BIOLOGICAL OVERVIEW

Cyclins and their partners, the cyclin dependent kinases (cdks), regulate progression of the cell cycle. For example Cyclin E can induce S phase in the absence of protein synthesis (Richardson, 1995). In vertebrates, up-regulation (activation) of inhibitors of cyclin dependent kinases accompanies, indeed, is integral to the switch from proliferation to differentiation. There are two families of vertebrate cdk inhibitiors: Ink and Cip/Kip. The characteristic feature of the Kip family is an N-terminal domain that mediates binding to the complex formed by cyclin dependent kinases with cyclins (Lane, 1996 and references). Dacapo, the subject of this overview, is also a member of the Cip/Kip family of cdk inhibitors.

Evidence for the existence of inhibitor regulation of cell cycle progression in Drosophila predates the cloning of dacapo. Human cdk inhibitor p21 was expressed in the Drosophila eye to discover its effect on eye differentiation. Expression of p21 in all cells posterior to the morphogenetic furrow can block the wave of S phase that immediately follows the furrow. When mitosis is blocked in mitotic eye cells by human p21, cell differentiation takes place according to each cell's predetermined fate. Nevertheless, there is a deficit of cell types that are determined last in temporal order: the pigment cells and bristle cells. This results in a rough eye phenotype. P21 expression does not alter cell fate, but it is able to block cell cycle progression. This suggests that the mechanism of inhibition of cell cycle progression by p21 is general and conserved across species, and that similar mechanisms act to promote differentiation in the fly (de Nooij, 1995).

With the cloning of dacapo (similar to the musical term, da capo, cueing the performer to repeat a passage "from the beginning"), its effects on cell cycle progression in the eye could be tested directly. In flies bearing up to two copies of a daptransgene, eyes appear to be wild-type, but in flies bearing four copies of a dap transgene, the eyes are slightly rough (de Nooij, 1996).

The effects of dap overexpression are dramatically enhanced by overexpression of the Drosophila Retinoblastoma-family protein) (Rbf) (Du, 1996). Discussion of the relation of DAP and RBF is incomplete without mentioning E2F, a transcription factor responsible for activation of S phase gene. It is unclear what the relationship is between cyclin E and dE2F in Drosophila. In Drosophila, cyclin E transcription appears to be downstream of dE2F during some phases of development (in endocycling cells of the midgut); during other phases, cyclin E is required for dE2F-dependent transcription and appears to act upstream of dE2F (in the CNS) (Duronio, 1995 and Sauer, 1995). In mammals RB and related proteins associate with E2F and repress E2F dependent transcription.

While flies carrying either two copies of dap or Rbf transgenes have wild-type eyes, the combination results in extremely rough eyes. Many pigment cells are missing consistent with a marked deficit of precursor cells, and the stripe of DNA synthesizing cells posterior to the morphogenetic furrow is completely blocked. Thus dap and Rbf exhibit significant synergy in arresting cell cycle entry in vivo. This synergy is likely to involve binding of Rbf to E2F and interference of cyclin E/cdk involvement with E2F (de Nooij, 1996).

dap expression during embryogenesis is sufficient to arrest cell proliferation. dap was expressed in seven epidermal stripes using a paired promoter. DNA synthetic phase 16 is largely inhibited in the prd-expressing regions, and subsequent mitosis 16 (early in embryogenesis) is also inhibited. Epidermal cell counts during stage 14 (late in embryogenesis), by which time epidermal cell proliferation is long over, reveals a reduction in cell density in these regions. Mitosis 15 is refractory to inhibition by DAP (Lane, 1996).

DAP is not the only protein to limit cell cycle progression in developing embryos. The G1 arrest observed after the terminal division of the epidermal cells is dependent on the inactivation of cyclin E/cdk2 activity. In addition to the up-regulation of DAP, down-regulation of cyclin E transcription appears to contribute to the timely inactivation of cyclin E/cdk2 activity (Knoblich, 1994). This cyclin E down-regulation also occurs normally in dap mutants. In addition to cyclin E, other cell cycle regulators that have been analyzed (cyclin A, cyclin B, cyclin B3, cdc2 and string) are also transcriptionally down regulated, when epidermal cells become postmitotic (Lane, 1996 and references). Thus cell cycle exit is subject to tight regulation by means of multiple regulatory events. This makes sense when considering how crucial cell quiescence is, to avoid unwanted cell proliferation.

PROTEIN STRUCTURE

Amino Acids - 245

Structural Domains

The DAP protein shares 20-27% amino acid identity with vertebrate Kip inhibitors of the p21/p27 family and a 28% identity to a C. elegans gene. The few N-terminal motifs that are identical in all these Kip family members are required for cyclin/cdk binding in the analyzed inhibitors. With the C-terminal region, DAP contains basic clusters characteristic of bipartite nuclear targeting signals that are also present in the vertebrate inhibitors. Two additional regions have been shown to be necessary for cdk inhibition (Lane, 1996 and deNooij, 1996).

date revised: 5 JAN 96 

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