Inhibition of Cdc7/Dbf4 kinase activity affects specific phosphorylation sites on MCM2 in cancer cells.

Novartis Institute of Biomedical Research, Oncology, Emeryville, California 94608, USA.
Journal of Cellular Biochemistry (Impact Factor: 3.37). 07/2008; 104(3):1075-86. DOI: 10.1002/jcb.21698
Source: PubMed

ABSTRACT The Cdc7/Dbf4 kinase is required for initiation of DNA replication and also plays a role in checkpoint function in response to replication stress. Exactly how Cdc7/Dbf4 mediates those activities remains to be elucidated. Cdc7/Dbf4 physically interacts with and phosphorylates the minichromosome maintenance complex (MCM), such as MCM2, MCM4 and MCM6. Cdc7/Dbf4 activity is required for association of Cdc45 followed by recruitment of DNA polymerase on the chromatin. Using high resolution mass spectrometry, we identified six phosphorylation sites on MCM2, two of them have not been described before. We provide evidence that Cdc7/Dbf4 mediates phosphorylation on serine 108 and serine 40 on human MCM2 in vitro and in vivo in cancer cells in the absence of DNA damage. Antibodies specific to pS108 or pS40 confirmed the sites and established useful read-outs for inhibition of Cdc7/Dbf4. This report demonstrates the utility of an in vitro to in vivo workflow utilizing immunoprecipitation and mass spectrometry to map phosphorylation sites on endogenous kinase substrates. The approach can be readily generalized to identify target modulation read-outs for other potential kinase cancer targets.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The pre-sensor 1 (PS1) hairpin is found in ring-shaped helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding of archaeal mini-chromosome maintenance (MCM) and superfamily 3 viral replicative helicases. To determine whether the PS1 hairpin is required for the function of the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the putative PS1 hairpin motif in each of the Saccharomyces cerevisiae Mcm2-7 subunits to alanine. Interestingly, only the PS1 hairpin of Mcm3 was essential for viability. While mutation of the PS1 hairpin in the remaining MCM subunits resulted in minimal phenotypes, with the exception of Mcm7 which showed slow growth under all conditions examined, the viable alleles were synthetic lethal with each other. Reconstituted Mcm2-7 containing Mcm3 with the PS1 mutation (Mcm3K499A) had severely decreased helicase activity. The lack of helicase activity provides a probable explanation for the inviability of the mcm3 K499A strain. The ATPase activity of Mcm2-73K499A was similar to the wild type complex, but its interaction with single-stranded DNA in an electrophoretic mobility shift assay and its associations in cells were subtly altered. Together, these findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions, most evident by the essential nature of the Mcm3 PS1 hairpin in DNA unwinding.
    PLoS ONE 12/2013; 8(12):e82177. · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Many patients with brain cancer experience seizures or epilepsy and tumor-associated epilepsy (TAE) significantly decreases their quality of life. This study aimed to achieve a better understanding of the mechanisms of TAE. The differentially expressed genes (DEG) between epilepsy patients with or without brain tumor were firstly screened using the Linear Models for Microarray Data package using GSE4290 datasets from the USA National Center for Biotechnology Information Gene Expression Omnibus database. Then the protein-protein interaction (PPI) network, using data from the Human Protein Reference Database and the Biological General Repository for Interaction Datasets, was constructed. For further analysis, the PPI network structure and clusters in this PPI network were identified by ClusterOne. Meanwhile, gene set enrichment analysis was performed to illuminate the biological pathways and processes which generally affect patients with TAE. A total of 5113 DEG were identified and a PPI network, which contained 114 DEG and 21 normal genes, was established. Proteins, which mainly belonged to the mini chromosome maintenance and collagen families, were discovered to be enriched in the three identified clusters in the PPI network. Finally, several biological pathways (including cell cycle, DNA replication and transforming growth factor β1 signaling pathways) and processes (such as nucleocytoplasmic transport, nuclear transport and regulation of phosphorylation) were identified. Proteins in these three clusters may become new targets for TAE treatment. Our results provide some potential underlying biomarkers for understanding the pathogenesis of epilepsy in patients with brain tumor.
    Journal of Clinical Neuroscience 11/2013; · 1.32 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A series of pyrrolopyridinone derivatives as specific inhibitors towards the cell division cycle 7 (Cdc7) was taken into account, and the efficacy of these compounds was analyzed by QSAR and docking approaches to gain deeper insights into the interaction mechanism and ligands selectivity for Cdc7. By regression analysis the prediction models based on Grid score and Zou-GB/SA score were found, respectively with good quality of fits (r(2)=0.748, 0.951; rcv(2)=0.712, 0.839). The accuracy of the models was validated by test set and the deviation of the predicted values in validation set using Zou-GB/SA score was smaller than that using Grid score, suggesting that the model based on Zou-GB/SA score providesa moreeffective method for predicting potencies of Cdc7 inhibitors.
    European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences 08/2013; · 2.61 Impact Factor