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Scientific Report 2005
Molecular Biology
Control of Cell Division
S.I. Reed, C. Baskerville, L.-C. Chuang, B. Grünenfelder, M. Henze, J. Keck, V. Liberal, K. Luo, B. Olson,
S. Ekholm-Reed, S. Rudyak, O. Sangfelt, A. Smith, C. Spruck, D. Tedesco, F. van Drogen, J. Wohlschlegel, V. Yu
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 use 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 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 Cks1, a 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 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 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 previously that a cyclin E
transgene expressed in 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 mitotic checkpoint proteins.
Our interest
in cyclin E deregulation in cancer led us to examine the pathway for turnover of
cyclin E. We showed that phosphorylation-dependent proteolysis of cyclin E depends
on a protein-ubiquitin ligase known as SCFhCdc4. 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. We are also investigating SCFhCdc4 ubiquitylation of
other important cellular proteins.
Because of
the functional relationship between hCdc4 and cyclin E, we are studying the role
of mutations of hCDC4, the gene that encodes
hCdc4, in carcinogenesis. We found that hCDC4 is mutated and most likely
is a tumor suppressor in endometrial cancer and breast cancer. In endometrial cancer,
tumors with mutations in hCDC4 are more aggressive than tumors without mutations
in this gene. Because we showed that loss of hCdc4 leads to deregulation of cyclin
E through the cell cycle, these results confirm the observation that in some cancers
deregulation of cyclin E is associated with aggressive disease and poor outcome.
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 transcriptional
elongation and is linked to chromatin remodeling, as in yeast.
Publications
Huisman, S.M., Bales, O.A.M., Bertrand, M., Smeets, M.F.M.A., Reed, S.I., Segal, M. Differential contribution of Bud6p and Kar9p in microtubule capture and spindle
orientation in S. cerevisiae. J. Cell Biol. 167:231, 2004.
Reed, S.I. Cell cycle. In: Cancer: Principles and Practice of Oncology, 7th ed. DeVita V.T., Jr., Hellman,
S., Rosenberg, S.A. (Eds.). Lippincott Williams & Wilkins, Philadelphia, 2004, p. 83.
Reed, S.I., Rothman, J.H. Cell division, growth and death [editorial]. Curr. Opin. Cell Biol. 16:599, 2004.
Spruck C.H., Smith, A.P.L., Ekholm-Reed, S., Sangfelt, O., Keck, J., Strohmaier, H., Méndez,
J., Widschwendter, M., Stillman, B., Zetterberg A., Reed, S.I. Deregulation of cyclin E and genomic Instability. In: Hormonal Carcinogenesis
IV. Li, J.J., Li, S.A., Llombart-Bosch, A. (Eds.). Springer, New York, 2004, p.
98.
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. Global analysis of protein sumoylation in Saccharomyces cerevisiae. J. Biol.
Chem. 279:45662, 2004.
Yu, V.P.C.C., Baskerville, C., Grünenfelder, B., Reed, S.I. A kinase-independent function of Cks1 and Cdk1 in regulation of transcription. Mol.
Cell 17:145, 2005.
Yu, V.P.C.C., Reed, S.I. Cks1 is dispensable for survival in Saccharomyces cerevisiae. Cell Cycle 3:1402,
2004.
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