Abstract AMP-activated protein kinase (AMPK) regulates metabolism in response to metabolic stress that includes stimulating glucose uptake in skeletal muscle independently of the canonical insulin signalling pathway, positioning it as an attractive therapeutic target for insulin resistance and type 2 diabetes mellitus (T2DM). AMPK is an αβγ heterotrimer, with multiple isoforms for each subunit enabling the formation of 12 different complexes with distinct tissue expression profiles. Among these, the α2β2γ3 complex is predominantly expressed in skeletal muscle, the major site of glucose disposal and a highly desirable therapeutic target for T2DM. Here, we characterise the functional role of a unique, 182 residue N-terminal extension (NTE) within γ3 subunit. Deletion of the γ3-NTE from α2β2γ3 complex increases basal AMPK activity without affecting activation by AMP or pharmacological AMPK activators, demonstrating the γ3-NTE performs an autoinhibitory function. Using complementary biophysical techniques, including hydrogen-deuterium exchange-mass spectrometry, surface plasmon resonance, chemical crosslinking and co-pulldowns, we identified a 39-residue sequence in the γ3-NTE (residues 129-168), that directly interacts with the αC-helix of the AMPK kinase domain small lobe, a key regulatory element in many protein kinases. Using AlphaFold3, we probe the interaction predicted to take place between a γ3-NTE α-helix (γ3-iHelix; ∼T142-E154) and the αC-helix in the α2β2γ3 complex. These findings provide the groundwork for developing novel T2DM therapies that target AMPK activation selectively in skeletal muscle involving reversal of the γ3 autoinhibition.

Protein ubiquitination is a critical post-translational modification that regulates protein stability and cellular homeostasis. KLHDC3 is a substrate recognition receptor within the recently identified C-terminal degron-mediated DesCEND ubiquitination pathway. It has been characterised to selectively bind proteins with C-terminal RxxxG motifs, targeting them for degradation. Unlike the well-characterised N-terminal degron pathway, the physiological roles of C-terminal degrons remain poorly understood. To investigate KLHDC3 function in vivo , we generated Klhdc3-deficient ( Klhdc3-/- ) mice. These mice exhibited sub-Mendelian birth rates and progressive postnatal lethality, with a median survival of 136 days and maximum survival observed of ∼1 year of age. Early growth retardation was apparent, followed by a normalisation of body mass with age. However, as Klhdc3-/- mice aged they developed pronounced obesity at the expense of lean mass, with some individuals reaching fat mass exceeding 50% of total body weight. Combined transcriptomic and proteomic analyses of Klhdc3-/- embryonic fibroblasts revealed significant changes in protein expression with minimal impact on transcript levels, consistent with KLHDC3’s role in post-translational regulation. Among the upregulated proteins, HINT1 was identified as a novel KLHDC3 substrate, possessing a C-terminal degron motif. Protein stability assays and immunoblotting confirmed HINT1 as a direct target of KLHDC3. These findings establish a new in vivo physiological role for the DesCEND pathway and highlight KLHDC3 as a key regulator of development, survival, and adiposity in mice. ### Competing Interest Statement The authors have declared no competing interest. National Health and Medical Research Council, GNT2018098 Australian Government, MRF2007435 Government of Victoria, Operational Infrastructure Support Scheme to St Vincent’s Institute and Hudson Institute of Medical Research

The nutrient-sensitive protein kinases AMPK and mTORC1 form a fundamental negative feedback loop that governs cell growth and proliferation. mTORC1 phosphorylates α2-S345 in the AMPK αβγ heterotrimer to suppress its activity and promote cell proliferation under nutrient stress conditions. Whether AMPK contains other functional mTORC1 substrates is unknown. Using mass spectrometry, we generated precise stoichiometry profiles of phosphorylation sites across all twelve AMPK complexes expressed in proliferating human cells and identified seven sites displaying sensitivity to pharmacological mTORC1 inhibition. These included the abundantly phosphorylated residues β1-S182 and β2-S184, which were confirmed as mTORC1 substrates on purified AMPK, and four residues in the unique γ2 N-terminal extension. β-S182/184 phosphorylation was elevated in α1-containing complexes relative to α2, an effect attributed to the α-subunit serine/threonine-rich loop. Mutation of β1-S182 to non-phosphorylatable Ala had no effect on basal and ligand-stimulated AMPK activity; however, β2-S184A mutation increased nuclear AMPK activity, enhanced cell proliferation under nutrient stress and altered expression of genes implicated in glucose metabolism and Akt signalling. Our results indicate that mTORC1 directly or indirectly phosphorylates multiple AMPK residues that may contribute to metabolic rewiring in cancerous cells.

