Oncogenic RAC1 and NRAS drive resistance to endoplasmic reticulum stress through MEK/ERK signalling

Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases.     

Kinetics of ERK1/2 activation determine sensitivity of acute myeloid leukaemia cells to the induction of apoptosis by the novel small molecule ingenol 3-angelate (PEP005)

The novel small molecule ingenol 3-angelate (PEP005) has been shown previously to induce apoptosis in leukaemic cell lines and primary AML cells, an effect that requires the expression of protein kinase C-delta (PKCδ). Here we have investigated signalling events downstream of PKCδ that determine sensitivity of AML cells to PEP005. We show that activation of ERK1/2 MAP kinase occurred in both sensitive and resistant cells and that induction of apoptosis required sustained signalling through the ERK1/2 pathway. Inhibition of ERK1/2 signalling using the MEK inhibitor PD98059 inhibited PEP005-induced apoptosis and activation of ERK1/2 was shown to occur downstream of PKC activation. The data show that PEP005-induced apoptosis is both PKC and ERK1/2 dependent and indicate that chronic activation of ERK1/2 in leukaemic cells delivers a pro-apoptotic rather than a proliferative or survival signal.     

Mechanisms of regulating the Raf kinase family

The MAP Kinase pathway is a key signalling mechanism that regulates many cellular functions such as cell growth, transformation and apoptosis. One of the essential components of this pathway is the serine/threonine kinase, Raf. Raf (MAPKK kinase, MAPKKK) relays the extracellular signal from the receptor/Ras complex to a cascade of cytosolic kinases by phosphorylating and activating MAPK/ERK kinase (MEK; MAPK kinase, MAPKK) that phosphorylates and activates extracellular signal regulated kinase (ERK; mitogen-activated protein kinase, MAPK), which phosphorylates various cytoplasmic and nuclear proteins. Regulation of both Ras and Raf is crucial in the proper maintenance of cell growth as oncogenic mutations in these genes lead to high transforming activity. Ras is mutated in 30% of all human cancers and B-Raf is mutated in 60% of malignant melanomas. The mechanisms that regulate the small GTPase Ras as well as the downstream kinases MEK and extracellular signal regulated kinase (ERK) are well understood. However, the regulation of Raf is complex and involves the integration of other signalling pathways as well as intramolecular interactions, phosphorylation, dephosphorylation and protein-protein interactions. From studies using mammalian isoforms of Raf, as well as C. elegans lin45-Raf, common patterns and unique differences of regulation have emerged. This review will summarize recent findings on the regulation of Raf kinase.     

The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G1 Phase Progression in Proliferating Hepatocytes

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G(1)/S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12- and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G(1) phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.     

Rapid and transient activation of the ERK MAPK signalling pathway by macrophage migration inhibitory factor (MIF) and dependence on JAB1/CSN5 and Src kinase activity

Macrophage migration inhibitory factor (MIF) is a 12.5 kD polypeptide that serves as a critical regulator of cell functions such as gene expression, proliferation or apoptosis. However, the signal transduction pathways through which MIF takes part in cellular regulation are only incompletely understood. MIF leads to CD74-dependent "sustained" activation of ERK1/2 MAPK, but MIF's role in "transient" ERK activation and the involved upstream pathways are unknown. Here we report that the transient ERK pathway was markedly activated by MIF. This effect involved the phosphorylation and activation of Raf-1, MEK, ERK, and Elk-1. Of note, rapid and transient ERK phosphorylation by MIF was measurable in MIF-deficient cells, suggesting that MIF acted in a non-autocrine fashion. Applying the inhibitor genistein, a tyrosine kinase (TPK) activity was identified as a critical upstream signalling event in MIF-induced transient ERK signalling. Experiments using the Src kinase inhibitor PP2 indicated that the involved TPK was a Src-type tyrosine kinase. A role for an upstream Src kinase was proven by applying Src-deficient cells which did not exhibit transient ERK activation upon treatment with MIF, but in which MIF-induced ERK signalling could be restored by re-expressing Src. Intriguingly, JAB1/CSN5, a signalosome component, cellular binding protein of MIF and regulator of cell proliferation and survival, had a marked, yet dual, effect on MIF-induced ERK signalling. JAB1 overexpression inhibited sustained, but not transient, ERK phosphorylation. By contrast, JAB1-knock-down by siRNA revealed that minimum JAB1 levels were necessary for transient activation of ERK by MIF. In conclusion, MIF rapidly and transiently activates the ERK pathway, an effect that has not been recognized previously. This signalling pathway involves the upstream activation of a Src-type kinase and is co-regulated by the cellular MIF binding protein JAB1/CSN5. Our study thus has unravelled a novel MIF-driven signalling pathway and an intricate regulatory system involving extra- and possibly intracellular MIF, and which likely critically participates in controlling cell proliferation and survival.     

