogenous DLK. These observations support a role for MLKs in the neuronal death mechanism. With respect to ordering the death pathway, dominant-negative forms of MKK4 and -7 and c-Jun are protective against death induced by MLK overexpression, placing MLKs upstream of these kinases. Additional findings place the MLKs upstream of mitochondrial cytochrome c release and caspase activation.
Highlights
There has been much progress in defining the general mechanisms by which neurons and other cell types die in response to apoptotic stimuli
We show here that members of the mixed-lineage kinase (MLK) family are expressed in neuronal cells and are likely to act between Rac1/Cdc42 and MKK4 and -7 in death signaling
CEP-1347 (KT7515), which blocks neuronal death caused by nerve growth factor (NGF) deprivation and a variety of additional apoptotic stimuli and which selectively inhibits the activities of MLKs, effectively protects neuronal PC12 cells from death induced by overexpression of MLK family members
Summary
There has been much progress in defining the general mechanisms by which neurons and other cell types die in response to apoptotic stimuli. A well-defined downstream target of activated JNKs in neuronal cells that plays an obligatory part in death is c-Jun [14, 15, 22]. Several upstream members of the death-related JNK/c-Jun pathway have been defined. Overexpression of constitutively active forms of Rac and Cdc (Rac V12 [mutated at position 12 to V] and Cdc V12) leads to activation of the JNK pathway and to death of Jurkat T lymphocytes, PC12 cells, and sympathetic neurons; overexpression of dominant-negative mutants of Cdc and Rac (Cdc N17 and Rac N17) in sympathetic neurons prevents elevation of c-Jun and death evoked by nerve growth factor (NGF) withdrawal [2, 7]. We show in particular that MLKs are mediators of JNK activation in neuronal cells
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