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Innate or acquired resistance to small molecule BRAF or MEK1/2 inhibitors (BRAFi or MEKi) typically arises through mechanisms that sustain or reinstate ERK1/2 activation. This has led to the development of a range of ERK1/2 inhibitors (ERKi) that either inhibit kinase catalytic activity (catERKi) or additionally prevent the activating pT-E-pY dual phosphorylation of ERK1/2 by MEK1/2 (dual-mechanism or dmERKi). Here, we show that eight different ERKi (both catERKi or dmERKi) drive the turnover of ERK2, the most abundant ERK isoform, with little or no effect on ERK1. Thermal stability assays show that ERKi do not destabilise ERK2 (or ERK1) in vitro, suggesting that ERK2 turnover is a cellular consequence of ERKi binding. ERK2 turnover is not observed upon treatment with MEKi alone, suggesting it is ERKi binding to ERK2 that drives ERK2 turnover. However, MEKi pre-treatment, which blocks ERK2 pT-E-pY phosphorylation and dissociation from MEK1/2, prevents ERK2 turnover. ERKi treatment of cells drives the poly-ubiquitylation and proteasome-dependent turnover of ERK2 and pharmacological or genetic inhibition of Cullin-RING E3 ligases prevents this. Our results suggest that ERKi, including current clinical candidates, act as ‘kinase degraders’, driving the proteasome-dependent turnover of their major target, ERK2. This may be relevant to the suggestion of kinase-independent effects of ERK1/2 and the therapeutic use of ERKi. [ABSTRACT FROM AUTHOR]

Inhibitor of kappa B (IκB) kinase ß (IKKß) has long been viewed as the dominant IKK in the canonical nuclear factor-κB (NF-κB) signalling pathway, with IKKa being more important in non-canonical NF-κB activation. Here we have investigated the role of IKKa and IKKß in canonical NF-κB activation in colorectal cells using CRISPR-Cas9 knock-out cell lines, siRNA and selective IKKß inhibitors. IKKa and IKKß were redundant for IκBa phosphorylation and turnover since loss of IKKa or IKKß alone had little (SW620 cells) or no (HCT116 cells) effect. However, in HCT116 cells IKKa was the dominant IKK required for basal phosphorylation of p65 at S536, stimulated phosphorylation of p65 at S468, nuclear translocation of p65 and the NF-κB-dependent transcriptional response to both TNFa and IL-1a. In these cells, IKKß was far less efficient at compensating for the loss of IKKa than IKKa was able to compensate for the loss of IKKß. This was confirmed when siRNA was used to knock-down the non-targeted kinase in single KO cells. Critically, the selective IKKß inhibitor BIX02514 confirmed these observations in WT cells and similar results were seen in SW620 cells. Notably, whilst IKKa loss strongly inhibited TNFa- dependent p65 nuclear translocation, IKKa and IKKß contributed equally to c-Rel nuclear translocation indicating that different NF-κB subunits exhibit different dependencies on these IKKs. These results demonstrate a major role for IKKa in canonical NF-κB signalling in colorectal cells and may be relevant to efforts to design IKK inhibitors, which have focused largely on IKKß to date. [ABSTRACT FROM AUTHOR]

Disruption of protein folding in the endoplasmic reticulum (ER) causes ER stress. Activation of the unfolded protein response (UPR) acts to restore protein homeostasis or, if ER stress is severe or persistent, drive apoptosis, which is thought to proceed through the cell intrinsic, mitochondrial pathway. Indeed, cells that lack the key executioner proteins BAX and BAK are protected from ER stress-induced apoptosis. Here we show that chronic ER stress causes the progressive inhibition of the extracellular signal-regulated kinase (ERK1/2) signalling pathway. This is causally related to ER stress since reactivation of ERK1/2 can protect cells from ER stress-induced apoptosis whilst ERK1/2 pathway inhibition sensitises cells to ER stress. Furthermore, cancer cell lines harbouring constitutively active BRAFV600E are addicted to ERK1/2 signalling for protection against ER stress-induced cell death. ERK1/2 signalling normally represses the pro-death proteins BIM, BMF and PUMA and it has been proposed that ER stress induces BIM-dependent cell death. We found no evidence that ER stress increased the expression of these proteins; furthermore, BIM was not required for ER stress-induced death. Rather, ER stress caused the PERK-dependent inhibition of cap-dependent mRNA translation and the progressive loss of pro-survival proteins including BCL2, BCLXL and MCL1. Despite these observations, neither ERK1/2 activation nor loss of BAX/BAK could confer long-term clonogenic survival to cells exposed to ER stress. Thus, ER stress induces cell death by at least two biochemically and genetically distinct pathways: a classical BAX/BAK-dependent apoptotic response that can be inhibited by ERK1/2 signalling and an alternative ERK1/2- and BAX/BAK-independent cell death pathway. [ABSTRACT FROM AUTHOR]

