Mitosis Entry Research Articles

Activation of protein kinase C alters p34cdc2 phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients

The p34(cdc2) protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34(cdc2) are induced by Ca(2+) and prevented by Ca(2+) chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca(2+) transients may play an important physiological role in the control of p34(cdc2) kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca(2+) concentration ([Ca(2+)](i)) in early sea urchin embryos without altering the normal resting level of Ca(2+). We have used this finding to investigate whether [Ca(2+)](i) transients control p34(cdc2) kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34(cdc2). In the present study we show that the elimination of [Ca(2+)](i) transients during interphase blocks p34(cdc2) activation and entry into mitosis, while the elimination of mitotic [Ca(2+)](i) transients prevents p34(cdc2) inactivation and exit from mitosis. Moreover, we find that [Ca(2+)](i) transients are not required for the synthesis of cyclin B, its binding to p34(cdc2) or its destruction during anaphase. However, in the absence of interphase [Ca(2+)](i) transients p34(cdc2) does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca(2+)](i) transients p34(cdc2) does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca(2+)](i) transients trigger the dephosphorylation of p34(cdc2) at key regulatory sites, thereby controlling the timing of mitosis entry and exit.

Activation of protein kinase C alters p34cdc2 phosphorylation state and kinase activity in early sea urchin embryos by abolishing intracellular Ca2+ transients

The p34cdc2 protein kinase, a universal regulator of mitosis, is controlled positively and negatively by phosphorylation, and by association with B-type mitotic cyclins. In addition, activation and inactivation of p34cdc2 are induced by Ca2+ and prevented by Ca2+ chelators in permeabilized cells and cell-free systems. This suggests that intracellular Ca2+ transients may play an important physiological role in the control of p34cdc2 kinase activity. We have found that activators of protein kinase C can be used to block cell cycle-related alterations in intracellular Ca2+ concentration ([Ca2+]i) in early sea urchin embryos without altering the normal resting level of Ca2+. We have used this finding to investigate whether [Ca2+]i transients control p34cdc2 kinase activity in living cells via a mechanism that involves cyclin B or the phosphorylation state of p34cdc2. In the present study we show that the elimination of [Ca2+]i transients during interphase blocks p34cdc2 activation and entry into mitosis, while the elimination of mitotic [Ca2+]i transients prevents p34cdc2 inactivation and exit from mitosis. Moreover, we find that [Ca2+]i transients are not required for the synthesis of cyclin B, its binding to p34cdc2 or its destruction during anaphase. However, in the absence of interphase [Ca2+]i transients p34cdc2 does not undergo the tyrosine dephosphorylation that is required for activation, and in the absence of mitotic [Ca2+]i transients p34cdc2 does not undergo threonine dephosphorylation that is normally associated with inactivation. These results provide evidence that intracellular [Ca2+]i transients trigger the dephosphorylation of p34cdc2 at key regulatory sites, thereby controlling the timing of mitosis entry and exit.

Different kinases phosphorylate nucleophosmin/B23 at different sites during G 2 and M phases of the cell cycle

The recombinant GST-nucleophosmin/B23 and the truncated mutants were tested for phosphorylation in cell-free extracts of G 2 and M phases or by purified kinases. Our results indicated that a threonine residue at amino acids (a.a.) 185–240 was phosphorylated by cdc2 kinase during the entry of mitosis while the serine phosphorylation site at the middle acidic portion of the molecule (a.a. 83–152) was phosphorylated by casein kinase II during G 2 phase. Our results also showed that there was possibly another serine phosphorylation at site other than the middle portion of nucleophosmin/B23 (a.a. 83–152) during the entry of cells into mitosis. The demonstration of the characteristic changes in phosphorylation of nucleophosmin/B23 during the cell cycle implicates important role of nucleophosmin/B23 in the control of the fate of nucleoli and cell growth.

Down-Regulation of Nucleophosmin/B23 mRNA Delays the Entry of Cells into Mitosis

In investigating the regulation of nucleophosmin/B23 mRNA expression at the entry of mitosis, the results of Northern gel blot analysis showed that the nucleophosmin/B23 mRNA levels significantly increased in prometaphase (nocodazole-arrested) or metaphase (colchicine-arrested) cells collected by mitotic shake-off. A higher level of nucleophosmin/B23 mRNA was detected in all the collected mitotic cells arrested by treatment with nocodazole for 10-18h as compared to that in G2cells. An attempt was then made to determine whether the regulation of nucleophosmin/B23 mRNA plays a role in the control of entry into mitosis. Down-regulation of nucleophosmin/B23 mRNA by transfection of its antisense construct resulted in the delay of cells entering mitosis. The demonstration of the characteristic changes in the mRNA level of nucleophosmin/B23 during the entry of cells into mitosis implicates the importance of nucleophosmin/B23 in the control of the mitotic fate of nucleoli and cell growth.

