Scripps Research Logo

The Reed Lab

Control of Cell Division

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 their selective elimination by mutation 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

Most of our work in recent years has 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 central to a number of aspects of control of cell-cycle progression.  

We have made considerable progress in understanding the regulation of the Cdk1 kinase in terms of its function at 2 points in the cell cycle: the transition from G1 to S and the transition from G2 to M. It appears that the same protein kinase catalytic subunit performs these different functions by associating with other specific regulatory proteins known as cyclins to form active protein kinase complexes. One current area of interest in this context is regulation of cellular morphogenesis by Cdk1 kinase.   The activity of Cdk1 driven by mitotic cyclins modulates polarized growth in yeast cells.  Specifically these activities depolarize growth by altering the actin cytoskeleton.  Since many of the proteins required for polarized growth have been identified in yeast, this is an ideal system for investigating the role of Cdks in morphogenesis. 

A second major area of interest is in the regulation of mitosis.  One key aspect of mitotic regulation in yeast is the accumulation of Cdc20, which triggers the metaphase-anaphase transition.  Cdc20 is an essential co-factor 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.  The coupling between cell cycle progression and Cdc20 accumulation is complex.  Cdc20 is expressed periodically at the G2/M transition.  To a large extent this is due to periodic transcription of the CDC20 gene.  We are investigating the role of Cdk1 and associated cyclins in CDC20 transcription.  We are also investigating the regulation of Cdc20 by proteolysis.  This form of regulation appears to be particularly critical when cells are arrested by the S phase checkpoint, the mechanism whereby completion of DNA replication is coupled to initiation of mitosis.  We have shown that cells are prevented from entering mitosis when DNA replication is blocked by the drug hydroxyurea by the destabilization and resultant downregulation of Cdc20.    

Downstream of Cdc20 accumulation in the regulation of anaphase is the anaphase inhibitor securin/Pds1, and its target separase (Esp1 in yeast).    Separase is a protease whose activity is required for anaphase.  One key target is a component of cohesin, a complex of proteins that maintains cohesion between sister chromatids.  The cleavage of the Scc1 subunit of cohesin allowing sister chromatid separation is a critical event in the triggering of anaphase.  However, Esp1 also regulates anaphase spindle elongation.  We are currently investigating the underlying mechanism of Esp1-mediated spindle elongation.   

In the course of investigation of mitosis, we found that a small Cdk1-associated protein, known as Cks1, appears to regulate the proteasome.  Proteasomes are complex proteases that target ubiquitylated proteins, including important cell-cycle regulatory proteins. Surprisingly, we found that Cks1 regulation of the proteasome is for a nonproteolytic function, the transcriptional activation of Cdc20.   Specifically, Cks1 is required to recruit proteasomes to the CDC20 gene 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.  A major current research focus is the elucidation of the mechanism whereby Cks1 recruits proteasomes and facilitates transcriptional elongation.  A recurrent theme in our research is the function of ubiquitin-mediated proteolysis and other forms of ubiquitin-mediated regulation in cell cycle control.  We are currently utilizing proteomic approaches in yeast to investigate the role of modification of proteins by covalent attachment of ubiquitin and other related small proteins in cell cycle control.   

Control In Mammalian Cells

We have demonstrated 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. We found that in human cells, agents that have positive or negative effects on proliferation exert these effects at the level of regulating cyclins and cyclin-dependent kinases.   A principle research focus of the laboratory is the positive regulator of Cdk2, cyclin E.  We are investigating cyclin E in two contexts:  its normal functions in regulation of entry into S phase and the mechanism(s) whereby its deregulation can lead to carcinogenesis.  In the first context, we are attempting to determine how cyclin E/Cdk2 influences pre-replication complex assembly. It has been observed that cyclin E has a positive role on pre-replication complex assembly as cells enter the cell cycle from quiescence.  However, cyclin E has a negative role on pre-replication complex assembly in cycling cells.  We are attempting to elucidate the basis for this differential regulation.  We are also investigating the role of Cdk2 in initiation of DNA replication by identifying the relevant phosphorylation targets. 

Cyclin E is often found overexpressed and/or deregulated in human cancer.    We have shown using a tissue culture model 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 (LOH) at tumor suppressor loci.   We are testing this hypothesis in transgenic mouse models.  We have shown that a cyclin E transgene expressed in the mammary epithelium significantly increases LOH at the p53 locus, leading to enhanced mammary carcinogenesis.    We are extending these investigations using mouse prostate, testis and skin models. 

We are currently investigating how deregulation of cyclin E affects both S phase and mitosis in an attempt to understand cyclin E-mediated genomic instability.   Our recent data suggest that deregulation of cyclin E impairs DNA replication by interfering with pre-replication complex assembly.   Cyclin E deregulation also impairs the metaphase-anaphase transition.  Experiments to determine the basis for this effect are ongoing. 

Our interest in cyclin E deregulation in cancer has led us to investigate the pathway whereby cyclin E is turned over.  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.  Analysis of the hCDC4 gene and its transcripts has led to the discovery that it encodes three alternatively spliced isoforms.  We are currently performing both RNAi and mouse knock-out experiments to determine the functions of the individual isoforms.   

Because of its functional relationship to cyclin E, we have been investigating the role of hCDC4 mutation in carcinogenesis.  We have found that hCDC4 is mutated and likely to be a tumor suppressor in endometrial cancer and breast cancer.  In endometrial cancer, tumors with hCDC4 mutations are more aggressive than those without mutations.  Since we have shown that loss of hCdc4 leads to deregulation of cyclin E through the cell cycle, these results confirm the observation that in some cancers, at least, deregulation of cyclin E is associated with aggressive disease and poor outcome.  We are continuing and extending these investigations.  

Another area of interest is the regulation and function of the pRb-related protein, p130.  p130 is a transcriptional repressor as well as a Cdk2 inhibitor.  Unlike pRb, p130 undergoes cell-cycle dependent proteolysis, important in regulation of Cdk2 activity.  We have demonstrated that turnover of p130 is mediated by phosphorylation by Cdk4 and subsequent ubiquitylation by SCFSkp2.    However, degradation is delayed until S phase, after another SCFSkp2 target p27Kip1 is already degraded.  We are investigating the relationship between p130 and p27 turnover and the mechanisms for the delay in p130 degradation.   

A major research interest is the role of Cks proteins in mammals, complementing our work in yeast.  Mammals express two orthologs of yeast Cks1, known as Cks1 and Cks2.   Knockout mouse experiments have revealed that each ortholog has a specialized function.  Cks1 is required as a cofactor for Skp2-mediated ubiquitylation and turnover of inhibitors p27 and p130.   Cks2 is required for the metaphase-anaphase transition in both male and female meiosis I.  Nevertheless, mice nullizygous at the individual loci are viable.  However doubly nullizygous mice have not been observed, consistent with an essential redundant function.  We using both mouse genetic and RNAi approaches to elucidate this function.  In line with our finding that Cks1 has an important transcriptional role in yeast, we are investigating if Cks1 and Cks2 have similar functions in mammalian cells.   Finally, the observation that Cks1 and Cks2 are often overexpressed in cancer has led us to investigate the role of Cks proteins in carcinogenesis.

___________

Home
___________

Research
___________

Publications
___________

Group Members
___________

Alumni
___________

Photo Gallery
___________

Contact Us
___________