These authors contributed equally to this work. SUMMARY SNF1-related protein kinase 1 (SnRK1) is the plant orthologue of the evolutionarily-conserved SNF1/AMPK/ SnRK1 protein kinase family that contributes to cellular energy homeostasis. Functional as heterotrimers, family members comprise a catalytic a subunit and non-catalytic b and c subunits; multiple isoforms of each subunit type exist, giving rise to various isoenzymes. The Arabidopsis thaliana genome contains homologues of each subunit type, and, in addition, two atypical subunits, b 3 and bc, with unique domain architecture, that are found only amongst plants, suggesting atypical heterotrimers. The AtSnRK1 subunit structure was determined using recombinant protein expression and endogenous co-immunoprecipitation, and six unique isoenzyme combinations were identified. Each heterotrimeric isoenzyme comprises a catalytic a subunit together with the unique bc subunit and one of three non-catalytic b subunits: b 1 , b 2 or the plant-specific b 3 isoform. Thus, the AtSnRK1 heterotrimers contain the atypical bc subunit rather than a conventional c subunit. Mammalian AMPK heterotrimers are phosphorylated on the T-loop (pThr175/176) within both catalytic a subunits. However, AtSnRK1 is insensitive to AMP and ADP, and is resistant to T-loop dephosphorylation by protein phosphatases, a process that inactivates other SNF1/AMPK family members. In addition, we show that SnRK1 is inhibited by a heat-labile, >30 kDa, soluble proteinaceous factor that is present in the lysate of young rosette leaves. Finally, none of the three SnRK1 carbohydrate-binding modules, located in the b 1 , b 2 and bc subunits, associate with various carbohydrates, including starch, the plant analogue of glycogen to which AMPK binds in vitro. These data clearly demonstrate that AtSnRK1 is an atypical member of the SNF1/AMPK/SnRK1 family.

Highlights d De novo synthesized glycogen accumulates in the nucleus of non-small cell lung cancers d Nuclear glycogen provides a carbon pool for histone acetylation d Nuclear glycogenolysis is dependent on translocation of glycogen phosphorylase d Glycogen phosphorylase translocation is regulated by the E3 ubiquitin ligase malin

Edited by Judit OvádiKeywords: AMP-activated protein kinase b-Subunit Carbohydrate-binding module Glycogen Oligosaccharide a b s t r a c t AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (a) and regulatory (b and c) subunits with at least two isoforms for each subunit. AMPK b1 is widely expressed whilst AMPK b2 is highly expressed in muscle and both b isoforms contain a mid-molecule carbohydrate-binding module (b-CBM). Here we show that b2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single a-1,6 branched residue. Deletion of Thr-101 reduces affinity for single a-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the b1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

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Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans.

Sucrose non-fermenting 1-related protein kinase 1 (SnRK1) is a central metabolic regulator and the plant orthologue of the mammalian AMP-activated protein kinase (AMPK); both are energy-sensing heterotrimeric enzymes comprising a catalytic α- and regulatory β- and γ-subunits. α-Subunits contain a serine/threonine kinase domain (KD) at their N-terminus that is immediately followed by a small regulatory domain termed the auto-inhibitory domain (AID) in AMPK and the ubiquitin-associated domain (UBA) in SnRK1. Association of the AID with the AMPK KD inhibits activating phosphorylation of the KD by upstream kinases and promotes dephosphorylation, as well as inhibiting AMPK catalytic activity. Despite these mechanistic insights regarding the AMPK AID, the SnRK1 UBA regulatory implications have not been investigated. Using recombinant protein comprising either the KD-only or KD-AID/KD-UBA, we found that the UBA of SnRK1 acts in a distinct regulatory manner to its orthologous AID of AMPK. Firstly, the plant upstream kinase GRIK2 preferentially phosphorylates the SnRK1 KD-UBA. Secondly, the SnRK1 KD in the absence of the UBA shows near identical initial catalytic activity to the KD-UBA, but in comparison a rapid loss of catalytic activity is observed. Our findings indicate that the role of the UBA in SnRK1 regulation may be more akin to that of the UBA in the mammalian AMPK-related kinases rather than its immediate functional orthologue, AMPK. This study adds to a growing body of work demonstrating the divergent regulatory mechanisms of the orthologous plant SnRK1 and mammalian AMPK.

The role of cellular metabolites in the direct control of epigenetic modifications is an emerging and rapidly evolving field. Herein, we identify a key function for nuclear glycogen in epigenetic regulation through compartmentalized acetyl‐CoA production and histone acetylation. In this study, we report glycogen accumulation in the nucleus of human non‐small cell lung cancer (NSCLC) specimens. To study this striking phenotype, we developed an innovative assay combining high‐purity sub‐cellular organelle isolation and stable isotope tracer technology to define the origin and metabolic fate of nuclear glycogen. We demonstrate that the nucleus is capable of carrying out glycogen synthesis, glycogenolysis and glycolysis, and both glycogen synthesis and glycolysis pathways require glucose‐6‐phosphate as the substrate. Nuclear glycogenolysis is dependent on ubiquitination and translocation of glycogen phosphorylase (GP) into the nucleus by malin, an E3 ubiquitin ligase. GP translocation is required for nuclear glycogen degradation and subsequent glycolysis to generate substrates for histone acetylation. Inhibition of nuclear glycogenolysis as a result of malin suppression is the cause of NSCLC nuclear glycogen accumulation. Re‐introduction of malin in model NSCLC cell lines restores nuclear glycogenolysis, results in increased histone acetylation, and delays cancer cell growth in vivo. This study uncovers a previously unknown foundational role for glycogen metabolism in the nucleus and elucidates another mechanism by which cellular metabolites control epigenetic regulation.Support or Funding InformationThe study was supported by the National Institute of Neurological Disorders and Stroke (grant ID: R01 NS070899‐06, P01 NS097197‐01), National Science Foundation (Grant ID: MCB‐1817414) and by the University of Kentucky Center for Cancer and Metabolism, National Institute of general medical sciences COBRE program (grant ID: P20 GM121327).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.