A Biotin Switch-Based Proteomics Approach Identifies 14-3-3ζ as a Target of Sirt1 in the Metabolic Regulation of Caspase-2

Abstract

While lysine acetylation in the nucleus is well characterized, comparatively little is known about its significance in cytoplasmic signaling. Here we show that inhibition of the Sirt1 deacetylase, which is primarily cytoplasmic in cancer cell lines, sensitizes these cells to caspase-2-dependent death. To identify relevant Sirt1 substrates, we developed a novel proteomics strategy, enabling the identification of a range of putative substrates, including 14-3-3ζ, a known direct regulator of caspase-2. We show here that inhibition of Sirtuin activity accelerates caspase activation and overrides caspase-2 suppression by nutrient abundance. Furthermore, 14-3-3ζ is acetylated prior to caspase activation, and supplementation of Xenopus egg extract with glucose-6-phosphate, which promotes caspase-2/14-3-3ζ binding, enhances 14-3-3ζ-directed Sirtuin activity. Conversely, inhibiting Sirtuin activity promotes 14-3-3ζ dissociation from caspase-2 in both egg extract and human cell lines. These data reveal a role for Sirt1 in modulating apoptotic sensitivity, in response to metabolic changes, by antagonizing 14-3-3ζ acetylation.

Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells

“…11,37 Consistent with previous reports, 17,18 however, including LMB during isotonic digitonin fractionation did not preserve GFP-SIRT1 in the nuclear fraction (Fig. 3C), suggesting the fractionation-induced leakage does not recapitulate the nuclear exportation of SIRT1.…”

“…Cumulative evidence suggests that SIRT1 could promote both tumor-suppressive and oncogenic activities and such bipolar functions of SIRT1 in tumorigenesis could be achieved by deacetylating different substrates with different subcellular localization. 14,15 While most known and potential SIRT1 substrates are nuclear proteins, 3,16 several studies also showed that SIRT1 is mainly enriched in the cytoplasm in a wide range of cancer cell lines but localizes to the nucleus in normal cells, [17][18][19][20] indicating that cytoplasm-localized SIRT1 could contribute to the cancer-specific function. 18 However, the subcellular localization of SIRT1 in cancer and transformed cells remains controversial in the literatures, even with the same cell line.…”

“…18 However, the subcellular localization of SIRT1 in cancer and transformed cells remains controversial in the literatures, even with the same cell line. 2,19,21,22 For example, live cell imaging suggests that GFP-SIRT1 mainly localizes to the nucleus in 293T cells in several studies, 21,23,24 whereas cell fractionation often shows cytoplasmic SIRT1 enrichment [17][18][19] and immunofluorescence gives diversified localizations depending on antibody and protocol that were used. 18 The reason underlying this controversy is largely unknown.…”

Impaired antioxidant defence and accumulation of oxidative stress in caspase-2-deficient mice

“…30 In particular, caspase-2 is required for the regulation of the pentose phosphate pathway as an important antioxidant defence mechanism. 30,31 However, it remains unclear whether this role would explain our observations in casp2 À/À animals. Analysis of mitochondrial dysfunction in MEFs indicates that loss of caspase-2 does not affect mitochondrial membrane potential or respiration during the oxidative stress response.…”

Caspase-2 at a glance

“…Nutrient depletion promotes the release of caspase-2 from 14-3-3f, which, in turn, allows the dephosphorylation of Ser135 by PP1, relieving caspase-2 inhibition and causing cell death ). The ability of 14-3-3f to interact with and inhibit caspase-2 in oocytes and human cancer cell lines (Andersen et al, 2011) is regulated by acetylation. The control of the acetylation status of 14-3-3f is exerted partly through the activity of the deacetylase sirtuin 1 (SIRT1) that can respond to glucose 6-phosphate by an unknown mechanism (Andersen et al, 2011).…”