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1.
Proc Natl Acad Sci U S A ; 97(6): 2579-84, 2000 Mar 14.
Article in English | MEDLINE | ID: mdl-10716991

ABSTRACT

Two paradigms exist for maintaining order during cell-cycle progression: intrinsic controls, where passage through one part of the cell cycle directly affects the ability to execute another, and checkpoint controls, where external pathways impose order in response to aberrant structures. By studying the mitotic inhibitor Mik1, we have identified evidence for an intrinsic link between unperturbed S phase and mitosis. We propose a model in which S/M linkage can be generated by the production and stabilization of Mik1 protein during S phase. The production of Mik1 during unperturbed S phase is independent of the Rad3- and Cds1-dependent checkpoint controls. In response to perturbed S phase, Rad3-Cds1 checkpoint controls are required to maintain high levels of Mik1, probably indirectly by extending the S phase period, where Mik1 is stable. In addition, we find that Mik1 protein can be moderately induced in response to irradiation of G(2) cells in a Chk1-dependent manner.


Subject(s)
Mitosis , Protein-Tyrosine Kinases/metabolism , S Phase , Schizosaccharomyces pombe Proteins , Checkpoint Kinase 1 , Enzyme Inhibitors/pharmacology , Epitopes/metabolism , Flow Cytometry , G2 Phase , Hydroxyurea/pharmacology , Immunoblotting , Microscopy, Fluorescence , Models, Biological , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation , Protein Kinases/metabolism , Schizosaccharomyces/enzymology , Time Factors
3.
EMBO J ; 17(24): 7239-49, 1998 Dec 15.
Article in English | MEDLINE | ID: mdl-9857181

ABSTRACT

UNLABELLED: Eukaryotic cells respond to DNA damage and S phase replication blocks by arresting cell-cycle progression through the DNA structure checkpoint pathways. In Schizosaccharomyces pombe, the Chk1 kinase is essential for mitotic arrest and is phosphorylated after DNA damage. During S phase, the Cds1 kinase is activated in response to DNA damage and DNA replication blocks. The response of both Chk1 and Cds1 requires the six 'checkpoint Rad' proteins (Rad1, Rad3, Rad9, Rad17, Rad26 and Hus1). We demonstrate that DNA damage-dependent phosphorylation of Chk1 is also cell-cycle specific, occurring primarily in late S phase and G2, but not during M/G1 or early S phase. We have also isolated and characterized a temperature-sensitive allele of rad3. Rad3 functions differently depending on which checkpoint pathway is activated. Following DNA damage, rad3 is required to initiate but not maintain the Chk1 response. When DNA replication is inhibited, rad3 is required for both initiation and maintenance of the Cds1 response. We have identified a strong genetic interaction between rad3 and cds1, and biochemical evidence shows a physical interaction is possible between Rad3 and Cds1, and between Rad3 and Chk1 in vitro. Together, our results highlight the cell-cycle specificity of the DNA structure-dependent checkpoint response and identify distinct roles for Rad3 in the different checkpoint responses. KEYWORDS: ATM/ATR/cell-cycle checkpoints/Chk1/Rad3


Subject(s)
Adenosine Triphosphatases/metabolism , DNA Helicases/metabolism , Interphase/physiology , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Adenosine Triphosphatases/genetics , Amino Acid Sequence , Checkpoint Kinase 1 , Checkpoint Kinase 2 , DNA Damage , DNA Helicases/genetics , DNA Replication , G2 Phase/physiology , Gene Dosage , Hydroxyurea/pharmacology , Molecular Sequence Data , Mutation , Phosphorylation , Protein Binding , Radiation Tolerance , S Phase/physiology , Saccharomyces cerevisiae Proteins , Schizosaccharomyces pombe Proteins , Selection, Genetic , Suppression, Genetic , Ultraviolet Rays
4.
Mol Biol Cell ; 7(11): 1771-88, 1996 Nov.
Article in English | MEDLINE | ID: mdl-8930899

ABSTRACT

Cell surface heparan sulfate proteoglycans (HSPGs) participate in molecular events that regulate cell adhesion, migration, and proliferation. The present study demonstrates that soluble heparin-binding proteins or cross-linking antibodies induce the aggregation of cell surface HSPGs and their distribution along underlying actin filaments. Immunofluorescence and confocal microscopy and immunogold and electron microscopy indicate that, in the absence of ligands, HSPGs are irregularly distributed on the fibroblast cell surface, without any apparent codistribution with the actin cytoskeleton. In the presence of ligand (lipoprotein lipase) or antibodies against heparan sulfate, HSPGs aggregate and colocalize with the actin cytoskeleton. Triton X-100 extraction and immunoelectron microscopy have demonstrated that in this condition HSPGs were clustered and associated with the actin filaments. Crosslinking experiments that use biotinylated lipoprotein lipase have revealed three major proteoglycans as binding sites at the fibroblast cell surface. These cross-linked proteoglycans appeared in the Triton X-100 insoluble fraction. Platinum/carbon replicas of the fibroblast surface incubated either with lipoprotein lipase or antiheparan sulfate showed large aggregates of HSPGs regularly distributed along cytoplasmic fibers. Quantification of the spacing between HSPGs by confocal microscopy confirmed that the nonrandom distribution of HSPG aggregates along the actin cytoskeleton was induced by ligand binding. When cells were incubated either with lipoprotein lipase or antibodies against heparan sulfate, the distance between immunofluorescence spots was uniform. In contrast, the spacing between HSPGs on fixed cells not incubated with ligand was more variable. This highly organized spatial relationship between actin and proteoglycans suggests that cortical actin filaments could organize the molecular machinery involved in signal transduction and molecular movements on the cell surface that are triggered by heparin-binding proteins.


Subject(s)
Actin Cytoskeleton/metabolism , Cell Membrane/metabolism , Heparitin Sulfate/metabolism , Proteoglycans/metabolism , Binding Sites , Cells, Cultured , Heparan Sulfate Proteoglycans , Heparitin Sulfate/immunology , Humans , Ligands , Lipoprotein Lipase/metabolism , Membrane Proteins/metabolism , Microscopy, Confocal , Microscopy, Electron , Octoxynol , Solubility
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