Dephosphorylated C/EBPα Accelerates Cell Proliferation through Sequestering Retinoblastoma Protein

Partial hepatectomy (PH) in animals is a classical model for the investigations of biochemical pathways that control liver proliferation after surgeries (4). A commonly useful model of liver regeneration includes a removal of 70% of liver. The remaining liver proliferates and restores its original size (4, 6, 10, 12). The initiation of cell cycle in regenerating livers has been intensively investigated, and the major pathways of the initiation have been elucidated (4, 6). In addition to this step, liver proliferation requires removing a negative control, which is supported by certain liver-specific proteins and by retinoblastoma protein (Rb) family proteins (pocket proteins). These pocket proteins display their growth-inhibitory activities via interaction with E2F transcription factors and through the formation of complexes that occupy promoters and repress expression of S-phase and mitotis-specific genes (3). Quiescence in the liver is also supported by growth-inhibitory activity of a transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPα). C/EBPα is a strong inhibitor of proliferation of cultured cells, and it is also required for the inhibition of liver growth. Ablation of C/EBPα in animals leads to an increased rate of proliferation in the liver and in cultured hepatocytes derived from C/EBPα−/− livers (5, 21, 23, 24). Although C/EBPα is a transcription factor, C/EBPα displays its inhibitory activity through interactions with cell cycle proteins such as cyclin-dependent kinase 2 (cdk2), cdk4, Rb, E2F, and Brm (9, 13, 15, 24-27). A number of recent papers revealed that the ability of C/EBPα to cause growth arrest is regulated on several levels. In addition to transcriptional regulation of C/EBPα mRNA and regulation of protein stability (20), certain cellular transduction pathways are able to activate or inhibit activities of C/EBPα without changing the protein levels of C/EBPα. Ross et al. have recently found that the extracellular signal-regulated kinases 1 and 2 inhibit C/EBPα-mediated differentiation of granulocytes through phosphorylation of C/EBPα at Ser21 (17). The authors demonstrated that this pathway regulates C/EBPα activities in a tissue-specific manner; it operates only in myeloid cells but does not affect C/EBPα activities in other cells such as adipocytes (17). We have recently found a pathway that blocks growth-inhibitory activity of C/EBPα in hepatoma cells and in liver tumors. The activation of phosphatidylinositol 3-kinase (PI3K)/Akt in liver tumors leads to accumulation of protein phosphatase 2A (PP2A) in nuclei where PP2A dephosphorylates C/EBPα on Ser193 and blocks its growth-inhibitory activity (25). This PI3K/Akt-mediated block of C/EBPα inhibition leads to the lack of negative control of proliferation in liver and to development of tumors (25). These examples clearly demonstrate that certain signal transduction pathways regulate C/EBPα activities on the level of posttranslational modifications. In this paper, we identified a new function of C/EBPα: acceleration of proliferation, which is opposite to that previously described for this protein. We found that the choice of a biological function of C/EBPα is achieved by phosphorylation-dephosphorylation of a single Ser193 residue within the C/EBPα growth-inhibitory region. Phosphorylated C/EBPα binds to cdk2 and Brm and inhibits proliferation, while Ser193-dephosphorylated C/EBPα accelerates proliferation via sequestering Rb. These findings show that phosphorylation-dependent switch of biological activities of C/EBPα not only eliminates C/EBPα-mediated negative control of proliferation but also neutralizes growth-inhibitory activity of Rb through sequestering Rb from E2F-Rb complex repressors. This phosphorylation-dependent neutralization of two negative regulators of liver proliferation, C/EBPα and Rb, contributes to liver proliferation after surgical resections and to development of liver tumors.