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J L Maller

Publications and source records attributed to J L Maller.

At least 37 records · Page 2Linked to original sources
PubMed1998

Cdc2-cyclin B phosphorylates p70 S6 kinase on Ser411 at mitosis.

The carboxyl terminus of p70 S6 kinase (p70(s6k)) has a set of Ser and Thr residues (Ser411, Ser418, Ser424, and Thr421) phosphorylated in vivo by an unidentified kinase(s). These Ser/Thr sites are immediately followed by proline, a motif that is commonly seen in the substrates of cyclin-dependent kinases (Cdk) and mitogen-activated protein kinases. A previous study has shown that Cdc2 (Cdk1) indeed phosphorylates these p70(s6k) Ser/Thr residues in vitro. Here, we demonstrate that Cdc2-cyclin B complex phosphorylates Ser411 in the KIRSPRR sequence, whereas other Cdk-cyclin complexes including those containing Cdk2, Cdk4, or Cdk6 do not. Additionally, Ser411 phosphorylation in vivo was increased at mitosis in parallel with Cdc2 activation, and it was suppressed by a dominant negative form of Cdc2. These data indicate that p70(s6k) is a physiological substrate of Cdc2-cyclin B in mitosis. Since the activity of p70(s6k) is low during mitosis, Cdc2-cyclin B may play a role in inactivating p70(s6k) during mitosis, where protein synthesis is suppressed.

Amino Acid Sequence↗
PubMed1998

Developmental regulation of MCM replication factors in Xenopus laevis.

At the midblastula transition (MBT) during Xenopus laevis development, zygotic transcription begins [1], and the rapid, early cleavage cycles are replaced by cell-division cycles that lengthen and acquire G (gap) phases [2] and checkpoints [3-5]. This cell-cycle remodeling may result from either a loss of maternal products, the transcription of zygotic genes, or the replacement of maternal proteins by zygotic gene products. We have identified an example of the third possibility: distinct maternal and zygotic genes encoding a member of the minichromosome maintenance (MCM) protein family. The mcm genes were identified in yeast by mutations that blocked replication of artificial chromosomes or perturbed the G1/S transition in the cell cycle [6,7]. In Xenopus eggs, the MCM2-MCM7 proteins assemble as multimeric complexes at chromosomal origins of replication [8-14]. The sequential, cell-cycle-dependent assembly of the origin replication complex (ORC), CDC6 protein and the MCM complex at origins of replication ensures that DNA replicates only once per cell cycle [15,16]. The periodic association of the MCM complex with chromatin may be regulated via phosphorylation by cyclin-dependent kinases (Cdks) [11]. We have cloned the first example of a developmentally regulated mcm gene, zygotic mcm6 (zmcm6), expressed only after gastrulation when the cell cycle is remodeled. The zMCM6 protein assembles into MCM complexes and differs from maternal MCM6 (mMCM6) in having a carboxy-terminal extension and a consensus cyclin-Cdk phosphorylation site. There may also be maternal-zygotic pairs of other MCMs. These data suggest that MCMs are critical for cell-cycle remodeling during early Xenopus development.

Animals↗
PubMed1998

Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis.

Entry into mitosis depends upon activation of the dual-specificity phosphatase Cdc25C, which dephosphorylates and activates the cyclin B-Cdc2 complex. Previous work has shown that the Xenopus polo-like kinase Plx1 can phosphorylate and activate Cdc25C in vitro. In the work presented here, we demonstrate that Plx1 is activated in vivo during oocyte maturation with the same kinetics as Cdc25C. Microinjection of wild-type Plx1 into Xenopus oocytes accelerated the rate of activation of Cdc25C and cyclin B-Cdc2. Conversely, microinjection of either an antibody against Plx1 or kinase-dead Plx1 significantly inhibited the activation of Cdc25C and cyclin B-Cdc2. This effect could be reversed by injection of active Cdc25C, indicating that Plx1 is upstream of Cdc25C. However, injection of Cdc25C, which directly activates cyclin B-Cdc2, also caused activation of Plx1, suggesting that a positive feedback loop exists in the Plx1 activation pathway. Other experiments show that injection of Plx1 antibody into early embryos, which do not require Cdc25C for the activation of cyclin B-Cdc2, resulted in an arrest of cleavage that was associated with monopolar spindles. These results demonstrate that in Xenopus laevis, Plx1 plays important roles both in the activation of Cdc25C at the initiation of mitosis and in spindle assembly at late stages of mitosis.

