Search

Glycogen synthase kinase 3β (GSK-3β) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component β-catenin for phosphorylation by GSK-3β and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3β bound to a phosphoprimed β-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of β-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3β with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3β active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3β and facilitate β-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution. Editor's summary: The kinase GSK-3β is a potential therapeutic target in various neurodegenerative diseases. After phosphorylation of the Wnt signaling component β-catenin by an upstream kinase (phosphopriming), β-catenin is subsequently phosphorylated by GSK-3β and targeted for degradation. Enos et al. determined structures of GSK-3β bound to phosphoprimed or phosphomimetic β-catenin and to the scaffold Axin, which showed that substrate positioning from these interactions was not sufficient for optimal catalysis. However, phosphorylation of phosphoprimed β-catenin was more rapid than that of the phosphomimetic peptide, highlighting the importance of native substrate phosphopriming for catalytic function. —John F. Foley [ABSTRACT FROM AUTHOR]

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a "dual anchor" mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors. [ABSTRACT FROM AUTHOR]

In cells, signal transduction heavily relies on the intricate regulation of protein kinases, which provide the fundamental framework for modulating most signaling pathways. Dysregulation of kinase activity has been implicated in numerous pathological conditions, particularly in cancer. The druggable nature of most kinases positions them into a focal point during the process of drug development. However, a significant challenge persists, as the role and biological function of nearly one third of human kinases remains largely unknown. Within this diverse landscape, cyclin-dependent kinases (CDKs) emerge as an intriguing molecular subgroup. In human, this kinase family encompasses 21 members, involved in several key biological processes. Remarkably, 13 of these CDKs belong to the category of understudied kinases, and only 5 having undergone broad investigation to date. This knowledge gap underscores the pressing need to delve into the study of these kinases, starting with a comprehensive review of the less-explored ones. Here, we will focus on the PCTAIRE subfamily of CDKs, which includes CDK16, CDK17, and CDK18, arguably among the most understudied CDKs members. To contextualize PCTAIREs within the spectrum of human pathophysiology, we conducted an exhaustive review of the existing literature and examined available databases. This approach resulted in an articulate depiction of these PCTAIREs, encompassing their expression patterns, 3D configurations, mechanisms of activation, and potential functions in normal tissues and in cancer. We propose that this effort offers the possibility of identifying promising areas of future research that extend from basic research to potential clinical and therapeutic applications. [ABSTRACT FROM AUTHOR]

Conformational changes of catalytically-important structural elements are a key feature of the regulation mechanisms of protein kinases and are important for dictating inhibitor binding modes and affinities. The lack of widely applicable methods for tracking kinase conformational changes in solution has hindered our understanding of kinase regulation and our ability to design conformationally selective inhibitors. Here we provide an overview of two recently developed methods that detect conformational changes of the regulatory activation loop and αC-helix of kinases and that yield complementary information about allosteric mechanisms. An intramolecular Förster resonance energy transfer-based approach provides a scalable platform for detecting and classifying structural changes in high-throughput, as well as quantifying ligand binding cooperativity, shedding light on the energetics governing allostery. The pulsed electron paramagnetic resonance technique double electron-electron resonance provides lower throughput but higher resolution information on structural changes that allows for unambiguous assignment of conformational states and quantification of population shifts. Together, these methods are shedding new light on kinase regulation and drug interactions and providing new routes for the identification of novel kinase inhibitors and allosteric modulators. [ABSTRACT FROM AUTHOR]

The study of phosphorylase kinase (PhK)-regulated glycogen metabolism has contributed to the fundamental understanding of protein phosphorylation; however, the molecular mechanism of PhK remains poorly understood. Here we present the high-resolution cryo-electron microscopy structures of human muscle PhK. The 1.3-megadalton PhK α4β4γ4δ4 hexadecamer consists of a tetramer of tetramer, wherein four αβγδ modules are connected by the central β4 scaffold. The α- and β-subunits possess glucoamylase-like domains, but exhibit no detectable enzyme activities. The α-subunit serves as a bridge between the β-subunit and the γδ subcomplex, and facilitates the γ-subunit to adopt an autoinhibited state. Ca2+-free calmodulin (δ-subunit) binds to the γ-subunit in a compact conformation. Upon binding of Ca2+, a conformational change occurs, allowing for the de-inhibition of the γ-subunit through a spring-loaded mechanism. We also reveal an ADP-binding pocket in the β-subunit, which plays a role in allosterically enhancing PhK activity. These results provide molecular insights of this important kinase complex. High-resolution cryo-EM study of human muscle phosphorylase kinase reveals its complex structure and how calcium ions activate it, offering insights into glycogen metabolism and kinase regulation. [ABSTRACT FROM AUTHOR]

