Group 3 innate lymphoid cells (ILC3s) are tissue-resident cells that sense environmental cues, control infections, and promote tissue homeostasis at mucosal surfaces. The metabolic sensor liver kinase B1 (LKB1) integrates intracellular stress, metabolism, and mitochondrial function to promote the development and effector functions of a variety of immune cells; however, the role of LKB1 in ILC3 function was unknown. Here, we show that LKB1 is crucial for adult ILC3 homeostasis, cytokine production, and mitochondrial function. ILC3-specific LKB1 deletion resulted in a reduced number of ILC3s and interleukin-22 (IL-22) production. LKB1-deficient ILC3s had decreased survival, mitochondrial dysfunction, cytoplasmic lipid accumulation, and altered bioenergetics. Using LKB1 downstream kinase modulators, we found that LKB1 regulation of ILC3 survival and IL-22 production requires signaling through microtubule affinity-regulating kinases (MARKs). Mechanistically, LKB1 deficiency resulted in increased reactive oxygen species (ROS) production and NFAT2 and PD-1 expression. Our work reveals that metabolic regulation of enteric ILC3 function by an LKB1-dependent signaling network is crucial for intestinal immunity and tissue homeostasis.

Solute carrier (SLC) family is essential for immune responses; nevertheless, whether and how SLCs regulate macrophage inflammation remains unclear. Here, we demonstrate that K636 acetylation mediates high abundance of SLC6A14 in inflammatory macrophages. Notably, the pharmacological inhibition or genetic modulation of SLC6A14 reduces macrophage interleukin-1β (IL-1β) secretion dependently of lower asparagine uptake and subsequently enhanced nuclear LKB1. Mechanistically, nuclear LKB1 lessens MAPK pathway-mediated NLRP3 inflammasome activation by increased histone 3 S10/28 phosphorylation-dependent cyclin O transcription. Moreover, myeloid Slc6a14 deficiency alleviates pulmonary inflammation via suppressing inflammatory macrophage responses. Overall, these results uncover a network by which SLC6A14-mediated asparagine uptake orchestrates macrophage inflammation through histone phosphorylation, providing a crucial target for modulation of inflammatory diseases.

The extracellular-activated kinases 1 & 2 (Erk1/2) are catalytically active when dually phosphorylated on a TEY motif located at the activation loop. In human patients with cardiac hypertrophy Erk1/2 are phosphorylated on yet another activation loop's residue, T207/188. Intrinsically active variants of Erk1/2, mutated at R84/65, are also (auto)phosphorylated on T207/188. It is not known whether T207/188 phosphorylation is restricted to these cases nor how it affects Erks' activity. We report that T207/188 phosphorylation is not rare, as we found that: 1) All known auto-activated Erk1/2 variants are phosphorylated on T207/188. 2) It occurs in various cell lines and mouse tissues. 3) It is extremely high in patients with skeletal muscle atrophies or myopathies. We propose that T207/188 controls the permissiveness of the TEY motif for phosphorylation because T207/188-mutated Erk1/2 and the yeast Erk/Mpk1 were efficiently dually phosphorylated when expressed in HEK293 or yeast cells, respectively. The T207/188-mutated Mpk1 was not TEY-phosphorylated in cells knocked out for MEKs, suggesting that its enhanced phosphorylation in wildtype cells is MEK-dependent. Thus, as T207/188-mutated Erk1/2 and Mpk1 recruit MEKs the role of T207/188 is to impede MEKs' ability to phosphorylate Erks. T207/188 also impedes autophosphorylation as recombinant Erk2 mutated at T188 is spontaneously autophosphorylated, although exclusively on Y185. The role of T207/188 in regulating activation loop phosphorylation may be common to most Ser/Thr kinases as 86% of them (in the human kinome) possess T207/188 orthologs, and 160 of them were already reported to be phosphorylated on this residue.

In eukaryotes, protein kinase signaling is regulated by a diverse array of post-translational modifications, including phosphorylation of Ser/Thr residues and oxidation of cysteine (Cys) residues. While regulation by activation segment phosphorylation of Ser/Thr residues is well understood, relatively little is known about how oxidation of cysteine residues modulate catalysis. In this study, we investigate redox regulation of the AMPK-related brain-selective kinases (BRSK) 1 and 2, and detail how broad catalytic activity is directly regulated through reversible oxidation and reduction of evolutionarily conserved Cys residues within the catalytic domain. We show that redox-dependent control of BRSKs is a dynamic and multilayered process involving oxidative modifications of several Cys residues, including the formation of intramolecular disulfide bonds involving a pair of Cys residues near the catalytic HRD motif and a highly conserved T-loop Cys with a BRSK-specific Cys within an unusual CPE motif at the end of the activation segment. Consistently, mutation of the CPE-Cys increases catalytic activity in vitro and drives phosphorylation of the BRSK substrate Tau in cells. Molecular modeling and molecular dynamics simulations indicate that oxidation of the CPE-Cys destabilizes a conserved salt bridge network critical for allosteric activation. The occurrence of spatially proximal Cys amino acids in diverse Ser/Thr protein kinase families suggests that disulfide-mediated control of catalytic activity may be a prevalent mechanism for regulation within the broader AMPK family.

Peutz-Jeghers syndrome (PJS), is a rare autosomal dominant hereditary disease characterized by an elevated risk of various cancers. Serine/Threonine Kinase 11 (STK11) gene is a major tumor suppressor crucial for immune evasion with and beyond tumorigenic cells. It has garnered increasing attention in the realm of oncology treatment, particularly in the context of immunotherapy development.

This study aimed to assess the suitability of polyps obtained from individuals with PJS, resulting from germline STK11 deficiency, for immunotherapy. Additionally, we seek to identify potential shared mechanisms related to immune evasion between PJS polyps and cancers. To achieve this, we examined PJS polyps alongside familial adenomatous polyposis (FAP) and sporadic polyps.