The mTOR [mammalian (or mechanistic) target of rapamycin] protein kinase co-ordinates catabolic and anabolic processes in response to growth factors and nutrients and is a validated anticancer drug target. Rapamycin and related allosteric inhibitors of mTORC1 (mTOR complex 1) have had some success in specific tumour types, but have not exhibited broad anticancer activity, prompting the development of new ATP-competitive mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2. In common with other targeted kinase inhibitors, tumours are likely to adapt and acquire resistance to mTOR inhibitors. In the present article, we review studies that describe how tumour cells adapt to become resistant to mTOR inhibitors. mTOR is a central signalling hub which responds to an array of signalling inputs and activates a range of downstream effector pathways. Understanding how this signalling network is remodelled and which pathways are invoked to sustain survival and proliferation in the presence of mTOR inhibitors can provide new insights into the importance of the various mTOR effector pathways and may suggest targets for intervention to combine with mTOR inhibitors. Finally, since chronic mTOR inhibition by rapamycin can increase lifespan and healthspan in nematodes, fruitflies and mice, we contrast these studies with tumour cell responses to mTOR inhibition. [ABSTRACT FROM AUTHOR]

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim−/− fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-xL and Mcl-1. Upon serum withdrawal, newly expressed BimEL associates with Bcl-xL and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed BimEL/Mcl-1 and BimEL/Bcl-xL complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for BimEL and requires ERK1/2-dependent phosphorylation of BimEL at Ser65. Finally, ERK1/2-dependent dissociation of BimEL from Mcl-1 and Bcl-xL may play a role in regulating BimEL degradation, since mutations in the BimEL BH3 domain that disrupt binding to Mcl-1 cause increased turnover of BimEL. These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway. [ABSTRACT FROM AUTHOR]

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 ?MEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38a 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 p21CIP1 expression is silenced by methylation, ?MEKK3: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 ?MEKK3:ER*-induced G1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated ?MEKK3: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 p38aß pathways cooperated to induce p21CIP1 expression and inhibition of p38aß caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by ?Raf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by ?MEKK1: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.Oncogene (2004) 23, 3284-3295. doi:10.1038/sj.onc.1207467 Published online 23 February 2004 [ABSTRACT FROM AUTHOR]

Elevation of cellular cyclic AMP (cAMP) levels inhibits cell cycle reentry in a variety of cell types. While cAMP can prevent the activation of Raf-1 and extracellular signal-regulated kinases 1 and 2 (ERK½) by growth factors, we now show that activation of ERK½ by ΔRaf-1:ER is insensitive to cAMP. Despite this, ΔRaf-1:ER-stimulated DNA synthesis is still inhibited by cAMP, indicating a cAMP-sensitive step downstream of ERK½. Although cyclin D1 expression has been proposed as an alternative target for cAMP, we found that cAMP could inhibit ΔRaf-1:ER-induced cyclin D1 expression only in Rat-1 cells, not in CCl39 or NIH 3T3 cells. ΔRaf-1:ER-stimulated activation of CDK2 was strongly inhibited by cAMP in all three cell lines, but cAMP had no effect on the induction of p21[sup CIP1]. cAMP blocked the fetal bovine serum (FBS)-induced degradation of p27[sup KIP1]; however, loss of p27[sup KIP1] in response to ΔRaf-1:ER was less sensitive in CC139 and Rat-1 cells and was completely independent of cAMP in NIH 3T3 cells. The most consistent effect of cAMP was to block both FBSand ΔRaf-1:ER-induced expression of Cdc25A and cyclin A, two important activators of CDK2. When CDK2 activity was bypassed by activation of the ER-E2F1 fusion protein, cAMP no longer inhibited expression of Cdc25A or cyclin A but still inhibited DNA synthesis. These studies reveal multiple points of cAMP sensitivity during cell cycle reentry. Inhibition of Raf-1 and ERK½ activation may operate early in G[sub 1], but when this early block is bypassed by ΔRaf-1:ER, cells still fail to enter S phase due to inhibition of CDK2 or targets downstream of E2F1. [ABSTRACT FROM AUTHOR]

