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W G Dunphy

Publications and source records attributed to W G Dunphy.

At least 37 records · Page 2Linked to original sources
PubMed1996

Cell cycle control by Xenopus p28Kix1, a developmentally regulated inhibitor of cyclin-dependent kinases.

We have isolated Xenopus p28Kix1, a member of the p21CIP1/p27KIP1/p57KIP2 family of cyclin-dependent kinase (Cdk) inhibitors. Members of this family negatively regulate cell cycle progression in mammalian cells by inhibiting the activities of Cdks. p28 shows significant sequence homology with p21, p27, and p57 in its N-terminal region, where the Cdk inhibition domain is known to reside. In contrast, the C-terminal domain of p28 is distinct from that of p21, p27, and p57. In co-immunoprecipitation experiments, p28 was found to be associated with Cdk2, cyclin E, and cyclin A, but not the Cdc2/cyclin B complex in Xenopus egg extracts. Xenopus p28 associates with the proliferating cell nuclear antigen, but with a substantially lower affinity than human p21. In kinase assays with recombinant Cdks, p28 inhibits pre-activated Cdk2/cyclin E and Cdk2/cyclin A, but not Cdc2/cyclin B. However, at high concentrations, p28 does prevent the activation of Cdc2/cyclin B by the Cdk-activating kinase. Consistent with the role of p28 as a Cdk inhibitor, recombinant p28 elicits an inhibition of both DNA replication and mitosis upon addition to egg extracts, indicating that it can regulate multiple cell cycle transitions. The level of p28 protein shows a dramatic developmental profile: it is low in Xenopus oocytes, eggs, and embryos up to stage 11, but increases approximately 100-fold between stages 12 and 13, and remains high thereafter. The induction of p28 expression temporally coincides with late gastrulation. Thus, although p28 may play only a limited role during the early embryonic cleavages, it may function later in development to establish a somatic type of cell cycle. Taken together, our results indicate that Xenopus p28 is a new member of the p21/p27/p57 class of Cdk inhibitors, and that it may play a role in developmental processes.

Amino Acid Sequence↗
PubMed1995

Myt1: a membrane-associated inhibitory kinase that phosphorylates Cdc2 on both threonine-14 and tyrosine-15.

Cdc2 is the cyclin-dependent kinase that controls entry of cells into mitosis. Phosphorylation of Cdc2 on threonine-14 and tyrosine-15 inhibits the activity of the enzyme and prevents premature initiation of mitosis. Although Wee1 has been identified as the kinase that phosphorylates tyrosine-15 in various organisms, the threonine-14-specific kinase has not been isolated. A complementary DNA was cloned from Xenopus that encodes Myt1, a member of the Wee1 family that was discovered to phosphorylate Cdc2 efficiently on both threonine-14 and tyrosine-15. Myt1 is a membrane-associated protein that contains a putative transmembrane segment. Immunodepletion studies suggested that Myt1 is the predominant threonine-14-specific kinase in Xenopus egg extracts. Myt1 activity is highly regulated during the cell cycle, suggesting that this relative of Wee1 plays a role in mitotic control.

Amino Acid Sequence↗
PubMed1995

Cell cycle regulation of a Xenopus Wee1-like kinase.

Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase.

Amino Acid Sequence↗
PubMed1995

Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors.

Proliferating eukaryotic cells possess checkpoint mechanisms that block cell division in the presence of unreplicated or damaged DNA. Using cell-free extracts from Xenopus eggs, we have investigated the mechanisms underlying the inability of a recombinant Cdc2/cyclin B complex to induce mitosis in the presence of incompletely replicated DNA. We found that the activities of the kinases and phosphatases that regulate the major phosphorylation sites on Cdc2 (e.g., tyrosine 15, threonine 14, and threonine 161) are not altered significantly under conditions where Xenopus extracts remain stably arrested in interphase due to the presence of the replication inhibitor aphidicolin. However, at threshold concentrations, a Cdc2/cyclin B complex containing a mutant Cdc2 subunit that cannot be phosphorylated on either tyrosine 15 or threonine 14 displays a markedly reduced capacity to induce mitosis in the presence of aphidicolin. This observation indicates that the replication checkpoint in Xenopus egg extracts functions without the inhibitory tyrosine and threonine phosphorylation of Cdc2. We provide evidence that the checkpoint-dependent suppression of the Cdc2/cyclin B complex involves a titratable inhibitor that is regulated by the presence of unreplicated DNA.

