MOLECULAR MECHANISMS GOVERNING EUKARYOTIC DNA REPLICATION AND CELL CYCLE PROGRESSION

THOMAS R. COLEMAN, Ph.D., Associate Member

Cells grow and divide by progressing through an ordered series of events referred to as the cell division cycle. A network of regulatory proteins ensures that the cell cycle program is followed in an orderly and timely fashion. Protein components that regulate the cell cycle have been identified largely through yeast genetic studies. Our laboratory uses complementary biochemical techniques with Xenopus laevis cell-free egg extracts to characterize these components in a direct, mechanistic, and practical manner.

Eggs are ideal for studying the basic processes of the cell cycle because they are specialized for rapid cell division. Moreover, relatively crude extract preserves many of the normal cell cycle processes, and can complete several full cell cycles in a test tube. Each in vitro cell cycle consists of nuclear assembly, semi-conservative DNA replication, chromosomal segregation, and nuclear envelope breakdown.

The faithful duplication of the genome is one of the fundamental control points of the cell cycle. Considerable evidence implicates the cell division cycle Cdc6 protein as a key regulator in initiating DNA replication. It is an essential component of the DNA replication machinery. It is modified in a cell cycle-dependent manner and its destruction may play a role in keeping the cell from deleteriously re-replicating its genome. Recently, we have identified a Xenopus Cdc6 (Xcdc6) homologue. A major focus of the laboratory is to determine how it functions and how it is regulated.

The remarkable degree of conservation in the cell cycle machinery throughout evolution makes it highly likely that our discoveries will be applicable to human biology. For example, since Xenopus and human Cdc6 are approximately 80% identical, what we learn in biochemically-tractable systems, like frog egg extracts, will advance our understanding of human cell cycle control. Only through understanding these regulatory pathways can we devise effective strategies to combat uncontrolled cell division, which is the hallmark of cancer.

By comparing the amino acid sequences of the known Cdc6-related proteins, we have identified conserved domains and potential functional motifs. Xcdc6 shares significant amino acid identity with human Cdc6 (77% identical residues), budding yeast Cdc6 (30%), and fission yeast Cdc18 (34%). Like the other Cdc6-related proteins, Xcdc6 contains three prominent motifs: 1) a consensus purine nucleotide binding site (GXXGXGKT, residues 196-203); 2) a putative bipartite nuclear localization signal (KKTSQTLAKEVSRAKSK, residues 17-33), and 3) multiple Serine-Proline or Threonine-Proline motifs that reside in potential recognition sites for cell cycle regulated kinases such as the cyclin-dependent kinases.

We propose that Xcdc6 is a bifunctional molecule, composed of a N-terminal regulatory and a C-terminal functional domain. In support of this contention, both the putative nuclear localization signal (NLS) and a majority of the potential phosphorylation sites (8 of 10) reside in the N-terminus. In contrast, the putative purine nucleotide-binding site resides in a highly conserved midportion of the protein. Thus, the N-terminus, which comprises the NLS and multiple phosphorylation sites, may regulate the localization and activity of Xcdc6, while the midportion contains the highly conserved putative functional domain(s). To address these structure/function issues, we will express recombinant domains of Xcdc6 and characterize their activity and localization in the Xenopus egg extract.

In our initial characterization, we observed that the electrophoretic mobility of Xcdc6 varied in a cell cycle-dependent manner. Specifically, the interphase form migrated at the predicted molecular weight (61 kilodaltons [kDa]), while the mitotic form migrated with a slower electrophoretic mass (63 kDa). We have recapitulated this finding in vitro by introducing purified recombinant Xcdc6 protein into Xenopus egg extracts arrested at different stages of the cell cycle. Xcdc6 incubated in mitotic extracts was efficiently converted to a slower electrophoretic mobility form, whereas Xcdc6 was not altered in apparent molecular weight upon incubation in interphase extracts. To determine if this mitotic modification is the result of a cell cycle-dependent phosphorylation of Xcdc6, we treated the 63 kDa mitotically-modified recombinant Xcdc6 with a variety of phosphatases. When treated with alkaline phosphatase, mitotic Xcdc6 increased its mobility to that of 61 kDa, suggesting that the mobility shift at mitosis is due to phosphorylation.

Our working hypothesis is that phosphorylation is one means by which the cell regulates Xcdc6 activity. To pinpoint which residues on Xcdc6 are phosphorylated during mitosis, we are analyzing the posttranslational modifications of Xcdc6 using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS). To perform these experiments, we mimicked the cyclical changes in Xcdc6 phosphorylation by incubating recombinant, tagged Xcdc6 in either mitotic or interphase egg extracts, after which it was re-isolated for analysis. Following SDS-gel electrophoresis and staining, the mitotic (63 kDa) and interphase (61 kDa) Xcdc6, protein bands were excised and processed for trypsin digestion. Extracted tryptic peptides were then subjected to mass spectrometry analysis to identify peptides whose mass was shifted in a manner consistent with a cell cycle-dependent phosphorylation.

After establishing stringent criteria with which to assess the data derived from our preliminary MS analysis, we have identified several potential phosphorylation sites. Once we have unequivocally identified the modified residue(s), we will change each to the corresponding nonphosphorylatable amino acid. We will then express Xcdc6 mutants in insect cells using recombinant baculoviruses and purify the recombinant mutant proteins. Finally, we will characterize their activity in Xenopus egg extracts. By analyzing how these mutant Xcdc6 proteins affect cell cycle progression, we will determine how phosphorylation regulates the activity of Xcdc6.

Entry into mitosis is controlled by Mphase promoting factor (MPF), a complex of cyclin B and cdc2 kinase. A complex kinase/phosphatase cascade regulates MPF activity. For example, the wee1 kinase suppresses entry into mitosis by mediating the inhibitory phosphorylation of cdc2, while the nim1 kinase acts as a positive regulator of mitosis by down-regulating the wee1 kinase. To date, metazoan homologues of nim1 have not been identified. We searched for Xenopus nim1 homologues using a degenerate polymerase chain reaction (PCR) approach. PCR primers were designed based on the sequence similarity between the budding and fission yeast nim1 homologues. Xenopus cDNA was prepared by reverse transcription of messenger RNA isolated from Xenopus tissue culture cells. The cDNA was then used as a template for PCR using the degenerate nim1 primers. Unfortunately, this search only identified kinases with no nim1 homology.

^§   Fox Chase researcher

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998