Animals↗
PubMed1998

Recurring themes in oocyte maturation.

I am pleased to contribute to this special issue of Biology of the Cell in honor of Yoshio Masui. Oocyte maturation remains a small enough and young enough field that the authors assembled for this issue can trace the entire development of the field over the last 30 years, beginning with the early demonstration by Masui (1967) and others, that steroids can induce complete maturation of denuded oocytes in vitro. Not very long after that Masui and Markert (1971) published the seminal paper that identified MPF and CSF activity. In this article I intend to highlight recurring themes in oocyte maturation that continue to be actively investigated, almost all of which derive from studies pursued at some point in time by Masui and his colleagues.

Animals↗
PubMed1997

Zygotic transcription is required to block a maternal program of apoptosis in Xenopus embryos.

At the midblastula transition during Xenopus development, the cell cycle is remodeled, and zygotic transcription is initiated. Additionally, cyclin E1 is degraded at the midblastula transition independently of protein synthesis, the number of cell cycles, and the nuclear-to-cytoplasmic ratio. In the studies reported here, cell cycles were delayed by transient inhibition of protein synthesis with cycloheximide (100 microg/ml) prior to the midblastula transition. Even after reaccumulation of mitotic cyclins and resumption of cell divisions, cycloheximide-treated embryos did not resume DNA synthesis, failed to initiate transcription, and synchronously became apoptotic before the gastrula stage. These results were independent of the stage at which embryos were treated or the duration of treatment. Inhibition of zygotic transcription with alpha-amanitin also induced apoptosis. These data suggest that a developmental checkpoint at the midblastula transition is maternally regulated and can trigger apoptosis. Apoptosis induced by cycloheximide or alpha-amanitin was blocked by injection of RNA encoding Xenopus Bcl-2, suggesting that this maternal program is normally blocked by expression of an apoptotic inhibitor. Embryos pulsed with lower doses of cycloheximide (10 microg/ml) delayed development prior to the midblastula transition but resumed DNA synthesis, initiated transcription, and gastrulated normally. This indicates that the apoptotic response is initiated only when delayed embryos are unable to support initiation of zygotic transcription.

Amanitins↗
PubMed1997

A role for cyclin E/Cdk2 in the timing of the midblastula transition in Xenopus embryos.

During Xenopus development, the early cell cycles consist of rapid oscillations between DNA synthesis and mitosis until completion of the 12th mitotic division. Then the cycle lengthens and becomes asynchronous, zygotic transcription begins, and G phases are established, a period known as the midblastula transition (MBT). Some aspects of the MBT, such as zygotic transcription, depend on acquisition of a threshold nuclear to cytoplasmic (N/C) ratio, whereas others, such as maternal cyclin E degradation, are independent of nuclear events and appear to be controlled by an autonomous maternal timer. To investigate the function of cyclin E during the early cycles, cyclin E/Cdk2 kinase activity was specifically inhibited in fertilized eggs by a truncated form of the Xenopus Cdk inhibitor, Xic1 (Delta34Xic1). Delta34Xic1 caused lengthening of the embryonic cell cycles that correlated with increased levels of mitotic cyclins. However, DNA synthesis was not inhibited. Several hallmarks of the MBT were delayed for several hours in Delta34Xic1-injected embryos, including the disappearance of cyclins E and A, the initiation of zygotic transcription, and the reappearance of phosphotyrosine on Cdc2. In both control and Delta34Xic1-injected embryos, cyclin E was degraded after the 12th mitotic division as zygotic transcription began, but experiments with alpha-amanitin show that cyclin E degradation is not dependent on zygotic transcription. Thus, the length of the early cycles and the timing of maternal cyclin degradation depend upon cyclin E/Cdk2 activity. Neither oscillations in cyclin E/Cdk2 activity during the early cycles nor the disappearance of cyclin E at the MBT were dependent on protein synthesis. These data suggest that cyclin E/Cdk2 is directly linked to an autonomous maternal timer that drives the early embryonic cell cycles until the MBT.