Polyps were compared among themselves and with either the paracancerous tissues or colon cancers. Pathological and gene expression profiling approaches were employed to characterize infiltrating immune cells and assess the expression of immune checkpoint genes.

Our findings revealed that PJS polyps exhibited a closer resemblance to cancer tissues than other polyps in terms of their immune microenvironment. Notably, PJS polyps displayed heightened expression of the immune checkpoint gene CD80 and an accumulation of myeloid cells, particularly myeloid-derived suppressor cells (MDSCs).

The findings suggest an immunobiological foundation for the increased cancer susceptibility in PJS patients, paving the way for potential immune therapy applications in this population. Furthermore, utilizing PJS as a model may facilitate the exploration of immune evasion mechanisms, benefiting both PJS and cancer patients.

Albumin-bound paclitaxel (nab-PTX) is an important chemotherapeutic drug used for the treatment of advanced and metastatic non-small cell lung cancer (NSCLC). One critical issue in its clinical application is the development of resistance; thus, a deeper understanding of the mechanisms underlying the primary resistance to nab-PTX is expected to help to develop effective therapeutic strategies to overcome resistance. In this study, we made an unexpected discovery that NSCLC with wild-type (WT) Liver kinase B1 (LKB1), an important tumor suppressor and upstream kinase of AMP-activated protein kinase (AMPK), is more resistant to nab-PTX than NSCLC with mutant LKB1. Mechanistically, LKB1 status does not alter the intracellular concentration of nab-PTX or affect its canonical pharmacological action in promoting microtubule polymerization. Instead, we found that LKB1 mediates AMPK activation, leading to increased expression of SLC7A11, a key amino acid transporter and intracellular level of glutathione (GSH), which then attenuates the production of reactive oxygen species (ROS) and apoptotic cell death induced by nab-PTX. On the other hand, genetic or pharmacological inhibition of AMPK in LKB1-WT NSCLC reduces the expression of SLC7A11 and intracellular GSH, increases ROS level, and eventually promotes the apoptotic cell death induced by nab-PTX in vitro. Consistently, the combination of nab-PTX with an AMPK inhibitor exhibits a greater therapeutic efficacy in LKB1-WT NSCLC using xenograft models in vivo. Taken together, our data reveal a novel role of LKB1-AMPK-SLC7A11-GSH signaling pathway in the primary resistance to nab-PTX, and provide a therapeutic strategy for the treatment of LKB1-WT NSCLC by targeting the LKB1-AMPK-SLC7A11-GSH pathway.

Stress adaptation is an evolutionarily conserved mechanism that promotes survival in the face of adverse conditions. AMP-activated protein kinase (AMPK) is a highly conserved energy-sensing kinase found in nearly all eukaryotic cells. It maintains energy homeostasis by promoting catabolism and inhibiting anabolism. In the context of cancer, the role of AMPK is controversial. It was initially touted as a tumor suppressor due to its association with Liver Kinase B1 (LKB1) (an upstream regulator and a known tumor suppressor) and ensuing growth-suppressive actions. However, emerging studies across a variety of cancer types unambiguously reveal AMPK's pro-survival and, thus, tumor-promoting activity, especially under cancer-associated stresses such as hypoxia, nutrient deprivation, oxidative stress, matrix detachment, and chemotherapy. In cancer cells, AMPK is activated in response to stress-induced increases in the levels of adenosine monophosphate (AMP), Ca2+, or reactive oxygen species (ROS). Upon activation, AMPK engages in metabolic rewiring and crosstalk with signaling molecules to mobilize resources toward survival while compromising proliferation. Here, we posit that AMPK is a non-genetic "reversible switch," allowing cancer cells' phenotype to switch to dormant, stem-like, and drug-resistant states, thereby enabling tumor cell survival, pathological progression, and therapy resistance. This review underscores the critical role of AMPK in driving cancer cell stress resilience and survival, advocating for the strategic use of AMPK inhibitors to improve cancer treatment outcomes.

Critical evaluation of computational tools for predicting variant effects is important considering their increased use in disease diagnosis and driving molecular discoveries. In the sixth edition of the Critical Assessment of Genome Interpretation (CAGI) challenge, a dataset of 28 STK11 rare variants (27 missense, 1 single amino acid deletion), identified in primary non-small cell lung cancer biopsies, was experimentally assayed to characterize computational methods from four participating teams and five publicly available tools. Predictors demonstrated a high level of performance on key evaluation metrics, measuring correlation with the assay outputs and separating loss-of-function (LoF) variants from wildtype-like (WT-like) variants. The best participant model, 3Cnet, performed competitively with well-known tools. Unique to this challenge was that the functional data was generated with both biological and technical replicates, thus allowing the assessors to realistically establish maximum predictive performance based on experimental variability. Three out of the five publicly available tools and 3Cnet approached the performance of the assay replicates in separating LoF variants from WT-like variants. Surprisingly, REVEL, an often-used model, achieved a comparable correlation with the real-valued assay output as that seen for the experimental replicates. Performing variant interpretation by combining the new functional evidence with computational and population data evidence led to 16 new variants receiving a clinically actionable classification of likely pathogenic (LP) or likely benign (LB). Overall, the STK11 challenge highlights the utility of variant effect predictors in biomedical sciences and provides encouraging results for driving research in the field of computational genome interpretation.

