Martin Dickens

Opposing Effects of ERK and JNK-p38 MAP Kinases on Apoptosis

Apoptosis plays an important role during neuronal development, and defects in apoptosis may underlie various neurodegenerative disorders. To characterize molecular mechanisms that regulate neuronal apoptosis, the contributions to cell death of mitogen-activated protein (MAP) kinase family members, including ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38, were examined after withdrawal of nerve growth factor (NGF) from rat PC-12 pheochromocytoma cells. NGF withdrawal led to sustained activation of the JNK and p38 enzymes and inhibition of ERKs. The effects of dominant-interfering or constitutively activated forms of various components of the JNK-p38 and ERK signaling pathways demonstrated that activation of JNK and p38 and concurrent inhibition of ERK are critical for induction of apoptosis in these cells. Therefore, the dynamic balance between growth factor-activated ERK and stress-activated JNK-p38 pathways may be important in determining whether a cell survives or undergoes apoptosis.

Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3

ABSTRACT The c-Jun NH2-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPKs) is activated in response to the treatment of cells with inflammatory cytokines and by exposure to environmental stress. JNK activation is mediated by a protein kinase cascade composed of a MAPK kinase and a MAPK kinase kinase. Here we describe the molecular cloning of a putative molecular scaffold protein, JIP3, that binds the protein kinase components of a JNK signaling module and facilitates JNK activation in cultured cells. JIP3 is expressed in the brain and at lower levels in the heart and other tissues. Immunofluorescence analysis demonstrated that JIP3 was present in the cytoplasm and accumulated in the growth cones of developing neurites. JIP3 is a member of a novel class of putative MAPK scaffold proteins that may regulate signal transduction by the JNK pathway.

c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis.

ABSTRACT Recent studies have shown that during apoptosis protein synthesis is inhibited and that this is in part due to the proteolytic cleavage of eukaryotic initiation factor 4G (eIF4G). Initiation of translation can occur either by a cap-dependent mechanism or by internal ribosome entry. The latter mechanism is dependent on a complex structural element located in the 5′ untranslated region of the mRNA which is termed an internal ribosome entry segment (IRES). In general, IRES-mediated translation does not require eIF4E or full-length eIF4G. In order to investigate whether cap-dependent and cap-independent translation are reduced during apoptosis, we examined the expression of c-Myc during this process, since we have shown previously that the 5′ untranslated region of the c-myc proto-oncogene contains an IRES. c-Myc expression was determined in HeLa cells during apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand. We have demonstrated that the c-Myc protein is still expressed when more than 90% of the cells are apoptotic. The presence of the protein in apoptotic cells does not result from either an increase in protein stability or an increase in expression of c-myc mRNA. Furthermore, we show that during apoptosis initiation of c-myc translation occurs by internal ribosome entry. We have investigated the signaling pathways that are involved in this response, and cotransfection with plasmids which harbor either wild-type or constitutively active MKK6, a specific immediate upstream activator of p38 mitogen-activated protein kinase (MAPK), increases IRES-mediated translation. In addition, the c-myc IRES is inhibited by SB203580, a specific inhibitor of p38 MAPK. Our data, therefore, strongly suggest that the initiation of translation via the c-myc IRES during apoptosis is mediated by the p38 MAPK pathway.

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3′ UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

Differentiation of PC12 cells in response to a cAMP analogue is accompanied by sustained activation of mitogen-activated protein kinase: Comparison with the effects of insulin, growth factors and phorbol esters

It has been proposed previously that the sustained activation of mitogen‐activated protein kinase may be necessary for the differentiation of PC12 cells. Differentiation of PC12 cells is induced by many extracellular agonists including nerve growth factor (NGF) and cyclicAMP analogues, but not epidermal growth factor (EGF), insulin or phorbol esters. Our results demonstrate that: (i) 8‐(4‐chlorophenylthio)‐cyclicAMP (CPT‐cAMP) activates MAP kinase; this raises the possibility that the MAP kinase pathway may be activated by agents that act through adenylate cyclase; (ii) NGF and CPT‐cAMP as well as phorbol esters promote sustained activation of MAP kinase. This suggests that while sustained MAP kinase activation may be associated with differentiation it may not be sufficient, and that other as yet unidentified parallel pathways may be involved.