Investigates the promotion of phosphorylation and proteasome-dependent degradation of the BH-3 only protein bim by activating the extracellular signal-regulated kinase 1 and the phosphatidylinositol 3'-kinase signaling pathways. Cell protection from apoptosis following withdrawal of survival factors; Increase in the turnover rate of the Bim[sub EL] protein; Accumulation of polyubiquitinated forms of Bim.

CC139 fibroblasts are one of several model systems in which the Raf?MEK?ERK1/2 pathway can inhibit apoptosis independently of the PI3K pathway; however, the precise mechanism for this protective effect is not known. Serum withdrawal from CC139 fibroblasts resulted in the rapid onset of apoptosis, which was prevented by actinomycin D or cycloheximide. Serum withdrawal promoted the rapid, de novo accumulation of BimEL, a proapoptotic 'BH3-only' member of the Bcl-2 protein family. BimEL expression was an early event, occurring several hours prior to caspase activation. In contrast to studies in neurons, activation of the JNK?c-Jun pathway was neither necessary nor sufficient to induce BimEL expression. Selective inhibition of either the ERK pathway (with U0126) or the PI3K pathway (with LY294002) caused an increase in the expression of BimEL. Furthermore, selective activation of the ERK1/2 pathway by ?Raf-1:ER* substantially reduced BimEL expression, abolished conformational changes in Bax and blocked the appearance of apoptotic cells. The ability of ?Raf-1:ER* to repress BimEL expression required the ERK pathway but was independent of the PI3K?PDK?PKB pathway. Thus, serum withdrawal-induced expression of BimEL occurs independently of the JNK?c-Jun pathway and can be repressed by the ERK pathway independently of the PI3K pathway. This may contribute to Raf- and Ras-induced cell survival at low serum concentrations.Oncogene (2003) 22, 1281-1293. doi:10.1038/sj.onc.1206261 [ABSTRACT FROM AUTHOR]

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 ΔMEKK3: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 ΔMEKK3: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 ΔMEKK3:ER* resulted in the up-regulation of p21CIP1 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. ΔMEKK3:ER*-mediated CDK inhibition and G2 arrest did not absolutely require p21CIP1, since both events were observed in Rat-1 cells in which p21CIP1 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 p38α/β2 pathway can promote a G2 cell cycle arrest.Oncogene (2002) 21, 8089–8104. doi:10.1038/sj.onc.1206000 [ABSTRACT FROM AUTHOR]

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21Cip1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 and cell cycle re-entry. The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent... [ABSTRACT FROM AUTHOR]

The pharmacokinetics and pharmacodynamics of prolonged oral etoposide chemotherapy were investigated in 15 women with metastatic breast cancer who received oral etoposide 100 mg as a single daily dose for up to 15 days. There was considerable interpatient variability in the day 1 pharmacokinetic parameters: area under the plasma concentration time curve (AUC) (0-24 h) 1.95±0.87 mg/ml per min (mean ± SD), apparent oral clearance 60.9±21.7 ml/min per 1.73 m, peak plasma concentration 5.6±2.5 μg/ml, time to peak concentration 73±35 min and half-life 220±83 min. However, intrapatient variability in systemic exposure to etoposide was much less with repeated doses. The intrapatient coefficient of variation (CV) of AUC for day 8 relative to day 1 was 20% and for day 15 relative to day 1 was 15%, compared to the day 1 interpatient CV of 45%. Neutropenia was the principal toxicity. Day 1 pharmacokinetic parameters were related to the percentage decrease in absolute neutrophil count using the sigmoidal E equation. A good fit was found between day 1 AUC and neutrophil toxicity ( R=0.77). All patients who had a day 1 AUC>2.0 mg/ml per min had WHO grade III or IV neutropenia. The predictive performance of the models for neutrophil toxicity was better for AUC (percentage mean predictive error 5%, percentage root mean square error 18.1%) than apparent oral clearance, peak plasma concentration, or daily dose (mg/m). A limited sampling strategy was developed to predict AUC using a linear regression model incorporating a patient effect. Data sets were divided into training and test sets. The AUC could be estimated using a model utilizing plasma etoposide concentration at only two time points, 4 h and 6 h after oral dosing ( R=98.9%). The equation AUC=−0.376+0.631×C+0.336×C was validated on the test set with a relative mean predictive error of −0.88% and relative root mean square error of 6.4%. These results suggest monitoring of AUC to predict subsequent myelosuppression as a strategy for future trials with oral etoposide. [ABSTRACT FROM AUTHOR]

