title: Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways.
abstract: SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spk1p during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spk1p is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S- and G2-phase cdc mutants arrest with hyperphosphorylated Spk1p, damage-induced phosphorylation of Spk1p can occur in G1 and M as well. Hydroxyurea (HU) induces phosphorylation of kinase-defective forms of Spk1p, demonstrating that this regulated phosphorylation of Spk1p occurs in trans. HU-induced phosphorylation is associated with increased catalytic activity of Spk1p. Furthermore, overexpression of wild-type SPK1, but not checkpoint-defective alleles, delays progression through the G1/S boundary. Damage-dependent phosphorylation of Spk1p requires both MEC1 and MEC3, whereas MEC1 but not MEC3, is required for replication block-induced phosphorylation. These data support the model that Spk1p is an essential intermediate component in a signal transduction pathway coupling damage and checkpoint functions to cell cycle arrest. This regulation is mediated through a protein kinase cascade that potentially includes Mec1p and Tel1p as the upstream kinases.
authors.affiliation: Department of Pathology, Yale University of Medicine, New Haven Connecticut 06520-8023, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Sun
authors.forename: D S
authors.initials: DS
authors.lastname: Fay
authors.forename: F
authors.initials: F
authors.lastname: Marini
authors.forename: M
authors.initials: M
authors.lastname: Foiani
authors.forename: D F
authors.initials: DF
authors.lastname: Stern
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002455:Cell Division; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D004261:DNA Replication; D004271:DNA, Fungal / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D006918:Hydroxyurea / Q000494:pharmacology; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D016296:Mutagenesis / Q000235:genetics; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013696:Temperature
chemical_list: D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; D008741:Methyl Methanesulfonate; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D000469:Alkaline Phosphatase; D006918:Hydroxyurea