Table of contents
MAP kinase and meiotic progression
The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 (See Drosophila Cyclin B and cdc2) complexes (prematuration-promoting factor [MPF]) be kept in an inactive form, due largely to the inhibitory phosphorylation of this pre-MPF. The role of mitogen-activated protein (MAP) kinase has been investigated in the activation of this pre-MPF. Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of either G2-arrested starfish or Xenopus oocytes is prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation is much reduced. Both effects are suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation (Abrieu, 1997a).
Down-regulation of MAP kinase (MAPK) is a universal consequence of fertilization in the animal kingdom, although its role is not known. MAPK inactivation is essential for embryos, both vertebrate and invertebrate, to enter first mitosis. Suppressing down-regulation of MAPK at fertilization, for example by constitutively activating the upstream MAPK cascade, specifically suppresses cyclin B-cdc2 kinase activation and its consequence, entry into first mitosis. It thus appears that MAPK functions in meiotic maturation by preventing unfertilized eggs from proceeding into parthenogenetic development. The most general effect of artificially maintaining MAPK activity after fertilization is prevention of the G2 to M-phase transition in the first mitotic cell cycle, even though inappropriate reactivation of MAPK after fertilization may lead to metaphase arrest in vertebrates. Advancing the time of MAPK inactivation in fertilized eggs does not, however, speed up their entry into first mitosis. Thus, sustained activity of MAPK during part of the first mitotic cell cycle is not responsible for late entry of fertilized eggs into first mitosis (Abrieu, 1997b).
MAP kinase activation occurs during meiotic maturation of oocytes from all animals, but the requirement for MAP kinase activation in reinitiation of meiosis appears to vary between different organisms. In particular, it has become accepted that MAP kinase activation is necessary for progesterone-stimulated meiotic maturation of Xenopus oocytes, while this is clearly not the case in other systems. While MAP kinase increases the efficiency of Cdc2 activation, it is the intrinsic sensitivity of oocytes to levels of Cdc2 kinase that determines whether or not oocytes undergo initiation of meiotic maturation in the absence of MAP kinase activity. MAP kinase activation in Xenopus oocytes is an early response to progesterone and can be temporally dissociated from MPF activation. MAP kinase activation can be suppressed by treatment with geldanamycin or by overexpression of the MAP kinase phosphatase Pyst1. A transient and low-level early activation of MAP kinase increases the efficiency of cell cycle activation later on, when MAP kinase activity is no longer essential. Many oocytes can still undergo reinitiation of meiosis in the absence of active MAP kinase. Suppression of MAP kinase activation does not affect the formation or activation of Cdc2-cyclin B complexes, but reduces the level of active Cdc2 kinase. Even in species that do not require cyclin B synthesis for germinal vesicle breakdown (GVBD), MAP kinase regulates cyclin B synthesis after GVBD. For example, oocytes from mos -/- mice can undergo initiation of meiotic maturation but do not activate MAP kinase, indicating that other activators of the MAP kinase cascade, such as Raf-1, cannot replace Mos function. The rate of formation of Cdc2 kinase activity after GVBD in mouse oocytes is controlled by cyclin B1 synthesis, while cyclin B does not accumulate in mouse oocytes injected with Mos antisense oligonucleotides. The results in Xenopus oocytes (this paper) show striking parallels to these results and lead to a suggestion that oocyte meiotic initiation might indeed be controlled in a universally similar way. Quantitative differences may well exist between different oocyte systems, such as the sensitivity of GVBD to Cdc2 kinase levels, which means that meiosis is organized along the same principles but quantitatively different ways (Fisher, 1999).
The Mos protein kinase is a key regulator of vertebrate oocyte maturation. Oocyte-specific Mos protein expression is subject to translational control. In the frog Xenopus, the translation of Mos protein requires the progesterone-induced polyadenylation of the maternal Mos mRNA, which is present in the oocyte cytoplasm. Both the Xenopus p42 mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) signaling pathways have been proposed to mediate progesterone-stimulated oocyte maturation. In this study, the relative contributions of the MAPK and MPF signaling pathways to Mos mRNA polyadenylation have been determined. Progesterone-induced Mos mRNA polyadenylation is attenuated in oocytes expressing the MAPK phosphatase rVH6. Moreover, inhibition of MAPK signaling blocks progesterone-induced Mos protein accumulation. Activation of the MAPK pathway by injection of RNA encoding Mos is sufficient to induce both the polyadenylation of synthetic Mos mRNA substrates and the accumulation of endogenous Mos protein in the absence of MPF signaling. Activation of MPF, by injection of cyclin B1 RNA or purified cyclin B1 protein, also induces both Mos protein accumulation and Mos mRNA polyadenylation. However, this action of MPF requires MAPK activity. By contrast, the cytoplasmic polyadenylation of maternal cyclin B1 mRNA is stimulated by MPF in a MAPK-independent manner, thus revealing a differential regulation of maternal mRNA polyadenylation by the MAPK and MPF signaling pathways. It is propose that MAPK-stimulated Mos mRNA cytoplasmic polyadenylation is a key component of the positive-feedback loop, which contributes to the all-or-none process of oocyte maturation (Howard, 1999).
Stage VI Xenopus oocytes are suspended at the G2/M transition of meiosis I, and represent an excellent system for the identification and examination of cell cycle regulatory proteins. Essential cell cycle regulators such as MAPK, cyclins and and the oncogene mos have the ability to induce oocyte maturation, causing the resumption of the cell cycle from its arrested state. The product of a novel Xenopus gene, Speedy or Spy1, is able to induce rapid maturation of Xenopus oocytes, resulting in the induction of germinal vesicle breakdown (GVBD) and activation of M-phase promoting factor (MPF). Spy1 activates the MAPK pathway in oocytes, and its ability to induce maturation is dependent on this pathway. Spy1-induced maturation occurs much more rapidly than maturation induced by other cell cycle regulators, including progesterone, mos or Ras, and does not require any of these proteins or hormones, indicating that Spy1-induced maturation proceeds through a novel regulatory pathway. In addition, Spy1 physically interacts with cdk2, and prematurely activates cdk2 kinase activity. Therefore, Spy1 represents a novel cell cycle regulatory protein, inducing maturation through the activation of MAPK and MPF, and also leading to the premature activation of cdk2 (Lenormand, 1999).