Inhibition of JNK by Overexpression of the JNK Binding Domain of JIP-1 Prevents Apoptosis in Sympathetic Neurons

Studies in non-neuronal cells show that c-Jun N-terminal kinases (JNK) play a key role in apoptotic cell death. In some neurons JNK is also thought to initiate cell death by the activation of c-Jun. JNK inhibition has been achieved pharmacologically by inhibiting upstream kinases, but there has been no direct demonstration that inhibition of JNK can prevent neuronal death. We have therefore examined whether the JNK binding domain (JBD) of JNK-interacting protein-1 (JIP-1, a scaffold protein and specific inhibitor of JNK) can inhibit c-Jun phosphorylation and support the survival of sympathetic neurons deprived of NGF. We show that expression of the JBD in >80% of neurons was sufficient to prevent the phosphorylation of c-Jun and its nuclear accumulation as well as abrogate neuronal cell death induced by NGF deprivation. JBD expression also preserved the capacity of mitochondria to reduce MTT. Interestingly, although the PTB domain of JIP was reported to interact with rhoGEF, expression of the JBD domain was sufficient to localize the protein to the membrane cortex and growth cones. Hence, JNK activation is a key event in apoptotic death induced by NGF withdrawal, where its point of action lies upstream of mitochondrial dysfunction.

Central role for phosphatidylinositide 3-kinase in the repression of glucose-6-phosphatase gene transcription by insulin

Transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase) is stimulated by glucocorticoids and strongly repressed by insulin. We have explored the signaling pathways by which insulin mediates the repression of G6Pase transcription in H4IIE cells. Wortmannin, a phosphatidylinositide 3-kinase (PtdIns 3-kinase) inhibitor blocked the repression of G6Pase mRNA expression by insulin. However, both rapamycin, which inhibits p70S6 kinase activation, and PD98059, an inhibitor of mitogen-activated protein kinase activation, were without effect. Insulin inhibited dexamethasone-induced luciferase expression from a transiently transfected plasmid that places the luciferase gene under the control of the G6Pase promoter. This effect of insulin was mimicked by the overexpression of a constitutively active PtdIns 3-kinase but not by a constitutively active protein kinase B. Taken together, these data demonstrate that PtdIns 3-kinase activation is both necessary and at least partly sufficient for the repression of G6Pase expression by insulin, but neither mitogen-activated protein kinase nor p70S6 kinase are involved. In addition, activation of protein kinase B alone is not sufficient for repression of the G6Pase gene. These results imply the existence of a novel signaling pathway downstream of PtdIns 3 kinase that is involved in the regulation of G6Pase expression by insulin.

JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: Differential requirements for caspase activation

Activation of the stress-activated mitogen-activated protein kinases (MAP kinases), c-Jun N-terminal kinase (JNK) and p38, is necessary for the induction of apoptosis in neuronal cells; however, in other cell types their involvement may be stimulus-dependent. In the present study we investigate the activation of JNK and p38 in a single non-neuronal cell type, undergoing receptor-mediated (tumour necrosis factor-related apoptosis-inducing ligand and CD95) or chemically-induced (lactacystin) apoptosis. In Jurkat T-cells, receptor-mediated and chemically-induced apoptosis resulted in a time-dependent activation of the initiator caspases-8 and -9, respectively. Both types of stimuli resulted in a significant activation of JNK and p38, which closely paralleled the time-dependent induction of apoptosis. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.FMK) inhibited receptor-mediated apoptosis and suppressed JNK and p38 activation. In contrast, inhibition of lactacystin-induced apoptosis with z-VAD.FMK, as assessed by phosphatidylserine exposure and poly(ADP-ribose) polymerase cleavage, did not inhibit activation of JNK or p38, demonstrating that during chemically-induced apoptosis, activation of JNK and p38 is independent of effector caspases. The role of p38 in apoptosis was assessed using the specific p38 inhibitor, SB203580. No effect on the induction of apoptosis or caspase activation was observed, although activation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), an immediate downstream target of p38, was inhibited. Therefore neither p38 activation nor activation of MAPKAPK-2 is critical for induction of either receptor- or chemically-induced apoptosis. Thus, within a single cell type, (1) the mechanism of p38 and JNK activation during apoptosis is stimulus-dependent and (2) activation of the p38 pathway is not required for caspase activation or apoptosis, assessed by phosphatidylserine exposure, but may still be required to elicit other features of the apoptotic phenotype.

Orientation of skeletal muscle actin in strong magnetic fields

Actin Magnetic orientation Birefringence α‐Helix Muscle contraction