The G1 phase of the cell cycle represents a critical stage where cells can respond to extracellular cues either to commit to another round of cell division, to withdraw temporarily from the cell cycle, or to terminally differentiate. Studies from both yeast and mammalian systems suggest that progression through G1 is regulated in response to extra- and intracellular signals that act directly on the cell cycle machinery. I am interested in exploring the mechanisms involved in regulating G1 progression in vivo,and the requirement for G1 in the developmental decision to proliferate or to differentiate. To this end, I have been studying a gene, roughex (rux),which is required to arrest cells in G1 in the developing compound eye of Drosophila. A striking feature of development in the Drosophila eye is the simultaneous synchronization of cell cycle progression in G1 and the onset of pattern formation mediated by the Hedgehog/Patched (Ptc) signaling cascade. A genetic screen for loci that interact with ruxin a dosage-sensitive fashion identified both Ptc and the known G2 cell cycle molecule Cyclin A (CycA) as a dominant suppresser of the ruxmutant phenotype, suggesting that Rux functions to inhibit CycA expression and/or activity. The CycA gene was previously identified as a Hepatitis B viral insertion site in a hepatocellular carcinoma, and recent findings show that CycA expression highly correlates with tumor progression and severity in a variety of cancers. Therefore, understanding the regulation of CycA activity has important implications for oncogenesis. We have shown that the Rux protein physically interacts with CycA and can inhibit CycA-dependent kinase activity in vitro. Overexpression of Rux can drive all of the cellular CycA into the nucleus, where it is then degraded, possibly via the anaphase-promoting complex or cyclosome (APC/C). In addition, Rux itself is destabilized in S phase cells that express the G1 cyclin, Cyclin E (CycE). Rux degradation is dependent on the presence of four consensus sites for phosphorylation by cyclin-dependent kinases. We have demonstrated that Rux can bind CycE and is phosphorylated by CycE/Cdk complexes in vitro. We have also identified a novel eye-specific suppresser of rux, S(rux)2B,which may play a role in regulating S phase entry. These data support a model whereby Rux-mediated inhibition of CycA in the nucleus is relieved by CycE as cells re-enter S phase, releasing CycA for its S/G2 role. Current efforts in the lab are directed towards three overlapping and complementary areas. First, we will characterize the mechanism by which Rux protein is degraded in S phase cells. We will pursue this directly, through an analysis of the interaction between CycE and Rux in vitro and in a two-hybrid assay in mammalian cells, and through the development of a tissue culture cell system to study CycE-dependent proteolysis of Rux protein. We will also characterize an eye-specific suppresser of the rux mutant phenotype, S(rux)2B,which may play a role in regulating CycE expression and/or activity. Second, we will characterize a mutation, shtd,in the largest subunit of the APC/C, APC1. We will use the shtdmutation to screen for novel genes that interact with the APC/C. In this manner, we hope to identify genes involved in regulating the APC/C during development, as well as targets for APC/C activity. Third, we will characterize the pathway whereby Hh and Ptc regulate Rux activity. The high degree of conservation of cell cycle components between species makes it likely that many of the pathways for cell cycle regulation during development will be conserved during evolution.
| Avedisov, S N; Krasnoselskaya, I; Mortin, M et al. (2000) Roughex mediates G(1) arrest through a physical association with cyclin A. Mol Cell Biol 20:8220-9 |