Fertilization releases the brake on the cell cycle and the egg completes meiosis and enters into S phase of the mitotic cell cycle. The MAP kinase pathway has been implicated in this process, but the precise role of MAP kinase in meiosis and the first mitotic cell cycle remains unknown and may differ according to species. Unlike the eggs of most animals, sea urchin eggs have completed meiosis prior to fertilization and are arrested at the pronuclear stage. Using both phosphorylation-state-specific antibodies and a MAP kinase activity assay, it has been observed that MAP kinase is phosphorylated and active in unfertilized sea urchin eggs and then dephosphorylated and inactivated by 15 min post-insemination. Further, Ca2+ is both sufficient and necessary for this MAP kinase inactivation. Treatment of eggs with the Ca2+ ionophore A23187 causes MAP kinase inactivation and triggers DNA synthesis. When the rise in intracellular Ca2+ is inhibited by injection of a chelator, BAPTA or EGTA, the activity of MAP kinase remains high. Inhibition of the MAP kinase signaling pathway by the specific MEK inhibitor PD98059 triggers DNA synthesis in unfertilized eggs. Thus, whenever MAP kinase activity is retained, DNA synthesis is inhibited while inactivation of MAP kinase correlates with initiation of DNA synthesis (Carroll, 2000).
The eukaryotic cell cycle is regulated by oscillations in the activity of M-phase Promoting Factor (MPF) consisting of a catalytic subunit, p24cdc2 and a regulatory subunit, cyclin B. Entry into metaphase is driven by the activation of MPF; anaphase is correlated with cyclin B destruction and decreased MPF activity. Oscillations in MPF activity also drives meiosis in mammalian oocytes. Oocytes of almost all vertebrates become arrested at metaphase II to await fertilization. Arrest is achieved with the participation of a protein complex known as cytostatic factor (CSF), which stabilizes histone H1 kinase activity. MOS and mitogen-activated protein kinase (MAPK) are important components of CSF. MOS is essential for MAPK activity since there is little or no MAPK activity detectable in oocytes of Mos mutant mice. Thus MOS probably functions to maintain metaphase II arrest by promoting MAPK activity, which in turn may either inactivate the cyclin B degradation system, prevent an increased rate of degradation, or both. The critical consequence of either mechanism is sustained high MPF activity and maintenance of metaphase II arrest. Strain LT/Sv mice, and strains related to LT/Sv, produce a high percentage of atypical oocytes that are arrested at metaphase I when normal oocytes have progressed to metaphase II. The potential role of MOS in metaphase I arrest was investigated using strain LT/Sv and LT-related recombinant inbred strains, LTXBO and CX8-4. MOS and MAPK are produced and functional in maturing LT oocytes. Two experimental paradigms were used to reduce or delete MOS in LT oocytes and assess effects on metaphase I arrest: (1) sense and antisense Mos oligonucleotides were microinjected into metaphase I-arrested oocytes. Antisense (but not sense) Mos oligonucleotides promote the activation of metaphase I-arrested oocytes. (2) Mice carrying a Mos null mutation were crossed with LT mice. The null mutation was backcrossed three times to LT mice, and Mos(+/-) N3 mice were intercrossed to produce Mos(-/-), Mos(+/-) and Mos(+/+) N3F1 mice. Oocytes of all three Mos genotypes of N3F1 mice sustain meiotic arrest for 17 hours, indicating that metaphase I arrest is not initiated by a MOS-dependent mechanism. However, unlike Mos(+/+) and Mos(+/-) CX8-4 N3F1 oocytes, metaphase I arrest of Mos(-/-) CX8-4 N3F1 oocytes is not sustained after 17 hours and becomes reversed gradually. These results, like the antisense Mos oligonucleotide microinjection experiments, suggest that MOS participates in sustaining metaphase I arrest in LT oocytes (Hirao, 1997).
Although MAP kinase is an important regulatory enzyme in many somatic cells, almost nothing is known about its functions during meiosis, except in frog and mouse oocytes. MAPK activation and function were investigated in oocytes of the marine worm Urechis caupo that are fertilized at meiotic prophase. Activity is first detected at 4-6 min after fertilization in immunoblots with antibody to active MAPK, prior to germinal vesicle breakdown (GVBD). MAPK activation does not require new protein synthesis and is dependent on the increases in both intracellular pH and intracellular Ca(2+) that normally occur during activation. When MAPK activation is inhibited with PD98059 or U0126, GVBD still occurs, but meiosis is abnormal and there is a dramatic premature enlargement of sperm asters, which normally do not appear until second polar body formation. Failure of polar body formation and premature sperm aster enlargement also occurs when MAPK activation is inhibited by an entirely different treatment, which involves lowering the pH of external seawater to interrupt the normal cytoplasmic pH increase. Thus, in Urechis, active MAPK appears to be required for (1) normal meiotic divisions and (2) suppressing the paternal centrosome until after the egg completes meiosis, a general phenomenon whose mechanism has been unknown (Gould, 1999).
These results identify a new role for MAPK during meiosis: suppressing the development of the sperm centrosome until after meiosis is completed. This delay in sperm centrosome activation is a classic observation in many species, but the data presented here provide the first evidence for a MAPK regulated mechanism. This function for MAPK could explain some previously unexplained observations on the timing of inactivation of cytostatic factor (CSF), the product of the c-mos proto-oncogene. CSF and MAPK inactivation occur after fertilization. In Xenopus oocytes, although mos (and therefore presumably active MAPK) is one of the components of CSF required to maintain the metaphase II arrest prior to fertilization, it (and CSF) is not inactivated until after second polar body formation. Why CSF should remain active after it is no longer needed to maintain metaphase has, until now, had no functional explanation. The results with Urechis suggest that active MAPK persists to suppress sperm aster formation until after meiosis is completed. In agreement with this hypothesis, sperm asters begin to grow at about the time of second polar body formation in Xenopus eggs. In mouse oocytes, MAPK is inactivated well after second polar body formation and the decline in activity is correlated with growth of asters in the cytoplasm. Mice are unusual in that their centrosomes are apparently maternally inherited, so the hypothesis concerning MAPK function could be extended to include maternal mitotic asters (Gould, 1999).