Animals↗
PubMed1997

Ionizing radiation induces apoptosis and elevates cyclin A1-Cdk2 activity before but not after the midblastula transition in Xenopus.

After the twelfth cell division in Xenopus embryos, zygotic gene transcription is activated, cells become motile, and cell division becomes asynchronous. This developmental change is termed the midblastula transition. High doses of gamma-irradiation (gamma-IR) before the midblastula transition induced apoptotic cell death and increased the levels of cyclin A1 and cyclin A1-Cdk2 activity. The addition of recombinant cyclin A1-Cdk2 induced the formation of apoptotic nuclei in Xenopus egg extracts, suggesting a role for cyclin A1-Cdk2 in apoptosis. Hallmarks of apoptosis, such as internucleosomal DNA fragmentation, pyknotic and uniformly condensed nuclei, and loss of intercellular attachments, were evident in embryos exposed to gamma-IR before the midblastula transition. Apoptotic cells accumulated in the blastocoel, suggesting that before the midblastula transition Xenopus embryos use apoptosis to eliminate cells containing damaged DNA. However, embryos treated with the same dose of gamma-IR after the midblastula transition developed normally and exhibited no signs of apoptosis, no change in cyclin A1 level, and no increase in cyclin A1-Cdk2 activity. These results indicate that there is a change in the response to DNA damage at the midblastula transition in Xenopus embryos.

Animals↗
PubMed1996

Cloning and characterization of a novel serine/threonine protein kinase expressed in early Xenopus embryos.

We have cloned from a Xenopus ovary cDNA library a novel protein kinase gene whose expression peaks in the oocyte and unfertilized egg, begins to decrease gradually after fertilization, and disappears during the gastrulation stage of embryogenesis. The cloned gene, termed XEEK1 (for Xenopus egg and embryo kinase), encodes a protein with a predicted molecular mass of 49 kDa. Bacterially expressed XEEK1 migrates at 57 kDa upon polyacrylamide gel electrophoresis analysis, and a XEEK1-specific antibody recognizes a protein of 57 kDa in Xenopus oocyte and egg extracts. The XEEK1 kinase domain shares 35% identity (approximately 65% similarity) with the yeast SNF1 kinase and related kinases. However, expression of XEEK1 does not complement a snf1 deletion mutation in yeast, which suggests that it is probably not a Xenopus homolog of SNF1. Recombinant XEEK1 protein autophosphorylates on threonine residues in vitro in a reaction that prefers Mn2+ to Mg2+ ions. Site-directed mutagenesis of the conserved lysine residue (Lys-81) within the kinase domain to isoleucine totally abolishes kinase activity, and threonine 192 has been identified as the autophosphorylation site. This site is distinct from the conserved threonine (Thr-215 in XEEK1) present in the protein kinase activation loop that is the site of autophosphorylation for many protein kinases. XEEK1 is a substrate for the cyclic AMP-dependent protein kinase both in vitro and in vivo, suggesting a possible mode of regulation of XEEK1. An immunoprecipitate of oocyte/egg extracts with anti-XEEK1 serum contains a protein of approximately 155 kDa that may be a substrate and/or a regulatory component of the kinase.

Amino Acid Sequence↗
PubMed1996

Mos proto-oncogene function during oocyte maturation in Xenopus.

