Coordinated execution of numerous mitotic events is crucial for the proper completion of cell division. The transient inactivation of most sequence-specific DNA-binding proteins, in synchrony with chromosome condensation and segregation, is a critical part of mitosis. However, we know very little about the signaling pathways that regulate this process and its restriction to the duration of mitosis. We have recently identified the serine/threonine kinase TOPK (aka PBK) as a critical mitotic regulator of transcription factors. We showed that TOPK phosphorylates a conserved motif in the DNA-binding domain of hundreds of C2H2 zinc-finger proteins resulting in the mitosis-specific inactivation of their DNA-binding activity. It is critical that this massive phosphorylation event occurs exclusively during mitosis. However, it is not yet clear how the cell activates TOPK only in mitosis. To address this fundamental biological question we propose to: 1) Investigate the mitosis-specific activation mechanism of TOPK. Our preliminary data indicate that only mitotically- phosphorylated TOPK can phosphorylate the conserved motif of C2H2 transcription factors. Using mass spectrometry, we mapped the TOPK amino acid residues that are phosphorylated in vivo. We will determine which of these phosphosites are required for the kinase activity of TOPK towards its mitotic substrates, which of these sites occur only in mitosis, and what is their physiological relevance to the completion of mitosis and to cell proliferation. 2) Identify upstream signaling pathways needed for the mitosis-specific activation of TOPK. Our preliminary data strongly implicate the Polo-like kinase 1 (Plk1) as a potential mitotic activator of TOPK. We will elucidate the role of Plk1 in the regulation of TOPK, and examine a possible collaborative role of cyclin- dependent kinase 1 (cdk1). Completion of these proposed tasks will advance our understanding of the regulation of an important mitotic mechanism. Moreover, TOPK is an oncogene that is highly overexpressed in most types of cancer cells and its high expression correlates with poor patient prognosis. Although TOPK has been shown to be important for tumorigenesis, its mitotic activation mechanism and the significance of this activity to cancer cell proliferation are not yet fully understood. Therefore, the proposed research will further our knowledge of the functions and regulation of an important oncogene. The findings that will be generated upon completion of this research proposal will very likely have a positive impact the design of future therapeutic approaches targeting this kinase.
Cancer is primarily a proliferative disease. The proposed research aims to characterize the activation mechanism(s) and function of an oncogenic kinase that is required for cancer cell proliferation. The results of this study will enhance our knowledge of the mechanisms that contribute to tumorigenesis, and will directly and positively impact the future design of novel strategies for cancer therapy.