During oocyte maturation in Xenopus, progesterone induces entry into meiosis I, and the M phases of meiosis I and II occur consecutively without an intervening S phase. The mitogen-activated protein (MAP) kinase is activated during meiotic entry, and it has been suggested that the linkage of M phases reflects activation of the MAP kinase pathway and the failure to fully degrade cyclin B during anaphase I. To analyze the function of the MAP kinase pathway in oocyte maturation, U0126, a potent inhibitor of MAP kinase kinase, and a constitutively active mutant of the protein kinase p90Rsk, a MAP kinase target, were used. Even with complete inhibition of the MAP kinase pathway by U0126, up to 90% of oocytes were able to enter meiosis I after progesterone treatment, most likely through activation of the phosphatase Cdc25C by the polo-like kinase Plx1. Subsequently, however, U0126-treated oocytes fail to form metaphase I spindles, fail to reaccumulate cyclin B to a high level and fail to hyperphosphorylate Cdc27, a component of the anaphase-promoting complex (APC) that controls cyclin B degradation. Such oocytes enter S phase rather than meiosis II. U0126-treated oocytes expressing a constitutively active form of p90Rsk are able to reaccumulate cyclin B, hyperphosphorylate Cdc27 and form metaphase spindles in the absence of detectable MAP kinase activity. It is concluded that the MAP kinase pathway is not essential for entry into meiosis I in Xenopus but is required during the onset of meiosis II to suppress entry into S phase, to regulate the APC so as to support cyclin B accumulation, and to support spindle formation. Moreover, one substrate of MAP kinase, p90Rsk, is sufficient to mediate these effects during oocyte maturation (Gross, 2000).
Activation of mitogen-activated protein kinase (MAPK) in maturing mouse oocytes occurs after synthesis of Mos, a MAPKKK. To investigate whether Mos acts only through MEK1, constitutively active forms of MEK1 (MEK1S218D/S222D referred herein as MEK*) and Raf (DeltaRaf) were injected into mouse oocytes. In mos minus oocytes, which do not activate MAPK during meiosis and do not arrest in metaphase II, MEK* and DeltaRaf did not rescue MAPK activation and metaphase II arrest, whereas Mos induces a complete rescue. MEK* and DeltaRaf induce cleavage arrest of two-cell blastomeres. They induced MAPK activation when protein phosphatases are inhibited by okadaic acid, suggesting that Mos may inhibit protein phosphatases. Finally, in mos minus oocytes, MEK* induces the phosphorylation of Xp42(mapk)D324N, a mutant less sensitive to dephosphorylation, showing that a MAPK phosphatase activity is present in mouse oocytes. Active MAPKK or MAPKKK cannot substitute for Mos to activate MAPK in mouse oocytes. A phosphatase activity inactivates MAPK, and Mos can overcome this inhibitory activity. Thus Mos activates MAPK through two opposite pathways: activation of MEK1 and inhibition of a phosphatase (Verlhac, 2000).
Fully grown starfish oocytes are arrested at prophase of meiosis I. The hormonal stimulation of 1-methyladenine (1-MA) induces meiosis reinitiation and germinal vesicle breakdown (GVBD). Optimal development occurs when maturing oocytes are fertilized between GVBD and first polar body emission. In the absence of sperm, oocytes complete both meiotic divisions to yield haploid interphase-arrested eggs. Spontaneous and synchronous activation of caspase-3 in starfish eggs occurs 9-12 h after 1-MA stimulation. Then, caspase-dependent membrane blebbing and egg fragmentation occur, indicating that mature eggs undergo apoptosis if not fertilized. Activation of caspase-3 and induction of apoptosis are blocked both by a MEK inhibitor and by emetine treatment which inhibits MEK kinase (Mos) synthesis. Conversely, when recombinant GST-Mos is injected into the emetine-treated eggs, apoptosis is induced. These results indicate that persistent activation of the Mos/MEK/MAP kinase cascade gives the death-activating signal in starfish eggs. Fertilization inactivates the MAP kinase pathway and suppresses apoptosis, followed by normal development (Sasaki, 2001).
Chromosome condensation during the G2/M progression of mouse pachytene spermatocytes induced by the phosphatase inhibitor okadaic acid (OA) requires the activation of the MAPK Erk1. In many cell systems, p90Rsks (Ribosomal-S6-kinases) are the main effectors of Erk1/2 function. p90Rsk2 has been identified as the isoform that is specifically expressed in mouse spermatocytes; it is activated during the OA-triggered meiotic G2/M progression. By using the MEK inhibitor U0126, it has been demonstrated that activation of p90Rsk2 during meiotic progression requires activation of the MAPK pathway. Immunofluorescence analysis indicates that activated Erks and p90Rsk2 are tightly associated with condensed chromosomes during the G2/M transition in meiotic cells. Active p90Rsk2 is able to phosphorylate histone H3 at Ser10 in vitro, but the activation of the Erk1/p90Rsk2 pathway is not necessary for phosphorylation of H3 in vivo. Furthermore, phosphorylation of H3 is not sufficient to cause condensation of meiotic chromosomes in mouse spermatocytes. Other proteins known to associate with chromatin may represent effectors of Erk1 and p90Rsk2 during chromosome condensation. Nek2 (NIMA-related kinase 2), which associates with chromosomes, plays an active role in chromatin condensation and is stimulated by treatment of pachytene spermatocytes with okadaic acid. Inhibition of the MAPK pathway by preincubation of spermatocytes with U0126 suppresses Nek2 activation, and incubation of spermatocyte cell extracts with activated p90Rsk2 causes stimulation of Nek2 kinase activity. Furthermore, the Nek2 kinase domain is a substrate for p90Rsk2 phosphorylation in vitro. These data establish a connection between the Erk1/p90Rsk2 pathway, Nek2 activation and chromosome condensation during the G2/M transition of the first meiotic prophase (Di Agostino, 2002).
Mammalian eggs are arrested in metaphase II of meiosis until fertilization. Arrest is maintained by cytostatic factor (CSF) activity, which is dependent on the MOS-MEK-MAPK pathway. Inhibition of MEK1/2 with a specific inhibitor, U0126, parthenogenetically activates mouse eggs, producing phenotypes similar to Mos-/- parthenogenotes (premature, unequal cleavages and large polar bodies). U0126 inactivates MAPK in eggs within 1 h, in contrast to the 5 h required after fertilization, while the time course of MPF inactivation is similar in U0126-activated and fertilized eggs. Inactivation of MPF by the cdc2 kinase inhibitor roscovitine induces parthenogenetic activation. Inactivation of MPF by roscovitine results in the subsequent inactivation of MAPK with a time course similar to that following fertilization. Notably, roscovitine also produces some Mos-/--like phenotypes, indistinguishable from U0126 parthenogenotes. Simultaneous inhibition of both MPF and MAPK in eggs treated with roscovitine and U0126 produces a very high proportion of eggs with the more severe phenotype. These findings confirm that MEK is a required component of CSF in mammalian eggs and imply that the sequential inactivation of MPF followed by MAPK inactivation is required for normal spindle function and polar body emission (Philips, 2002).