Calcium/Calmodulin-dependent Phosphorylation and Activation of Human Cdc25-C at the G2/M Phase Transition in HeLa Cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).

Zeatin is indispensable for the G 2-M transition in tobacco BY-2 cells

The importance of N 6-isoprenoid cytokinins in the G 2-M transition of Nicotiana tabacum BY-2 cells was investigated. Both cytokinin biosynthesis and entry in mitosis were partially blocked by application at early or late G 2 of lovastatin (10 μM), an inhibitor of mevalonic acid synthesis. LC-MS/MS quantification of endogenous cytokinins proved that lovastatin affects cytokinin biosynthesis by inhibiting HMG-CoA reductase. Out of eight different aminopurines and a synthetic auxin tested for their ability to override lovastatin inhibition of mitosis, only zeatin was active. Our data point to a key role for a well-defined cytokinin (here, zeatin) in the G 2-M transition of tobacco BY-2 cells.

Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization.

Sperm incorporation and the conversion of the sperm-derived components into zygotic structures during in vitro fertilization of bovine oocytes was explored by combining ultrastructural studies with observations of the fertilizing sperm tagged with a mitochondrion-specific vital dye MitoTracker green FM. The zygotes fertilized by the MitoTracker-labeled sperm were fixed at various times after fertilization and then processed for immunocytochemistry to examine the distribution of DNA, microtubules, and sperm tail components, including the fibrous sheath and axonemal microtubules. We show here that the complete incorporation of the sperm, but not sperm-oocyte binding and oocyte activation, depends upon the integrity of oocyte microfilaments and is inhibited by the microfilament disrupter cytochalasin B. After sperm incorporation, the mitochondria are displaced from the sperm's connecting piece, and the sperm centriole is exposed to the egg cytoplasm. This event is followed by the formation of the microtubule-based sperm aster, which is responsible for the union of male and female pronuclei. Concomitantly, the major structure of the sperm principal piece, the fibrous sheath, disappears. After the first mitosis, the compact mitochondrial sheath can be seen in one of the blastomeres. An aggregate of the sperm mitochondria is observed at the entry of the second mitosis, although they remain in the vicinity of the nucleus and can later be seen at one pole of the metaphase spindle. The mitochondrial cluster is occasionally found in one of the blastomeres in the early-stage four-cell embryos, but it is no longer detected by the beginning of the third mitotic cycle. These data suggest that the disassembly of the sperm tail during bovine fertilization occurs as a series of precisely orchestrated events involving the destruction (fibrous sheath and mitochondrial sheath) and transformation (DNA, sperm centriole) of particular sperm structures into zygotic and embryonic components.

Local perinuclear calcium signals associated with mitosis-entry in early sea urchin embryos.

Using calcium-sensitive dyes together with their dextran conjugates and confocal microscopy, we have looked for evidence of localized calcium signaling in the region of the nucleus before entry into mitosis, using the sea urchin egg first mitotic cell cycle as a model. Global calcium transients that appear to originate from the nuclear area are often observed just before nuclear envelope breakdown (NEB). In the absence of global increases in calcium, confocal microscopy using Calcium Green-1 dextran indicator dye revealed localized calcium transients in the perinuclear region. We have also used a photoinactivatable calcium chelator, nitrophenyl EGTA (NP-EGTA), to test whether the chelator-induced block of mitosis entry can be reversed after inactivation of the chelator. Cells arrested before NEB by injection of NP-EGTA resume the cell cycle after flash photolysis of the chelator. Photolysis of chelator triggers calcium release. TreatmenT with caFfeine to enhance calcium-induced calcium release increases the amplitude of NEB-associated calcium transients. These results indicate that calcium increases local to the nucleus are required to trigger entry into mitosis. Local calcium transients arise in the perinuclear region and can spread from this region into the cytoplasm. Thus, cell cycle calcium signals are generated by the perinuclear mitotic machinery in early sea urchin embryos.

The roles of DNA topoisomerase II during the cell cycle.

DNA topoisomerase II (topo II) is essential for survival of all eukaryotic cells. Topo II is both an enzyme and a structural component of the nuclear matrix. It regulates the topological states of DNA by transient cleavage, strand passing and re-ligation of double-stranded DNA resulting in decatenation of intertwined DNA molecules and relaxation of supercoiled DNA. Topo II plays an important role in DNA replication and is required for condensation and segregation of chromosomes. The expression of topo II is cell cycle dependent with both protein levels and catalytic activity peaking at G2/M. Phosphorylation/dephosphorylation of topo II may be a part of regulatory checkpoints at the entry and progression of mitosis.

