The Polo-like Kinase Plx1 Is Required for Activation of the Phosphatase Cdc25C and Cyclin B-Cdc2 in Xenopus Oocytes

In the Xenopus oocyte system mitogen treatment triggers the G(2)/M transition by transiently inhibiting the cAMP-dependent protein kinase (PKA); subsequently, other signal transduction pathways are activated, including the mitogen-activated protein kinase (MAPK) and polo-like kinase pathways. To study the interactions between these pathways, we have utilized a cell-free oocyte extract that carries out the signaling events of oocyte maturation after addition of the heat-stable inhibitor of PKA, PKI. PKI stimulated the synthesis of Mos and activation of both the MAPK pathway and the Plx1/Cdc25C/cyclin B-Cdc2 pathway. Activation of the MAPK pathway alone by glutathione S-transferase (GST)-Mos did not lead to activation of Plx1 or cyclin B-Cdc2. Inhibition of the MAPK pathway in the extract by the MEK1 inhibitor U0126 delayed, but did not prevent, activation of the Plx1 pathway, and inhibition of Mos synthesis by cycloheximide had a similar effect, suggesting that MAPK activation is the only relevant function of Mos. Immunodepletion of Plx1 completely inhibited activation of Cdc25C and cyclin B-Cdc2 by PKI, indicating that Plx1 is necessary for Cdc25C activation. In extracts containing fully activated Plx1 and Cdc25C, inhibition of cyclin B-Cdc2 by p21(Cip1) had no significant effect on either the phosphorylation of Cdc25C or the activity of Plx1. These results demonstrate that maintenance of Plx1 and Cdc25C activity during mitosis does not require cyclin B-Cdc2 activity.     

Mos mediates the mitotic activation of p42 MAPK in Xenopus egg extracts

The ERK1/ERK2 MAP kinases (MAPKs) are transiently activated during mitosis, and MAPK activation has been implicated in the spindle assembly checkpoint and in establishing the timing of an unperturbed mitosis. The MAPK activator MEK1 is required for mitotic activation of p42 MAPK in Xenopus egg extracts; however, the identity of the kinase that activates MEK1 is unknown. Here we have partially purified a Cdc2-cyclin B-induced MEK-activating protein kinase from mitotic Xenopus egg extracts and identified it as the Mos protooncoprotein, a MAP kinase kinase kinase present at low levels in mitotic egg extracts, early embryos, and somatic cells. Immunodepletion of Mos from interphase egg extracts was found to abolish Delta90 cyclin B-Cdc2-stimulated p42 MAPK activation. In contrast, immunodepletion of Raf-1 and B-Raf, two other MEK-activating kinases present in Xenopus egg extracts, had little effect on cyclin-stimulated p42 MAPK activation. Immunodepletion of Mos also abolished the transient activation of p42 MAPK in cycling egg extracts. Taken together, these data demonstrate that Mos is responsible for the mitotic activation of the p42 MAPK pathway in Xenopus egg extracts.     

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.     

Raf-1/MEK/MAPK pathway is necessary for the G2/M transition induced by nocodazole

The dynamic balance between polymerization and depolymerization of microtubules is critical for cells to enter and exit mitosis, and drugs that disrupt this balance, such as taxol, colchicine, and nocodazole, arrest the cell cycle in mitosis. Although the Raf/MEK/MAPK pathway can be activated by these drugs, its role in mitosis has not been addressed. Here, we characterize activation of Raf/MEK/MAPK by nocodazole when mitosis is induced. We find that at early time points (up to 3 h) in nocodazole induction, Raf/MEK/MAPK is activated, and inhibition of MAPK activation by a MEK inhibitor, PD98059 or U0126, reduces the number of cells entering mitosis by creating a block at G(2). At later time points and in mitosis, activation of MEK/MAPK is severely inhibited, even though Raf-1 activity remains high and can be further increased by growth factor. This inhibition is reversed when cells are released from metaphase and enter G(0)/G(1) phase. In addition, we find that binding of Raf-1 to 14-3-3 is progressively induced by nocodazole, reaching a maximum in mitosis, and that this binding is necessary to maintain mitotic Raf-1 activity. Our present study indicates that activation of the Raf/MEK/MAPK pathway is necessary for the G(2)/M progression.     

Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis

Mitogen-activated protein (MAP) kinase and phosphoinositide 3-kinase (PI3K) pathways are necessary for cell cycle progression into S phase; however the importance of these pathways after the restriction point is poorly understood. In this study, we examined the regulation and function of extracellular signal-regulated kinase (ERK) and PI3K during G(2)/M in synchronized HeLa and NIH 3T3 cells. Phosphorylation and activation of both the MAP kinase kinase/ERK and PI3K/Akt pathways occur in late S and persist until the end of mitosis. Signaling was rapidly reversed by cell-permeable inhibitors, indicating that both pathways are continuously activated and rapidly cycle between active and inactive states during G(2)/M. The serum-dependent behavior of PI3K/Akt versus ERK pathway activation indicates that their mechanisms of regulation differ during G(2)/M. Effects of cell-permeable inhibitors and dominant-negative mutants show that both pathways are needed for mitotic progression. However, inhibiting the PI3K pathway interferes with cdc2 activation, cyclin B1 expression, and mitotic entry, whereas inhibiting the ERK pathway interferes with mitotic entry but has little effect on cdc2 activation and cyclin B1 and retards progression from metaphase to anaphase. Thus, our study provides novel evidence that ERK and PI3K pathways both promote cell cycle progression during G(2)/M but have different regulatory mechanisms and function at distinct times.     

Spatiotemporal regulations of Wee1 at the G2/M transition

Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.     

Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in Cycling Xenopus Egg Extracts

We have added constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrested the cell cycle in metaphase, as has been shown previously. Unexpectedly, p42MAPK activation during interphase inhibited entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remained low, Cdc2 was tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest was overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibited Cdc2 activation by cyclin B. PD098059, a specific inhibitor of MEK, blocked the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK did not inhibit DNA replication. These results indicate that, in addition to the established role of p42MAPK activation in M-phase arrest, the inappropriate activation of p42MAPK during interphase prevents normal entry into M-phase.     

Regulation of Cdc25C by ERK-MAP kinases during the G2/M transition

Induction of G(2)/M phase transition in mitotic and meiotic cell cycles requires activation by phosphorylation of the protein phosphatase Cdc25. Although Cdc2/cyclin B and polo-like kinase (PLK) can phosphorylate and activate Cdc25 in vitro, phosphorylation by these two kinases is insufficient to account for Cdc25 activation during M phase induction. Here we demonstrate that p42 MAP kinase (MAPK), the Xenopus ortholog of ERK2, is a major Cdc25 phosphorylating kinase in extracts of M phase-arrested Xenopus eggs. In Xenopus oocytes, p42 MAPK interacts with hypophosphorylated Cdc25 before meiotic induction. During meiotic induction, p42 MAPK phosphorylates Cdc25 at T48, T138, and S205, increasing Cdc25's phosphatase activity. In a mammalian cell line, ERK1/2 interacts with Cdc25C in interphase and phosphorylates Cdc25C at T48 in mitosis. Inhibition of ERK activation partially inhibits T48 phosphorylation, Cdc25C activation, and mitotic induction. These findings demonstrate that ERK-MAP kinases are directly involved in activating Cdc25 during the G(2)/M transition.     

Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1

During mitosis the Xenopus polo-like kinase 1 (Plx1) plays key roles in the activation of Cdc25C, in spindle assembly, and in cyclin B degradation. Previous work has shown that the activation of Plx1 requires phosphorylation on serine and threonine residues. In the present work, we demonstrate that replacement of Ser-128 or Thr-201 with a negatively charged aspartic acid residue (S128D or T201D) elevates Plx1 activity severalfold and that replacement of both Ser-128 and Thr-201 with Asp residues (S128D/T201D) increases Plx1 activity approximately 40-fold. Microinjection of mRNA encoding S128D/T201D Plx1 into Xenopus oocytes induced directly the activation of both Cdc25C and cyclin B-Cdc2. In egg extracts T201D Plx1 delayed the timing of deactivation of Cdc25C during exit from M phase and accelerated Cdc25C activation during entry into M phase. This supports the concept that Plx1 is a "trigger" kinase for the activation of Cdc25C during the G(2)/M transition. In addition, during anaphase T201D Plx1 reduced preferentially the degradation of cyclin B2 and delayed the reduction in Cdc2 histone H1 kinase activity. In early embryos S128D/T201D Plx1 resulted in arrest of cleavage and formation of multiple interphase nuclei. Consistent with these results, Plx1 was found to be localized on centrosomes at prophase, on spindles at metaphase, and at the midbody during cytokinesis. These results demonstrate that in Xenopus laevis activation of Plx1 is sufficient for the activation of Cdc25C at the initiation of mitosis and that inactivation of Plx1 is required for complete degradation of cyclin B2 after anaphase and completion of cytokinesis.     

