ERK1/2 and p38 cooperate to delay progression through G1 by promoting cyclin D1 protein turnover.

The conditional kinase DeltaMEKK3:ER allows activation of JNK, p38 and ERK1/2 without overt cellular stress or damage and has proved useful in understanding how these pathways regulate apoptosis and cell cycle progression. We have previously shown that activation of DeltaMEKK3:ER causes a sustained G(1) cell cycle arrest which requires p21(CIP1), with ERK1/2 and p38 cooperating to promote p21(CIP1) expression. In cells lacking p21(CIP1), DeltaMEKK3:ER causes only a transient delay in cell cycle re-entry. We now show that this delay in cell cycle re-entry is due to a reduction in cyclin D1 levels. Activation of DeltaMEKK3:ER promotes the proteasome-dependent turnover of cyclin D1; this requires phosphorylation of threonine 286 (T(286)) and expression of cyclin D1T(286)A rescues the delay in G(1)/S progression. DeltaMEKK3:ER-dependent phosphorylation of T(286) does not appear to be mediated by GSK3beta but requires activation of the ERK1/2 and p38 pathways. ERK1/2 can physically associate with cyclin D1 but activation of ERK1/2 alone is not sufficient for phosphorylation of T(286). Rather, cyclin D1 phosphorylation appears to require coincident activation of ERK1/2 and p38. Thus activation of DeltaMEKK3:ER promotes a sustained G(1) cell cycle arrest by a bipartite mechanism involving the rapid destruction of cyclin D1 and the slower more prolonged expression of p21(CIP1). This has parallels with the bipartite response to ionizing radiation and p53-independent mechanisms of G(1) cell cycle arrest in simple organisms such as yeast.

The conditional kinase DeltaMEKK1:ER* selectively activates the JNK pathway and protects against serum withdrawal-induced cell death.

The conditional protein kinase DeltaMEKK3:ER* allows activation of the mitogen-activated and stress-activated protein kinases (MAPKs and SAPKs) without imposing a primary cellular stress or damage. Such separation of stress from stress-induced signalling is particularly important in the analysis of apoptosis. Activation of DeltaMEKK3:ER* in cycling CCl39 cells caused a rapid stimulation of the ERK1/2, JNK and p38 pathways but resulted in a slow, delayed apoptotic response. Paradoxically, activation of the same pathways inhibited the rapid expression of Bim(EL) and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of Bim(EL) but caused only a partial reversion of the cyto-protective effect of DeltaMEKK3:ER*. In contrast, inhibition of p38 had no effect, raising the possibility that activation of JNK might also exert a protective effect. To test this we used CCl39 cells expressing DeltaMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IkappaB kinase. Activation of DeltaMEKK1:ER* inhibited serum withdrawal-induced conformational changes in Bax and apoptosis. These results suggest that in the absence of any overt cellular damage or chemical stress activation of JNK can act independently of the ERK1/2 or PKB pathways to inhibit serum withdrawal-induced cell death.

Selective activation of the c-Jun N-terminal kinase (JNK) pathway fails to elicit Bax activation or apoptosis unless the phosphoinositide 3'-kinase (PI3K) pathway is inhibited.

c-Jun N-terminal kinase (JNK) is activated when cells are exposed to noxious stimuli. The role of JNK in apoptosis is subject to considerable debate; for example, JNK activation may promote or inhibit apoptosis depending on the cell type and stimulus involved. These conflicting results have arisen in part because few studies have successfully separated JNK activation from the primary stress-induced damage or from other stress-induced signalling pathways. Here we describe a conditional mutant, deltaMEKK1:ER*, which allows selective activation of the JNK cascade in the absence of any cellular stress. Activation of deltaMEKK1:ER* in CC139 fibroblasts resulted in the rapid and sustained activation of JNK without activating ERK or p38 or promoting IkappaBalpha phosphorylation. Activation of deltaMEKK1:ER* caused a reversible halt in cell growth but failed to induce apoptosis. In contrast, treatment of cells with LY294002, to inhibit phosphoinositide 3-kinase (PI3K), caused downregulation of Bcl-2 and Mcl-1 and allowed deltaMEKK1:ER* to elicit a robust apoptotic response characterized by activation of Bax and caspases. This PI3K-inhibitable, JNK-induced death response was not impeded, but actually accelerated, by cycloheximide. This suggests that JNK-induced activation of Bax and cell death does not require the upregulation of pro-death genes such as Bim or FasL, but rather proceeds through pre-existing components. However, if the PI3K cell survival pathway is not inhibited, even sustained activation of JNK exerts no overt proapoptotic effect in CC139 cells.