The function of the Xenopus c-mos proto-oncogene product (Mos(xe)) has been investigated during oocyte maturation. Experiments with a new antibody able to immunoblot Mos(xe) demonstrated the time course of MAP kinase (MAP K) activation in oocytes paralleled Mos(xe) accumulation, and in activated eggs the deactivation of MAP K paralleled the degradation of Mos(xe). Ablation of Mos synthesis by microinjection of antisense oligodeoxynucleotides abolished activation of MAP K by progesterone, but microinjection of GST-Mos fully restored both MAP K activation and germinal vesicle breakdown (GVBD). The Mos(xe) level at metaphase of Meiosis I (MI) was 2 - 3-fold less than that at metaphase of Meiosis II (MII), but MAP K activation was maximal at metaphase in both MI and MII. In the transition between MI and MII, both cyclin B and Mos(xe) levels rapidly declined in the presence of cycloheximide and injection of exogenous GST-Mos(xe) did not prevent degradation of either protein, although MAP K was activated. Microinjection of GST-Mos(xe) into oocytes was able to activate MAP K before GVBD and H1 kinase activation, and microinjection of constitutively-activated thiophosphorylated MAP K induced de novo synthesis of Mos(xe) before H1 kinase activation, suggesting the existence of a positive feedback loop between MAP K and Mos(xe) accumulation.

Animals↗
PubMed1996

In vivo regulation of the early embryonic cell cycle in Xenopus.

We report here the first extensive in vivo study of cell cycle regulation in the Xenopus embryo. Cyclin A1, B1, B2, and E1 levels, Cdc2 and Cdk2 kinase activity, and Cdc25C phosphorylation states were monitored during early Xenopus embryonic cell cycles. Cyclin B1 and B2 protein levels were high in the unfertilized egg, declined upon fertilization, and reaccumulated to the same level during the first cell cycle, a pattern repeated during each of the following 11 divisions. Cyclin A1 showed a similar pattern, except that its level was lower in the egg than in the cell cycles after fertilization. Cyclin B1/Cdc2 kinase activity oscillated, peaking before each cleavage, and Cdc25C alternated between a highly phosphorylated and a less phosphorylated form that correlated with high and low cyclin B1/Cdc2 kinase activity, respectively. Unlike the mitotic cyclins, the level of cyclin E1 did not oscillate during embryogenesis, although its associated Cdk2 kinase activity cycled twice for each oscillation of cyclin B1/Cdc2 activity, consistent with a role for cyclin E1 in both S-phase and mitosis. Although the length of the first embryonic cycle is regulated by both the level of cyclin B and the phosphorylation state of Cdc2, cyclin accumulation alone was rate-limiting for later cycles, since overexpression of a mitotic cyclin after the first cycle caused cell cycle acceleration. The activity of Cdc2 closely paralleled the accumulation of cyclin B2, but cell cycle acceleration caused by cyclin B overexpression was not associated with elevation of Cdc2 activity to higher than metaphase levels. Tyrosine phosphorylation of Cdc2, absent during cycles 2-12, reappeared at the midblastula transition coincident with the disappearance of cyclin E1. Cyclin A1 disappeared later, at the beginning of gastrulation. Our results suggest that the timing of the cell cycle in the Xenopus embryo evolves from regulation by accumulation of mitotic cyclins to mechanisms involving periodic G1 cyclin expression and inhibitory tyrosine phosphorylation of Cdc2.

Animals↗
PubMed1996

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Animals↗
PubMed1995

Cloning and characterization of the Xenopus cyclin-dependent kinase inhibitor p27XIC1.

We have isolated a gene encoding Xic-1, a 27-kDa cyclin-dependent kinase (Cdk) inhibitor from Xenopus ovary that shares significant homology with both mammalian CIP1 and Kip1/Kip2. The N- and C-terminal halves of Xic-1 are sufficient for interacting with Cdks and proliferating cell nuclear antigen, respectively. Recombinant Xic-1 inhibits Xenopus cyclin E/Cdk2, cyclin A/Cdk2 and cyclin B/Cdc2 activities, although with quite different IC50 values. Truncation of the N terminus of Xic-1 increases the IC50 value for cyclin A/Cdk2 50-fold with no effect on the inhibition of cyclin E/Cdk2 or cyclin B/Cdc2.Xic-1 inhibits both single-stranded and nuclear DNA synthesis in egg extracts, an effect reversed by proliferating cell nuclear antigen or cyclin E/Cdk2, respectively. These results suggest a function for Xic-1 in the control of DNA synthesis by cyclin E/Cdk2.

