Identification of IRS4 and TOP2A as novel CDC7 kinase interacting proteins

CDC7 is an essential serine/threonine kinase required for the initiation of DNA replication in eukaryotic cells, through phosphorylating the MCM helicase complex. Formation of an active kinase requires the binding to either of its regulatory subunits, DBF4 or DBF4B. The main aim of this project was to identify novel roles of CDC7 kinase through the identification of its interacting proteins. To aid their purification, we have generated human cell lines that conditionally express epitope-tagged CDC7, DBF4, and DBF4B, and we first describe the characterisation of these cell lines. We found that expression of DBF4 or DBF4B was able to increase MCM2 phosphorylation, but did not impair DNA replication or cell proliferation. Epitope-tagging also allowed us to determine the localisation of CDC7 kinase by immunofluorescence, and we present evidence of novel DBF4B localisation to centrosomes and microtubules in interphase cells. Using a quantitative proteomics approach, we identified Insulin Receptor Substrate 4 (IRS4) as a novel CDC7-DBF4B interacting protein, and we confirmed this interaction through immunoprecipitation experiments. IRS4 associates with receptor tyrosine kinases, where it mediates the activation of key signalling pathways, including the PI3K/AKT pathway. The CDC7 kinase inhibitor, XL413, attenuated the PI3K-dependent phosphorylation of AKT S473 in untransformed fibroblasts, but this was not reproduced by siRNA depletion of CDC7, suggesting that the effect was independent of CDC7 kinase activity. We then investigated a potential role for IRS4 in mediating the activation of CDC7 kinase, but IRS4 depletion did not affect CDC7-dependent MCM2 phosphorylation or DNA replication either. Topoisomerase 2-α (TOP2A) was identified through mass spectrometry analysis of proteins co-immunoprecipitated with DBF4. TOP2A mediates the decatenation of double stranded DNA molecules, and is therefore required for maintenance of proper chromatin structure, chromosome condensation, and accurate segregation of DNA into daughter cells. We showed that the DBF4-TOP2A interaction occurs early in S-phase, and that it is abolished by treatment with XL413, suggesting that it is dependent on CDC7 kinase activity. We have found that both DBF4 and TOP2A localise to centromeres in S-phase, and that depletion or chemical inhibition of CDC7-DBF4 affects the timing of centromeric TOP2A recruitment. We hypothesise that timely TOP2A recruitment may contribute to centromeric cohesion or replication, and this will be the subject for future investigation.2020-02-0