Previous liver regeneration studies demonstrated that the mouse forkhead box M1B (FoxM1B) transcription factor regulates hepatocyte proliferation through expression of cell cycle genes that stimulate cyclin-dependent kinase 2 (Cdk2) and Cdk1 activity. In this study, we demonstrated that disruption of the FoxM1B Cdk1/2 phosphorylation site at Thr residue 596 significantly reduced both FoxM1B transcriptional activity and Cdk phosphorylation of the FoxM1B T596A mutant protein in vivo. Retention of this FoxM1B 596 Cdk phosphorylation site was found to be essential for recruiting the histone acetyltransferase CREB binding protein (CBP) to the FoxM1B transcriptional activation domain. Consistent with these findings, dominant negative Cdk1 protein significantly reduced FoxM1B transcriptional activity and inhibited FoxM1B recruitment of the CBP coactivator protein. Likewise, Cdc25B-mediated stimulation of Cdk activity together with elevated levels of the CBP coactivator protein provided a 6.2-fold synergistic increase in FoxM1B transcriptional activity. Furthermore, mutation of the FoxM1B Leu 641 residue within an LXL motif (residues 639 to 641) inhibited recruitment of Cdk-cyclin complexes and caused significant reduction in both FoxM1B transcriptional activity and in vivo Cdk phosphorylation of the FoxM1B Thr 596 residue. We demonstrated that FoxM1B transcriptional activity requires binding of either S-phase or M-phase Cdk-cyclin complexes to mediate efficient Cdk phosphorylation of the FoxM1B Thr 596 residue, which is essential for recruitment of p300/CBP coactivator proteins.Cellular proliferation involves stimulation of the mitogenactivated protein kinase (MAPK) pathway, consisting of the Ras/Raf1/MEK/MAPK cascade (19,47), and activation of the phosphoinositol 3-kinase (PI3K) pathway, consisting of the PI3K/phosphoinositide-dependent kinase 1 (PDK1)/Akt cascade (5). However, cell division is tightly regulated at the G 1 /S (DNA replication) and G 2 /M (mitosis) transitions of the cell cycle by temporal activation of multiple cyclin-dependent kinases (Cdks) complexed with their corresponding cyclin regulatory subunits. Cdk2-cyclin E or A and Cdk1-cyclin B kinase activity is essential for progression through the G 1 /S and G 2 /M transitions of the cell cycle, respectively. Furthermore, Cdk activity is negatively regulated by phosphorylation of Thr 14 and Tyr 15 by the dual specific Myt1 kinase (6, 30, 31, 37). Both MAPK and PDK1 phosphorylate and activate the downstream p90 ribosomal S6 kinase (Rsk) (4, 21, 51), which provides inhibitory phosphorylation to the dual Myt1 kinase (44, 45). Simulation of Cdk1 or Cdk2 activity involves removal of inhibitory phosphates at Thr 14 and Tyr 15 by either Cdc25A phosphatase (G 1 /S phase), Cdc25B (late S phase), or Cdc25C phosphatase (G 2 /M phase) (7,41,62). In addition, Cdk activity requires phosphorylation of Thr 160 by the Cdk-activating kinase (CAK) protein, which is also a direct target for MAPK phosphorylation (14,28,55).Cdk1 and Cdk2 proteins are proline-directed kinase...

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SUMMARY BackgroundSerum alpha fetoprotein (AFP), ultrasound, computerized tomography scanning, and magnetic resonance imaging are commonly used to screen for hepatocellular carcinoma (HCC) in patients with cirrhosis.

The Forkhead Box (Fox) proteins are an extensive family of transcription factors that shares homology in the winged helix DNA binding domain. Liver regeneration studies with the -3 kb transthyretin (TTR) promoter-driven FoxM1B transgenic (TG) mice demonstrated that premature hepatocyte nuclear localization of the FoxM1B transgene protein at 16 h following partial hepatectomy (PHx) caused an 8-h acceleration in the onset of hepatocyte DNA replication (S-phase) and mitosis by stimulating earlier expression of cell cycle genes. Whether the FoxM1B transgene protein participates in immediate early events during liver regeneration remains to be determined. Here, we found that the FoxM1B transgene protein translocated to hepatocyte nuclei immediately following PHx, that its nuclear staining persisted for the first 6 h after surgery, and that this translocation was associated with an increase in size of regenerating TG hepatocytes. However, regenerating TTR-FoxM1B liver did not exhibit altered expression of proteins that have been implicated in mediating increased cell size, including serum-and-gucocorticoid-inducible protein kinase (SGK), protein kinase-B/Akt, the tumor suppresser gene PTEN (negative regulator of the PI3K/Akt pathway), c-Myc, or peroxisome proliferation. Moreover, we demonstrated that hepatocyte nuclear translocation of the FoxM1B transgene protein was rapidly induced during the hepatic acute phase response, which occurs during the immediate early stages of liver regeneration. Analysis of cDNA expression arrays identified a number of genes such as immediate early transcription factors (ID-3, Stat3, Nur77), matrix metalloproteinase-9 (MMP-9), and several glutathione S-transferase (GST) isoforms and stress response genes, whose expression is elevated in regenerating TTR-FoxM1B TG livers compared with regenerating wild-type (WT) liver. These liver regeneration studies demonstrate that hepatocyte nuclear translocation of the FoxM1B transgene protein was sustained for the first 6 h after PHx, and was associated with transient hypertrophy of regenerating TG hepatocytes and increased expression of genes that may enhance hepatocyte proliferation.