Maternal embryonic leucine zipper kinase (MELK), a member of the adenosine monophosphate-activated protein kinase (AMPK) protein family, has been reported to be involved in the regulation of many cellular events. The aberrant expression of MELK is associated with tumorigenesis and malignant progression of various tumors. Moreover, MELK plays an essential role in the regulation of tumor microenvironment (TME), which affects the function of immune cells and the responsiveness to immunotherapy. Currently, small molecule inhibitors targeting MELK have been developed and evaluated in clinical trials. A comprehensive understanding of MELK may provide clues and confidence for subsequent basic research and scientific transformation. In this review, we provide a comprehensive overview of the structural features, molecular biological functions, and critical roles of MELK in tumors and TME, as well as the targeted agents under development for the treatment of tumors and discuss the perspective for MELK-targeted therapies for tumors.

The term "master regulator" emerged in the 1960s and 1970s and referred to autoregulatory transcription factors that sat atop a developmental lineage. Since that time, usage of the term has increased and broadened to the point where it has lost clear meaning. Here we discuss the term "master regulator" with the goals of developing a consensus view of its definition and stimulating discussion on use of similar terms. We propose that the designation "master regulator" be reserved for transcription factors that are: 1) positioned at the top of a regulatory hierarchy specifying a cell lineage (and potentially specific cell states, such as hypoxia); and 2) sufficient to drive the transcriptional program characterizing that lineage or state. It is hoped that this piece will provide a precedent for use of additional terms applied to incompletely understood biological processes, resulting in experimentation that sheds light on such processes.

This study was conducted to investigate the relationship between STK11 genetic alterations and the outcomes of patients with metastatic EGFR mutant lung cancer. Clinical characteristics and genomic data were downloaded from the cBioPortal database. The information of the case with STK11 mutation was collected from Jiangyin People's Hospital. Univariate and multivariate analyses were performed to distinguish the prognostic differences. Outcomes were analyzed before and after propensity score matching (PSM). A patient with STK11 mutation was insensitive to osimertinib and had an extremely poor prognosis. Further analysis showed that STK11 mutations had a strong mutual exclusion with EGFR mutations. A total of 960 patients with metastatic EGFR mutant lung adenocarcinoma were enrolled in the prognostic analysis. STK11 alternation was a significant predictor of worse outcomes in univariate or multivariate analyses. After PSM, patients with STK11 alternations still exhibited poor prognoses. Cell culture experiments also showed that the loss of STK11 could contribute to the resistance of osimertinib. Functionally, STK11 mutation was positively associated with metabolic signaling pathways and immune infiltrates negatively. Through drug screening, trametinib was identified to sensitize osimertinib in the STK11-deficient cell. This study found that STK11 genetic alterations portend a worse prognosis for patients with metastatic EGFR mutant lung cancer and led to osimertinib resistance potentially. MEK inhibitors could sensitize osimertinib in the STK11-deficient cell.

Sleep/wakefulness is regulated by intracellular signaling pathways composed of protein kinases such as salt-inducible kinase 3 (Sik3). Sik3-deficiency in neurons decreases nonrapid eye movement (NREM) sleep time and electroencephalogram (EEG) delta power during NREM sleep, while Sik3Slp mice lacking a protein kinase A (PKA)-phosphorylation site, S551, show hypersomnia phenotype. In this study, we examined how a phosphomimetic mutation of the 221st threonine residue (T221E), which provides a partial (weak) constitutive activity of the kinase, affects sleep/wakefulness and circadian behavior. We also examined the effect of T221E substitution on the hypersomnia phenotype of Sik3Slp mice.

We examined the sleep/wake behavior of heterozygous and homozygous Sik3T221E mice and Sik3T221E;Slp mice using EEG and electromyogram recording. We also examined the circadian behavior of Sik3T221E mice using a running wheel under the light-dark cycle and constant darkness.

Heterozygous and homozygous Sik3T221E mice showed normal sleep time and sleep homeostatic responses. Homozygous Sik3T221E mice exhibited a delayed onset of wakefulness at the early dark phase and longer circadian periods. Sik3T221E;Slp mice showed decreased NREM sleep time and homeostatic responses compared to Sik3Slp mice.

Our results suggest that the peak onset of wakefulness is sensitive to disturbed kinase activity of SIK3, and the relationship between phosphorylation at T221 and S551 is critical for regulating sleep need.

Salt-inducible kinases (SIKs), a member of the serine/threonine protein kinase family, have recently garnered considerable research interest as one of the emerging key regulators of metabolism. The 3 SIK isoforms-SIK1, SIK2, and SIK3-exhibit diverse roles both in central and peripheral physiological processes. While early studies focused on their role in inflammation, spurring the development of SIK inhibitors for chronic inflammatory diseases currently in clinical trials, emerging evidence highlights their broader functions in metabolism. In this review, we will summarize the current state of research on the central roles of SIKs in the brain, particularly in regulating energy balance and glucose homeostasis, alongside their peripheral functions in critical metabolic tissues such as the liver, adipose tissue, and pancreas. By integrating insights into their central and peripheral roles, this review underscores the importance of SIKs in maintaining metabolic homeostasis and highlights their therapeutic potential as novel targets for metabolic disease.

