Anna C. Maroney

JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis

Trophic factor deprivation (TFD) activates c-Jun N-terminal kinases (JNKs), culminating in coordinate AP1-dependent transactivation of the BH3-only BCL-2 proteins BIM(EL) and HRK, which in turn are critical for BAX-dependent cytochrome c release, caspase activation, and apoptosis. Here, we report that TFD caused not only induction but also phosphorylation of BIM(EL). Mitochondrially localized JNKs but not upstream activators, like mixed-lineage kinases (MLKs) or mitogen-activated protein kinase kinases (MKKs), specifically phosphorylated BIM(EL) at Ser65, potentiating its proapoptotic activity. Inhibition of the JNK pathway attenuated BIM(EL) expression, prevented BIM(EL) phosphorylation, and abrogated TFD-induced apoptosis. Conversely, activation of this pathway promoted BIM(EL) expression and phosphorylation, causing BIM- and BAX-dependent cell death. Thus, JNKs regulate the proapoptotic activity of BIM(EL) during TFD, both transcriptionally and posttranslationally.

The MLK Family Mediates c-Jun N-Terminal Kinase Activation in Neuronal Apoptosis

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 cytochromec release and caspase activation.

β‐Amyloid‐induced neuronal apoptosis requires c‐Jun N‐terminal kinase activation

β‐Amyloid (Aβ) has been strongly implicated in the pathophysiology of Alzheimers disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c‐Jun N‐terminal kinases (JNKs) and of their substrate, c‐Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated Aβ. The activities of JNK family members increased in neuronal PC12 cells within 2 h of Aβ treatment and reached 3–4‐fold elevation by 6 h. To test the role of these changes in death caused by Aβ, we examined the effects of CEP‐1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP‐1347 (100–300 nm) in the culture medium effectively blocked the increases in cellular JNK activity caused by Aβ and, at similar concentrations, protected both PC12 cells and sympathetic neurons from Aβ‐evoked‐death. Effective protection required addition of CEP‐1347 within 2 h of Aβ treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant‐negative c‐Jun construct also conferred protection from Aβ‐evoked death, supporting a model in which JNK activation contributes to death via activation of c‐Jun. Finally, CEP‐1347 blocked Aβ‐stimulated activation of caspase‐2 and ‐3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by Aβ and hence identify it as a potential therapeutic target in AD.

Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC50values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Discovery of CEP-1347/KT-7515, an inhibitor of the JNK/SAPK pathway for the treatment of neurodegenerative diseases.

Apoptosis has been proposed as a mechanism of cell death in Alzheimers, Huntingtons and Parkinsons diseases and the occurrence of apoptosis in these disorders suggests a common mechanism. Events such as oxidative stress, calcium toxicity, mitochondria defects, excitatory toxicity, and deficiency of survival factors are all postulated to play varying roles in the pathogenesis of the diseases. However, the transcription factor c-jun may play a role in the pathology and cell death processes that occur in Alzheimers disease. Parkinsons disease (PD) is also a progressive disorder involving the specific degeneration and death of dopamine neurons in the nigrostriatal pathway. In Parkinsons disease, dopaminergic neurons in the substantia nigra are hypothesized to undergo cell death by apoptotic processes. The commonality of biochemical events and pathways leading to cell death in these diseases continues to be an area under intense investigation. The current therapy for PD and AD remains targeting replacement of lost transmitter, but the ultimate objective in neurodegenerative therapy is the functional restoration and/or cessation of progression of neuronal loss. This chapter will describe a novel approach for the treatment of neurodegenerative diseases through the development of kinase inhibitors that block the active cell death process at an early transcriptional independent step in the stress activated kinase cascade. In particular, preclinical data will be presented on the c-Jun Amino Kinase pathway inhibitor, CEP-1347/KT-7515, with respect to its properties that make it a desirable clinical candidate for treatment of various neurodegenerative diseases.

c-Jun N-terminal kinase specifically phosphorylates p66ShcA at serine 36 in response to ultraviolet irradiation.