Radiometric kinase assays have been widely used due to their high sensitivity and dynamic range. The assay measures the transfer of 32Pi from [γ-32P]-ATP to specific substrates, typically synthetic peptides. The 32P-phosphorylated peptide product is captured by binding it to phosphocellulose paper, specifically P81. Unfortunately, GE Healthcare, the sole supplier of P81, has discontinued its manufacture. Recently, a replacement for P81, SVI-P cation-exchange filter paper, has become available. We have tested SVI-P in various kinase assays, including those for AMPK, Abl, CDK2, and ERK, and found that it performs comparably to P81 in capturing substrates. Additionally, a commercial kinase profiling assay using SVI-P successfully captured a range of peptide and protein substrates from 48 different protein kinases. One minor limitation of SVI-P was the higher background radioactivity; however, this can be addressed through optimisation and extended wash steps. Overall, SVI-P represents a viable alternative for radiometric kinase assays, ensuring continued reliability in both academic and industrial research settings.

RECQL4 is a member of the RecQ family of DNA helicases, essential for maintenance of genome integrity. Bi-allelic germline mutations of RECQL4 cause Rothmund-Thomson Syndrome Type 2 (RTS2), a rare inherited condition characterised by short stature, skeletal abnormalities, and elevated rates of cancers. In mice, loss or mutation of RECQL4 causes a rapid, fully penetrant bone marrow failure (BMF) phenotype. While we know RECQL4 is essential for DNA replication, how this occurs and the precise role of RECQL4 are poorly understood.To define pathways that can modify Recql4 loss of function mutations we performed a genome-wide loss of function suppressor screen in myeloid cells to identify genes, that when deleted, rescued the loss of proliferation and viability caused by RECQL4 mutation. The top candidate from the screen was Klhdc3, a substrate receptor of the Cullin2-RING E3 ubiquitin ligase (CRL2) complex. Loss of Klhdc3 restored near normal cellular viability and proliferation kinetics to cells with a RECQL4 point mutation or RECQL4 null cells, with no effect on RECQL4 wild-type cells. The re-expression of wild-type KLHDC3 in the Recql4D/D sgKlhdc3-/- myeloid cell lines caused rapid cell death. Importantly, re-expression of either a dominant-negative (DN) KLHDC3 mutant or a C-terminal deletion, both of which can still bind but not ubiquitinate their substrates, did not cause cell death, demonstrating that the rescue was specifically due to loss of KLHDC3 mediated substrate degradation.Ongoing experiments are seeking to identify the key substrates of KLHDC3 that can functionally rescue loss of RECQL4 and restore DNA replication in its absence. We are also testing if loss of KLHDC3 can prevent the BMF that characterises in vivo loss of RECQL4. These studies have identified a tractable target that can rescue and prevent phenotypes associated with the loss of RECQL4.

Excitotoxicity, a neuronal death process in neurological disorders such as stroke, is initiated by the overstimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbing synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src, protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ, undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, by identifying the neuronal proteins undergoing proteolysis during excitotoxicity, our findings offer new insights into excitotoxic neuronal death mechanisms and reveal potential neuroprotective targets for neurological disorders.

AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1) are metabolic kinases that co-ordinate nutrient supply with cell growth. AMPK negatively regulates mTORC1, and mTORC1 reciprocally phosphorylates S345/7 in both AMPK α-isoforms. We report that genetic or torin1-induced loss of α2-S345 phosphorylation relieves suppression of AMPK signaling; however, the regulatory effect does not translate to α1-S347 in HEK293T or MEF cells. Dephosphorylation of α2-S345, but not α1-S347, transiently targets AMPK to lysosomes, a cellular site for activation by LKB1. By mass spectrometry, we find that α2-S345 is basally phosphorylated at 2.5-fold higher stoichiometry than α1-S347 in HEK293T cells and, unlike α1, phosphorylation is partially retained after prolonged mTORC1 inhibition. Loss of α2-S345 phosphorylation in endogenous AMPK fails to sustain growth of MEFs under amino acid starvation conditions. These findings uncover an α2-specific mechanism by which AMPK can be activated at lysosomes in the absence of changes in cellular energy.

