Telomeres are critical for maintaining genomic stability and allowing cells escaping from crisis, and their deregulation has been implicated in cancer and premature aging. To elucidate how telomeres are maintained is therefore important for understanding physiology and pathology of cancer progression and may provide novel therapeutic targets. The major enzyme that replenishes telomeric DNA is telomerase, which is active in most immortalized and transformed/cancer cells, but not in most normal human cells. However, how telomerase activity is regulated is largely unknown. In addition, although telomere maintenance is tightly linked to mitotic progression, the underlying mechanism is unclear. We previously cloned Pini and Pin2 that are critical for mitotic regulation. Pin 1 is a prolyl isomerase that regulates protein conformation and function by isomerizing specific phosphorylated Ser/Thr-Pro bonds. Pin2 is a major telomeric protein almost identical to TRF1, which inhibits telomere elongation but does not directly inhibit telomerase activity. We have shown that Pin2/TPF1 is tightly regulated during the cell cycle and is important for mitotic checkpoint regulation. Our laboratory have further cloned four novel Pin2/TRF1-interacting proteins, PinXl-4, and discovered that PinX1 is the first endogenous telomerase catalytic inhibitor and is a putative tumor suppressor located at 8p23, a region with frequent loss of heterozygosity in a number of cancers. Our preliminary results indicate that Pin2/TRF1 is phosphorylated by Cdc2 in mitosis, which renders Pin2/TRF1 to be a Pini substrate and also disrupts its ability to bind telomeres or PinX3, a novel F-box-containing protein likely involved in ubiquitin-mediated proteolysis. These results led us to propose that Pin2/TRF1 and PinXl are important regulators in telomere maintenance, mitotic regulation and oncogenesis. To test this hypothesis, we will first characterize Pin2/TRF1 ubiquitination and phosphorylation, and determine their roles in mitotic regulation and telomere maintenance. Next, we will determine the role of PinX1 in mediating the inhibitory effect of Pin2/TRF1 on telomere elongation and elucidate how PinX1 inhibits telomerase activity. To evaluate the potential of PinXl as a novel tumor suppressor, we will examine PinX1 genetic mutations in human cancer and their effects on PinXl function, and generate PinXl-deficient mice to determine its in vivo function. Finally, given that PinXl is less potent than its telomerase inhibitory domain in vivo but not in vitro, we will examine how PinX 1 function is regulated. These studies should yield new insights into telomere maintenance, cell growth control and oncogenesis, and may lead to the development of new anticancer therapy.
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