Animals↗
PubMed1994

Cdc2 regulatory factors.

A growing family of kinases and phosphatases controls the activity of the cyclin-dependent kinase cdc2. The past year has seen the identification of the cdk activating kinase as well as considerable elucidation of the cdc25/wee1 regulatory pathways. Both cdc25 and wee1 appear to be regulated by upstream kinase/phosphatase networks. In addition, it is likely that other regulatory mechanisms cooperate with the wee1/cdc25 phosphorylation systems to control the action of cdc2. Together, these elaborate checks and balances ensure that cdc2 triggers mitosis at the appropriate time.

Animals↗
PubMed1994

The decision to enter mitosis.

The phosphotyrosine content of the cdc2 protein kinases, the catalytic component of maturation-promoting factor (MPF), is an important parameter of mitotic regulation in a variety of organisms. Recent studies have shed considerable light on how the cdc2-specific tyrosine kinase (wee1) and its competing phosphatase (cdc25) are regulated during the cell cycle. A goal for the future will be to obtain a comprehensive picture of how the wee1-cdc25 regulatory system collaborates with other steps in mitotic activation to ensure that cell division occurs at the appropriate time during the cell cycle.

Journal Article↗
PubMed1993

Negative regulation of the wee1 protein kinase by direct action of the nim1/cdr1 mitotic inducer.

The wee1 protein kinase suppresses the entry into mitosis by mediating the inhibitory tyrosine phosphorylation of p34cdc2. Genetic studies have suggested that the nim1 protein kinase (also known as cdr1) acts as a positive regulator of mitosis by down-regulating the wee1 pathway in yeast cells. We have overexpressed the nim1 protein in both bacteria and insect cells. The recombinant nim1 protein autophosphorylates on both tyrosine and serine residues and can phosphorylate the isolated wee1 protein directly in a cell-free system. The nim1-catalyzed phosphorylation of the wee1 protein occurs in its C-terminal region and leads to a substantial drop in its activity as a cdc2-specific tyrosine kinase. This nim1-dependent inhibition of the wee1 protein kinase can be reversed readily in vitro by treatment with a protein phosphatase. These experiments provide direct biochemical evidence that the wee1 protein is subject to negative regulation by phosphorylation and indicate that the nim1 protein acts as an inhibitory, wee1-specific kinase.

Animals↗
PubMed1993

Two distinct mechanisms for negative regulation of the Wee1 protein kinase.

The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase.

Amino Acid Sequence↗
PubMed1992

Regulation of the cdc25 protein during the cell cycle in Xenopus extracts.

The cdc25 protein is a highly specific tyrosine phosphatase that triggers mitosis by dephosphorylating the cdc2 protein kinase. Using Xenopus extracts, we have found that the cdc25 protein is active at a low level throughout interphase. Near the onset of mitosis, the cdc25 protein undergoes a marked elevation in phosphatase activity that coincides with an extensive phosphorylation of the protein in its N-terminal region. In vitro dephosphorylation of this hyperphosphorylated form of cdc25 reduces its phosphatase activity back to the interphase level. Moreover, treatment of interphase Xenopus extracts with okadaic acid, a phosphatase inhibitor that accelerates the entry into mitosis, elicits both the premature hyperphosphorylation of cdc25 and the stimulation of its cdc2-specific tyrosine phosphatase activity. These experiments demonstrate the existence of a cdc25 regulatory system consisting of both a stimulatory kinase that phosphorylates a putative regulatory domain of the cdc25 protein and an inhibitory serine/threonine phosphatase that counteracts this kinase activity.

