A PP2A-dependent gateway to quiescence operating in G2/M

Quiescence is one of three stable non-proliferative cell fates, along with senescence and terminal differentiation. Unlike the other two, it is fully reversible and characterized by a unique gene expression pattern not seen during other types of cell cycle arrest. The main molecular players involved in establishing and maintaining quiescence are the pocket proteins, retinoblastoma protein and its two close relatives p107 and p130. Whereas it has long been assumed that these regulators operate during the G1 phase of the cell cycle, exciting unpublished data described in this proposal find that an essential decision point for the establishment of quiescence seems to operate in the G2 phase of the cell cycle involving the action of a major cellular serin-threonine phosphatase, PP2A. Inhibition of PP2A function specifically in G2 led to release of pocket proteins from chromatin and inability of cells to enter quiescence in the subsequent G1 phase. The specific PP2A complex responsible for this action was shown to contain the B56 subunit, which was previously demonstrated to contribute to transformation of mammalian cells, if functionally inhibited. The exact molecular mechanism behind the G2-centered PP2A- dependent quiescence decision point, however, remains unclear. This project proposal aims to unravel the molecular players involved in the G2 quiescence decision process and clarify their functional interaction with PP2A. First, the recently described p130/DREAM complex will be tested for its involvement in the G2-centered quiescence pathway. This complex comprising the pocket protein p130, E2F4 and mammalian homologues of the C. elegans synMUVB complex has previously been suggested to operate in quiescence maintenance. Specifically, I will ask whether PP2A can influence p130/DREAM complex assembly or disassembly, and whether it does so by dephosphorylating certain DREAM subunits during G2. Finally, I will ask whether a failure to properly assemble the complex translates into the inability to establish quiescence upon growth factor deprivation. In parallel, to gain insight into the nature of proteins that participate in G2-mediated quiescence and, therefore, into the mechanisms that underlie this process I will initiate a screen employing a bar-coded lentiviral shRNA library and a recently described system of fluorescently tagged proteins, which allows specific labeling of S/G2 cells versus G1/G0 cells. More specifically, the screen will be performed in an effort to identify proteins essential for the establishment of quiescence and to further elucidate their particular role in quiescence establishment and/or maintenance. In addition, their potential involvement in the PP2A-dependent G2 quiescence decision process will be assessed.