ABSTRACT
Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle Proteins/antagonists & inhibitors , DNA Replication/drug effects , DNA/drug effects , Piperidones/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyrroles/pharmacology , Animals , Antineoplastic Agents/chemistry , Cell Cycle/drug effects , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Cell Death/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , DNA/biosynthesis , Dose-Response Relationship, Drug , Fibroblasts/drug effects , HeLa Cells , Humans , Mice , Mice, Nude , Mice, SCID , Minichromosome Maintenance Complex Component 2 , Molecular Structure , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/chemistry , Phosphorylation , Piperidones/chemistry , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Pyrroles/chemistry , Rats , Small Molecule Libraries , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Cdc7 kinase promotes and regulates DNA replication in eukaryotic organisms. Multiple mechanisms modulating kinase activity in response to DNA replication stress have been reported, supporting the opposing notions that Cdc7 either plays an active role under these conditions or, conversely, is a final target inactivated by a checkpoint response. We have developed new immnunological reagents to study the properties of human Cdc7 kinase in cells challenged with the ribonucleotide reductase inhibitor hydroxyurea or with the DNA topoisomerase II inhibitor etoposide. We show that Cdc7.Dbf4 and Cdc7.Drf1 complexes are stable and active in multiple cell lines upon drug treatment, with Cdc7.Dbf4 accumulating on chromatin-enriched fractions. Cdc7 depletion by small interfering RNA in hydroxyurea and etoposide impairs hyper-phosphorylation of Mcm2 at specific Cdc7-dependent phosphorylation sites and drug-induced hyper-phosphorylation of chromatin-bound Mcm4. Furthermore, sustained inhibition of Cdc7 in the presence of these drugs increases cell death supporting the notion that the Cdc7 kinase plays a role in maintaining cell viability during replication stress.
Subject(s)
Cell Cycle Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Apoptosis , Cell Cycle Proteins/physiology , Cell Survival , Chromatin/chemistry , DNA/chemistry , Etoposide/chemistry , Etoposide/pharmacology , HeLa Cells , Humans , Hydroxyurea/chemistry , Hydroxyurea/pharmacology , Minichromosome Maintenance Complex Component 2 , Nuclear Proteins/physiology , Phosphorylation , Protein Binding , RNA Interference , RNA, Small Interfering/metabolism , Ribonucleotide Reductases/antagonists & inhibitorsABSTRACT
Minichromosome maintenance 2-7 proteins play a pivotal role in replication of the genome in eukaryotic organisms. Upon entry into S-phase several subunits of the MCM hexameric complex are phosphorylated. It is thought that phosphorylation activates the intrinsic MCM DNA helicase activity, thus allowing formation of active replication forks. Cdc7, Cdk2, and ataxia telangiectasia and Rad3-related kinases regulate S-phase entry and S-phase progression and are known to phosphorylate the Mcm2 subunit. In this work, by in vitro kinase reactions and mass spectrometry analysis of the products, we have mapped phosphorylation sites in the N terminus of Mcm2 by Cdc7, Cdk2, Cdk1, and CK2. We found that Cdc7 phosphorylates Mcm2 in at least three different sites, one of which corresponds to a site also reported to be phosphorylated by ataxia telangiectasia and Rad3-related. Three serine/proline sites were identified for Cdk2 and Cdk1, and a unique site was phosphorylated by CK2. We raised specific anti-phosphopeptide antibodies and found that all the sites identified in vitro are also phosphorylated in cells. Importantly, although all the Cdc7-dependent Mcm2 phosphosites fluctuate during the cell cycle with kinetics similar to Cdc7 kinase activity and Cdc7 protein levels, phosphorylation of Mcm2 in the putative cyclin-dependent kinase (Cdk) consensus sites is constant during the cell cycle. Furthermore, our analysis indicates that the majority of the Mcm2 isoforms phosphorylated by Cdc7 are not stably associated with chromatin. This study forms the basis for understanding how MCM functions are regulated by multiple kinases within the cell cycle and in response to external perturbations.
Subject(s)
Cell Cycle Proteins/physiology , Nuclear Proteins/physiology , S-Phase Kinase-Associated Proteins/metabolism , Amino Acid Sequence , Binding Sites , Blotting, Western , CDC2 Protein Kinase/metabolism , Casein Kinase II/metabolism , Cell Cycle , Cell Cycle Proteins/metabolism , Chromatin/chemistry , Chromatography, Liquid , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinases/metabolism , DNA Helicases/chemistry , Electrophoresis, Polyacrylamide Gel , Fibroblasts/metabolism , HeLa Cells , Humans , Ions , Luciferases/metabolism , Mass Spectrometry , Microscopy, Fluorescence , Minichromosome Maintenance Complex Component 2 , Molecular Sequence Data , Nuclear Proteins/metabolism , Peptides/chemistry , Phosphorylation , Proline/chemistry , Protein Isoforms , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Serine/chemistry , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Thymidine/chemistry , Transfection , Trypsin/pharmacology , Cyclin-Dependent Kinase-Activating KinaseABSTRACT
Cdc7 is an evolutionarily conserved kinase that regulates S phase by promoting replication origin activation. Down-regulation of Cdc7 by small interfering RNA in a variety of tumor cell lines causes an abortive S phase, leading to cell death by either p53-independent apoptosis or aberrant mitosis. Unlike replication fork blockade, Cdc7-depleted tumor cells do not elicit a robust checkpoint response; thus, inhibitory signals preventing additional cell cycle progression are not generated. In normal fibroblasts, however, a p53-dependent pathway actively prevents progression through a lethal S phase in the absence of sufficient Cdc7 kinase. We show that in this experimental system, p53 is required for the lasting maintenance of this checkpoint and for cell viability. With this work we reveal and begin to characterize a novel mechanism that regulates DNA synthesis in human cells, and we suggest that inhibition of Cdc7 kinase represents a promising approach for the development of a new generation of anticancer agents.
Subject(s)
Cell Cycle Proteins/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , S Phase/physiology , Tumor Suppressor Protein p53/physiology , Apoptosis/physiology , Cell Cycle/physiology , Cell Cycle Proteins/genetics , Disease Progression , Down-Regulation , HeLa Cells , Humans , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/genetics , RNA, Small Interfering/genetics , TransfectionABSTRACT
Signaling from receptor tyrosine kinases (RTKs)* requires the sequential activation of the small GTPases Ras and Rac. Son of sevenless (Sos-1), a bifunctional guanine nucleotide exchange factor (GEF), activates Ras in vivo and displays Rac-GEF activity in vitro, when engaged in a tricomplex with Eps8 and E3b1-Abi-1, a RTK substrate and an adaptor protein, respectively. A mechanistic understanding of how Sos-1 coordinates Ras and Rac activity is, however, still missing. Here, we demonstrate that (a) Sos-1, E3b1, and Eps8 assemble into a tricomplex in vivo under physiological conditions; (b) Grb2 and E3b1 bind through their SH3 domains to the same binding site on Sos-1, thus determining the formation of either a Sos-1-Grb2 (S/G) or a Sos-1-E3b1-Eps8 (S/E/E8) complex, endowed with Ras- and Rac-specific GEF activities, respectively; (c) the Sos-1-Grb2 complex is disrupted upon RTKs activation, whereas the S/E/E8 complex is not; and (d) in keeping with the previous result, the activation of Ras by growth factors is short-lived, whereas the activation of Rac is sustained. Thus, the involvement of Sos-1 at two distinct and differentially regulated steps of the signaling cascade allows for coordinated activation of Ras and Rac and different duration of their signaling within the cell.