Mice lacking expression of the p66 isoform of the ShcA adaptor protein (p66ShcA) are less susceptible to oxidative stress and have an extended life span. Specifically, phosphorylation of p66ShcA at serine 36 is critical for the cell death response elicited by oxidative damage. We sought to identify the kinase(s) responsible for this phosphorylation. Utilizing the SH-SY5Y human neuroblastoma cell model, it is demonstrated that p66ShcA is phosphorylated on serine/threonine residues in response to UV irradiation. Both c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases are activated by UV irradiation, and we show that both are capable of phosphorylating serine 36 of p66ShcA in vitro. However, treatment of cells with a multiple lineage kinase inhibitor, CEP-1347, that blocks UV-induced JNK activation, but not p38, phosphatidylinositol 3-kinase, or MEK1 inhibitors, prevented p66ShcAphosphorylation in SH-SY5Y cells. Consistent with this finding, transfected activated JNK1, but not the kinase-dead JNK1, leads to phosphorylation of serine 36 of p66ShcA in Chinese hamster ovary cells. In conclusion, JNKs are the kinases that phosphorylate serine 36 of p66ShcA in response to UV irradiation in SH-SY5Y cells, and blocking p66ShcA phosphorylation by intervening in the JNK pathway may prevent cellular damage due to light-induced oxidative stress.

CEP‐751 inhibits trk receptor tyrosine kinase activity in vitro and exhibits anti‐tumor activity

The present report describes the in vitro and in vivo profile of CEP‐751, a novel receptor tyrosine kinase inhibitor. CEP‐751 at 100 nM inhibits the receptor tyrosine kinase activity of the neurotrophin receptors trkA, trkB and trkC. CEP‐751 has no effect on activity of receptors for EGF, IGF‐I, insulin or on erbB2; inhibition of receptors for PDGF and bFGF was observed but occurred with lesser potency than inhibition of trk. CEP‐751 exhibited anti‐tumor efficacy against tumors derived from NIH3T3 cells transfected with trkA. Inhibition of trk phosphorylation could also be measured in these tumors, suggesting that anti‐tumor efficacy of CEP‐751 is related to inhibition of trk receptor tyrosine kinase activity. CEP‐751 was found to be without effect when administered to nude mice bearing SK‐OV‐3 tumors, which overexpress erbB2 receptors, providing further evidence that inhibition of tumor growth may be related to inhibition of trk receptor tyrosine kinase activity. Our data indicate that CEP‐751 is a potent trk inhibitor which possesses anti‐tumor activity. Int. J. Cancer 72:673–679, 1997.

Identification of JNK-dependent and -independent components of cerebellar granule neuron apoptosis.

Cerebellar granule neurons grown in high potassium undergo rapid apoptosis when switched to medium containing 5 mm potassium, a stimulus mimicking deafferentation. This cell death can be blocked by genetic deletion of Bax, a member of the pro‐apoptotic Bcl‐2 family, cycloheximide an inhibitor of macromolecular synthesis or expression of dominant‐negative c‐jun. These observations suggest that Bax activation is the result of c‐jun target gene(s) up‐regulation following trophic withdrawal. Candidate genes include the BH3‐only Bcl‐2 family members Dp5 and Bim. The molecular mechanisms underlying granule cell neuronal apoptosis in response to low potassium were investigated using CEP‐1347 (KT7515), an inhibitor of the MLK family of JNKKK. CEP‐1347 provided protection of potassium–serum‐deprived granule cells, but such neuroprotection was not long term. The incomplete protection was not due to incomplete blockade of the JNK signaling pathway because c‐jun phosphorylation as well as induction of c‐jun RNA and protein were completely blocked by CEP‐1347. Following potassium–serum deprivation the JNKK MKK4 becomes phosphorylated, an event blocked by CEP‐1347. Cells that die in the presence of CEP‐1347 activate caspases; and dual inhibition of caspases and MLKs has additive, not synergistic, effects on survival. A lack of synergism was also seen with the p38 inhibitor SB203580, indicating that the neuroprotective effect of the JNK pathway inhibitor cannot be explained by p38 activation. Activation of the JNK signaling pathway seems to be a key event in granule cell apoptosis, but these neurons cannot survive long term in the absence of sustained PI3 kinase signaling.

