Evidence for a Role of Mixed Lineage Kinases in Neuronal Apoptosis

Abstract

Superior cervical ganglion (SCG) sympathetic neurons die by apoptosis when deprived of nerve growth factor (NGF). It has been shown previously that the induction of apoptosis in these neurons at NGF withdrawal requires both the activity of the small GTP-binding protein Cdc42 and the activation of the c-Jun N-terminal kinase (JNK) pathway. The mixed lineage kinase 3 (MLK3) belongs to a family of mitogen-activated protein (MAP) kinase kinase kinases. MLK3 contains a Cdc42/Rac interactive-binding (CRIB) domain and activates both the JNK and the p38 MAP kinase pathways. In this study the role of MLK3 in the induction of apoptosis in sympathetic neurons has been investigated. Overexpression of an active MLK3 induces activation of the JNK pathway and apoptosis in SCG neurons. In addition, overexpression of kinase dead mutants of MLK3 blocks apoptosis as well as c-Jun phosphorylation induced by NGF deprivation. More importantly, MLK3 activity seems to increase by 5 hr after NGF withdrawal in both differentiated PC12 cells and SCG neurons. We also show that MLK3 lies downstream of Cdc42 in the neuronal death pathway. Regulation of MLK3 in neurons seems to be dependent on MLK3 activity and possibly on an additional cellular component, but not on its binding to Cdc42. These results suggest that MLK3, or a closely related kinase, is a physiological element of NGF withdrawal-induced activation of the Cdc42-c-Jun pathway and neuronal death. MLK3 therefore could be an interesting therapeutic target in a number of neurodegenerative diseases involving neuronal apoptosis. Key words: apoptosis; Cdc42; MLK3; signal transduction; sympathetic neurons; Jun kinaseActivation of the c-Jun transcriptional pathway has been shown to be an important regulator of apoptosis of sympathetic neurons induced by nerve growth factor (NGF) withdrawal by both microinjection of antibodies against c-Jun or expression of a c-Jun dominant negative mutant (Estus et al., 1994;Ham et al., 1995). Consistent with these observations, removal of survival factors leads to the activation of c-Jun N-terminal kinase (JNK), either alone or together with p38 mitogen-activated kinase, in a number of neuronal death paradigms, including PC12 cells (Xia et al., 1995), superior cervical ganglion (SCG) neurons (Ham et al., 1995;Eilers et al., 1998), and embryonic motoneurons (Maroney et al., 1998). In addition, phosphorylation of c-Jun and activation of JNK have been observed after neuronal injury in the adult rat brain (Herdegen et al., 1998) (for review, see Mielke and Herdegen, 2000). Taken together, these studies demonstrate that the pathways regulating both the level of c-Jun and its phosphorylation are crucial for the induction of neuronal cell death. Therefore, we were interested in identifying the upstream signaling pathways regulating the activation of the JNK-c-Jun pathway in neurons.Recently, we have shown that, in rat SCG neurons, the Rholike GTPases, Cdc42 and Rac1, are required for NGF withdrawal-induced death and that they induce apoptosis via activation of ...

Endoplasmic reticulum stress-induced apoptosis requires bax for commitment and Apaf-1 for execution in primary neurons

“…20,21 In these neurons, MLK activation by Cdc42/Rac is thought to phosphorylate MEKK4/7 which subsequently phosphorylates and activates JNK signaling. 22,23 JNK activation leads to the upregulation, phosphorylation and activation of the transcription factor c-Jun which is essential for sympathetic neuronal apoptosis in response to nerve growth factor (NGF) withdrawal. 24,25 We examined whether the JNK pathway was activated in sympathetic neurons in response to ER stress by using immunohistochemistry to assess the phosphorylation state of c-Jun.…”

A Scaffold Protein JIP-1b Enhances Amyloid Precursor Protein Phosphorylation by JNK and Its Association with Kinesin Light Chain 1

“…Thus, the JNK activation, which leads to neuronal death, could induce APP phosphorylation. Overexpression of MLK3, which induces the JNK activation leading to neuronal apoptosis (22,26), induced APP phosphorylation in PC12 cells, suggesting that APP phosphorylation is correlated with neuronal death induced by JNK. Although the functional significance of APP phosphorylation in neuronal apoptosis is not clear, it is tempting to speculate that APP phosphorylation is related to the pathogenesis of Alzheimer's disease.…”

“…It was recently shown that overexpression of MLK3 induced the activation of JNK in PC12 cells and effectively induced apoptotic cell death (22,26). Thus, to examine whether the JNK activity that leads to apoptotic cell death also induces APP phosphorylation, the HA-tagged MLK3 was transiently expressed in neuronally differentiated PC12 cells, and APP phosphorylation was detected by immunofluorescence staining.…”

Inhibition of mixed-lineage kinase (MLK) activity during G2-phase disrupts microtubule formation and mitotic progression in HeLa cells

“…The functional role of MLK proteins in activating the stress-activated protein kinases and apoptotic pathways in neuronal cells has been well characterized [6][7][8]. MLK3 is a potent activator of the c-Jun N-terminal kinase (JNK) MAP kinases by directly phosphorylating and activating the JNK kinases, MKK4 and 7 [9,10].…”

“…MLK proteins have been proposed to play a critical role in the progression of neurodegenerative diseases such as Parkinson's, Huntington's, and Alzheimer's through a mechanism involving MLK induced JNK activation, cytochrome c release, and caspase activation of apoptotic pathways [6,8]. Thus, several small molecule inhibitors of MLK activity belonging to the indolocarbazole family, which include CEP-1347 (KT7515) and CEP-11004 (KT-8138), are being tested in clinical trials and may prove beneficial in preventing premature neuronal cell death [6][7][8]11,12].…”