Amino Acid Sequence↗
PubMed1991

The cdc25 protein contains an intrinsic phosphatase activity.

Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase. We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides. The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators. Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis. These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.

4-Nitrophenylphosphatase↗
PubMed1991

The cdc25 protein controls tyrosine dephosphorylation of the cdc2 protein in a cell-free system.

As a prerequisite for the activation of MPF, the cdc2 protein kinase must undergo tyrosine dephosphorylation. Genetic studies have demonstrated that the cdc25 protein activates the cdc2 protein kinase once DNA replication has been completed. We have produced the cdc25 protein in bacteria and shown that it activates MPF in Xenopus extracts. In extracts that normally cannot enter mitosis owing to inhibition of DNA synthesis, the addition of active cdc25 protein efficiently elicits the mitotic state by inducing premature dephosphorylation of tyrosine on the cdc2 protein. The cdc25-dependent activation reaction can be reconstituted in a partially purified system lacking ATP. These biochemical experiments demonstrate that the cdc25 protein actively drives tyrosine dephosphorylation of the cdc2 protein and offer the prospect for characterizing the individual factors that regulate the activation of MPF during the progression from S phase to mitosis.

Animals↗
PubMed1990

A lamin-independent pathway for nuclear envelope assembly.

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.

Animals↗
PubMed1989

Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase.

It has been demonstrated that the Xenopus homolog of the fission yeast cdc2 protein is a component of M phase promoting factor (MPF). We show that the Xenopus cdc2 protein is phosphorylated on tyrosine in vivo, and that this tyrosine phosphorylation varies markedly with the stage of the cell cycle. Tyrosine phosphorylation is high during interphase (in Xenopus oocytes and activated eggs) but absent during M phase (in unfertilized eggs). In vitro activation of pre-MPF from Xenopus oocytes results in tyrosine dephosphorylation of the cdc2 protein and switching-on of its kinase activity. The product of the fission yeast suc1 gene (p13), which inhibits the entry into mitosis in Xenopus extracts, completely blocks tyrosine dephosphorylation and kinase activation. However, p13 has no effect on the activated form of the cdc2 kinase. These findings suggest that p13 controls the activation of the cdc2 kinase, and that tyrosine dephosphorylation is an important step in this process.

Animals↗
PubMed1988

The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis.

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.

Animals↗
PubMed1988

Mitosis-inducing factors are present in a latent form during interphase in the Xenopus embryo.

During the conversion to the mitotic state, higher eukaryotic cells activate a cascade of reactions which result in the disintegration of the nuclear envelope, the condensation of the DNA into chromosomes, and the reorganization of the cytoskeleton. In Xenopus, the induction of the mitotic state appears to be under the control of a cytoplasmic factor(s) known as mitosis-promoting factor or MPF. We have developed a rapid and highly sensitive version of an in vitro assay for MPF. The assay uses reconstituted nuclei in interphase cytoplasm from activated Xenopus eggs. The MPF-induced conversion from interphase to mitosis is conveniently monitored by the visual observation of the loss of the nuclear envelope from the substrate nuclei. At near saturating concentrations of MPF, nuclear breakdown requires 20-30 min. Preincubation experiments have revealed that the action of MPF requires only a few minutes and that the disassembly process itself takes up the remainder of the incubation period. Using this cell-free system, we have investigated the observation that protein synthesis is required for the progression through each successive mitotic cycle in the developing Xenopus embryo. A simple explanation for this finding would be that MPF is degraded after each mitosis and then resynthesized before the next mitotic cycle. However, using in vitro reactivation experiments, we have found that MPF is present in a latent, inactive form during interphase. These results suggest that the block in the cell cycle induced by inhibitors of protein synthesis is due to the lack of production of an activator of MPF.

Animals↗