Mammalian oocyte maturation depends on the translational activation of stored maternal mRNAs upon meiotic resumption. Cytoplasmic polyadenylation element binding protein-1 (CPEB1; see Drosophila Orb2) is a key oocyte factor that regulates maternal mRNA translation. However, the signal that triggers CPEB1 activation at the onset of mammalian oocyte maturation is not known. This study provides evidence that a mitogen-activated protein kinase (MAPK) cascade couples maternal mRNA translation to meiotic cell cycle progression in mouse oocytes, by triggering CPEB1 phosphorylation and degradation. Mutations of the phosphorylation sites or ubiquitin E3 ligase binding sites in CPEB1 have a dominant negative effect in oocytes, and mimic the phenotype of ERK1/2 (see Drosophila Rolled) knockout, by impairing spindle assembly and mRNA translation. Overexpression of the CPEB1-downstream translation activator DAZL (see Drosophila Boule) in ERK1/2-deficient oocytes partially rescued the meiotic defects, indicating that ERK1/2 is essential for spindle assembly, metaphase II arrest, and maternal-zygotic transition (MZT) primarily by triggering the translation of key maternal mRNAs. Taken together, ERK1/2-mediated CPEB1 phosphorylation/degradation is a major mechanism of maternal mRNA translational activation, and is crucial for mouse oocyte maturation and MZT (Sha, 2016).
MAP kinase, cell cycle and mitotic progression
Re-entry into the cell cycle from quiescence requires the activation of mitogen-activated protein (MAP) kinases of the extracellular-signal-regulated kinase (ERK) family. However, the relationship between ERK and cell-cycle control is complex, because ERK activation can also lead to terminal differentiation or a senescence-like growth arrest. Reversible cell-cycle exit induced by serum withdrawal in primary avian fibroblasts is associated with rapid deactivation of ERK, but ERK activity is subsequently regenerated and sustained at high levels in fully quiescent (G0) cells. As in proliferating cells, ERK activation during G0 requires the MAP kinase kinase MEK and is partially dependent on cell adhesion. Active, phosphorylated ERK is concentrated in the nucleus in cycling cells, but is largely confined to the cytoplasm during G0. This was unexpected, since activatory phosphorylation mediated by MEK is thought to play an important role in promoting nuclear translocation. These results indicate that transient deactivation of ERK signaling can be sufficient for stable cell-cycle exit, and that MEK-mediated phosphorylation is not sufficient for nuclear translocation of active ERK in G0. Cytoplasmic sequestration may prevent active ERK from accessing critical nuclear cell-cycle targets, thus allowing quiescent or post-mitotic cells to retain ERK activity for other physiological functions (Black, 2000).
The spindle assembly checkpoint prevents cells whose spindles are defective or whose chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. An investigation was carried out to see whether MAP kinase function is required for mitotic progression and/or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Purified recombinant XCL100, a Xenopus MAP kinase phosphatase, was microinjected into XTC cells in various stages of mitosis to interfere with MAP kinase activation. Mitotic progression is unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. In cells injected with phosphatase at prometaphase or metaphase succeed in exiting mitosis in the presence of nocodazole. In such cells the chromosomes decondense and the nuclear envelope re-forms, whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remain arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonizes the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells (Wang, 1997).
In Xenopus laevis egg cell cycle extracts that mimic early embryonic cell cycles, activation of MAP kinase and MAP kinase kinase occurs in M phase, slightly behind that of maturation promoting factor. To examine the possible role of MAP kinase in the in vitro cell cycle, the extracts of MAP kinase were depleted by using anti-Xenopus MAP kinase antibody. As in the mock-treated extracts, the periodic activation and deactivation of MPF occurs normally in the MAP kinase-depleted extracts, suggesting that MAP kinase is dispensable for the normal M phase entry and exit in vitro. It has recently been reported that microtubule depolymerization by nocodazole treatment can block exit from mitosis in the extracts if enough sperm nuclei are present, and that the addition of MAP kinase-specific phosphatase MKP-1 overcomes this spindle assembly checkpoint, suggesting the involvement of MAP kinase in the checkpoint signal transduction. It has been shown that the spindle assembly checkpoint mechanism cannot operate in the MAP kinase-depleted extracts. But, adding recombinant Xenopus MAP kinase to the MAP kinase-depleted extracts restores the spindle assembly checkpoint. These results indicate unambiguously that classical MAP kinase is required for the spindle assembly checkpoint in the cell cycle extracts. In addition, strong activation of MAP kinase by the addition of a constitutively active MAP kinase kinase kinase in the absence of sperm nuclei and nocodazole, induces mitotic arrest in the extracts. Therefore, activation of MAP kinase alone is sufficient for inducing the mitotic arrest in vitro (Takenaka, 1997).
In somatic cells, MAP kinase activation seems to be triggered after exit from a quiescent state only (in G0 or G2) and then inactivated by entry into a proliferative state. In oocytes of various species, a one-time activation of MAP kinase that is apparently not repeated during the succeeding mitotic cycles occurs after meiotic activation. However, several reports suggest that a myelin basic protein (MBP) kinase activity, unrelated to that of maturation promoting factor, can sometimes be detected during mitotic divisions in various types of cells and oocytes. This problem was investigated in order to determine the origin and the role of MBP kinase that is stimulated at time of mitosis in the fertilized eggs of the sea urchin Paracentrotus lividus. Anti-ERK1 antibodies or substrates specific for different MAP kinases were used, and in-gel phosphorylation experiments were performed. An ERK1-like protein was found to be responsible for part of the MBP kinase activity that is stimulated during the first mitotic divisions. Wortmannin, an inhibitor of PI 3-kinase that arrests the fertilized sea urchin eggs at the prometaphase stage, inhibits the inactivation of MAP kinase normally observed when the eggs divide, suggesting a role for PI 3-kinase in the deactivation process of MAP kinase. The activities of maturation-promoting factor (MPF), which consists of cdc2 (a catalytic subunit) and cyclin B (a regulatory subunit), and MAP kinase may be interconnected to regulate the first mitotic divisions of the early sea urchin embryo (Chiri, 1998).