Acquired resistance to MEK1/2 inhibitors (MEKi) arises through amplification of BRAFV600E or KRASG13D to reinstate ERK1/2 signalling. Here we show that BRAFV600E amplification and MEKi resistance are reversible following drug withdrawal. Cells with BRAFV600E amplification are addicted to MEKi to maintain a precise level of ERK1/2 signalling that is optimal for cell proliferation and survival, and tumour growth in vivo. Robust ERK1/2 activation following MEKi withdrawal drives a p57KIP2-dependent G1 cell cycle arrest and senescence or expression of NOXA and cell death, selecting against those cells with amplified BRAFV600E. p57KIP2 expression is required for loss of BRAFV600E amplification and reversal of MEKi resistance. Thus, BRAFV600E amplification confers a selective disadvantage during drug withdrawal, validating intermittent dosing to forestall resistance. In contrast, resistance driven by KRASG13D amplification is not reversible; rather ERK1/2 hyperactivation drives ZEB1-dependent epithelial-to-mesenchymal transition and chemoresistance, arguing strongly against the use of drug holidays in cases of KRASG13D amplification. Colorectal cancer cells can acquire resistance to MEK inhibition due to BRAF or KRAS amplification. Here, the authors show that while MEK inhibitor withdrawal in BRAF mutant cells restores sensitivity to the inhibitor through the loss of BRAF amplification mediated by a p57-dependent mechanism, drug withdrawal from KRAS mutant cells does not restore sensitivity but results in EMT and chemoresistance. [ABSTRACT FROM AUTHOR]

Abstract: The pro-apoptotic BH3 only protein BIMEL is phosphorylated by ERK1/2 and this targets it for proteasome-dependent degradation. A recent study has shown that ERK5, an ERK1/2-related MAPK, is activated during mitosis and phosphorylates BIMEL to promote cell survival. Here we show that treatment of cells with nocodazole or paclitaxel does cause phosphorylation of BIMEL, which is independent of ERK1/2. However, this was not due to ERK5-catalysed phosphorylation, since it was not reversed by the MEK5 inhibitor BIX02189 and proceeded normally in ERK5−/− fibroblasts. Indeed, although ERK5 is phosphorylated at multiple sites in the C-terminal transactivation region during mitosis, these do not include the activation-loop and ERK5 kinase activity does not increase. Mitotic phosphorylation of BIMEL occurred at proline-directed phospho-acceptor sites and was abolished by selective inhibition of CDK1. Furthermore, cyclin B1 was able to interact with BIM and cyclin B1/CDK1 complexes could phosphorylate BIM in vitro. Finally, we show that CDK1-dependent phosphorylation of BIMEL drives its polyubiquitylation and proteasome-dependent degradation to protect cells during mitotic arrest. These results provide new insights into the regulation of BIMEL and may be relevant to the therapeutic use of agents such as paclitaxel. [Copyright &y& Elsevier]

Abstract: The conditional protein kinase ΔMEKK3: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 ΔMEKK3: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 BimEL and apoptosis following withdrawal of serum. Inhibition of the ERK1/2 pathway prevented the down-regulation of BimEL but caused only a partial reversion of the cyto-protective effect of ΔMEKK3: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 ΔMEKK1:ER* which activates JNK but not ERK1/2, p38, PKB or IκB kinase. Activation of ΔMEKK1: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. [Copyright &y& Elsevier]

