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Biomedical subjects

W G Dunphy

Publications and source records attributed to W G Dunphy.

At least 19 recordsLinked to original sources
PubMed2001

Regulation of Op18 during spindle assembly in Xenopus egg extracts.

Oncoprotein 18 (Op18) is a microtubule-destabilizing protein that is negatively regulated by phosphorylation. To evaluate the role of the three Op18 phosphorylation sites in Xenopus (Ser 16, 25, and 39), we added wild-type Op18, a nonphosphorylatable triple Ser to Ala mutant (Op18-AAA), and to mimic phosphorylation, a triple Ser to Glu mutant (Op18-EEE) to egg extracts and monitored spindle assembly. Op18-AAA dramatically decreased microtubule length and density, while Op18-EEE did not significantly affect spindle microtubules. Affinity chromatography with these proteins revealed that the microtubule-destabilizing activity correlated with the ability of Op18 to bind tubulin. Since hyperphosphorylation of Op18 is observed upon addition of mitotic chromatin to extracts, we reasoned that chromatin-associated proteins might play a role in Op18 regulation. We have performed a preliminary characterization of the chromatin proteins recruited to DNA beads, and identified the Xenopus polo-like kinase Plx1 as a chromatin-associated kinase that regulates Op18 phosphorylation. Depletion of Plx1 inhibits chromatin-induced Op18 hyperphosphorylation and spindle assembly in extracts. Therefore, Plx1 may promote microtubule stabilization and spindle assembly by inhibiting Op18.

Animals↗
PubMed2001

Positive regulation of Wee1 by Chk1 and 14-3-3 proteins.

Wee1 inactivates the Cdc2-cyclin B complex during interphase by phosphorylating Cdc2 on Tyr-15. The activity of Wee1 is highly regulated during the cell cycle. In frog egg extracts, it has been established previously that Xenopus Wee1 (Xwee1) is present in a hypophosphorylated, active form during interphase and undergoes down-regulation by extensive phosphorylation at M-phase. We report that Xwee1 is also regulated by association with 14-3-3 proteins. Binding of 14-3-3 to Xwee1 occurs during interphase, but not M-phase, and requires phosphorylation of Xwee1 on Ser-549. A mutant of Xwee1 (S549A) that cannot bind 14-3-3 is substantially less active than wild-type Xwee1 in its ability to phosphorylate Cdc2. This mutation also affects the intranuclear distribution of Xwee1. In cell-free kinase assays, Xchk1 phosphorylates Xwee1 on Ser-549. The results of experiments in which Xwee1, Xchk1, or both were immunodepleted from Xenopus egg extracts suggested that these two enzymes are involved in a common pathway in the DNA replication checkpoint response. Replacement of endogenous Xwee1 with recombinant Xwee1-S549A in egg extracts attenuated the cell cycle delay induced by addition of excess recombinant Xchk1. Taken together, these results suggest that Xchk1 and 14-3-3 proteins act together as positive regulators of Xwee1.

14-3-3 Proteins↗
PubMed2000

Activation of Xenopus Chk1 by mutagenesis of threonine-377.

Xenopus Chk1 (Xchk1) is required for the checkpoint-associated delay of the cell cycle in frog egg extracts containing unreplicated or UV-damaged DNA. Phosphorylation of Xchk1 at multiple sites in the SQ/TQ domain (residues 314-366) in response to unreplicated or UV-damaged DNA results in elevation of its kinase activity. We have found that mutagenesis of Thr-377 in the conserved Thr-Arg-Phe (TRF) motif of Xchk1 also leads to a substantial increase in kinase activity. Thr-377 does not appear to be a site of phosphorylation in Xchk1. These findings suggest that Thr-377 may play a role in suppressing the activity of Xchk1.

Amino Acid Sequence↗
PubMed2000

Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.