Amino Acid Sequence↗
PubMed1995

Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase.

Earlier work demonstrated that cyclins A1, B1, and B2 are not associated with Cdk2 from unfertilized Xenopus eggs. As a potential Cdk2 partner during meiosis, a cyclin E homolog was cloned from a Xenopus oocyte cDNA library and found to be 60% identical at the amino acid level to human cyclin E. Cyclin E1 protein was detected in resting oocytes, and the level increased severalfold in meiosis II, concomitant with the appearance of forms with decreased electrophoretic mobility. During oocyte maturation, the patterns of cyclin E1-associated kinase activity and Cdk2 activity were identical, with activity low until after germinal vesicle breakdown, peaking during meiosis II. Cyclin E1 complexes immunoprecipitated from unfertilized Xenopus eggs contained Cdk2 but not Cdc2. In cycling egg extracts Cdk2-cyclin E1-associated kinase activity oscillated, but the level of cyclin E1 protein and its association with Cdk2 did not vary appreciably; complex activity appeared to be regulated neither by the synthesis and destruction of the cyclin subunit nor by association/disassociation of the two subunits. During the early cleavage divisions in embryos, cyclin E1 and Cdk2 remained associated. The data indicate that the Cdk2-cyclin E complex functions during meiotic and embryonic cell cycles in addition to performing its established role during G1 in somatic cells.

Amino Acid Sequence↗
PubMed1995

Cloning and expression of a Xenopus gene that prevents mitotic catastrophe in fission yeast.

In fission yeast the Wee1 kinase and the functionally redundant Mik1 kinase provide a regulatory mechanism to ensure that mitosis is initiated only after the completion of DNA synthesis. Yeast in which both Wee1 and Mik1 kinases are defective exhibit a mitotic catastrophe phenotype, presumably due to premature entry into mitosis. Because of the functional conservation of cell cycle control elements, the expression of a vertebrate wee1 or mik1 homolog would be expected to rescue such lethal mutations in yeast. A Xenopus total ovary cDNA library was constructed in a fission yeast expression vector and used to transform a yeast temperature-dependent mitotic catastrophe mutant defective in both wee1 and mik1. Here we report the identification of a Xenopus cDNA clone that can rescue several different yeast mitotic catastrophe mutants defective in Wee1 kinase function. The expression of this clone in a wee1/mik1-deficient mutant causes an elongated cell phenotype under non-permissive growth conditions. The 2.0 kb cDNA clone contains an open reading frame of 1263 nucleotides, encoding a predicted 47 kDa protein. Bacterially expressed recombinant protein was used to raise a polyclonal antibody, which specifically recognizes a 47 kDa protein from Xenopus oocyte nuclei, suggesting the gene encodes a nuclear protein in Xenopus. The ability of this cDNA to complement mitotic catastrophe mutations is independent of Wee1 kinase activity.

Amino Acid Sequence↗
PubMed1995

Induction of Xenopus oocyte meiotic maturation by MAP kinase.

Mitogen-activated protein kinase (MAPK) is one of the protein kinases activated during meiotic maturation of Xenopus laevis oocytes. The c-Mosxe protein kinase, which has been shown to be sufficient to promote germinal vesicle breakdown (GVBD) in meiosis I, can directly activate MAP kinase kinase in vitro and leads to the activation of MAPK in vivo. Recently we have shown that constitutively activated MAPK induces metaphase arrest when injected into one blastomere of a two-cell embryo. This arrest mimics the natural arrest of vertebrate unfertilized eggs in second meiotic metaphase due to cytostatic factor and c-Mosxe activity. We show here that microinjection of constitutively activated thiophosphorylated MAPK into resting oocytes is able to activate maturation-promoting factor (MPF) and promote GVBD. These results strongly support the hypothesis that MAPK plays an important role in the pathway that links c-Mosxe to the activation of MPF.

Animals↗
PubMed1995

Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

Amino Acid Sequence↗