Scientific Report 2006

Molecular Biology

Control of Cell Division

S.I. Reed, C. Baskerville, L.-C. Chuang, S. Ekholm-Reed, M. Henze, J. Keck, V. Liberal, K. Luo, B. Olson, S. Rudyak, D. Tedesco, F. van Drogen, J. Wohlschlegel

Biological processes of great complexity can be approached by beginning with a systematic genetic analysis in which the relevant components are first identified and the consequences of selectively eliminating the components via mutations are investigated. We have used yeast, which is uniquely tractable to this type of analysis, to investigate control of cell division. In recent years, it has become apparent that the most central cellular processes throughout the eukaryotic phylogeny are highly conserved in terms of both the regulatory mechanisms used and the proteins involved. Thus, it has been possible in many instances to generalize from yeast cells to human cells.

Control in Yeast

In recent years, we have focused on the role and regulation of the Cdc28 protein kinase (Cdk1). Initially identified by means of a mutational analysis of the yeast cell cycle, this protein kinase and its analogs are ubiquitous in eukaryotic cells and are central to a number of aspects of control of cell-cycle progression.

One current area of interest is regulation of cellular morphogenesis by Cdk1. The activity of Cdk1 driven by mitotic cyclins modulates polarized growth in yeast cells. Specifically, these activities depolarize growth by altering the actin cytoskeleton. We found that several proteins that modulate actin structure are targeted by Cdk1, and we are investigating whether these phosphorylation events control actin depolarization.

A second major area of interest is in the regulation of mitosis. A key aspect of mitotic regulation in yeast is the accumulation of Cdc20, which triggers the transition from metaphase to anaphase. Cdc20 is an essential cofactor of the protein-ubiquitin ligase known as the anaphase-promoting complex or APC/C. It is through the ubiquitin-mediated proteolysis of a specific anaphase inhibitor, securin (Pds1 in yeast), that anaphase is initiated. We found that cells are prevented from entering mitosis when DNA replication is blocked by the drug hydroxyurea, which causes the destabilization of Cdc20 and inhibition of Cdc20 translation.

While investigating mitosis, we found that a Cks1, small Cdk1-associated protein, appears to regulate the proteasome. Proteasomes are complex proteases that target ubiquitylated proteins, including important cell-cycle regulatory proteins. Surprisingly, we found that Cks1 regulates a nonproteolytic function of proteasomes, the transcriptional activation of Cdc20. Specifically, Cks1 is required to recruit proteasomes to the gene CDC20 for efficient transcriptional elongation. Our investigations of CDC20 have led to the conclusion that Cks1 is required for recruitment of proteasomes to and transcriptional elongation of many other genes as well. Currently, we are elucidating the mechanism whereby Cks1 recruits proteasomes and facilitates transcriptional elongation. Our most recent results suggest that Cks1 and proteasomes in conjunction with Cdk1 mediate remodeling of chromatin.

Control in Mammalian Cells

We showed previously that the human homologs of the Cdc28 protein kinase are so highly conserved, structurally and functionally, relative to the yeast protein kinase, that they can function and be regulated properly in a yeast cell. Analyzing control of the cell cycle in mammalian cells, we produced evidence for the existence of regulatory schemes, similar to those elucidated in yeast, that use networks of both positive and negative regulators.

A principal research focus is the positive regulator of Cdk2, cyclin E. Cyclin E is often overexpressed and/or deregulated in human cancers. Using a tissue culture model, we showed that deregulation of cyclin E confers genomic instability, probably explaining the link to carcinogenesis. The observation that deregulation of cyclin E confers genomic instability has led us to hypothesize a mechanism of cyclin E–mediated carcinogenesis based on accelerated loss of heterozygosity at tumor suppressor loci. We are testing this hypothesis in transgenic mouse models. We showed that a cyclin E transgene expressed in the mammary epithelium markedly increases loss of heterozygosity at the p53 locus, leading to enhanced mammary carcinogenesis. We are extending these investigations by using mouse prostate, testis, and skin models.

