Post-translational modification of CUX1 during the cell cycle and regulation of DNA replication initiation by the p110 CUX1 isoform

CDP/Cux/Cut proteins are expressed as a 200 kDa protein that show transient interaction with DNA and repress transcription. In G1/S of the cell cycle p200 CUX1 is cleaved to p110 CUX1 and activates transcription of DNA replication and cell cycle progression genes. Following completion of S-phase, CUX1 is phosphorylated by cyclin A/CDK1 resulting in inhibition of DNA binding. Constitutive over-expression of p110 CUX, as seen in may tumor and cancer cell lincs, shortens the time that cells spend in G1 phase. In one part of this study we investigated the post-translational regulation of CUX1 through mitosis and the subsequent G1 phase. Using SDS-PAGE, EMSA and mutational analysis we showed that CUX1 is hyper-phosphorylated on 10 or more serine residues in mitosis by cyclin B/CDK1 resulting in complete inhibition of DNA binding. Following cell division and formation of the nuclear membrane residual CUX1 molecules are de-phosphorylated and imported back into the nucleus. In another part of this study we investigated mechanism of how p110 CUX1 shortens the G1 phase of the cell cycle. Using a cell free replication assay we demonstrated that over-expression of p110 CUX1 reduces the time needed for pre-RC complex formation thus, shortening the G1 phase. We hypothesize that the effect of p110 CUX1 is mediated through global over-expression of genes encoding both the protein subunits that form the pre-RC complex and their activating kinases.