Histone deacetylase 6 (HDAC6) promotes the pro-survival activity of 14-3-3ζ via deacetylation of lysines within the 14-3-3ζ binding pocket.

The phospho-binding protein 14-3-3ζ acts as a signaling hub controlling a network of interacting partners and oncogenic pathways. We show here that lysines within the 14-3-3ζ binding pocket and protein-protein interface can be modified by acetylation. The positive charge on two of these lysines, Lys(49) and Lys(120), is critical for coordinating 14-3-3ζ-phosphoprotein interactions. Through screening, we identified HDAC6 as the Lys(49)/Lys(120) deacetylase. Inhibition of HDAC6 blocks 14-3-3ζ interactions with two well described interacting partners, Bad and AS160, which triggers their dephosphorylation at Ser(112) and Thr(642), respectively. Expression of an acetylation-refractory K49R/K120R mutant of 14-3-3ζ rescues both the HDAC6 inhibitor-induced loss of interaction and Ser(112)/Thr(642) phosphorylation. Furthermore, expression of the K49R/K120R mutant of 14-3-3ζ inhibits the cytotoxicity of HDAC6 inhibition. These data demonstrate a novel role for HDAC6 in controlling 14-3-3ζ binding activity.

Acylation of Superoxide Dismutase 1 (SOD1) at K122 Governs SOD1-Mediated Inhibition of Mitochondrial Respiration.

In this study, we employed proteomics to identify mechanisms of posttranslational regulation on cell survival signaling proteins. We focused on Cu-Zn superoxide dismutase (SOD1), which protects cells from oxidative stress. We found that acylation of K122 on SOD1, while not impacting SOD1 catalytic activity, suppressed the ability of SOD1 to inhibit mitochondrial metabolism at respiratory complex I. We found that deacylase depletion increased K122 acylation on SOD1, which blocked the suppression of respiration in a K122-dependent manner. In addition, we found that acyl-mimicking mutations at K122 decreased SOD1 accumulation in mitochondria, initially hinting that SOD1 may inhibit respiration directly within the intermembrane space (IMS). However, surprisingly, we found that forcing the K122 acyl mutants into the mitochondria with an IMS-targeting tag did not recover their ability to suppress respiration. Moreover, we found that suppressing or boosting respiration levels toggled SOD1 in or out of the mitochondria, respectively. These findings place SOD1-mediated inhibition of respiration upstream of its mitochondrial localization. Lastly, deletion-rescue experiments show that a respiration-defective mutant of SOD1 is also impaired in its ability to rescue cells from toxicity caused by SOD1 deletion. Together, these data suggest a previously unknown interplay between SOD1 acylation, metabolic regulation, and SOD1-mediated cell survival.

Metabolic-stress-induced rearrangement of the 14-3-3ζ interactome promotes autophagy via a ULK1- and AMPK-regulated 14-3-3ζ interaction with phosphorylated Atg9.

14-3-3ζ promotes cell survival via dynamic interactions with a vast network of binding partners, many of which are involved in stress regulation. We show here that hypoxia (low glucose and oxygen) triggers a rearrangement of the 14-3-3ζ interactome to favor an interaction with the core autophagy regulator Atg9A. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3ζ docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3ζ interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These data suggest a novel mechanism whereby the level of autophagy induction can be modulated by AMPK/ULK1-mediated phosphorylation of mammalian Atg9A.