Identification and Investigation of the Centrosome Localization Domain of Mps1

Abstract

The centrosome is a single copy organelle that is responsible for organizing the microtubule network throughout the cell cycle. The centrosome also regulates the cell cycle, organizes the mitotic spindle, and is required for cytokinesis. In fact, the organelle is a major factor in the conservation of genomic integrity. Mps1 is a family of protein kinases originally identified in Saccharomyces cerevisiae. Much is known about the functions of the human orthologue hMps1; it is involved in the mitotic spindle checkpoint, centrosome duplication, and cytokinesis. It is localized to the centrosome throughout the cell cycle, but the method by which it is targeted to centrosomes is unknown. In this study we identify a conserved domain of hMps1 and propose that is involved in targeting the protein to the centrosome. We show that it is sufficient for centrosome localization and are working on determining its necessity. Within the identified domain is a tyrosine residue (Y91) that is completely conserved in vertebrate species. Tyrosine residues are often important because they can be phosphorylated and are sometimes involved in the regulation of a protein’s function. Here we demonstrate that phosphorylation of Y91 is indeed involved in the regulation of centrosome localization and propose methods to determine how it might regulate that event. We also utilize the two-hybrid system to determine proteins that bind to the centrosome localization domain (CLD). We have identified five proteins from the screen and further investigated one of them, VDAC3. Using GFP-fusion proteins we provide evidence that VDAC3 can indeed localize to centrosomes and are testing its interaction with hMps1. This work is significant because centrosome misregulation can lead to serious cellular defects. In fact, abnormal centrosome numbers have been found in a number of tumors, and are especially prevalent in breast cancer. Therefore, centrosomal defects could potentially be a catalyst for the progression of cancer; and because of its function in centrosome duplication and cytokinesis, defects in Mps1 itself could be a factor in the development of human tumors. It is therefore essential to understand the mechanism of Mps1 centrosome localization to explain problems that occur in cancers containing chromosome defects.

Advisor: Harold A. Fisk