Substrate selection in ubiquitination reactions is achieved by ubiquitin ligases, which simultaneously bind both the target protein and a ubiquitin conjugating enzyme. We have developed a phosphopeptide based approach to facilitate the identification of ubiquitin ligases (e.g. F-box proteins) that recognize regulatory proteins in a phosphorylation-dependent manner. Thus far, we have used this approach to identify substrates of two F-box proteins, Fbw7 and beta-TRCP. Cyclin E associates with Fbw7 through a phosphodegron near its C-terminus. Experiments in vitro and in vivo have identified critical phosphorylation sites in this degron that are required for interaction with Fbw7. Interestingly, this motif is found in a number of other unstable oncogenic proteins, including c-myc, c-jun, and SREBP. In a second series of studies, we have identified the cell cycle regulatory protein Cdc25A as a target of the beta-TRCP protein. We have found that Chk1 phosphorylates Cdc25A in response to DNA damage to generate a priming event that facilitates phosphorylation of Cdc25A on a motif that then binds to beta-TRCP. Using biochemical and genetic techniques, we demonstrate that this interaction is required for regulated proteolysis of Cdc25A.

Substrate selection in ubiquitination reactions is achieved by ubiquitin ligases, which simultaneously bind both the target protein and a ubiquitin conjugating enzyme. While recent data indicates that there are a large number of distinct ubiquitin ligases that are responsible for the destruction of hundreds or perhaps thousands of proteins, there are currently no general methods to identify proteins whose levels are controlled by this mechanism. The development of general methods to systematically identify proteins whose abundance is regulated has major implications for: (1) elucidating proteolytic components of signaling pathways such as those activated in response to oncogenes and growth inhibitory factors, (2) identifying candidate drug targets whose altered destruction leads to therapeutic benefit, and (3) identifying genes that are required for the proper destruction of particular proteins of interest. We propose to merge our expertise in gene discovery approaches in yeast and mammalian cells with our experience in the molecular biology of regulated proteolysis to devise techniques that will facilitate the systematic identification of proteins derived from breast cell cDNA libraries whose levels are regulated.

The C. elegans sex determination hierarchy specifies either one of two sexes: the hermaphrodite or the male. The tra-1 gene is the global terminal regulator of the somatic sex determination pathway. Active TRA-1 promotes female fates in the hermaphrodite by repressing the transcription of genes that direct male development. The objective of the work presented here is to understand how the activity of TRA-1 is regulated. The tra-1 gene encodes two transcripts, tra-1a and tra-1b. Males and hermaphrodites have similar levels of tra-1 transcripts, suggesting that tra-1 is regulated post transcriptionally. Using a new antibody against TRA-1 in whole mount immunostaining I observed that TRA-1 appears to be much more abundant in hermaphrodites than in males. Western blot analysis revealed similar levels of TRA-1A and TRA-1B in lysates from males and hermaphrodites. Additional TRA-1-specific species (90-110 kDa) were present in the hermaphrodite lysates but absent from male lysates. These isoforms are the most abundant form of TRA-1 protein in hermaphrodites. The hermaphrodite-specific TRA-1 isoforms appear to be phosphoisoforms of a single TRA-1 band. Here I also describe the identification of proteins that specifically interact with FEM-2 in affinity chromoatography experiments. Loss-of-function mutants and RNAi silencing of three of these genes appear to partially phenocopy fem-2 mutants. I show evidence that the hermaphrodite-specific isoform is generated by proteolytic processing of TRA-1A. Since the truncated TRA-1A isoforms are present in embryos before sexual differentiation is apparent, they are unlikely to be a result of female differentiation. Therefore I propose that these isoforms are responsible for the feminizing activity of the tra-1 gene. The truncation mapped to the region between amino acids 842 and 921. The observations that both a nonsense mutation that results in the production of a C-terminally truncated TRA-1A protein and truncated TRA-1A transgenes are feminizing, support my hypothesis that the truncated TRA-1 isoforms are responsible for the feminizing activity of the tra-1 gene. Genetic analyses indicate that the fem gene products regulate, directly or indirectly, the activity of TRA-1. Therefore, characterization of these putative FEM-2 binding proteins may identify the mechanism by which the FEMs regulate TRA-1 activity.

In spite of the fact that the process of meiosis is fundamental to inheritance, surprisingly little is understood about how it actually occurs. There has recently been a flurry of research activity in this area and this volume summarizes the advances coming from this work. All authors are recognized and respected research scientists at the forefront of research in meiosis. Of particular interest is the emphasis in this volume on meiosis in the context of gametogenesis in higher eukaryotic organisms, backed up by chapters on meiotic mechanisms in other model organisms. The focus is on modern molecular and cytological techniques and how these have elucidated fundamental mechanisms of meiosis. Authors provide easy access to the literature for those who want to pursue topics in greater depth, but reviews are comprehensive so that this book may become a standard reference. Key Features* Comprehensive reviews that, taken together, provide up-to-date coverage of a rapidly moving field* Features new and unpublished information* Integrates research in diverse organisms to present an overview of common threads in mechanisms of meiosis* Includes thoughtful consideration of areas for future investigation

In recent years, the study of the plant cell cycle has become of major interest, not only to scientists working on cell division sensu strictu , but also to scientists dealing with plant hormones, development and environmental effects on growth. The book The Plant Cell Cycle is a very timely contribution to this exploding field. Outstanding contributors reviewed, not only knowledge on the most important classes of cell cycle regulators, but also summarized the various processes in which cell cycle control plays a pivotal role. The central role of the cell cycle makes this book an absolute must for plant molecular biologists.