The checkpoint kinase Xchk1 becomes phosphorylated in Xenopus egg extracts in response to DNA replication blocks or UV-damaged DNA. Xchk1 is also required for the cell cycle delay that is induced by unreplicated or UV-damaged DNA. In this report, we have removed the Xenopus homolog of ATR (Xatr) from egg extracts by immunodepletion. In Xatr-depleted extracts, the checkpoint-associated phosphorylation of Xchk1 is abolished, and the cell cycle delay induced by replication blocks is strongly compromised. Xatr from egg extracts phosphorylated recombinant Xchk1 in vitro, but not a mutant form of Xchk1 (Xchk1-4AQ) containing nonphosphorylatable residues in its four conserved SQ/TQ motifs. Recombinant human ATR, but not a kinase-inactive mutant, phosphorylated the same sites in Xchk1. Furthermore, the Xchk1-4AQ mutant was found to be defective in mediating a checkpoint response in egg extracts. These findings suggest that Xchk1 is a functionally important target of Xatr during a checkpoint response to unreplicated or UV-damaged DNA.

Amino Acid Motifs↗
PubMed2000

Negative cell cycle regulation and DNA damage-inducible phosphorylation of the BRCT protein 53BP1.

In a screen designed to discover suppressors of mitotic catastrophe, we identified the Xenopus ortholog of 53BP1 (X53BP1), a BRCT protein previously identified in humans through its ability to bind the p53 tumor suppressor. X53BP1 transcripts are highly expressed in ovaries, and the protein interacts with Xp53 throughout the cell cycle in embryonic extracts. However, no interaction between X53BP1 and Xp53 can be detected in somatic cells, suggesting that the association between the two proteins may be developmentally regulated. X53BP1 is modified via phosphorylation in a DNA damage-dependent manner that correlates with the dispersal of X53BP1 into multiple foci throughout the nucleus in somatic cells. Thus, X53BP1 can be classified as a novel participant in the DNA damage response pathway. We demonstrate that X53BP1 and its human ortholog can serve as good substrates in vitro as well as in vivo for the ATM kinase. Collectively, our results reveal that 53BP1 plays an important role in the checkpoint response to DNA damage, possibly in collaboration with ATM.

Amino Acid Sequence↗
PubMed2000

Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts.

We have identified Claspin, a novel protein that binds to Xenopus Chk1 (Xchk1). Binding of Claspin to Xchk1 is highly elevated in the presence of DNA templates that trigger a checkpoint arrest of the cell cycle in Xenopus egg extracts. Xchk1 becomes phosphorylated during a checkpoint response, and we demonstrate directly that this phosphorylation results in the activation of Xchk1. Immunodepletion of Claspin from egg extracts abolishes both the phosphorylation and activation of Xchk1. Furthermore, Claspin-depleted extracts are unable to arrest the cell cycle in response to DNA replication blocks. Taken together, these findings indicate that Claspin is an essential upstream regulator of Xchk1.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Response of Xenopus Cds1 in cell-free extracts to DNA templates with double-stranded ends.

Although homologues of the yeast checkpoint kinases Cds1 and Chk1 have been identified in various systems, the respective roles of these kinases in the responses to damaged and/or unreplicated DNA in vertebrates have not been delineated precisely. Likewise, it is largely unknown how damaged DNA and unreplicated DNA trigger the pathways that contain these effector kinases. We report that Xenopus Cds1 (Xcds1) is phosphorylated and activated by the presence of some simple DNA molecules with double-stranded ends in cell-free Xenopus egg extracts. Xcds1 is not affected by aphidicolin, an agent that induces DNA replication blocks. In contrast, Xenopus Chk1 (Xchk1) responds to DNA replication blocks but not to the presence of double-stranded DNA ends. Immunodepletion of Xcds1 (and/or Xchk1) from egg extracts did not attenuate the cell cycle delay induced by double-stranded DNA ends. These results imply that the cell cycle delay triggered by double-stranded DNA ends either does not involve Xcds1 or uses a factor(s) that can act redundantly with Xcds1.

Amino Acid Sequence↗
PubMed1999

The xenopus Suc1/Cks protein promotes the phosphorylation of G(2)/M regulators.