Nonalcoholic fatty liver disease (NAFLD) is a progressive condition characterized by ectopic fat accumulation in the liver, for which no FAD-approved drugs currently exist. Emerging evidence highlights the role of liver kinase B1 (LKB1), a key metabolic regulator, has been proposed in NAFLD, particularly in response to excessive nutrient levels. However, few agents have been identified that can prevent the progression of nonalcoholic steatohepatitis (NASH) by targeting LKB1 deacetylation. Through comprehensive screening of our in-house chemical library, we identified tranilast, a small molecule with remarkable inhibitory efficacy against lipid deposition induced by palmitic acid/oleic acid (PO). In this study, we investigated the novel biological function and mechanism of tranilast in regulating hepatic lipid response in NAFLD, focusing on its role in LKB1 deacetylation within hepatocytes. Our findings demonstrate that tranilast effectively reduced hepatic steatosis, inflammation, and fibrosis in NASH models induced by high-fat and high-cholesterol (HFHC) and methionine choline-deficient (MCD) diets. Mechanistic analysis using RNA sequencing revealed that tranilast mitigated hepatic lipid response by promoting LKB1 deacetylation and activating AMPK. Notably, in vivo experiments showed that the beneficial effects of tranilast in MCD diet-induced NASH model were reversed by the compound C (C-C), a known AMPK inhibitor, confirming that tranilast's effects on hepatic lipid response are mediated through the AMPK pathway. In summary, tranilast inhibits hepatic lipid response in NAFLD through LKB1 deacetylation, providing robust experimental evidence for the role of LKB1 in NAFLD. These findings position tranilast as a promising therapeutic candidate for the pharmacological management of metabolic diseases.

The cAMP response element binding protein (CREB) is required for regulation of daily sleep amount, whereas gain of function of CREB-regulated transcription coactivator 1 (CRTC1) causes severe insomnia in mice. However, the physiological functions of CRTCs and their downstream target genes in the regulation of sleep amount remain unclear. Here, we use an adult brain chimeric (ABC)-expression/knock-out platform for somatic genetic analysis of sleep in adult male mice. ABC expression of constitutively active mutant CRTC1/2CA in the mouse brain neurons significantly reduces the amount of non-rapid eye movement sleep (NREMS) and/or rapid eye movement sleep (REMS). Consistent with the fact that SIK3 phosphorylates and inhibits CRTCs, ABC expression of CRTC1/2/3CA rescues the hypersomnia phenotype of Sleepy (Sik3Slp ) mice. While ABC-Crtc2KO or Crtc3KO causes no sleep phenotype, ABC-Crtc1KO or ABC expression of dominant-negative CRTC (dnCRTC) results in a modest reduction of NREMS amount accompanied with elevated NREMS delta power. Moreover, ABC expression of CRTC1CA or dnCRTC in the excitatory neurons causes bidirectional changes of NREMS/REMS amount and/or NREMS delta power. The ability of CRTC1CA to regulate sleep requires its transactivation domain and CREB-binding domain and is dependent on CREB. Furthermore, we showed that inducible ABC expression of corticotropin-releasing hormone (Crh) and brain-derived neurotrophic factor (Bdnf)-two target genes of CRTCs-significantly reduces daily sleep amount. Notably, ABC-CrhKO , but not BdnfKO , rescues the insomnia phenotype of ABC-CRTC1CA mice. Taken together, these results indicate that the CREB-CRTC1 complex regulates daily sleep amount by modulating the transcription of Crh in the mouse brain neurons.

Research into mechanisms underlying sleep traditionally relies on electrophysiology and genetics. Because sleep can only be measured on whole animals by behavioral observations and physical means, no sleep research was initiated by biochemical and chemical biological approaches. We used phosphorylation sites of kinases important for sleep as targets for biochemical and chemical biological approaches. Sleep was increased in mice carrying a threonine-to-alanine substitution at residue T469 of salt-inducible kinase 3 (SIK3). Our biochemical purification and photo-crosslinking revealed calcineurin (CaN) dephosphorylation, both in vitro and in vivo, of SIK3 at T469 and S551, but not T221. Knocking down CaN regulatory subunit reduced daily sleep by more than 5 h, exceeding all known mouse mutants. Our work uncovered a critical physiological role for CaN in sleep and pioneered biochemical purification and chemical biology as effective approaches to study sleep.

Cancer immunotherapy, alongside surgery, radiation therapy, and chemotherapy, has emerged as a key treatment modality. Immune checkpoint inhibitors (ICIs) represent a promising immunotherapy that plays a critical role in the management of various solid tumors. However, the limited efficacy of ICI monotherapy and the development of primary or secondary resistance to combination therapy remain a challenge. Consequently, identifying molecular markers for predicting ICI efficacy has become an area of active clinical research. Notably, the correlation between DNA damage repair (DDR) mechanisms and the effectiveness of ICI treatment has been established. This review outlines the two primary pathways of DDR, namely, the homologous recombination repair pathway and the mismatch repair pathway. The relationship between these key genes and ICIs has been discussed and the potential of these genes as molecular markers for predicting ICI efficacy summarized.

Amyloid-β peptide (Aβ) is a critical cause of Alzheimer's disease (AD). It is generated from amyloid precursor protein (APP) through cleavages by β-secretase and γ-secretase. γ-Secretase, which includes presenilin, is regulated by several stimuli. Tau protein has also been identified as a significant factor in AD. In particular, Tau phosphorylation is crucial for neuronal impairment, as phosphorylated Tau detaches from microtubules, leading to the formation of neurofibrillary tangles and the destabilization of the microtubule structure. This instability in microtubules damages axons and dendrites, resulting in neuronal impairment. Notably, Aβ is linked to Tau phosphorylation. Another crucial factor in AD is neuroinflammation, primarily occurring in the microglia. Microglia possess several receptors that bind with Aβ, triggering the expression and release of an inflammatory factor, although their main physiological function is to phagocytose debris and pathogens in the brain. NF-κB activation plays a major role in neuroinflammation. Additionally, the production of reactive oxygen species (ROS) in the microglia contributes to this neuroinflammation. In microglia, superoxide is produced through NADPH oxidase, specifically NOX2. Rho GTPases play an essential role in regulating various cellular processes, including cytoskeletal rearrangement, morphology changes, migration, and transcription. The typical function of Rho GTPases involves regulating actin filament formation. Neurons, with their complex processes and synapse connections, rely on cytoskeletal dynamics for structural support. Other brain cells, such as astrocytes, microglia, and oligodendrocytes, also depend on specific cytoskeletal structures to maintain their unique cellular architectures. Thus, the aberrant regulation of Rho GTPases activity can disrupt actin filaments, leading to altered cell morphology, including changes in neuronal processes and synapses, and potentially contributing to brain diseases such as AD.

Protein kinases regulate many intracellular processes, and their dysregulation causes cancers and other diseases. This review focuses on the atypical alpha-kinase 1 (ALPK1), which is activated in mammalian cells by nucleoside diphosphate heptoses (ADP-heptose, UDP-heptose, and CDP-heptose) produced by microbial pathogens but not by mammalian cells. Mutations in human ALPK1 cause ROSAH syndrome and spiradenoma, which result from an alteration in its specificity for nucleoside diphosphate heptoses, causing aberrant activation by mammalian nucleoside diphosphate sugars without microbial infection. These may be the first diseases caused by altered specificity of an enzyme for its allosteric activators and has suggested ways in which selective drugs could be developed to treat them without compromising the innate immune system.

Nuak1 (NUAK family SnF1-like kinase-1) is a serine-threonine kinase and a member of the AMPK family. Interest in Nuak1 has increased over the years due to the role it plays in several biological processes, from tumor cell invasion and proliferation to Tau stabilization. Nuak1 is expressed in many cancer cell lines and many reports describe this target as an oncogene, the inhibition of which is hypothesized to be valuable for treating various cancer types including glioma. We report here the discovery of Nuak1 inhibitors originating from HTS hit 9 with excellent selectivity and the subsequent medicinal chemistry optimization program, supported by structural information from the first crystal structures of a Nuak1 chimeric protein which provided insights into the binding modes of our compounds. These efforts yielded a nanomolar cell potent, highly selective and brain penetrant Nuak1 inhibitor UCB9386 (56) suitable for in vivo pharmacological studies for central nervous system (CNS) disorders.

Macropinocytosis has emerged as a scavenging pathway that cancer cells exploit to survive in a nutrient-deprived microenvironment. Tumor cells are especially reliant on glutamine for their survival, and in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deficiency can enhance the stimulation of macropinocytosis. Here, we identify the atypical protein kinase C (aPKC) enzymes, PKCζ and PKCι, as regulators of macropinocytosis. In normal epithelial cells, aPKCs associate with the scaffold proteins Par3 and Par6 to regulate cell polarity, affecting several targets, including the Par1 kinases and we find that each of these proteins is required for macropinocytosis. Mechanistically, aPKCs are regulated by EGFR signaling or by the transcription factor CREM to promote the Par3 relocation to microtubules, facilitating macropinocytosis in a dynein-dependent manner. Importantly, cell fitness impairment caused by aPKC depletion is rescued by the restoration of macropinocytosis and aPKCs support PDAC growth in vivo. Our findings enhance our understanding of the mechanistic underpinnings that control macropinocytic uptake in the context of metabolic stress.

Salt‑induced kinase 1 (SIK1) is a serine/threonine protein kinase that is a member of the AMP‑activated protein kinase family. SIK is catalytically activated through its phosphorylation by the upstream kinase LKB1. SIK1 has been reported to be associated with numerous types of cancer. The present review summarizes the structure, regulatory factors and inhibitors of SIK1, and also describes how SIK1 is a signal regulatory factor that fulfills connecting roles in various signal regulatory pathways. Furthermore, the anti‑inflammatory effects of SIK1 during the early stage of tumor occurrence and its different regulatory effects following tumor occurrence, are summarized, and through collating the tumor signal regulatory mechanisms in which SIK1 participates, it has been demonstrated that SIK1 acts as a necessary node in cancer signal transduction. In conclusion, SIK1 is discussed independent of the SIKs family, its research results and recent progress in oncology are summarized in detail with a focus on SIK1, and its potential as a therapeutic target is highlighted, underscoring the need for SIK1‑targeted regulatory strategies in future cancer therapy.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved regulator of energy metabolism. AMPK is sensitive to acute perturbations to cellular energy status and leverages fundamental bioenergetic pathways to maintain cellular homeostasis. AMPK is a heterotrimer comprised of αβγ-subunits that in humans are encoded by seven individual genes (isoforms α1, α2, β1, β2, γ1, γ2 and γ3), permitting formation of at least 12 different complexes with personalised biochemical fingerprints and tissue expression patterns. While the canonical activation mechanisms of AMPK are well-defined, delineation of subtle, as well as substantial, differences in the regulation of heterogenous AMPK complexes remain poorly defined.

Here, taking advantage of multidisciplinary findings, we dissect the many aspects of isoform-specific AMPK function and links to health and disease. These include, but are not limited to, allosteric activation by adenine nucleotides and small molecules, co-translational myristoylation and post-translational modifications (particularly phosphorylation), governance of subcellular localisation, and control of transcriptional networks. Finally, we delve into current debate over whether AMPK can form novel protein complexes (e.g., dimers lacking the α-subunit), altogether highlighting opportunities for future and impactful research.

Baseline activity of α1-AMPK is higher than its α2 counterpart and is more sensitive to synergistic allosteric activation by metabolites and small molecules. α2 complexes however, show a greater response to energy stress (i.e., AMP production) and appear to be better substrates for LKB1 and mTORC1 upstream. These differences may explain to some extent why in certain cancers α1 is a tumour promoter and α2 a suppressor. β1-AMPK activity is toggled by a 'myristoyl-switch' mechanism that likely precedes a series of signalling events culminating in phosphorylation by ULK1 and sensitisation to small molecules or endogenous ligands like fatty acids. β2-AMPK, not entirely beholden to this myristoyl-switch, has a greater propensity to infiltrate the nucleus, which we suspect contributes to its oncogenicity in some cancers. Last, the unique N-terminal extensions of the γ2 and γ3 isoforms are major regulatory domains of AMPK. mTORC1 may directly phosphorylate this region in γ2, although whether this is inhibitory, especially in disease states, is unclear. Conversely, γ3 complexes might be preferentially regulated by mTORC1 in response to physical exercise.

Cell cycle progression is dependent upon cell growth. Cells must therefore translate growth into a proportional signal that can be used to determine when there has been sufficient growth for cell cycle progression. In budding yeast, the protein kinase Gin4 is required for normal control of cell growth and undergoes gradual hyperphosphorylation and activation that are dependent upon growth and proportional to the extent of growth, which suggests that Gin4 could function in mechanisms that measure cell growth. However, the molecular mechanisms that drive hyperphosphorylation of Gin4 are poorly understood. Here, we used biochemical reconstitution and genetic analysis to test hypotheses for the mechanisms that drive phosphorylation of Gin4. We ruled out a previous model in which phosphatidylserine delivered to sites of plasma membrane growth binds Gin4 to initiate autophosphorylation. Instead, we show that Elm1, a homolog of the mammalian Lkb1 tumor suppressor kinase, is sufficient to promote hyperphosphorylation of Gin4 in vitro, likely via initiation of Gin4 autophosphorylation. Furthermore, we show that casein kinase I is required for growth-dependent hyperphosphorylation of Gin4 and also for normal regulation of Elm1. Together, these discoveries lead to new insight into mechanisms that link cell cycle progression to cell growth.

In eukaryotic cells, AMP-activated protein kinase (AMPK) plays a central role in responding to nutrient limitation by switching-off ATP-consuming (anabolic) pathways and switching-on ATP generating (catabolic) pathways. Over the last 30 years or so, a considerable body of research has been carried out that has provided us with a wealth of knowledge regarding the regulation and role of AMPK. Despite this, there is still much to learn about AMPK and the field remains highly active, with many groups around the world continuing to explore new roles for AMPK, providing insight into its biological function. This review series was inspired by recent AMPK-focused meetings in Scotland (2022) and Australia (2023) and draws on some of the research presented at those meetings.

NUAK1 and NUAK2 belong to a family of kinases related to the catalytic α-subunits of the AMP-activated protein kinase (AMPK) complexes. Despite canonical activation by the tumour suppressor kinase LKB1, both NUAKs exhibit a spectrum of activities that favour tumour development and progression. Here, we review similarities in structure and function of the NUAKs, their regulation at gene, transcript and protein level, and discuss their phosphorylation of specific downstream targets in the context of the signal transduction pathways and biological activities regulated by each or both NUAKs.

Late-stage epithelial ovarian cancer (EOC) involves the widespread dissemination of malignant disease throughout the peritoneal cavity, often accompanied by ascites. EOC metastasis relies on the formation of multicellular aggregates, called spheroids. Given that Liver Kinase B1 (LKB1) is required for EOC spheroid viability and LKB1 loss in EOC cells decreases tumor burden in mice, we investigated whether the LKB1 complex controls the invasive properties of human EOC spheroids. LKB1 signalling was antagonized through the CRISPR/Cas9 genetic knockout of LKB1 and/or the RNAi-dependent targeting of STE20-related kinase adaptor protein (STRAD, an LKB1 activator). EOC spheroids expressing nuclear GFP (green) or mKate2 (red) constructs were embedded in Matrigel for real-time live-cell invasion monitoring. Migration and invasion were also assessed in spheroid culture using Transwell chambers, spheroid reattachment, and mesothelial clearance assays. The loss of LKB1 and STRAD signalling decreased cell invasion through Matrigel and Transwell membranes, as well as mesothelial cell clearance. In the absence of LKB1, zymographic assays identified a loss of matrix metalloproteinase (MMP) activity, whereas spheroid reattachment assays found that coating plates with fibronectin restored their invasive potential. A three-dimensional EOC organoid model demonstrated that organoid area was greatly reduced by LKB1 loss. Overall, our data indicated that LKB1 and STRAD facilitated EOC metastasis by promoting MMP activity and fibronectin expression. Given that LKB1 and STRAD are crucial for EOC metastasis, targeting LKB1 and/or STRAD could disrupt the dissemination of EOC, making inhibitors of the LKB1 pathway an alternative therapeutic strategy for EOC patients.

A wide range of cells respond to fibroblast growth factor 2 (FGF2) by proliferation via activation of the Ras/ERK1/2 pathway. In this study, the potential involvement of salt inducible kinase SIK2) in this cascade within retinal Müller glia is explored. It is found that SIK2 phosphorylation status and activity are modulated in an FGF2-dependent manner, possibly via ERK1/2. With SIK2 downregulation, enhanced ERK1/2 activation with delayed attenuation and increased cell proliferation is observed, while SIK2 overexpression hampers FGF2-dependent ERK1/2 activation. In vitro kinase and site-directed mutagenesis studies indicate that SIK2 targets the pathway element GRB2-associated-binding protein 1 (Gab1) on Ser266. This phosphorylation event weakens Gab1 interactions with its partners growth factor receptor-bound protein 2 (Grb2) and Src homology region 2 domain containing phosphatase 2 (Shp2). Collectively, these results suggest that during FGF2-dependent proliferation process ERK1/2-mediated activation of SIK2 targets Gab1, resulting in downregulation of the Ras/ERK1/2 cascade in a feedback loop.

STK11 (serine-threonine kinase 11), also known as LKB1 (liver kinase B1) is a highly conserved master kinase that regulates cellular metabolism and polarity through a complex signaling network involving AMPK and 12 other AMPK-related kinases. Germline mutations in LKB1 have been causatively linked to Peutz-Jeghers Syndrome (PJS), an autosomal dominant hereditary disease with high cancer susceptibility. The identification of inactivating somatic mutations in LKB1 in different types of cancer further supports its tumor suppressive role. Deleterious mutations in LKB1 are frequently observed in patients with epithelial ovarian cancer. However, its inconsistent effects on tumorigenesis and cancer progression suggest that its functional impact is genetic context-dependent, requiring cooperation with other oncogenic lesions. In this review, we summarize the pleiotropic functions of LKB1 and how its altered activity in cancer cells is linked to oncogenic proliferation and growth, metastasis, metabolic reprogramming, genomic instability, and immune modulation. We also review the current mechanistic understandings of this master kinase as well as therapeutic implications with particular focus on the effects of LKB1 deficiency in ovarian cancer pathogenesis. Lastly, we discuss whether LKB1 deficiency can be exploited as an Achilles heel in ovarian cancer.

Microtubule affinity-regulating kinase 2 (MARK2) is a Ser/Thr protein kinase that regulates cell polarity and immune responses. Here, we report that Orf9b, one of the accessory proteins encoded in the SARS-CoV-2 genome, increases MARK2 activity via interaction with the autoinhibitory KAI domain. We found that co-expression of Orf9b enhances the kinase activity of MARK2 in HEK293 cells. Orf9b does not bind to or enhance the activity of the mutant form of MARK2 lacking the KA1 domain. Orf9b lowers inhibitory phosphorylation of MARK2 at T595 while mutation experiments indicate that this site is dispensable for Orf9b-mediated enhancement of MARK2 activity. Our results suggest that Orf9b enhances MARK2 activity by binding the autoinhibitory KA1 domain, which closely interacts with the kinase domain.

Tuberous sclerosis complex (TSC) is a rare multisystem disorder caused by heterozygous loss-of-function pathogenic variants in the tumour suppressor genes TSC1 and TSC2 encoding the tuberin and hamartin proteins, respectively. Both TSC1 and TSC2 inhibit the mammalian target of rapamycin (mTOR) complexes pathway, which is crucial for cell proliferation, growth, and differentiation, and is stimulated by various energy sources and hormonal signaling pathways. Pathogenic variants in TSC1 and TSC2 lead to mTORC1 hyperactivation, producing benign tumours in multiple organs, including the brain and kidneys, and drug-resistant epilepsy, a typical sign of TSC. Brain tumours, sudden unexpected death from epilepsy, and respiratory conditions are the three leading causes of morbidity and mortality. Even though several therapeutic options are available for the treatment of TSC, there is further need for a better understanding of the pathophysiological basis of the neurologic and other manifestations seen in TSC, and for novel therapeutic approaches. This review provides an overview of the main current therapies for TSC and discusses recent studies highlighting the repurposing of approved drugs and the emerging role of novel targets for future drug design.

Calcium dyshomeostasis, oxidative stress, autophagy and apoptosis are the pathogenesis of selective dopaminergic neuronal loss in Parkinson's disease (PD). Earlier, we reported that A2A R modulates IP3-dependent intracellular Ca2+ signalling via PKA. Moreover, A2A R antagonist has been reported to reduce oxidative stress and apoptosis in PD models, however intracellular Ca2+ ([Ca2+]i) dependent autophagy regulation in the 6-OHDA model of PD has not been explored. In the present study, we investigated the A2A R antagonists mediated neuroprotective effects in 6-OHDA-induced primary midbrain neuronal (PMN) cells and unilateral lesioned rat model of PD. 6-OHDA-induced oxidative stress (ROS and superoxide) and [Ca2+]i was measured using Fluo4AM, DCFDA and DHE dye respectively. Furthermore, autophagy was assessed by Western blot of p-m-TOR/mTOR, p-AMPK/AMPK, LC3I/II, Beclin and β-actin. Apoptosis was measured by Annexin V-APC-PI detection and Western blot of Bcl2, Bax, caspase3 and β-actin. Dopamine levels were measured by Dopamine ELISA kit and Western blot of tyrosine hydroxylase. Our results suggest that 6-OHDA-induced PMN cell death occurred due to the interruption of [Ca2+]i homeostasis, accompanied by activation of autophagy and apoptosis. A2A R antagonists prevented 6-OHDA-induced neuronal cell death by decreasing [Ca2+]i overload and oxidative stress. In addition, we found that A2A R antagonists upregulated mTOR phosphorylation and downregulated AMPK phosphorylation thereby reducing autophagy and apoptosis both in 6-OHDA induced PMN cells and 6-OHDA unilateral lesioned rat model. In conclusion, A2A R antagonists alleviated 6-OHDA toxicity by modulating [Ca2+]i signalling to inhibit autophagy mediated by the AMPK/mTOR pathway.

Exo70, a key exocyst complex component, is crucial for cell motility and extracellular matrix (ECM) remodeling in cancer metastasis. Despite its potential as a drug target, Exo70's post-translational modifications (PTMs) are poorly characterized. Here, we report that Exo70 is transamidated on Gln5 with Lys56 of cystatin A by transglutaminases TGM1 and TGM3, promoting tumor metastasis. This modification enhances Exo70's association with other exocyst subunits, essential for secreting matrix metalloproteinases, forming invadopodia, and delivering integrins to the leading edge. Tumor suppressor liver kinase B1 (LKB1), whose inactivation accelerates metastasis, phosphorylates TGM1 and TGM3 at Thr386 and Thr282, respectively, to inhibit their interaction with Exo70 and the following transamidation. Cantharidin, a US Food and Drug Administration (FDA)-approved drug, inhibits Exo70 transamidation to restrain tumor cell migration and invasion. Together, our findings highlight Exo70 transamidation as a key molecular mechanism and target and propose cantharidin as a therapeutic strategy with direct clinical translational value for metastatic cancers, especially those with LKB1 loss.

In Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase, which catalyzes the Mg2+-dependent dephosphorylation of PA to produce diacylglycerol, plays a key role in utilizing PA for the synthesis of the neutral lipid triacylglycerol and thereby controlling the PA-derived membrane phospholipids. The enzyme function is controlled by its subcellular location as regulated by phosphorylation and dephosphorylation. Pah1 is initially inactivated in the cytosol through phosphorylation by multiple protein kinases and then activated via its recruitment and dephosphorylation by the protein phosphatase Nem1-Spo7 at the nuclear/endoplasmic reticulum membrane where the PA phosphatase reaction occurs. Many of the protein kinases that phosphorylate Pah1 have yet to be characterized with the identification of the target residues. Here, we established Pah1 as a bona fide substrate of septin-associated Hsl1, a protein kinase involved in mitotic morphogenesis checkpoint signaling. The Hsl1 activity on Pah1 was dependent on reaction time and the amounts of protein kinase, Pah1, and ATP. The Hsl1 phosphorylation of Pah1 occurred on Ser-748 and Ser-773, and the phosphorylated protein exhibited a 5-fold reduction in PA phosphatase catalytic efficiency. Analysis of cells expressing the S748A and S773A mutant forms of Pah1 indicated that Hsl1-mediated phosphorylation of Pah1 promotes membrane phospholipid synthesis at the expense of triacylglycerol, and ensures the dependence of Pah1 function on the Nem1-Spo7 protein phosphatase. This work advances the understanding of how Hsl1 facilitates membrane phospholipid synthesis through the phosphorylation-mediated regulation of Pah1.

The gene encoding the NUAK family kinase 1 (NUAK1) is frequently amplified and its expression is upregulated, activating oncogenic signaling in various cancers. However, little is known about its role in gastric cancer (GC). We investigate the mechanistic links among NUAK1, Hedgehog signaling, and tumorigenesis in GC. NUAK1 overexpression is validated in local and public GC cohorts. Patient-derived xenograft and transgenic mouse models demonstrate that NUAK1 depletion or inhibition dramatically ameliorates gastric tumorigenesis. NUAK1 upregulates GLI1 expression by activating STAT5-mediated transcription and stabilizing GLI1 protein. NUAK1 depletion or inhibition impairs cancer cell expansion, tumor formation, and chemotherapy resistance in in vitro and in vivo models. Clinicopathological analysis confirms that upregulated NUAK1 expression correlates with poor prognosis and chemotherapy resistance in human GC. Our findings demonstrate that the signaling axis NUAK1/STAT5/GLI1 promotes cancer cell expansion and tumorigenesis and indicate that NUAK1 is an attractive therapeutic target and prognostic factor in GC.

NUAK2 is a member of the AMP-activated protein kinase (AMPK) family, which plays an essential role in cellular processes such as apoptosis, proliferation, and cell fate. Recent studies have already shown that silencing of NUAK2 blocks proliferation and promotes apoptosis of human melanoma cells and liver cancer cells. In addition, NUAK2 is involved in the development of glioblastoma via regulating the expression of cancer stem cell-related genes, and it promotes the cell cycle entry in the glioblastoma cells. However, the expression and the role of NUAK2 in the progress of peripheral nerve regeneration after injury are yet to be elucidated. We observed that NUAK2 was upregulated following distal sciatic nerve crush (SNC). Interestingly, we discovered that NUAK2 showed co-localization with S100 (Schwann cell marker). Furthermore, we found that the NUAK2 had a spatiotemporal protein expression, which was consistent with proliferating cell nuclear-antigen (PCNA). The protein level of NUAK2 and YAP was upregulated in the model of TNF-α-induced Schwann cell (SC) proliferation. Furthermore, flow cytometry analysis, CCK-8, transwell assays, and wound healing assays were all performed with the purpose of exploring the role of NUAK2 in the regulation of SC proliferation and migration. More importantly, we found that NUAK2-deficient SCs showed significantly reduced expression of Yes-associated protein (YAP). Bioinformatic analysis identified upstream regulators of NUAK2 and NUAK2-associated genes (e.g., YAP1). Finally, we investigated the recovery changes during regeneration progress through the walking track analysis. Thus, we speculated that NUAK2 was involved in biochemical and physiological responses of SCs after SNC via YAP-driven proliferation and migration, and this study determined the importance of NUAK2 as a potential target in peripheral nerve regeneration.

Critical evaluation of computational tools for predicting variant effects is important considering their increased use in disease diagnosis and driving molecular discoveries. In the sixth edition of the Critical Assessment of Genome Interpretation (CAGI) challenge, a dataset of 28 STK11 rare variants (27 missense, 1 single amino acid deletion), identified in primary non-small cell lung cancer biopsies, was experimentally assayed to characterize computational methods from four participating teams and five publicly available tools. Predictors demonstrated a high level of performance on key evaluation metrics, measuring correlation with the assay outputs and separating loss-of-function (LoF) variants from wildtype-like (WT-like) variants. The best participant model, 3Cnet, performed competitively with well-known tools. Unique to this challenge was that the functional data was generated with both biological and technical replicates, thus allowing the assessors to realistically establish maximum predictive performance based on experimental variability. Three out of the five publicly available tools and 3Cnet approached the performance of the assay replicates in separating LoF variants from WT-like variants. Surprisingly, REVEL, an often-used model, achieved a comparable correlation with the real-valued assay output as that seen for the experimental replicates. Performing variant interpretation by combining the new functional evidence with computational and population data evidence led to 16 new variants receiving a clinically actionable classification of likely pathogenic (LP) or likely benign (LB). Overall, the STK11 challenge highlights the utility of variant effect predictors in biomedical sciences and provides encouraging results for driving research in the field of computational genome interpretation.