In addition to the well-known protein kinase CDC2, a novel protein kinase, NIMA, plays an essential role in regulating mitosis in the lower eukaryotic Aspergillus nidulans. I previously demonstrated that a putative NIMA-like pathway also is required for entry into mitosis in human cells. Recently, I isolated three human genes, PIN1-3, encoding proteins that physically interact with NIMA and functionally suppress its function. Characterization of PIN1 and PIN3 has demonstrated that they encode important mitotic regulators, further providing strong support for the existence of a NIMA-like pathway in human cells. This proposal will focus on determining the function and regulation of Pin2. Preliminary results show that Pin2 is specifically associated with a putative protein kinase, Pink1, which is a strong candidate for the human NIMA functional homologue. Interestingly, Pin2 is also specifically interacts with double- stranded repeat DNA located in human telomeres. Telomeres, DNA- protein complexes capping chromosome ends, are essential for maintaining the genomic stability and the proliferative capacity of the cell. Furthermore, deregulation of telomere length has been shown to be implicated in cancer and aging. Although these results suggest a link between telomere length and growth control, little is known about the regulatory molecules and mechanisms involved. Our findings that the major telomeric DNA-binding protein Pin2 interacts with the Aspergillus mitotic kinase NIMA suggests that Pin2 and the putative human NIMA homologue Pink1 may be key molecules connecting the cell cycle and telomere function. To test this hypothesis, we first plan to determine the role of Pin2 in the cell cycle by examining the cell cycle-specific expression and localization of Pin2 and determining the effects of upregulating and downregulating the Pin2 function on cell cycle progression and the putative NIMH mitotic pathway. Next, we will determine the role of Pin2 in regulating telomere length by determining the relationship between the Pin2 concentration and telomere length during senescence, examining the effects of altering the Pin2 function on telomere length and cellular senescence, and elucidating the role of Pin2 in regulating telomerase activity. Finally, we will study the regulation of Pin2 by determining Pin2 phosphorylation sites and their functions, as well as purifying and cloning the Pin2-associated kinase Pink1. Once the Pink1 cDNA is isolated, we will determine whether it is indeed the human NIMA homologue and how it regulates Pin2 and the cell cycle. These studies should help elucidate the role and molecular mechanisms of the mammalian NIMA-like pathway in regulation of mitosis and telomere homeostasis, and may have novel implications for understanding mechanisms of aging and carcinogenesis.
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