The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF). In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1. At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself. The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown. We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex. Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts. We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B. This observation could explain why overexpression of Pin1 inhibits mitotic initiation. These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Binding of 14-3-3 proteins and nuclear export control the intracellular localization of the mitotic inducer Cdc25.

Binding of 14-3-3 proteins near the nuclear localization sequence of Xenopus Cdc25 suppresses its ability to induce entry into mitosis. We have examined the intracellular localization of green fluorescent protein (GFP)-tagged wild-type Cdc25 or a mutant (S287A) that cannot bind 14-3-3 proteins. Upon coexpression with Myc-14-3-3epsilon, GFP-Cdc25-WT was predominantly cytoplasmic, whereas GFP-Cdc25-S287A was exclusively nuclear. Leptomycin B, an inhibitor of nuclear export, elicited a prompt redistribution of GFP-Cdc25-WT to the nucleus. Mutagenesis experiments demonstrated that Cdc25 contains multiple nuclear export sequences. These studies indicate that the binding of 14-3-3 proteins and nuclear export regulate the intracellular localization of Cdc25.

14-3-3 Proteins↗
PubMed1998

The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts.

We have analyzed the role of the protein kinase Chk1 in checkpoint control by using cell-free extracts from Xenopus eggs. Recombinant Xenopus Chk1 (Xchk1) phosphorylates the mitotic inducer Cdc25 in vitro on multiple sites including Ser-287. The Xchk1-catalyzed phosphorylation of Cdc25 on Ser-287 is sufficient to confer the binding of 14-3-3 proteins. Egg extracts from which Xchk1 has been removed by immunodepletion are strongly but not totally compromised in their ability to undergo a cell cycle delay in response to the presence of unreplicated DNA. Cdc25 in Xchk1-depleted extracts remains bound to 14-3-3 due to the action of a distinct Ser-287-specific kinase in addition to Xchk1. Xchk1 is highly phosphorylated in the presence of unreplicated or damaged DNA, and this phosphorylation is abolished by caffeine, an agent which attenuates checkpoint control. The checkpoint response to unreplicated DNA in this system involves both caffeine-sensitive and caffeine-insensitive steps. Our results indicate that caffeine disrupts the checkpoint pathway containing Xchk1.

14-3-3 Proteins↗
PubMed1998

Identification of a novel 81-kDa component of the Xenopus origin recognition complex.

The Xenopus origin recognition complex is essential for chromosomal DNA replication in cell-free extracts. We have immunopurified the Xenopus origin recognition complex with anti-Xorc2 antibodies and analyzed its composition and properties. Xorc2 (p63) is specifically associated with Xorc1 (p115) and up to four additional polypeptides (p81, p78, p45, and p40). The cDNA encoding p81 is highly homologous to various expressed sequence tags from humans and mice encoding a protein of previously unknown function. Immunodepletion of p81 from Xenopus egg extracts, which also results in the removal of Xorc2, completely abolishes chromosomal DNA replication. Thus, p81 appears to play a crucial role at S phase in higher eukaryotes.

Amino Acid Sequence↗
PubMed1998

Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase- promoting complex at mitosis.

Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in mitotic cyclin destruction in Xenopus egg extracts. Specifically, if p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, we depleted p9 from extracts that had already entered M phase and thus contained an active APC. We found that ubiquitin-mediated proteolysis of cyclin B was not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase showed that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. Our results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase.

Anaphase↗
PubMed1998

14-3-3 proteins act as negative regulators of the mitotic inducer Cdc25 in Xenopus egg extracts.

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2-cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3epsilon and to a lesser extent with 14-3-3zeta. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2-M transition.

14-3-3 Proteins↗
PubMed1996

The Xenopus Cdc6 protein is essential for the initiation of a single round of DNA replication in cell-free extracts.