A myelin basic protein (MBP) kinase activity increases at mitosis during the first two embryonic cell cycles of the sea urchin embryo. The activity profile of the MBP kinase is the same both in whole cell extracts and after immunoprecipitation with an anti-MAP kinase antibody (2199). An in-gel assay of MBP activity also shows the same activity profile. The activity is associated with the 44 kDa protein that cross-reacts with anti-MAP kinase antibodies. The 44 kDa protein shows cross-reactivity to anti-phosphotyrosine and MAP kinase-directed anti-phosphotyrosine/phosphothreonine antibodies at the times that MBP kinase activity is high. The 2199 antibody co-precipitates some histone H1 kinase activity, but the MBP kinase activity cannot be accounted for by histone H1 kinase-dependent phosphorylation of MBP. The MAP kinase 2199 antibody was used to purify the MBP kinase activity. Peptide sequencing after partial digestion shows the protein to be homologous to MAP kinases from other species. These data demonstrate that MAP kinase activation during nuclear division is not confined to meiosis, but also occurs during mitotic cell cycles. MAP kinase activity in immunoprecipitates also increases immediately after fertilization, which in the sea urchin egg occurs at interphase of the cell cycle. Treating unfertilized eggs with the calcium ionophore A23187 stimulates the increase in MAP kinase activity, demonstrating that a calcium signal can activate MAP kinase and suggesting that the activation of MAP kinase at fertilization is due to the fertilization-induced increase in cytoplasmic free calcium concentration. This signaling pathway must differ from the pathway responsible for calcium-induced inactivation of MAP kinase activity that is found in eggs that are fertilized in meiotic metaphase (Philipova, 1998).
Constitutively active MAP kinase/ERK kinase (MEK), an activator of the mitogen-activated protein kinase (MAPK) signaling pathway, was added to cycling Xenopus egg extracts at various times during the cell cycle. p42MAPK activation during entry into M-phase arrests the cell cycle in metaphase. p42MAPK activation during interphase inhibits entry into M-phase. In these interphase-arrested extracts, H1 kinase activity remains low; Cdc2 is tyrosine phosphorylated, and nuclei continued to enlarge. The interphase arrest is overcome by recombinant cyclin B. In other experiments, p42MAPK activation by MEK or by Mos inhibits Cdc2 activation by cyclin B. A specific inhibitor of MEK blocks the effects of MEK(QP) and Mos. Mos-induced activation of p42MAPK does 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 (Bitangcol, 1998).
To investigate possible involvement of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 (extracellular signal-regulated kinases) in somatic cell mitosis, indirect immunofluorescence with a highly specific phospho-MAP kinase antibody was used. A portion of the active MAP kinase is found to be localized during mitosis at kinetochores, asters, and the midbody. Although the aster labeling is constant from the time of nuclear envelope breakdown, the kinetochore labeling first appears at early prometaphase, started to fade during chromosome congression, and then disappears at midanaphase. At telophase, active MAP kinase localizes at the midbody. Based on colocalization and the presence of a MAP kinase consensus phosphorylation site, the kinetochore motor protein CENP-E has been identified as a candidate mitotic substrate for MAP kinase. CENP-E is phosphorylated in vitro by MAP kinase on sites that are known to regulate its interactions with microtubules. CENP-E is found to associate in vivo preferentially with the active MAP kinase during mitosis. Therefore, the presence of active MAP kinase at specific mitotic structures and its interaction with CENP-E suggest that MAP kinase could play a role in mitosis at least in part by altering the ability of CENP-E to mediate interactions between chromosomes and microtubules (Zecevic, 1998).
The mitogen-activated protein (MAP) kinase pathway, which includes extracellular signal-regulated protein kinases 1 and 2 (ERK1, ERK2) and MAP kinase kinases 1 and 2 (MKK1, MKK2), is well-known to be required for cell cycle progression from G1 to S phase, but its role in somatic cell mitosis has not been clearly established. The regulation of ERK and MKK was examined in mammalian cells during mitosis using antibodies selective for active phosphorylated forms of these enzymes. In NIH 3T3 cells, both ERK and MKK are activated within the nucleus during early prophase; they localize to spindle poles between prophase and anaphase, and to the midbody during cytokinesis. During metaphase, active ERK is localized in the chromosome periphery, in contrast to active MKK, which shows clear chromosome exclusion. Prophase activation and spindle pole localization of active ERK and MKK are also observed in PtK1 cells. Discrete localization of active ERK at kinetochores is apparent by early prophase and during prometaphase with decreased staining on chromosomes aligned at the metaphase plate. The kinetochores of chromosomes displaced from the metaphase plate, or in microtubule-disrupted cells, still react strongly with the active ERK antibody. This pattern resembles that reported for the 3F3/2 monoclonal antibody, which recognizes a phosphoepitope that disappears with kinetochore attachment to the spindles, and has been implicated in the mitotic checkpoint for anaphase onset. The 3F3/2 reactivity of kinetochores on isolated chromosomes decreases after dephosphorylation with protein phosphatase, and then increases after subsequent phosphorylation by purified active ERK or active MKK. These results suggest that the MAP kinase pathway has multiple functions during mitosis, helping to promote mitotic entry as well as targeting proteins that mediate mitotic progression in response to kinetochore attachment (Shapiro, 1998).
The propagation of pluripotent mouse embryonic stem (ES) cells depends on signals transduced through the cytokine receptor subunit gp130. Signaling molecules activated downstream of gp130 in ES cells include STAT3, the protein tyrosine phosphatase SHP-2, and the mitogen-activated protein kinases ERK1 and ERK2. A chimaeric receptor in which tyrosine 118 in the gp130 cytoplasmic domain was mutated does not engage SHP-2 and fails to activate ERKs. However, this receptor does support ES cell self-renewal. In fact, stem cell colonies form at 100-fold lower concentrations of cytokine than the unmodified receptor. Moreover, altered ES cell morphology and growth are observed at high cytokine concentrations. These indications of deregulated signaling in the absence of tyrosine 118 are substantiated by sustained activation of STAT3. Confirmation that ERK activation is not required for self-renewal was obtained by propagation of pluripotent ES cells in the presence of the MEK inhibitor PD098059. In fact, the growth of undifferentiated ES cells is enhanced by culture in PD098059. Thus activation of ERKs appears actively to impair self-renewal, suggesting that inhibitors of the Ras/MAPK pathway should promote the propagation of undifferentiated ES cells. These data imply that the self-renewal signal from gp130 is a finely tuned balance of positive and negative effectors (Burdon, 1999).
