Project Summary The cell cycle is an ordered set of cellular processes that produces two identical daughter cells. This process involves DNA replication and chromosome segregation leading to cell division. Cyclin dependent kinase (Cdk1) drives eukaryotic cell cycle progression and cell proliferation. Cdk1 is a serine/threonine kinase whose activity depends on its binding partner, cyclin. The cyclin/Cdk1 complex phosphorylates various substrates at S/TP consensus motifs to trigger a downstream signaling cascade. The cell cycle is a unidirectional process which is regulated by temporal degradation of the cyclins. This Cdk1-dependent phosphorylation event is reversed by the phosphatases PP2A and Cdc14 in S. cerevisiae. PP2A is a heterotrimer with two possible regulatory subunits in yeast: Cdc55 or Rts1, which direct the complex to distinct targets. PP2A-Cdc55 preferentially dephosphorylates Cdk1 substrates at threonine residues in mitosis. Here we propose that PP2A-Cdc55 positively regulates DNA replication through Cdc6 and negatively regulates mitotic spindle elongation through Pds1 in S. cerevisiae. This mechanism ensures unidirectional cell cycle progression to maintain genome integrity. Aberrant cell cycle control leads to chromosome instability which is a hallmark of cancer. Our long-term goal is to understand the role of phosphatase in control of cell proliferation and tumorigenesis. In this proposal, two aims will test my hypothesis that PP2A-Cdc55 promotes origin licensing to initiate DNA replication and negatively regulates spindle elongation in S. cerevisiae. Aim1 will define the role of PP2A-Cdc55 in origin licensing. The general strategy of Aim1 is to study how Cdc6 and other DNA replication proteins are regulated by PP2A-Cdc55. Aim2 will determine how PP2A-Cdc55 dependent Pds1 dephosphorylation inhibits spindle elongation. We will test if PP2A-Cdc55 controls spindle elongation through Pds1-Esp1 and other downstream targets. The PP2A is frequently found to be inactivated in cancer patients making PP2A a potential therapeutic target. In this study, we will use a single cell model organism, S. cerevisiae, to understand the basic mechanism of how PP2A controls the cell cycle. DNA replication and mitosis are well conserved cellular events in eukaryotes. The yeast components focused on in this project are well conserved in humans. The results obtained from this study can be applied to understanding how PP2A functions in higher eukaryotes.

Project narrative Failure of proper cell cycle control results in chromosome instability which is a hallmark of tumorigenesis. It has been extensively studied how Cyclin Dependent Kinase (Cdk1) promotes cell cycle progression, but little is known how the Cdk1-dependent phosphorylation events are reversed by phosphatase. In this study, we identify and investigate targets of the PP2A phosphatase during cell cycle progression.