SUMMARY Xenopus oocyte death is partly controlled by the apoptotic initiator, caspase-2. We reported previously that oocyte nutrient depletion activates caspase-2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. We now show that caspase-2 phosphorylated at S135 binds 14-3-3ζ, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3ζ from caspase-2 is controlled by metabolism and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3ζ is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death.

No abstract

Several lines of evidence suggest that protein lysine acetylation pathways are deregulated in cancer (1). Moreover, deacetylase inhibitors are emerging as important anti-tumor therapeutics, suggesting that the forced reprogramming of protein-lysine acetylation is toxic to tumor cells. In this study we show that Sirt1, an NAD+-dependent Sirtuin deacetylase that promotes cancer cell survival, is aberrantly mislocalized to the cytoplasm of breast tumor cells. Moreover, the depletion of cytosolic Sirt1 by siRNA sensitizes breast tumor cells to paclitaxel-induced death. Previously, we developed a biotin-switch proteomics approach to identify cytosolic Sirt1 substrates (2). This approach yielded a variety of substrates with roles in metabolism, survival, and oxidative stress signaling. Our current work focuses on three of the proteins identified as Sirt1 substrates: SOD1, DJ-1, and 14-3-3z. SOD1 and DJ-1 both suppress oxidative stress-induced death, and high levels of 14-3-3z expression suppress chemotherapy-induced apoptosis and correlate with negative patient outcomes in breast cancer. Our preliminary results suggest that acetylation of DJ-1 and SOD1 suppress their anti-oxidant functions, while acetylation of 14-3-3z disrupts its binding to pro-survival proteins. Taken together, our data support a model in which cytosolic Sirt1 activates multiple pathways that work together to promote tumor cell survival. Citation Format: Jeffrey B. Mortenson, Vajira K. Weerasekara, Josh Andersen. Sirt1-mediated suppression of cell death in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 850. doi:10.1158/1538-7445.AM2013-850

Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress‐induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi‐pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome‐level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock‐out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3‐lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock‐in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.