The proapoptotic protein Bim is expressed de novo following withdrawal of serum survival factors. Here, we show that Bim−/− fibroblasts and epithelial cells exhibit reduced cell death following serum withdrawal in comparison with their wild-type counterparts. In viable cells, Bax associates with Bcl-2, Bcl-xL and Mcl-1. Upon serum withdrawal, newly expressed BimEL associates with Bcl-xL and Mcl-1, coinciding with the dissociation of Bax from these proteins. Survival factors can prevent association of Bim with pro-survival proteins by preventing Bim expression. However, we now show that even preformed BimEL/Mcl-1 and BimEL/Bcl-xL complexes can be rapidly dissociated following activation of ERK1/2 by survival factors. The dissociation of Bim from Mcl-1 is specific for BimEL and requires ERK1/2-dependent phosphorylation of BimEL at Ser65. Finally, ERK1/2-dependent dissociation of BimEL from Mcl-1 and Bcl-xL may play a role in regulating BimEL degradation, since mutations in the BimEL BH3 domain that disrupt binding to Mcl-1 cause increased turnover of BimEL. These results provide new insights into the role of Bim in cell death and its regulation by the ERK1/2 survival pathway. [ABSTRACT FROM AUTHOR]

Abstract: The duration of ERK1/2 activation influences the nature of the biological response to agonist. Members of the AP-1 transcription factor family are well known targets of the ERK1/2 pathway and are expressed in a temporally coordinated fashion during cell cycle re-entry. In CCl39 fibroblasts, sustained ERK1/2 activation is required for the expression of Fra-1, Fra-2, c-Jun and JunB, whereas expression of c-Fos is still strongly induced even in response to transient ERK activation. However, the significance of this pattern of expression for AP-1 activity has not been addressed. Here we show that growth factor stimulated activation of the C-terminal c-Fos transactivation domain (TAD) serves as a sensor for ERK1/2 signal duration whereas the c-JunTAD is not responsive to growth factors. In addition, sustained ERK1/2 activation determines the duration of increases in AP-1 DNA binding complexes as well as their qualitative make up. Finally, this is reflected in both the duration and quantitative transcriptional output of stably integrated AP-1 reporter constructs, indicating that AP-1 activity is finely tuned to ERK1/2 signal duration. These results provide new insights into the importance of ERK1/2 signal duration in the regulation of AP-1 and provide an explanation for how differences in signal duration can lead to both quantitative and qualitative changes in gene expression. [Copyright &y& Elsevier]

Bim, a ‘BH3-only’ protein, is expressed de novo following withdrawal of serum survival factors and promotes cell death. We have shown previously that activation of the ERK½ pathway promotes phosphorylation of BimEL, targeting it for degradation via the proteasome. However, the nature of the kinase responsible for BimEL phosphorylation remained unclear. We now show that BimEL is phosphorylated on at least three sites in response to activation of the ERK½ pathway. By using the peptidylprolyl isomerase, Pin1, as a probe for proline-directed phosphorylation, we show that ERK½-dependent phosphorylation of BimEL occurs at (S/T)P motifs. ERK½ phosphorylates BimEL, but not BimS or BimL, in vitro, and mutation of Ser65 to alanine blocks the phosphorylation of BimEL by ERK½ in vitro and in vivo and prevents the degradation of the protein following activation of the ERK½ pathway. We also find that ERK½, but not JNK, can physically associate with GST-BimEL, but not GST-BimL or GST-BimS, in vitro. ERK½ also binds to full-length BimEL in vivo, and we have localized a potential ERK½ ‘docking domain’ lying within a 27-amino acid stretch of the BimEL protein. Our findings provide new insights into the post-translational regulation of BimEL and the role of the ERK½ pathway in cell survival signaling. [ABSTRACT FROM AUTHOR]

Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis. • Autophagy initiation is repressed during mitosis, even during nutrient deprivation • RAPTOR phosphorylation in mitosis prevents mTORC1 localization to lysosomes • mTORC1 is inhibited during mitosis • CDK1 phosphorylates autophagy regulators at mTORC1 sites to repress autophagy Odle and colleagues show that while autophagy is usually repressed by the nutrient-responsive mTORC1 kinase complex, this is not the case during mitosis. Instead, CCNB1-CDK1 catalyzes phosphorylation at the same repressive sites, taking over the role of repressing autophagy regulators. Thus, repression of autophagy is ensured regardless of nutrient availability. [ABSTRACT FROM AUTHOR]