By using cycling Xenopus egg extracts, it has been found that if mitogen-activated protein kinase (p42 MAPK) is activated on entry into mitosis (M-phase), the extract is arrested with condensed chromosomes and spindle microtubules. These arrested extracts have high levels of M-phase promoting factor (MPF, Cyclin B/Cdc2) activity, stabilized levels of Cyclin B, and sustained M-phase-specific phosphorylations. The role of p42 MAPK was examined in DNA damage checkpoint-arrested extracts that were induced to enter M-phase by the addition of Cdc25C protein. In these extracts, Cdc25C protein triggers the abrupt, premature activation of MPF and entry into M-phase. MPF activity then drops suddenly due to Cyclin B proteolysis, just as p42 MAPK is activated. Unexpectedly, however, M-phase is sustained, as judged by maintenance of M-phase-specific phosphorylations and condensed chromosomes. To determine if this M-phase arrest depends on p42 MAPK activation, PD98059 (PD), an inhibitor of p42 MAPK activation, was added to egg extracts with exogenous Cdc25. Both untreated and PD-treated extracts enter M-phase simultaneously, with a sharp peak of MPF activity. However, only PD-treated extracts subsequently exit from M-phase and enter interphase. In PD-treated extracts, p42 MAPK is not activated, and the transition to interphase is accompanied by the formation of decondensed nuclei and the disappearance of M-phase-specific phosphorylation of proteins. These results show that although entry into M-phase requires the activation of MPF, exit from M-phase even after cyclin destruction, is dependent on the inactivation of p42 MAPK (Chau, 1999).
The mitogen-activated protein kinase (MAPK) cascade is required for mitogenesis in somatic mammalian cells and is activated by a wide variety of oncogenic stimuli. Specific roles for this signaling module in growth were dissected by inhibiting MAPK kinase 1 MAPKK1) activity in highly synchronized NIH 3T3 cells. In addition to the known role of this kinase in cell-cycle entry from G(0), the level of MAPKK activity was observed to affect the kinetics of progression through both the G(1) and G(2) phases of the cell cycle in NIH 3T3 cells. Ectopic expression of dominant-negative forms of MAPKK1, which was previously shown to inhibit G(0)/G(1) progression, was found to also delay progression of cells through G(2). In addition, treatment of cells with the specific MAPKK inhibitor PD 98059 during a synchronous S phase arrests the cells in the following G(2) phase. These data demonstrate a novel role for the MAPK cascade in progression from G(2) into mitosis in NIH 3T3 cells (Wright, 1999).
Activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. (1) A biphasic MEK/ERK activation is evidenced in G(1) phase of hepatocytes from regenerating liver but not from sham-operated control animals. One activation phase occurs in early G(1) (30 min to 4 h), and the other occurs in mid-late G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activation peak is located just before cyclin D1 induction in both in vivo and in vitro models. (2) 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 is inhibited, DNA replication is totally abolished, and the MEK1 isoform is preferentially targeted by this inhibition. This effect is dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct results in increased cyclin D1 mRNA accumulation. (3) 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 are able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, is able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (1) 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 (2) 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 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 signaling pathway (Talarmin, 1999).
Xenopus oocytes and eggs provide a dramatic example of how the consequences of p42 mitogen-activated protein kinase (p42 MAPK) activation depend on the particular context in which the activation occurs. In oocytes, the activation of Mos, MEK, and p42 MAPK is required for progesterone-induced Cdc2 activation, and activated forms of any of these proteins can bring about Cdc2 activation in the absence of progesterone. However, in fertilized eggs, activation of the Mos/MEK/p42 MAPK pathway has the opposite effect, inhibiting Cdc2 activation and causing a G2 phase delay or arrest. The mechanism and physiological significance of the p42 MAPK-induced G2 phase arrest has been investigated using Xenopus egg extracts as a model system. Wee1-depleted extracts are unable to arrest in G2 phase in response to Mos, and adding back Wee1 to the extracts restores their ability to arrest. This finding formally places Wee1 downstream of Mos/MEK/p42 MAPK. Purified recombinant p42 MAPK phosphorylates recombinant Wee1 in vitro at sites that are phosphorylated in extracts. Phosphorylation by p42 MAPK results in a modest (approximately 2-fold) increase in the kinase activity of Wee1 toward Cdc2. Titration experiments in extracts have demonstrated that a twofold increase in Wee1 activity is sufficient to cause the delay in mitotic entry seen in Mos-treated extracts. Finally, evidence is presented that the negative regulation of Cdc2 by Mos/MEK/p42 MAPK contributes to the presence of an unusually long G2 phase in the first mitotic cell cycle. Prematurely inactivating p42 MAPK in egg extracts results in a corresponding hastening of the first mitosis. The negative effect of p42 MAPK on Cdc2 activation may help ensure that the first mitotic cell cycle is long enough to allow karyogamy to be accomplished successfully (Walter, 2000).
tob (Drosophila homolog: Tob) is a member of an emerging family of genes with antiproliferative function. Tob is rapidly phosphorylated at Ser 152, Ser 154, and Ser 164 by Erk1 and Erk2 upon growth-factor stimulation. Oncogenic Ras-induced transformation and growth-factor-induced cell proliferation are efficiently suppressed by mutant Tob which carries alanines but not glutamates, thereby mimicking phosphoserines at these sites. Wild-type Tob inhibits cell growth when the three serine residues are not phosphorylated but is less inhibitory when the serines are phosphorylated. Because growth of Rb-deficient cells is not affected by Tob, Tob appears to function upstream of Rb. Intriguingly, cyclin D1 expression is elevated in serum-starved tob-/- cells. Reintroduction of wild-type Tob and mutant Tob with serine-to-alanine but not to glutamate mutations on the Erk phosphorylation sites in these cells restores the suppression of cyclin D1 expression. Finally, the S-phase population is significantly increased in serum-starved tob-/- cells as compared with that in wild-type cells. Thus, Tob inhibits cell growth by suppressing cyclin D1 expression, which is canceled by Erk1- and Erk2-mediated Tob phosphorylation. It is proposed that Tob is critically involved in the control of early G1 progression (Suzuki, 2002).
Fertilization of sea urchin eggs results in a large, transient increase in intracellular free Ca2+ concentration that is responsible for re-initiation of the cell division cycle. Activation of ERK1, a Ca2+-dependent MAP kinase response, is required for both DNA synthesis and cell cycle progression after fertilization. Experiments on populations of cells were combined with analysis at the single cell level, and a proxy assay was developed for DNA synthesis in single embryos, using GFP-PCNA. The effects of low molecular weight inhibitors were compared with a recombinant approach targeting the same signalling pathway. Inhibition of the ERK pathway at fertilization using either recombinant ERK phosphatase or U0126, a MEK inhibitor, was found to prevent accumulation of GFP-PCNA in the zygote nucleus, and U0126 prevents incorporation of [3H]-thymidine into DNA. Abrogation of the ERK1 signalling pathway also prevents chromatin decondensation of the sperm chromatin after pronuclear fusion, nuclear envelope breakdown and formation of a bipolar spindle (Philipova, 2005).