K‐252a Induces Tyrosine Phosphorylation of the Focal Adhesion Kinase and Neurite Outgrowth in Human Neuroblastoma SH‐SY5Y Cells

Abstract: The protein kinase inhibitor K‐252a has been shown to promote cholinergic activity in cultures of rat spinal cord and neuronal survival in chick dorsal root ganglion cultures. To determine the mechanism by which K‐252a acts as a neurotrophic factor, we examined the effects of this molecule on a human neuroblastoma cell line, SH‐SY5Y. K‐252a induced neurite outgrowth in a dose‐dependent manner. Coincident with neurite outgrowth was the early tyrosine phosphorylation of 125‐ and 140‐kDa proteins. The phosphorylation events were independent of protein kinase C inhibition because down‐regulation of protein kinase C by long‐term treatment with phorbol ester did not prevent K‐252a‐induced tyrosine phosphorylation. Similarly, the protein kinase C inhibitors H7, GF‐109203X, and calphostin C did not induce the phosphorylation. We have identified one of the phosphosubstrates as the pp125 focal adhesion protein tyrosine kinase (Fak). Induction of phosphorylation coincided with increased Fak activity and appeared to be independent of ligand/integrin interaction. The induction of Fak phosphorylation by K‐252a was also observed in LA‐N‐5 cells and primary cultures of rat embryonic striatal cells but not in PC12 cells. The protein kinase C‐independent induction of tyrosine phosphorylation and the identification of Fak as a substrate of K‐252a‐induced tyrosine kinase activity suggest that this compound mediates neurotrophic effects through a novel signaling pathway.

Inhibition of mixed lineage kinase 3 attenuates MPP+-induced neurotoxicity in SH-SY5Y cells

The neuropathology of Parkinsons Disease has been modeled in experimental animals following MPTP treatment and in dopaminergic cells in culture treated with the MPTP neurotoxic metabolite, MPP(+). MPTP through MPP(+) activates the stress-activated c-Jun N-terminal kinase (JNK) pathway in mice and SH-SY5Y neuroblastoma cells. Recently, it was demonstrated that CEP-1347/KT7515 attenuated MPTP-induced nigrostriatal dopaminergic neuron degeneration in mice, as well as MPTP-induced JNK phosphorylation. Presumably, CEP-1347 acts through inhibition of at least one upstream kinase within the mixed lineage kinase (MLK) family since it has been shown to inhibit MLK 1, 2 and 3 in vitro. Activation of the MLK family leads to JNK activation. In this study, the potential role of MLK and the JNK pathway was examined in MPP(+)-induced cell death of differentiated SH-SY5Y cells using CEP-1347 as a pharmacological probe and dominant negative adenoviral constructs to MLKs. CEP-1347 inhibited MPP(+)-induced cell death and the morphological features of apoptosis. CEP-1347 also prevented MPP(+)-induced JNK activation in SH-SY5Y cells. Endogenous MLK 3 expression was demonstrated in SH-SY5Y cells through protein levels and RT-PCR. Adenoviral infection of SH-SY5Y cells with a dominant negative MLK 3 construct attenuated the MPP(+)-mediated increase in activated JNK levels and inhibited neuronal death following MPP(+) addition compared to cultures infected with a control construct. Adenoviral dominant negative constructs of two other MLK family members (MLK 2 and DLK) did not protect against MPP(+)-induced cell death. These studies show that inhibition of the MLK 3/JNK pathway attenuates MPP(+)-mediated SH-SY5Y cell death in culture and supports the mechanism of action of CEP-1347 as an MLK family inhibitor.