Alternative ERK5 regulation by phosphorylation during the cell cycle

ERK5 is a member of the mitogen-activated protein kinase (MAPK) family that, after stimulation, is activated selectively by dual phosphorylation in the TEY motif by MAPK kinase 5 (MEK5). ERK5 plays an important role in regulating cell proliferation, survival, differentiation and stress response. Moreover, it is involved in G2/M progression and timely mitotic entry. ERK5 is phosphorylated during mitosis, but the molecular mechanism by which it is regulated during this phase is still unclear. Here we show that although ERK5 is phosphorylated in mitosis, this does not occur on the activation motif (TEY), but at its C-terminal half. We have identified five sites of ERK5 phosphorylation in mitosis, two of them unknown. Furthermore, we demonstrate that ERK5 phosphorylation in mitosis is not MEK5-dependent, but rather, cyclin-dependent kinase (CDK)-dependent. Using a mutagenesis approach, we analysed the importance of the phosphorylated residues in ERK5 function; our evidence show that phosphorylation in mitosis of the residues identified inhibits ERK5 activity and regulates ERK5 shuttling from cytoplasm to the nucleus. These results reveal a previously unreported form of ERK5 regulation by phosphorylation and establish a link between CDK and ERK5 pathways during mitosis, which could be crucial for the correct progression of the cell cycle.     

PDK1 is required for the hormonal signaling pathway leading to meiotic resumption in starfish oocytes

Meiotic resumption is generally under the control of an extracellular maturation-inducing hormone. It is equivalent to the G2-M phase transition in somatic cell mitosis and is regulated by cyclin B-Cdc2 kinase. However, the complete signaling pathway from the hormone to cyclin B-Cdc2 is yet unclear in any organism. A model system to analyze meiotic resumption is the starfish oocyte, in which Akt/protein kinase B (PKB) plays a key mediator in hormonal signaling that leads to cyclin B-Cdc2 activation. Here we show in starfish oocytes that when PDK1 activity is inhibited by a neutralizing antibody, maturation-inducing hormone fails to induce cyclin B-Cdc2 activation at the meiotic G2-M phase transition, even though PDK2 activity becomes detectable. These observations assign a novel role to PDK1 for a hormonal signaling intermediate toward meiotic resumption. They further support that PDK2 is a molecule distinct from PDK1 and Akt, and that PDK2 activity is not sufficient for the full activation of Akt in the absence of PDK1 activity.     

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.     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.     

Initiation factor 2B activity is regulated by protein phosphatase 1, which is activated by the mitogen-activated protein kinase-dependent pathway in insulin-like growth factor 1-stimulated neuronal cells

We have previously demonstrated that insulin-like growth factor 1 (IGF1) induces eukaryotic initiation factor 2B (eIF2B) activation in neuronal cells through the phosphatidylinositol 3 kinase/glycogen synthase kinase 3 pathway as well as by activation of the mitogen-activated protein kinase (MAPK)-activating kinase (MEK)/MAPK signaling pathway (Quevedo, C., Alcázar, A., and Salinas, M. (2000) J. Biol. Chem. 275, 19192-19197). This paper addresses the mechanism involved in IGF1-induced eIF2B activation via the MEK/MAPK cascade in cultured neurons treated with IGF1 and demonstrates that extracellular signal-regulated MAP kinase 1 and 2 (ERK1 and -2) immunoprecipitates of IGF1-treated neuronal cells promote this activation. This effect did not directly result from eIF2B phosphorylation by ERK immunoprecipitates. In addition, recombinant ERK1 and -2 neither activate eIF2B nor phosphorylate it. Endogenous protein phosphatase 1 and 2A catalytic subunits (PP1C and PP2AC, respectively) were co-immunoprecipitated with ERK1 and -2, and the association of ERK with PP1C was stimulated by IGF1 treatment, resulting in increased PP1 activity. ERK immunoprecipitates incubated with PP1 inhibitors did not activate eIF2B, indicating that PP1C activates eIF2B. In vitro experiments with phosphorylated eIF2B showed that recombinant PP1C (alpha isoform) dephosphorylates and activates eIF2B. Paralleling eIF2B activation, IGF1 treatment induced PP1 activation in a MEK/MAPK-dependent fashion. Moreover, the treatment of neurons with the PP1 inhibitor tautomycin inhibited PP1 activation and prevented IGF1-induced eIF2B activation. These findings strongly suggest that IGF1-induced eIF2B activation in neurons is effected by PP1, the activation of which is mediated by the MEK/MAPK signaling pathway.     

Distinct regulators for Plk1 activation in starfish meiotic and early embryonic cycles

The Polo-like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B-Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. Here we have studied the interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles in starfish. Distinct kinases, cyclin B-Cdc2, MAPK along with cyclin B- and/or cyclin A-Cdc2 and cyclin A-Cdc2, were unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M-phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 was required for cyclin B-Cdc2 activation, the action of Plk1 was mediated primarily through suppression of Myt1 rather than through activation of Cdc25. We propose that Plk1 can be activated by either cyclin A- or cyclin B-Cdc2, and its primary target is Myt1.     

B-Raf is critical for MAPK activation during mitosis and is regulated in an M phase-dependent manner in Xenopus egg extracts

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.