POSTER ABSTRACTS “Interaction Of Insep5 And Plk1 In The Cohesin Complex”
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Gladys Morrison*1 and Debananda Pati, Ph.D2 1 Spelman College, Chemistry, Atlanta, GA 30314 2 Baylor College of Medicine, Pediatrics‐Hematology/Oncology Houston, TX 77030 Abstract Sister chromatid cohesion is formed during DNA replication and destroyed at the onset of metaphase‐to‐anaphase transition. During mitosis, errors in the regulation of sister chromatid cohesion could result in the mis‐segregation of chromosomes and aneuploidy. The overall focus of Dr Pati’s lab is mainly on the cohesin complex, which has four core subunits, Rad21, Smc1, Smc3 and SA1/SA2. In addition, his lab is interested in the proteins that interact with cohesin complex, such as Insep5. Polo‐like kinase 1 (PLK1) is a protein that regulates cohesin complex disassociation from sister chromatid arms at prophase by phosphorylating SA1/SA2 of the cohesin complex. However, it is not known how PLK1 executes this function. The purpose of this project is to study the role of Insep5 in the phosphorylation of SA1/SA2 by PLK1. My research this summer was studying the protein interaction between the Polo Box Domain (PBD) of PLK1 and Insep5. Insep5 protein has two Cdk1/cyclin B phosphorylation sites. One of them is the consensus sequence of the PLK1 binding site. When over‐expressed, the PLK1 binding site mutant of Insep5 abolishes the interaction with PLK1. In order to further study the interaction between Insep5 and PLK1, we obtained two stable transfected Tet‐On cell lines, HeLa S3 PBDWT and PBDAA (Hanisch et al, 2006). I induced several cell plates with different concentrations of doxycylin, an antibiotic that activates the Tet‐On cell lines and induced another batch of cells over different time course. Our western blot indicated that PBDWT and PBDAA could be efficiently induced with 156 fg/ml doxycylin in 8hrs. Using a microscope, we also found that PBDwt cells were arrested when PBDwt was induced over 8hrs but we did not see cell cycle arrest in PBDAA cells. So over different time course, Fluorescence Activated Cell Sorting (FACS) was used to determine the stages of cells when PBD was expressed. The FACS results indicated that most PBDWT cells were arrested at G2/M phase over 8hrs. It is possible that over‐expressed PBDWT displaces endogenous PLK1, which normally binds to Insep5 and disrupts the cell cycle. To prove this hypothesis, I transfected Flag‐Insep5 into the two cell lines. Some were induced with or without the presence of doxycyclin. Co‐immunoprecipitation (IP) results indicated that PBDWT interacts to Insep5 but not its mutant. 171