We have cloned a Xenopus Cdc6 homolog (Xcdc6) and characterized its role in DNA replication with Xenopus egg extracts. Immunodepletion of Xcdc6 abolishes chromosomal replication but not elongation on single-stranded DNA templates. Xcdc6 binds to chromatin at the beginning of interphase but disappears from chromatin upon initiation of replication. Immunodepletion studies indicate that binding of Xcdc6 to chromatin requires Xorc2, a component of the origin recognition complex. Moreover, Xmcm3 cannot bind to chromatin lacking Xcdc6, suggesting that Xorc2, Xcdc6, and Xmcm3 associate with the DNA sequentially. In postreplicative nuclei, Xcdc6 is associated with the nuclear envelope. These studies indicate that Xcdc6, is essential for initiation of replication in vertebrates and that interaction with the nuclear envelope may regulate its function.

Amino Acid Sequence↗
PubMed1996

Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts.

Cdc2, the cyclin-dependent kinase that controls mitosis, is negatively regulated by phosphorylation on its threonine-14 and tyrosine-15 residues. Cdc25, the phosphatase that dephosphorylates both of these residues, undergoes activation and phosphorylation by multiple kinases at mitosis. Plx1, a kinase that associates with and phosphorylates the amino-terminal domain of Cdc25, was purified extensively from Xenopus egg extracts. Cloning of its complementary DNA revealed that Plx1 is related to the Polo family of protein kinases. Recombinant Plx1 phosphorylated Cdc25 and stimulated its activity in a purified system. Cdc25 phosphorylated by Plx1 reacted strongly with MPM-2, a monoclonal antibody to mitotic phosphoproteins. These studies indicate that Plx1 may participate in control of mitotic progression.

Amino Acid Sequence↗
PubMed1996

Xe-p9, a Xenopus Suc1/Cks homolog, has multiple essential roles in cell cycle control.

The small Suc1/Cks protein is a ubiquitous subunit of Cdk/cyclin complexes, but its precise function has remained unclear. We have isolated a Xenopus homolog, Xe-p9, of the Suc1/Cks protein by virtue of its ability to rescue a fission yeast mutant that enters mitosis prematurely. To assess its functional role in cell cycle control, we have both overexpressed p9 in Xenopus egg extracts and immunodepleted the protein from these extracts. We found that addition of recombinant His6-p9 to egg extracts results in a pronounced delay of mitosis that can be attributed to an inhibition of the tyrosine dephosphorylation of the inactive Cdc2/cyclin B complex. In immunodepletion studies, we observed that the consequences of removing p9 from egg extracts depend on the stage of the cell cycle. Specifically, in the case of interphase extracts, the removal of p9 abolishes the entry into mitosis as a result of a failure in the activation of the Cdc2/cyclin B complex by tyrosine dephosphorylation. Furthermore, mitotic extracts lacking p9 fail to exit mitosis because of a defect in the destruction of cyclin B. Collectively, these results indicate that p9 has multiple essential roles in the cell cycle by governing the interaction of the Cdc2/cyclin B complex with both positive and negative regulators.

Amino Acid Sequence↗
PubMed1996

Role for a Xenopus Orc2-related protein in controlling DNA replication.

The six-subunit origin recognition complex (ORC) is essential for the initiation of DNA replication at specific origins in the budding yeast Saccharomyces cerevisiae. An important issue is whether DNA replication in higher eukaryotes, in which the characteristics of replication origins are poorly defined, occurs by an ORC-dependent mechanism. We have identified a Xenopus laevis Orc2-related protein (XORC2) by its ability to rescue a mitotic-catastrophe mutant of the fission yeast Schizosaccharomyces pombe. We show that immunodepletion of XORC2 from Xenopus egg extracts abolishes the replication of chromosomal DNA but not elongation synthesis on a single-stranded DNA template. Indirect immunofluorescence indicates that XORC2 binds to chromatin well before the commencement of DNA synthesis, and even under conditions that prevent the association of replication licensing factor(s) with the DNA. These findings suggest that Orc2 plays an important role at an early step of chromosomal replication in animal cells.

Amino Acid Sequence↗