Centrosomes in mammalian cells have been implicated in cytokinesis; however, their role in this process is poorly defined. A human coiled-coil protein, Cep55 (centrosome protein 55 kDa), is described that localizes to the mother centriole during interphase. Despite its association with TuRC anchoring proteins CG-NAP and Kendrin, Cep55 is not required for microtubule nucleation. Upon mitotic entry, centrosome dissociation of Cep55 is triggered by Erk2/Cdk1-dependent phosphorylation at S425 and S428. Furthermore, Cep55 locates to the midbody and plays a role in cytokinesis, as evidenced by the observation that its depletion by siRNA results in failure of this process. S425/428 phosphorylation is required for interaction with Plk1, enabling phosphorylation of Cep55 at S436. Cells expressing phosphorylation-deficient mutant forms of Cep55 undergo cytokinesis failure. These results highlight the centrosome as a site to organize phosphorylation of Cep55, enabling it to relocate to the midbody to function in mitotic exit and cytokinesis (Fabbro, 2005).
The centrosome is the principle microtubule organizing center of the mammalian cell, consisting of a pair of barrel-shaped microtubule assemblies which are nonidentical and are described as the mother and daughter centrioles. They (mainly the mother) are surrounded by pericentriolar material (PCM), which consists of a matrix of predominantly coiled-coil proteins. PCM is the main site for nucleation of cytoplasmic microtubules and microtubules that form the meiotic and mitotic spindles. The centrosome is structurally and functionally regulated in a cell cycle-dependent manner to form a bipolar spindle to ensure the proper segregation of replicated chromosomes into two daughter cells. Defects in the number, structure, and function of centrosomes can generate mono- or multipolar mitotic spindles and cytokinesis defects resulting in aneuploidy and chromosome instability: these are common characteristics of tumor cells. Therefore, it is not surprising that these centrosome abnormalities are frequently found in tumors and are usually associated with high cytological grade. Thus, it is critical to understand regulators of the centrosome cycle because it must be carefully coordinated with the cell cycle to complete cell division precisely (Fabbro, 2005).
Microtubule nucleation by the PCM requires the conserved complex, gamma-tubulin ring complex (gamma-TuRC), in metazoan organisms. In yeast, the large coiled-coil spindle pole body (SPB) protein Spc110p anchors gamma-tubulin ring complex, providing sites for microtubule nucleation. In mammalian cells, Kendrin, like its yeast homolog Spc110p, complexes with CG-NAP to provide a structural scaffold for gamma-TuRC (Takahashi, 2002). Nlp has also been implicated in gamma-TuRC anchorage (Casenghi, 2003). CG-NAP and Kendrin associate with several protein kinases and phosphatases, suggesting that microtubule nucleation may be regulated through phosphorylation of CG-NAP/Kendrin complexes or associated proteins thatremain to be defined (Fabbro, 2005).
In recent years, several studies have provided a link between centrosomes and cytokinesis. Acentrosomal cells have been shown to form mitotic spindles and progress through mitosis but fail to complete cytokinesis. The molecular understanding of centrosome function in cytokinesis is only beginning to emerge. During cytokinesis, the mother centriole has been shown to transiently reposition to the midbody correlating with bridge narrowing and microtubule depolymerization, while movement away from the midbody correlates with cell cleavage. It is proposed that the mother centriole regulates an as yet unidentified pathway anchored at the centrosome that is analogous to the mitotic exit pathways in budding yeast called the 'mitotic exit network,' which is anchored at the SPB and controls mitotic exit and cytokinesis. The siRNA silencing of a recently identified mother centriole component, centriolin, produces cytokinesis failure (Gromley, 2003), suggesting that it is a component of this pathway in mammalian cells. However, additional components and pathways that control cytokinesis will need to be identified to understand the precise role of centrosomes in this process (Fabbro, 2005).
This study reports the molecular characterization of a coiled-coil protein called Cep55. Cep55 localizes to the centrosome of interphase cells and to the midbody during cytokinesis. Characterization of Cep55-depleted cells reveals that Cep55 participates in membrane abscission to form two daughter cells. Furthermore, Cdk1, Erk2, and Plk1 cooperate in the mitotic phosphorylation of Cep55, and this modification is required for its correct mitotic localization and cytokinesis function to maintain genomic stability (Fabbro, 2005).
Interestingly, Cep55 does not remain associated with the centrosome during mitosis, and its displacement from the centrosome is triggered by its phosphorylation. Therefore, a model is proposed whereby the centrosome acts as a regulatory site to organize phosphorylation of Cep55 upon mitotic entry. This phosphorylated Cep55 is then able to locate to the midbody during the final stages of cell division to function in the signal transduction pathway(s) that results in mitotic exit and cytokinesis. Therefore, it is proposed that dephosphorylation of Cep55 may allow it to relocate to the centrosome upon entry into G1 (Fabbro, 2005).
Fluctuations in the activity of Cdks drive cells to progress through mitosis. Specifically, mitotic entry is promoted by elevated activity of Cdk1 when complexed with cyclin B1, while the exit from mitosis requires the inactivation of Cdk1 and the dephosphorylation of at least a subset of Cdk1 substrates. This study demonstrates that Cep55 is phosphorylated at S425 and S428 by Cdk1/cyclin B1 upon mitotic entry, which is when this kinase is found at the centrosome. Another kinase, Erk2, has been shown to localize to the mitotic centrosomes. S425 and S428 can also be phosphorylated by Erk2 upon mitotic entry. In addition to Cdk1 and Erk2, Cep55 is also phosphorylated by Plk1 and this phosphorylation event is dependent on prior phosphorylation by Cdk1 and/or Erk2 at S425 and S428. Plk1 locates to the midbody and its role during cytokinesis has been demonstrated in yeast, Drosophila, and mammals. These data strongly indicate that Cep55 and Plk1 colocalize at the midbody and that Plk1-dependent phosphorylation of Cep55 is required for completion of cytokinesis. In addition, the Plk1-dependent phosphorylation mutant S436A causes cytokinesis failure to the same extent as the Cdk1/Erk2-dependent mutant S425/428A, indicating that phosphorylation at S436 is absolutely required for the function of Cep55 during cytokinesis, whereas phosphorylation at S425 and S428 is not required for Cep55 cytokinesis function directly but is essential for Plk1-dependent phosphorylation at S436. These findings indicate that Cep55 and Plk1 may cooperate at the midbody to coordinate mitotic exit and cytokinesis. Thus, it is proposed that unphosphorylatable Cep55 may fail to bind or generate the required signal to downstream components of the mitotic exit pathway (Fabbro, 2005).