In an attempt to understand cyclin E–mediated genomic instability, we are investigating how deregulation of cyclin E affects both S phase and mitosis. Recent data suggest that deregulation of cyclin E impairs DNA replication by interfering with assembly of the prereplication complex. Cyclin E deregulation also impairs the transition from metaphase to anaphase by promoting the accumulation of inhibitors of anaphase.

Our interest in cyclin E deregulation in cancer led us to investigate the pathway for turnover of cyclin E. We showed that phosphorylation-dependent proteolysis of cyclin E depends on a protein-ubiquitin ligase known as SCF^hCdc4. The F-box protein hCdc4 is the specificity factor that targets phosphorylated cyclin E. We are investigating how ubiquitylation of cyclin E is coordinated with other processes required for its degradation, including prolyl isomerization. We are also investigating SCF^hCdc4 ubiquitylation of other important cellular proteins.

Recently, we began determining the role of SCF^hCdc4 in neurodegenerative disease. We found that parkin, a protein often mutated in hereditary Parkinson’s disease, regulates the stability of hCdc4, possibly leading to neuropathologic changes. Consistent with this idea, we found that SCF^hCdc4 targets peroxisome proliferator–activated receptor γ coactivator-1α which protects neurons from oxidative damage. In addition, we showed that SCF^hCdc4 regulates the turnover of presenilins in the brain, proteins strongly implicated in Alzheimer’s disease.

Another area of interest is the role of Cks proteins in mammals, complementing our research in yeast. Mammals express 2 orthologs of yeast Cks1, known as Cks1 and Cks2. Experiments in mice lacking the gene for Cks1 and Cks2 revealed that each ortholog has a specialized function. Cks1 is required as a cofactor for Skp2-mediated ubiquitylation and turnover of inhibitors p21, p27, and p130. Cks2 is required for the transition from metaphase to anaphase in both male and female meiosis I. Nevertheless, mice nullizygous at the individual loci are viable. However, doubly nullizygous mice have not been observed because embryos die at the morula stage, a finding consistent with an essential redundant function. We found that this function most likely is involved in regulation of transcription and is linked to chromatin remodeling, as in yeast.

Publications

Jackson, L.P., Reed, S.I., Haase, S.B. Distinct mechanisms control the stability of the related S-phase cyclins Clb5 and Clb6. Mol. Cell. Biol. 26:2456, 2006.

Reed, S.I. Skp’n with Cks1: revelations from the Skp1-Skp2-Cks1-p27 structure. Mol. Cell 20:1, 2005.

Reed, S.I. The ubiquitin-proteasome pathway in cell cycle control. Results Probl. Cell Differ. 42:147, 2006.

Smith, A.P.L., Henze, M., Lee, J.A., Osborn, K.G., Keck, J., Tedesco, D., Bortner, D.M., Rosenberg, M.P., Reed, S.I. Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland. Oncogene, in press.

Spruck, C., Sun, D., Fiegl, H., Marth C., Mueller-Holzner, E., Goebel, G., Widschwendter, M., Reed, S.I. Detection of low molecular weight derivatives of cyclin E1 is a function of cyclin E1 protein levels in breast cancer. Cancer Res. 66:7355, 2006.

van Drogen, F., Sangfelt, O., Malyukova, A., Matskova, L., Yeh, E., Means, A.R., Reed, S.I. Ubiquitylation of cyclin E requires the sequential function of SCF complexes containing distinct hCdc4 isoforms. Mol. Cell 23:37, 2006.

Wittenberg, C., Reed, S.I. Cell cycle-dependent transcription in yeast: promoters, transcription factors, and transcriptomes. Oncogene 24:2746, 2005.

Wohlschlegel, J.A., Johnson, E.S., Reed, S.I., Yates, J.R. III. Improved identification of SUMO attachment sites using C-terminal SUMO mutants and tailored protease digestion strategies. J. Proteome Res. 5:761, 2006.