| SIRT1, the mammalian ortholog of Sir2, is an NAD+-dependent deacetylase, and its gene expression can affect the lifespan of multiple organisms, as can compounds that pharmacologically activate it. It possesses a large and growing list of substrates, such as p53, NFkappaB, and FoxO, and influences cell survival, stress resistance, cell differentiation, glucose and fat metabolism. Importantly, SIRT1 may regulate longevity and has proved to be a significant factor in aging research. However several key questions have not been addressed, namely: (1) are SIRT1 levels regulated in the context of aging and senescence, and (2) is SIRT1 regulated post-translationally.;In order to address the first question, I investigated the changes in the level of SIRT1 with aging and senescence, and showed that levels of the SIRT1 protein decline as cells lose mitotic activity with age and that the decline is due to a post-transcriptional mechanism. These results are described in chapter 2.;Based on the above findings, I tested the hypothesis that SIRT1 is regulated post-translationally by phosphorylation conferred by cyclin-dependent kinase (Cdk) in chapter 3. I found that SIRT1 interacts with and is phosphorylated by cyclin B and Cdk1 in vivo. Furthermore, SIRT1 is a good substrate for cyclin B/Cdk1 in vitro and this complex phosphorylates SIRT1 at Thr530 and Ser540. These phosphorylation sites and recognition motifs for cyclin and Cdk are conserved among different species. Thus, SIRT1 is a substrate for cyclin B/Cdk1.;I also investigated how phosphorylation of SIRT1 affects its deacetylase activity. Treating SIRT1 with phosphatase reduces its NAD+-dependent deacetylase activity. I further explored the biological functions of SIRT1 phosphorylation at Thr530 and Ser540 by using SIRT1 with a double mutation for T530A/S540A (AA mutant). Wild-type SIRT1, but not the AA mutant, rescued the slower growth rate and the deficit in S-phase cells, which were observed in Sirt1-/- ES cells. In summary, SIRT1 is phosphorylated by Cyclin B/Cdk1, and this process regulates its deacetylase activity and affects cell proliferation.;Thus SIRT1 is regulated in the context of aging and senescence by Cdk1 and in return controls cell proliferation. |