Cep55 localizes to the centrosome via its C-terminus and this same region associates in vivo with the centrosome proteins CG-NAP and Kendrin. In contrast to CG-NAP and Kendrin, which gain affinity for the centrosome to participate in gamma-TuRC anchorage (Takahashi, 2002), Cep55 loses affinity for the centrosome upon mitotic entry coinciding with its dissociation from CG-NAP and Kendrin. Thus, it is not surprising that no requirement is found for Cep55 for microtubule nucleation. Loss of Cep55 from the centrosome coincides with its phosphorylation at the C-terminal residues S425 and S428. It is plausible to suggest that phosphorylation of Cep55 by either Erk2 or Cdk1 causes a conformational change in the protein causing it to lose affinity for CG-NAP and Kendrin, consequently becoming displaced from the centrosome at the G2/M boundary. This in turn may enable S436 of Cep55 to be accessible to Plk1 for phosphorylation. It is hypothesized that the displacement of Cep55 from the centrosome enables CG-NAP and Kendrin to strongly anchor themselves to the centrosome by binding calmodulin (Takahashi, 2002). Consistent with this idea, Cep55 and calmodulin bind the same region of CG-NAP, and the CG-NAP/Kendrin/calmodulin interaction is thought to occur only during mitosis, which is when calmodulin is observed at the centrosome (Fabbro, 2005).
MAP kinase and branching morphogenesis
Branching morphogenesis of epithelium is a common and important feature of organogenesis; it is, for example, responsible for development of renal collecting ducts, lung airways, milk ducts of mammary glands and seminal ducts of the prostate. In each case, epithelial development is controlled by a variety of mesenchyme-derived molecules, both soluble (e.g. growth factors) and insoluble (e.g. extracellular matrix). Little is known about how these varied influences are integrated to produce a coherent morphogenetic response, but integration is likely to be achieved at least partly by cytoplasmic signal transduction networks. Work in other systems (Drosophila tracheae, MDCK models) suggests that the mitogen-activated protein (MAP) kinase pathway might be important to epithelial branching. The role of the MAP kinase pathway in one of the best characterized mammalian examples of branching morphogenesis: the ureteric bud of the metanephric kidney. Erk MAP kinase is normally active in ureteric bud; the inhibition of Erk activation with PD98059, the MAP kinase kinase inhibitor, reversibly inhibits branching in a dose-dependent manner, while allowing tubule elongation to continue. When Erk activation is inhibited, ureteric bud tips show less cell proliferation than controls and the bud tips also produce fewer laminin-rich processes to penetrate the mesenchyme and fail to show the strong concentration of apical actin filaments typical of controls; apoptosis and expression of Ret and Ros, are, however, normal. The activity of the Erk MAP kinase pathway is dependent on at least two known regulators of ureteric bud branching; the GDNF-Ret signaling system and sulphated glycosaminoglycans. MAP kinase is therefore essential for normal branching morphogenesis of the ureteric bud, and lies downstream of significant extracellular regulators of ureteric bud development (Fisher, 2001).
MAP kinase and photoperiod response
Although the suprachiasmatic nucleus (SCN) is the major pacemaker in mammals, the peripheral cells or immortalized cells also contain a circadian clock. The SCN and the periphery may use different entraining signals -- light and some humoral factors, respectively. Induction of the circadian oscillation of gene expression is triggered by TPA treatment of NIH-3T3 fibroblasts. This induction is inhibited by a MEK inhibitor, and prolonged activation of the MAPK cascade is sufficient to trigger circadian gene expression. Therefore, prolonged activation of MAPK by entraining cues may be involved in the resetting of the circadian clock (Akashi, 2000).
Light-induced entrainment of the circadian clock is accompanied by the induction of some immediate-early genes in the SCN, and the serum shock, which triggers the induction of the circadian gene expression in cultured cells, also results in a transient and immediate induction of some genes such as mPer1. The acute induction of mPer1 mRNA in the SCN after light exposure is thought to be involved in light-induced phase shifting of the overt rhythm. Stimuli were sought that can induce the transient expression of mPer1 in mouse fibroblast NIH-3T3 cells and it was found that TPA treatment as well as a serum shock is able to induce the transient and strong expression of mPer1. The mRNA expression levels of mPer1 and mPer2 and albumin site D-binding protein (DBP), a clock-related gene encoding transcription factor, were monitored during 2 days. After the transient exposure to 50% serum, expression levels of all the three mRNAs oscillate with an approximate period length of 24 hr in confluently grown NIH-3T3 cells in the absence of serum. Thus, the serum shock is able to trigger the induction of a circadian oscillation of expression of clock and clock-related genes in NIH-3T3 cells as well as in Rat-1 cells. Remarkably, TPA treatment without serum also triggers the induction of a circadian oscillation of expression of the three genes, mPer1, mPer2, and DBP, with essentially the same period length as seen in serum-shocked cells. The TPA treatment is as effective as the serum shock in triggering the induction of circadian gene expression. Pretreatment with a specific inhibitor of protein kinase C (PKC) abolishes the TPA-induced circadian oscillation of gene expressions, confirming that TPA exerts its effect through activation of PKC. In contrast, the addition of the PKC inhibitor after TPA treatment failed to inhibit the triggering of the induction of circadian gene expression, suggesting that the inhibitor does not have a toxic effect. These results suggest that PKC activation is able to entrain the circadian rhythm of the gene expression (Akashi, 2000).
These results indicate that prolonged activation of the classic MAPK cascade (MEK/ERK) is able to induce immediate expression of mPer1 and trigger the induction of the circadian oscillation of expression of clock and clock-related genes in mammalian cultured cells and therefore suggest that the MAPK cascade has a key role in entrainment of the circadian rhythm in cultured cells. Previous studies have suggested that not only the SCN but also the periphery has a circadian clock, and light and some humoral factor(s) may act as an entraining cue in the SCN and the periphery, respectively; transcriptional activation is an essential event linking the cue and the circadian entrainment as well. In the SCN, light induces both ERK activation and immediate-early gene expression, which in general is mediated by the ERK pathway. It has also been reported that PKC activation in the SCN may have a role in rodent circadian rhythm and that treatment of the SCN with NGF induces the phase shift of circadian rhythm. Taken together, these results suggest that the MAPK cascade may function as a key mediator in common with the signal transduction pathways for entrainment of circadian rhythm in the SCN, the periphery, and immortalized cultured cells and that circadian entrainment by light and humoral factors may employ similar signal transduction mechanisms. These results also suggest that an unidentified humoral factor(s) that functions as an entraining cue in the periphery may induce the prolonged activation of the MAPK cascade in peripheral cells (Akashi, 2000 and references therein).
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