AMP-activated protein kinase (AMPK) is a master regulator of cellular energy homeostasis and a therapeutic target for metabolic diseases. Co/post-translational N-myristoylation of glycine-2 (Gly2) of the AMPK 0 subunit has been suggested to regulate the distribution of the kinase between the cytosol and membranes through a "myristoyl switch"mechanism. However, the relevance of AMPK myristoylation for metabolic signaling in cells and in vivo is unclear. Here, we generated knockin mice with a Gly2-to-alanine point mutation of AMPK01 (01-G2A). We demonstrate that non-myristoylated AMPK01 has reduced stability but is associated with increased kinase activity and phosphorylation of the Thr172 activation site in the AMPK a subunit. Using proximity ligation assays, we show that loss of 01 myristoylation impedes colocalization of the phosphatase PPM1A/B with AMPK in cells. Mice carrying the 01-G2A mutation have improved metabolic health with reduced adiposity, hepatic lipid accumulation, and insulin resistance under conditions of high-fat diet-induced obesity.

Excitotoxicity is a neuronal death process initiated by over-stimulation of ionotropic glutamate receptors. Although dysregulation of proteolytic signaling networks is critical for excitotoxicity, the identity of affected proteins and mechanisms by which they induce neuronal cell death remain unclear. To address this, we used quantitative N-terminomics to identify proteins modified by proteolysis in neurons undergoing excitotoxic cell death. We found that most proteolytically processed proteins in excitotoxic neurons are likely substrates of calpains, including key synaptic regulatory proteins such as CRMP2, doublecortin-like kinase I, Src tyrosine kinase and calmodulin-dependent protein kinase IIβ (CaMKIIβ). Critically, calpain-catalyzed proteolytic processing of these proteins generates stable truncated fragments with altered activities that potentially contribute to neuronal death by perturbation of synaptic organization and function. Blocking calpain-mediated proteolysis of one of these proteins, Src protected against neuronal loss in a rat model of neurotoxicity. Extrapolation of our N-terminomic results led to the discovery that CaMKIIα, an isoform of CaMKIIβ undergoes differential processing in mouse brains under physiological conditions and during ischemic stroke. In summary, our findings inform excitotoxic neuronal death mechanism and suggest potential therapeutic strategies for neuroprotection. In Brief Ameen, et al. used a proteomic method called N-terminomics to identify proteolytic events occurring in neurons during excitotoxicity. They found that most proteolytic processing is mediated by calpains, resulting in the generation of stable truncated fragments with the potential to induce synaptic dysfunction and loss, eventually leading to neuronal death. They further showed that some of these proteolytic processed proteins, such as the protein kinases Src and CaMKII, are potential targets for neuroprotection. Highlights Identification of over 300 neuronal proteins cleaved by calpains to form stable truncated fragments during excitotoxicity. The calpain cleavage sites of these proteins unveil for the first time the preferred cleavage sequences of calpains in neurons. These pathological proteolytic events potentially induce synaptic dysfunction and loss, which likely contribute to excitotoxic neuronal death. Some of the neuronal proteins proteolyzed by calpains are potential targets of neuroprotection. Graphical abstract: Pathological proteolytic events in neurons during excitotoxicity unveiled by N-terminomic analyses (A) N-terminomic and global proteomic analyses identified neo-N-terminal sites and neuronal proteins undergoing significant abundance changes during excitotoxicity. (B) Informatic analysis of the proteomic results predicted (i) the preferred sequences of proteolytic processing of neuronal proteins catalyzed by calpains during excitotoxicity and (ii) perturbation of synaptic organization and functions as the major consequence of calpain-mediated proteolytic events. (C) Validation of these predictions and further experimentations unveiled: (i) calpain-mediated cleavage of proteins associated with synaptic damage in excitotoxic neurons, (ii) a new mechanism of dysregulation of CaMKIIα and CaMKIIβ, which are key protein kinases governing synaptic dysfunctions and excitotoxic neuronal death and (iii) potential therapeutic targets such as the protein kinases Src and CaMKII for neuroprotection One Sentence Summary Proteolytic events in neurons during excitotoxicity inform neuronal death mechanism and potential therapeutic strategies for neuroprotection.