Delta MEKK3:ER* activation induces a p38 alpha/beta 2-dependent cell cycle arrest at the G2 checkpoint.

Whilst many studies have examined the role of the MAP Kinases in regulating the G1-->S transition, much less is known about the function of these pathways in regulating other cell cycle transitions. Stimulation of the conditional mutant Delta MEKK3:ER* in asynchronous hamster (CCl39) and rat (Rat-1) fibroblasts resulted in the strong activation of endogenous JNK and p38 but only a weak activation of ERK. Activation of Delta MEKK3:ER* inhibited cell proliferation through a combination of an initial G1 and G2 cell cycle arrest, followed by a delayed onset of apoptosis. When cells were synchronized in S phase with aphidicolin and then released, activation of Delta MEKK3:ER* resulted in the up-regulation of p21(CIP1) and a pronounced inhibition of cyclin A/CDK2 and cyclin B1/CDK1 kinase activity. Analysis of mitotic figures indicated that cells failed to enter mitosis, arresting late in G2. Delta MEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21(CIP1), since both events were observed in Rat-1 cells in which p21(CIP1) is transcriptionally silenced due to promoter methylation. Rather, CDK inhibition was associated with a down-regulation of cyclin A and B1 expression. Finally, application of the p38 inhibitor SB203580 partially restored cyclin B associated kinase activity and allowed cells to proceed through mitosis into the next G1 phase, suggesting that activation of the p38 alpha/beta 2 pathway can promote a G2 cell cycle arrest.

ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest.

To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of DeltaMEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38alpha but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21CIP1 and inhibition of CDK2 activity. In Rat-1 cells, in which p21(CIP1) expression is silenced by methylation, DeltaMEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G1 arrest. Furthermore, p21CIP1-/- 3T3 cells were defective for the DeltaMEKK3:ER*-induced G1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated DeltaMEKK3:ER* inhibits the G1 --> S progression by two kinetically distinct mechanisms, with expression of p21CIP1 being required to ensure a sustained G1 cell cycle arrest. The ERK1/2 and p38alphabeta pathways cooperated to induce p21CIP1 expression and inhibition of p38alphabeta caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by DeltaRaf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by DeltaMEKK1:ER* failed to inhibit cell cycle re-entry, even in cells that retained wild-type p53, arguing against a major role for JNK alone in antagonizing the G1 --> S transition.

Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3.

Inhibition of mixed lineage kinase 3 (MLK3) is a potential strategy for treatment of Parkinson's disease and HIV-1 associated neurocognitive disorders (HAND), requiring an inhibitor that can achieve significant brain concentration levels. We report here URMC-099 (1) an orally bioavailable (F = 41%), potent (IC50 = 14 nM) MLK3 inhibitor with excellent brain exposure in mouse PK models and minimal interference with key human CYP450 enzymes or hERG channels. The compound inhibits LPS-induced TNFα release in microglial cells, HIV-1 Tat-induced release of cytokines in human monocytes and up-regulation of phospho-JNK in Tat-injected brains of mice. Compound 1 likely functions in HAND preclinical models by inhibiting multiple kinase pathways, including MLK3 and LRRK2 (IC50 = 11 nM). We compare the kinase specificity and BBB penetration of 1 with CEP-1347 (2). Compound 1 is well tolerated, with excellent in vivo activity in HAND models, and is under investigation for further development.

Carbapenem antibiotic biosynthesis in Erwinia carotovora is regulated by physiological and genetic factors modulating the quorum sensing-dependent control pathway.

Erwinia carotovora produces the beta-lactam antibiotic, carbapenem, in response to a quorum sensing signalling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). We have mapped the OHHL-dependent promoter upstream of the first of the biosynthetic genes, carA. We have also analysed the effect on this promoter of the known genetic regulators of carbapenem expression, carR, carI (encoding homologues of LuxR and LuxI respectively) and hor (encoding a SlyA/MarR-like transcriptional regulator). We describe a previously unknown promoter located within the carA-H operon. This promoter does not respond to CarR and is required for quorum sensing-independent expression of the carbapenem resistance determinants encoded by the carFG genes. We have mapped the carR, carI and hor transcription start points, shown that CarR is positively autoregulated in the presence of OHHL, and have demonstrated negative feedback affecting transcription of carI. In addition, various environmental and physiological factors were shown to impinge on the transcription of the car biosynthetic genes. The nature of the carbon source and the temperature of growth influence carbapenem production by modulating the level of the OHHL signalling molecule, and thereby physiologically fine-tune the quorum sensing regulatory system.