A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.     

ERK5/MAPK is activated by TGFbeta in hepatocytes and required for the GSK-3beta-mediated Snail protein stabilization

Extracellular signal-regulated protein kinase 5 (ERK5) is a mitogen-activated protein kinase, specifically activated by MEK5, and involved in the regulation of many cellular functions including proliferation, survival, differentiation and apoptosis. MEK5/ERK5 module is an important element of different signal transduction pathways. The aim of this study was to investigate whether ERK5 participates to the signalling of the multifunctional cytokine TGFbeta, known to play an important role in the regulation of hepatic growth. Here, we reported that ERK5 is phosphorylated and activated by TGFbeta in hepatocytes, with a rapid and sustained kinetic, through a Src-dependent pathway. Moreover, we demonstrated that ERK5 participates to the TGFbeta-induced Snail protein regulation being required for its stabilization. We also found that the functional inactivation of ERK5 impedes the TGFbeta-mediated glycogen synthase kinase-3beta inactivation suggesting this as mechanism responsible for ERK5-mediated Snail stabilization. Thus, results presented in this study uncovered for the first time a role for ERK5 in the TGFbeta-induced cellular responses.     

Rac-PAK signaling stimulates extracellular signal-regulated kinase (ERK) activation by regulating formation of MEK1-ERK complexes

Utilizing mutants of extracellular signal-regulated kinase 2 (ERK2) that are defective for intrinsic mitogen-activated protein kinase or ERK kinase (MEK) binding, we have identified a convergent signaling pathway that facilitates regulated MEK-ERK association and ERK activation. ERK2-delta19-25 mutants defective in MEK binding could be phosphorylated in response to mitogens; however, signaling from the Raf-MEK pathway alone was insufficient to stimulate their phosphorylation in COS-1 cells. Phosphorylation of ERK2-delta19-25 but not of wild-type ERK2 in response to Ras V12 was greatly inhibited by dominant-negative Rac. Activated forms of Rac and Cdc42 could enhance the association of wild-type ERK2 with MEK1 but not with MEK2 in serum-starved adherent cells. This effect was p21-activated kinase (PAK) dependent and required the putative PAK phosphorylation sites T292 and S298 of MEK1. In detached cells placed in suspension, ERK2 was complexed with MEK2 but not with MEK1. However, upon replating of cells onto a fibronectin matrix, there was a substantial induction of MEK1-ERK2 association and ERK activation, both of which could be inhibited by dominant-negative PAK1. These data show that Rac facilitates the assembly of a mitogen-activated protein kinase signaling complex required for ERK activation and that this facilitative signaling pathway is active during adhesion to the extracellular matrix. These findings reveal a novel mechanism by which adhesion and growth factor signals are integrated during ERK activation.     

Truncated MEK1 is required for transient activation of MAPK signalling in G2 phase cells

The primary endpoint of signalling through the canonical Raf–MEK–ERK MAP kinase cascade is ERK activation. Here we report a novel signalling outcome for this pathway. Activation of the MAP kinase pathway by growth factors or phorbol esters during G2 phase results in only transient activations of ERK and p90RSK, then suppression to below control levels. A small peak of ERK and p90RSK activation in early G2 phase cells was identified, and inhibition of this delayed entry into mitosis. The previously identified, proteolytically cleaved form of MEK1 termed tMEK (truncated MEK1), is also induced with G2 phase MAPK pathway activation. We demonstrate that addition of recombinant mutants of MEK1 with an N-terminal truncation similar to that of tMEK also inhibited ERK and p90RSK activations and delayed progression into mitosis. Only catalytically inactive forms of tMEK were capable of these effects, but surprisingly, phosphorylation on the activating Ser218/222 sites was also required. A lack of MEK1 or ability to accumulate tMEK resulted in the absence of the feedback inhibition of ERK and p90RSK activations. tMEK is a novel output from the canonical MAP kinase signalling pathway, acting in a MAPK signalling-regulated dominant negative manner to inhibit ERK and p90RSK activations, acting as a dampening mechanism to reduce the magnitude or duration of MAPK pathway signalling in G2/M phase.     

