A signature feature of all living organisms is their utilization of proteins to construct molecular machineries that undertake the complex network of cellular activities. The abundance of a protein element is temporally and spatially regulated in two opposing aspects: de novo synthesis to manufacture the required amount of the protein, and destruction of the protein when it is in excess or no longer needed. One major route of protein destruction is coordinated by a set of conserved molecules, the F-box proteins, which promote ubiquitination in the ubiquitin-proteasome pathway. Here we discuss the functions of F-box proteins in several cellular scenarios including cell cycle progression, synapse formation, plant hormone responses, and the circadian clock. We particularly emphasize the mechanisms whereby F-box proteins recruit specific substrates and regulate their abundance in the context of SCF E3 ligases. For some exceptions, we also review how F-box proteins function through non-SCF mechanisms.

In the yeast Saccharomyces cerevisiae, Ste12p induces transcription of pheromone-responsive genes by binding to a DNA sequence designated the pheromone response element. We generated a series of hybrid proteins of Ste12p with the DNA-binding and activation domains of the transcriptional activator Gal4p to define a pheromone induction domain of Ste12p sufficient to mediate pheromone-induced transcription by these hybrid proteins. A minimal pheromone induction domain, delineated as residues 301 to 335 of Ste12p, is dependent on the pheromone mitogen-activated protein (MAP) kinase pathway for induction activity. Mutation of the three serine and threonine residues within the minimal pheromone induction domain did not affect transcriptional induction, indicating that the activity of this domain is not directly regulated by MAP kinase phosphorylation. By contrast, mutation of the two tyrosines or their preceding acidic residues led to a high level of transcriptional activity in the absence of pheromone and consequently to the loss of pheromone induction. This constitutively high activity was not affected by mutations in the MAP kinase cascade, suggesting that the function of the pheromone induction domain is normally repressed in the absence of pheromone. By two-hybrid analysis, this minimal domain interacts with two negative regulators, Dig1p and Dig2p (also designated Rst1p and Rst2p), and the interaction is abolished by mutation of the tyrosines. The pheromone induction domain itself has weak and inducible transcriptional activity, and its ability to potentiate transcription depends on the activity of an adjacent activation domain. These results suggest that the pheromone induction domain of Ste12p mediates transcriptional induction via a two-step process: the relief of repression and synergistic transcriptional activation with another activation domain.In the yeast Saccharomyces cerevisiae, mating between haploid a and ␣ cells is initiated by binding of the pheromone secreted by cells of the opposite mating type. This binding activates a signal transduction process whose components include a heterotrimeric G-protein, a cascade of protein kinases, and the transcriptional activator Ste12p (for reviews, see references 1, 26, and 44). Pheromone stimulation results, among other changes, in an increase in cell-type-specific gene expression mediated by Ste12p. The protein kinases acting upstream of Ste12p include Ste20p (28, 38) and four others, Ste11p, Ste7p, Fus3p, and Kss1p, which comprise a highly conserved regulatory module found in other organisms as well (4,19,25,35,51; for reviews, see references 18 and 23). Fus3p and Kss1p are homologs of mitogen-activated protein (MAP) kinases and function redundantly in promoting mating (15). Ste7p is related to the dual-specificity MAP kinase kinase (MAPKK or MEK) that activates MAP kinases (25, 42), and Ste11p is related to the MAPKK regulator (MAPKKK or MEKK) (27). Each of these four protein kinases can bind to Ste5p, which may act as a scaffold to ensure the specificity of...

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age-dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC–male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging.

Proneural basic helix-loop-helix (bHLH) proteins initiate neurogenesis in both vertebrates and invertebrates. The Drosophila Achaete (Ac) and Scute (Sc) proteins are among the first identified members of the large bHLH proneural protein family. phyllopod (phyl), encoding an ubiquitin ligase adaptor, is required for ac-and sc-dependent external sensory (ES) organ development. Expression of phyl is directly activated by Ac and Sc. Forced expression of phyl rescues ES organ formation in ac and sc double mutants. phyl and senseless, encoding a Zn-finger transcriptional factor, depend on each other in ES organ development. Our results provide the first example that bHLH proneural proteins promote neurogenesis through regulation of protein degradation.E3 ligase ͉ senseless ͉ basic helix-loop-helix ͉ neurogenesis T he basic helix-loop-helix (bHLH) proneural proteins promote neurogenesis from flies to mammals (for reviews, see refs. 1 and 2). In Drosophila, the proneural proteins Achaete (Ac), Scute (Sc), Atonal (Ato), and Amos are bHLH transcriptional factors that are essential for the generation of neural precursors in the central and peripheral nervous systems (3-5). In mammals, the bHLH proteins Mash1, homolog of Ac and Sc, and Neurogenins, homologs of Ato and Amos, are essential for the initiation of neurogenesis (6, 7). Proneural genes are expressed in small clusters of cells, called proneural clusters, and they endow cells the potential to adopt neural fate, such as sensory organ precursors (SOPs) in the Drosophila peripheral nervous system. However, lateral inhibition mediated by the ligand Delta and the receptor Notch restricts the expression of proneural genes to only one or a few cells that differentiate into neural precursors, and prevents neighboring cells of the selected neural precursors from adapting the same fate (8).The Drosophila proneural genes ac and sc function redundantly in the formation of external sensory (ES) organs; in ac and sc double mutants, formation of ES organs is disrupted, and misexpression of either ac or sc induces ectopic ES organs (9-12). The Ac and Sc proteins share 70% identity in their bHLH domains (3), and form heterodimers with the ubiquitously expressed bHLH protein Daughterless (Da) to activate transcription of downstream target genes (13,14). One target gene of Ac and Sc, asense (ase), also encodes a bHLH protein that is specifically expressed in SOPs and involved in SOP differentiation (15-17). Likewise, NeuroD, the mammalian homolog of Ase, also plays an important role in neuronal differentiation (18). In addition to the bHLH genes, a number of Ac and Sc target genes have been identified. For example, senseless (sens) is expressed in SOPs and is required to maintain high levels of proneural proteins in SOPs (19,20). Genes involved in lateral inhibition to select SOPs are also targets for Ac and Sc, including scabrous (sca), Delta (Dl), and those in the Enhancer of split [E(spl)] and Bearded (Brd) complexes (21,22). However, target genes essential for SOP differentiation ...