AMPK and mTORC1 are nutrient-sensitive protein kinases that form a fundamental negative feedback loop that governs cell growth and proliferation. AMPK is an αβγ heterotrimer that is directly phosphorylated by mTORC1 on α2S345 to suppress AMPK activity and promote cell proliferation under nutrient stress conditions. Using mass spectrometry, we generated precise phosphorylation profiles of all 12 AMPK complexes expressed in proliferating human cells. Of the 18 phosphorylation sites detected, seven were sensitive to pharmacological mTORC1 inhibition, including four in the AMPK γ2 isoform NH2-terminal domain and α2S377 which is located in the nucleotide-sensing motif. In particular, β1S182 and β2S184 were found to be mTORC1 substrates in vitro and near-maximally or substantially phosphorylated under cellular growth conditions. βS182 phosphorylation was elevated in α1-containing complexes, relative to α2, an effect partly attributable to the non-conserved α-subunit serine/threonine-rich loop. While mutation of β1S182 to a non-phosphorylatable Ala had no effect on basal and ligand-stimulated AMPK activity, β2-S184A mutation increased nuclear AMPK activity and enhanced cell proliferation under nutrient stress. We conclude that mTORC1 governs the nuclear activity of AMPK to regulate transcription factors involved in metabolism and cell survival during nutrient shortage. ### Competing Interest Statement The authors have declared no competing interest.

The calcium-calmodulin–dependent protein kinase kinase-2 (CaMKK2) is a key regulator of cellular and whole-body energy metabolism. It is known to be activated by increases in intracellular Ca2+, but the mechanisms by which it is inactivated are less clear. CaMKK2 inhibition protects against prostate cancer, hepatocellular carcinoma, and metabolic derangements induced by a high-fat diet; therefore, elucidating the intracellular mechanisms that inactivate CaMKK2 has important therapeutic implications. Here we show that stimulation of cAMP-dependent protein kinase A (PKA) signaling in cells inactivates CaMKK2 by phosphorylation of three conserved serine residues. PKA-dependent phosphorylation of Ser495 directly impairs calcium-calmodulin activation, whereas phosphorylation of Ser100 and Ser511 mediate recruitment of 14-3-3 adaptor proteins that hold CaMKK2 in the inactivated state by preventing dephosphorylation of phospho-Ser495. We also report the crystal structure of 14-3-3ζ bound to a synthetic diphosphorylated peptide that reveals how the canonical (Ser511) and noncanonical (Ser100) 14-3-3 consensus sites on CaMKK2 cooperate to bind 14-3-3 proteins. Our findings provide detailed molecular insights into how cAMP-PKA signaling inactivates CaMKK2 and reveals a pathway to inhibit CaMKK2 with potential for treating human diseases.

Background Eukaryotic elongation factor-2 kinase (eEF2K) is a Ca 2+ /calmodulin (CaM)-dependent protein kinase that inhibits protein synthesis. However, the role of eEF2K in cancer development was reported paradoxically and remains to be elucidated. Methods Herein, A549 cells with eEF2K depletion or overexpression by stably transfected lentivirus plasmids were used in vitro and in vivo study. MTT and colony assays were used to detect cell proliferation and growth. Extracellular glucose and lactate concentration were measured using test kit. Immunoblot and co-immunoprecipitation assays were used to examine the molecular biology changes and molecular interaction in these cells. LC-MS/MS analysis and [γ- 32 P] ATP kinase assay were used to identify combining protein and phosphorylation site. Nude mice was utilized to study the correlation of eEF2K and tumor growth in vivo. Results We demonstrated that eEF2K inhibited lung cancer cells proliferation and affected the inhibitory effects of EGFR inhibitor gefitinib. Mechanistically, we showed that eEF2K formed a complex with PKM2 and STAT3, thereby phosphorylated PKM2 at T129, leading to reduced dimerization of PKM2. Subsequently, PKM2 impeded STAT3 phosphorylation and STAT3-dependent c-Myc expression. eEF2K depletion promoted the nuclear translocation of PKM2 and increased aerobic glycolysis reflected by increased lactate secretion and glucose. Conclusions Our findings define a novel mechanism underlying the regulation of cancer cell proliferation by eEF2K independent of its role in protein synthesis, disclosing the diverse roles of eEF2K in cell biology, which lays foundation for the development of new anticancer therapeutic strategies.

Highly conserved signalling pathways controlled by mammalian target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) are central to cellular metabolism and cell proliferation1,2, and their dysregulation is implicated in the pathogenesis of major human diseases such as cancer and type 2 diabetes. AMPK pathways leading to reduced cell proliferation are well established and, in part, act through inhibition of TOR complex 1 (TORC1) activity. Here we demonstrate reciprocal regulation, specifically that TORC1 directly downregulates AMPK signalling by phosphorylating the evolutionarily conserved residue S367 in the fission yeast AMPK catalytic subunit Ssp2 and AMPK α1 S347 and α2 S345 in the mammalian homologs, which is associated with reduced phosphorylation of activation loop T172. Genetic or pharmacological inhibition of TORC1 signalling led to AMPK activation in the absence of increased AMP/ATP ratios, which under nutrient stress conditions was associated with growth limitation in both yeast and human cell cultures. Our findings reveal fundamental bidirectional regulation between two major metabolic signalling networks and uncover new opportunities for cancer treatment strategies aimed at suppressing cell proliferation in the nutrient-poor tumour microenvironment. This study establishes reciprocal regulation between the two key nutrient sensors in cells, mTORC1 and AMPK, showing that mTORC1 directly inhibits AMPK by phosphorylation at S345 in the AMPK catalytic subunit α2.

Long-chain fatty acids (LCFAs) play important roles in cellular energy metabolism, acting as both an important energy source and signalling molecules1. LCFA-CoA esters promote their own oxidation by acting as allosteric inhibitors of acetyl-CoA carboxylase, which reduces the production of malonyl-CoA and relieves inhibition of carnitine palmitoyl-transferase 1, thereby promoting LCFA-CoA transport into the mitochondria for β-oxidation2–6. Here we report a new level of regulation wherein LCFA-CoA esters per se allosterically activate AMP-activated protein kinase (AMPK) β1–containing isoforms to increase fatty acid oxidation through phosphorylation of acetyl-CoA carboxylase. Activation of AMPK by LCFA-CoA esters requires the allosteric drug and metabolite site formed between the α-subunit kinase domain and the β-subunit. β1 subunit mutations that inhibit AMPK activation by the small-molecule activator A769662, which binds to the allosteric drug and metabolite site, also inhibit activation by LCFA-CoAs. Thus, LCFA-CoA metabolites act as direct endogenous AMPK β1–selective activators and promote LCFA oxidation. Steinberg and colleagues show that long-chain fatty acyl-CoA esters are endogenous ligands for the drug-binding domain of AMPK β1–containing isoforms, and that such binding is critical for enhancement of fatty acid oxidation. These data may help explain how AMPK integrates responses to ketogenic diets, fasting or endurance exercise across distinct tissues in the absence of changes in adenine nucleotides.

Objectives Loss-of-function mutations in the gene encoding the calcium-calmodulin (Ca2+-CaM)-dependent protein kinase kinase-2 (CaMKK2) enzyme are linked to bipolar disorder. Recently, a de novo arginine to cysteine (R311C) mutation in CaMKK2 was identified from a whole exome sequencing study of bipolar patients and their unaffected parents. The aim of the present study was to determine the functional consequences of the R311C mutation on CaMKK2 activity and regulation by Ca2+-CaM. Methods The effects of the R311C mutation on CaMKK2 activity and Ca2+-CaM activation were examined using a radiolabeled adenosine triphosphate (ATP) kinase assay. We performed immunoblot analysis to determine whether the R311C mutation impacts threonine-85 (T85) autophosphorylation, an activating phosphorylation site on CaMKK2 that has also been implicated in bipolar disorder. We also expressed the R311C mutant in CaMKK2 knockout HAP1 cells and used immunoblot analysis and an MTS reduction assay to study its effects on Ca2+-dependent downstream signaling and cell viability, respectively. Results The R311C mutation maps to the conserved HRD motif within the catalytic loop of CaMKK2 and caused a marked reduction in kinase activity and Ca2+-CaM activation. The R311C mutation virtually abolished T85 autophosphorylation in response to Ca2+-CaM and exerted a dominant-negative effect in cells as it impaired the ability of wild-type CaMKK2 to initiate downstream signaling and maintain cell viability. Conclusions The highly disruptive, loss-of-function impact of the de novo R311C mutation in human CaMKK2 provides a compelling functional rationale for being considered a potential rare monogenic cause of bipolar disorder.