AKAP-Lbc enhances cyclic AMP control of the ERK1/2 cascade

Mitogen-activated protein kinase (MAPK) cascades propagate a variety of cellular activities. Processive relay of signals through RAF-MEK-ERK modulates cell growth and proliferation. Signalling through this ERK cascade is frequently amplified in cancers, and drugs such as sorafenib (which is prescribed to treat renal and hepatic carcinomas) and PLX4720 (which targets melanomas) inhibit RAF kinases. Natural factors that influence ERK1/2 signalling include the second messenger cyclic AMP. However, the mechanisms underlying this cascade have been difficult to elucidate. We demonstrate that the A-kinase-anchoring protein AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signalling network that efficiently relay signals from RAF, through MEK, and on to ERK1/2. AKAP-Lbc functions as an enhancer of ERK signalling by securing RAF in the vicinity of MEK1 and synchronizing protein kinase A (PKA)-mediated phosphorylation of Ser 838 on KSR-1. This offers mechanistic insight into cAMP-responsive control of ERK signalling events.     

Mitogen-activated protein kinase feedback phosphorylation regulates MEK1 complex formation and activation during cellular adhesion

Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.     

Activated MEK Stimulates Expression of AP-1 Components Independently of Phosphatidylinositol 3-Kinase (PI3-Kinase) but Requires a PI3-Kinase Signal To Stimulate DNA Synthesis

To investigate the contribution that ERK/mitogen-activated protein kinase signalling makes to cell cycle progression and gene expression, we have constructed cell lines to express an inducible version of activated MEK1. Using these cells, we show that activation of MEK leads to the expression of Fra-1 and Fra-2 but not c-Fos. Treatment of Ras-transformed cells with the MEK inhibitor PD098059 blocks expression of Fra-1 and Fra-2, showing that in Ras transformation ERK signalling is responsible for Fra-1 and Fra-2 expression. Activation of MEK1 in growth-arrested cells leads to DNA synthesis; however, ERK activation alone is insufficient because the induction of DNA synthesis is blocked by inhibition of phosphatidylinositol 3-kinase (PI3-kinase). Activation of PI3-kinase is indirect, perhaps through autocrine growth factors, and is required for the induction of cyclin D1.     

Cdc25A and ERK interaction: EGFR-independent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5

Extracellular signal-regulated kinase (ERK) plays a central role in regulating cell growth, differentiation, and apoptosis. We previously found that 2-(2-mercaptoethanol)-3-methyl-1,4-napthoquinone or Compound 5 (Cpd 5), is a Cdc25A protein phosphatase inhibitor and causes prolonged, strong ERK phosphorylation which is triggered by epidermal growth factor receptor (EGFR) activation. We now report that Cpd 5 can directly cause ERK phosphorylation by inhibiting Cdc25A activity independently of the EGFR pathway. We found that Cdc25A physically interacted with and de-phosphorylated phospho-ERK both in vitro and in cell culture. Inhibition of Cdc25A activity by Cpd 5 resulted in ERK hyper-phosphorylation. Transfection of Hep3B human hepatoma cells with inactive Cdc25A mutant enhanced Cpd 5 action on ERK phosphorylation, whereas over-expression of Cdc25A attenuated this Cpd 5 action. Furthermore, endogenous Cdc25A knock-down by Cdc25A siRNA resulted in a constitutive-like ERK phosphorylation and Cpd 5 treatment further enhanced it. In EGFR-devoid NR6 fibroblasts and MEK (ERK kinase) mutated MCF7 cells, Cpd 5 treatment also resulted in ERK phosphorylation, providing support for the idea that Cpd 5 can directly act on ERK phosphorylation by inhibiting Cdc25A activity. These data suggest that phospho-ERK is likely another Cdc25A substrate, and Cpd 5-caused ERK phosphorylation is probably regulated by both EGFR-dependent and EGFR-independent pathways.     

MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt pathways are involved in the regulatory mechanisms of several cellular processes including proliferation, differentiation and apoptosis. Here we show that during chick, mouse and zebrafish limb/fin development, a known MAPK/ERK regulator, Mkp3, is induced in the mesenchyme by fibroblast growth factor 8 (FGF8) signalling, through the PI3K/Akt pathway. This correlates with a high level of phosphorylated ERK in the apical ectodermal ridge (AER), where Mkp3 expression is excluded. Conversely, phosphorylated Akt is detected only in the mesenchyme. Constitutively active Mek1, as well as the downregulation of Mkp3 by small interfering RNA (siRNA), induced apoptosis in the mesenchyme. This suggests that MKP3 has a key role in mediating the proliferative, anti-apoptotic signalling of AER-derived FGF8.     

Requirement for ERK activation in cisplatin-induced apoptosis

Cisplatin activates multiple signal transduction pathways involved in coordinating cellular responses to stress. Here we demonstrate a requirement for extracellular signal-regulated protein kinase (ERK), a member of the mitogen-activated protein kinase family in mediating cisplatin-induced apoptosis of human cervical carcinoma HeLa cells. Cisplatin treatment resulted in dose- and time- dependent activation of ERK. That elevated ERK activity contributed to cell death by cisplatin was supported by several observations: 1) PD98059 and U0126, chemical inhibitors of the MEK/ERK signaling pathway, prevented apoptosis; 2) pretreatment of cells with TPA, an activator of the ERK pathway, enhanced their sensitivity to cisplatin; 3) suramin, a growth factor receptor antagonist that greatly suppressed ERK activation, likewise inhibited cisplatin-induced apoptosis; and, finally, 4) HeLa cell variants selected for cisplatin resistance showed reduced activation of ERK following cisplatin treatment. Cisplatin-induced apoptosis was associated with cytochrome c release and subsequent caspase-3 activation, both of which could be prevented by treatment with the MEK inhibitors. However, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone protected HeLa cells against apoptosis without affecting ERK activation. Taken together, our findings suggest that ERK activation plays an active role in mediating cisplatin-induced apoptosis of HeLa cells and functions upstream of caspase activation to initiate the apoptotic signal.     

Extracellular ATP stimulates an inhibitory pathway towards growth factor-induced cRaf-1 and MEKK activation in astrocyte cultures

ATP, acting via P2Y, G protein-coupled receptors (GPCRs), is a mitogenic signal and also synergistically enhances fibroblast growth factor-2 (FGF-2)-induced proliferation in astrocytes. Here, we have examined the effects of ATP and FGF-2 cotreatment on the main components of the extracellular-signal regulated protein kinase (ERK) cascade, cRaf-1, MAPK/ERK kinase (MEK) and ERK, key regulators of cellular proliferation. Surprisingly, ATP inhibited activation of cRaf-1 by FGF-2 in primary cultures of rat cortical astrocytes. The inhibitory effect did not diminish MEK and ERK activation; indeed, cotreatment resulted in a greater initial activation of ERK. ATP inhibition of cRaf-1 activation was not mediated by an increase in cyclic AMP levels or by protein kinase C activation. ATP also inhibited the activation of cRaf-1 by other growth factors, epidermal growth factor and platelet-derived growth factor, as well as other MEK1 activators stimulated by FGF-2, MEK kinase 1 (MEKK1) and MEKK2. Serotonin, an agonist of another GPCR coupled to ERK, did not inhibit FGF-2-induced cRaf-1 activation, thereby indicating specificity in the ATP-induced inhibitory cross-talk. These findings suggest that ATP stimulates an inhibitory activity that lays upstream of MEK activators and inhibits growth factor-induced activation of cRaf-1 and MEKKS: Such a mechanism might serve to integrate the actions of receptor tyrosine kinases and P2Y-GPCRS: