FUNCTIONAL ANALYSIS OF MUTATIONS IN NEK1 AND NEK7 IN CANCER AND IDENTIFICATION OF THEIR PHYSIOLOGICAL SUBSTRATES

Abstract

Members of the Nek family of kinases (NIMA related kinases) have been identified as important regulators of cell cycle checkpoint, especially in the G2/M transition. Although it is one of the least studied families of kinases, recent studies have shown that they can have crucial roles in mitosis, separation the centrosome and signaling pathways in DNA damage. These characteristics by the fact that the various members of the family are overexpressed in cancer or have high mutation rates make them interesting candidates targets in cancer diagnosis and therapy. Our group is currently studying all 11 human members of Nek kinases. Conducted structural and functional biochemical and cellular studies as well as high performance screening assays to identify inhibitors of commercial libraries, natural products and inhibitory peptides. Studies indicate that Nek1 is related to the checkpoint of DNA damage during interphase. In cells that have mutations in the gene of Nek1 an ineffective repair of DNA damage was observe. Since overexpression of Nek7 resulted in multinucleated cells with mitotic aberrations, in addition to being overexpressed and studies in colorectal cancer, breast, larynx, lung and non-Hodgkin lymphomas. However, not known so far in both cases are the physiological substrates of these protein kinases and functional consequences of point mutations (amino acid exchange) that occur in their genes in cancer. The central objective of this project is to describe the functional role of Nek1 and Nek7 in normal and tumor human cells. We will use the approach of "Shokat" for generating kinases which recognize ATP analogues for the in vitro identification of physiological substrates. We also will introduce mutations that occur in cancer to analyze functional changes of the two kinases in vitro and in vivo; In support of these sub-objectives we will analyze the profile of the protein expression of Nek1 in thyroid normal and tumor tissues and will also perform "High Throughput Screening" bioassays. Thus, the project aims to clarify the physiological role of Nek1 and 7 and functional changes that occur in cancer. Taken together, the data obtained will help in analyzing the potential of these protein kinases as therapeutic targets in cancer treatment.