Molecular Biology - The Scripps Research Institute

224 MOLECULAR BIOLOGY 2005
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 cellcycle
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
Published by TSRI Press ®. ©Copyright 2005,
The Scripps Research Institute. All rights reserved.
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 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.
We are also investigating SCF hCdc4 ubiquitylation
of other important cellular proteins.
Because of the functional relationship between
hCdc4 and cyclin E, we are studying the role of muta-