Highly specific antibody to Rous sarcoma virus src gene product recognizes nuclear and nucleolar antigens in human cells.

An antiserum to the Rous sarcoma virus-transforming protein pp60v-src, raised in rabbits immunized with the bacterially produced protein alpha p60 serum (M. D. Resh and R. L. Erikson, J. Cell Biol. 100:409-417, 1985) previously reported to detect very specifically a novel population of pp60v-src and pp60c-src molecules associated with juxtareticular nuclear membranes in normal and Rous sarcoma virus-infected cells of avian and mammalian origin, was used here to investigate by immunofluorescence microscopy localization patterns of Src molecules in human cell lines, either normal or derived from spontaneous tumors. We found that the alpha p60 serum reveals nuclear and nucleolar concentrations of antigens in all the human cell lines tested and in two rat and mouse hepatoma cell lines derived from adult tumorous tissues but not in any established rat and mouse cell lines either untransformed or transformed by the src and ras oncogenes. Both the nuclear and nucleolar stainings can be totally extinguished by preincubation of the serum with highly purified chicken c-Src. We show also that the partitioning of the alpha p60-reactive proteins among the whole nucleus and the nucleolus depends mostly on two different parameters: the position in the cell cycle and the degree of cell confluency. Our observations raise the attractive possibility that, in differentiated cells, pp60c-src and related proteins might be involved not only in mediating the transduction of mitogenic signals at the plasma membrane level but also in controlling progression through the cell cycle and entry in mitosis by interacting with cell division cycle regulatory components at the nuclear level.

In isolated human centrosomes, the associated kinases phosphorylate a specific subset of centrosomal proteins

Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP-dependent protein kinase type II and the mitotic kinase p34cdc2 with centrosomes from human lymphoblast cells has previously been shown (Keryer et al, 1993, Exp Cell Res 204, 230-240; Bailly et al, 1989, EMBO J 8, 3985-3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (M(r) 230-220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1-histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP-dependent kinase or of cyclinB-p34cdc2 kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (M(r) 100000 and 37000) were phosphorylated only by p34cdc2 kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule-organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP-dependent protein kinase could be regulated by the mitotic kinase at the entry of mitosis.

Calmodulin Regulates the Expression of CDKS, Cyclins and Replicative Enzymes During Proliferative Activation of Human T Lymphocytes

Cell cycle is regulated by the activation of complexes of cyclins and cyclin-dependent protein kinases at specific points. Quiescent cells lack both cyclins and cyclin-dependent kinases but their expression is induced after proliferative activation. Cyclin A/cdk2 complexes are involved in the onset of DNA replication whereas cyclin B/cdc2 trigger mitosis. We report here that Ca2+ and calmodulin regulate the expression of cdk2, cdc2, cyclin B and the proliferating cell nuclear antigen (a co-factor of DNA polymerase-δ) in human T lymphocytes. Likewise, the expression of cdk4, cyclin A and DNA polymerase-α is dependent of the sinergistic effect of both the Ca2+/calmodulin and the protein kinase C pathways. Thus, calmodulin controls DNA synthesis by regulating the levels of cdk2 and proliferating cell nuclear antigen and mitosis entry by modulating the expression of cyclin B and cdc2.

Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair.

In eukaryotes a cell-cycle control termed a checkpoint causes arrest in the S or G2 phases when chromosomes are incompletely replicated or damaged. Previously, we showed in budding yeast that RAD9 and RAD17 are checkpoint genes required for arrest in the G2 phase after DNA damage. Here, we describe a genetic strategy that identified four additional checkpoint genes that act in two pathways. Both classes of genes are required for arrest in the G2 phase after DNA damage, and one class of genes is also required for arrest in S phase when DNA replication is incomplete. The G2-specific genes include MEC3 (for mitosis entry checkpoint), RAD9, RAD17, and RAD24. The genes common to both S phase and G2 phase pathways are MEC1 and MEC2. The MEC2 gene proves to be identical to the RAD53 gene. Checkpoint mutants were identified by their interactions with a temperature-sensitive allele of the cell division cycle gene CDC13; cdc13 mutants arrested in G2 and survived at the restrictive temperature, whereas all cdc13 checkpoint double mutants failed to arrest in G2 and died rapidly at the restrictive temperature. The cell-cycle roles of the RAD and MEC genes were examined by combination of rad and mec mutant alleles with 10 cdc mutant alleles that arrest in different stages of the cell cycle at the restrictive temperature and by the response of rad and mec mutant alleles to DNA damaging agents and to hydroxyurea, a drug that inhibits DNA replication. We conclude that the checkpoint in budding yeast consists of overlapping S-phase and G2-phase pathways that respond to incomplete DNA replication and/or DNA damage and cause arret of cells before mitosis.

Calcium and cell cycle control

The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT. Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin. We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.