Identification of PP2A B-subunit That Directs PP2A to Cyclin E

Cell cycle regulation is one cellular process that malfunctions during cancer development. The cell cycle is regulated by cyclin dependent kinases (cdks) and cyclin binding partners that phosphorylate various proteins throughout the cell cycle. This experiment focuses on the regulation of G1 to S phase transition where cyclin E and cdk2 are the proteins of interest. C-terminal phosphorylation on cyclin E's T380 and S384 by GSK3 and cdk2 activate cyclin E ubiquitylation and degradation by enabling the E3 ubiquitin ligase Fbw7 to bind.  Recent research indicates that the phosphatase, PP2A dephosphorylates cyclin E at S384 which could have important consequences for cyclin E stability throughout the cell cycle. The PP2A holoenzyme consists of three different subunits: the A subunit which serves as a scaffold, the B subunit that enables the holoenzyme to be substrate specific, and the C subunit which is the catalytic subunit. To determine which B-subunit directs PP2A to cyclin E, U2OS cells were transduced with retroviral vectors expressing GFP fusion proteins for each B subunit. These cells were then analyzed using fluorescent microscopy to determine which B subunits localize in the nucleus, similar to cyclin E. Mass spectrometry and co-immunoprecipiation are also being used to identify the cyclin E-specific PP2A B subunit. These approaches will enable us to further define PP2A’s role in cell-cycle regulation through its ability to modulate cyclin E phosphorylation and possibly its stability.