Role for Human SIRT2 NAD-Dependent Deacetylase Activity in Control of Mitotic Exit in the Cell Cycle

Abstract

Studies of yeast have shown that the SIR2 gene family is involved in chromatin structure, transcriptional silencing, DNA repair, and control of cellular life span. Our functional studies of human SIRT2, a homolog of the product of the yeast SIR2 gene, indicate that it plays a role in mitosis. The SIRT2 protein is a NADdependent deacetylase (NDAC), the abundance of which increases dramatically during mitosis and is multiply phosphorylated at the G 2 /M transition of the cell cycle. Cells stably overexpressing the wild-type SIRT2 but not missense mutants lacking NDAC activity show a marked prolongation of the mitotic phase of the cell cycle. Overexpression of the protein phosphatase CDC14B, but not its close homolog CDC14A, results in dephosphorylation of SIRT2 with a subsequent decrease in the abundance of SIRT2 protein. A CDC14B mutant defective in catalyzing dephosphorylation fails to change the phosphorylation status or abundance of SIRT2 protein. Addition of 26S proteasome inhibitors to human cells increases the abundance of SIRT2 protein, indicating that SIRT2 is targeted for degradation by the 26S proteasome. Our data suggest that human SIRT2 is part of a phosphorylation cascade in which SIRT2 is phosphorylated late in G 2 , during M, and into the period of cytokinesis. CDC14B may provoke exit from mitosis coincident with the loss of SIRT2 via ubiquitination and subsequent degradation by the 26S proteasome.As the founding member of a vast gene family with members present in archaebacteria, eubacteria, and eukaryotes, SIR2 was first described in the budding yeast as a gene mediating the transcriptional silencing of the silent mating type (MAT) loci HML and HMR (14,19). Additional functions for SIR2 in budding have been described, including the silencing of subtelomeric genes (telomere position effect [TPE]) and the regulation of transcription and recombination in the multiple tandem copies of ribosomal DNA (rDNA) (for a review, see reference 12). Guarente, Sinclair, and coworkers have shown that the SIR2 gene may suppress aging in budding yeast, through a mechanism involving the suppression of extrachromosomal rDNA circles (ERCs) derived from errant intralocus recombination and suggested that SIR2-related genes in other organisms may be involved in the aging process as well, even in multicellular eukaryotes (13). The mechanism by which SIRT (an acronym for SIR2 related) genes retard aging in metazoans may involve caloric restriction (CR) instead of the ERCs found in yeast (22). Support for this hypothesis has recently come from the key finding that providing the nematode Caenorhabditis elegans with two copies of one of its SIR2-related genes (the gene normally found on chromosome IV) can extend the worm's life span by ϳ50% (36). This extension of life span function is seen only for one of the three SIR2-related genes, Sir-2.1, encoding a large nuclear protein orthologous to that coded for by the SIR2-related gene known as SIRT1 in humans and SIR2␣ in mice. Neither of the other two SIRT genes in the worm (ort...

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