Background: Muscle growth post-birth relies on muscle fiber number and size. Myofibre number, metabolic and contractile capacities are established pre-birth during prenatal myogenesis. The aim of this study was to identify genes involved in skeletal muscle development in cattle, sheep, and pigs - livestock. Results: The cattle analysis showed significant differences in 5043 genes during the 135–280 dpc period. In sheep, 444 genes differed significantly during the 70–120 dpc period. Pigs had 905 significantly different genes for the 63–91 dpc period.The biological processes and KEGG pathway enrichment results in each species individually indicated that DEGs in cattle were significantly enriched in regulation of cell proliferation, cell division, focal adhesion, ECM-receptor interaction, and signaling pathways (PI3K-Akt, PPAR, MAPK, AMPK, Ras, Rap1); in sheep - positive regulation of fibroblast proliferation, negative regulation of endothelial cell proliferation, focal adhesion, ECM-receptor interaction, insulin resistance, and signaling pathways (PI3K-Akt, HIF-1, prolactin, Rap1, PPAR); in pigs - regulation of striated muscle tissue development, collagen fibril organization, positive regulation of insulin secretion, focal adhesion, ECM-receptor interaction, and signaling pathways (PPAR, FoxO, HIF-1, AMPK). Among the DEGs common for studied animal species, 45 common genes were identified. Based on these, a protein-protein interaction network was created and three significant modules critical for skeletal muscle myogenesis were found, with the most significant module A containing four recognized hub genes - EGFR, VEGFA, CDH1, and CAV1. Using the miRWALK and TF2DNA databases, miRNAs (bta-miR-2374 and bta-miR-744) and transcription factors (CEBPB, KLF15, RELA, ZNF143, ZBTB48, and REST) associated with hub genes were detected. Analysis of GO term and KEGG pathways showed that such processes are related to myogenesis and associated with module A: positive regulation of MAP kinase activity, vascular endothelial growth factor receptor, insulin-like growth factor binding, focal adhesion, and signaling pathways (PI3K-Akt, HIF-1, Rap1, Ras, MAPK). Conclusions: The identified genes, common to the prenatal developmental period of skeletal muscle in livestock, are critical for later muscle development, including its growth by hypertrophy. They regulate valuable economic characteristics. Enhancing and breeding animals according to the recognized genes seems essential for breeders to achieve superior gains in high-quality muscle mass. [ABSTRACT FROM AUTHOR]

Ubiquitin modifications alter protein function and stability, thereby regulating cell homeostasis and viability, particularly under stress. Ischemic stroke induces protein ubiquitination at the ischemic periphery, wherein cells remain viable, however the identity of ubiquitinated proteins is unknown. Here, we employed a proteomics approach to identify these proteins in mice undergoing ischemic stroke. The data are available in a searchable web interface (https://hochrainerlab.shinyapps.io/StrokeUbiOmics/). We detected increased ubiquitination of 198 proteins, many of which localize to the postsynaptic density (PSD) of glutamatergic neurons. Among these were proteins essential for maintaining PSD architecture, such as PSD95, as well as NMDA and AMPA receptor subunits. The largest enzymatic group at the PSD with elevated post-ischemic ubiquitination were kinases, such as CaMKII, PKC, Cdk5, and Pyk2, whose aberrant activities are well-known to contribute to post-ischemic neuronal death. Concurrent phospho-proteomics revealed altered PSD-associated phosphorylation patterns, indicative of modified kinase activities following stroke. PSD-located CaMKII, PKC, and Cdk5 activities were decreased while Pyk2 activity was increased after stroke. Removal of ubiquitin restored kinase activities to pre-stroke levels, identifying ubiquitination as the responsible molecular mechanism for post-ischemic kinase regulation. These findings unveil a previously unrecognized role of ubiquitination in the regulation of essential kinases involved in ischemic injury. This study identifies postsynaptic proteins, many being kinases known to participate in ischemic injury, as major ubiquitination targets after cerebral ischemic stroke. The authors also show that ubiquitination regulates activities of these kinases. [ABSTRACT FROM AUTHOR]

The normal ageing process affects resistance arteries, leading to various functional and structural changes. Systolic hypertension is a common occurrence in human ageing, and it is associated with large artery stiffening, heightened pulsatility, small artery remodeling, and damage to critical microvascular structures. Starting from young adulthood, a progressive elevation in the mean arterial pressure is evidenced by clinical and epidemiological data as well as findings from animal models. The myogenic response, a protective mechanism for the microcirculation, may face disruptions during ageing. The dysregulation of calcium entry channels (L-type, T-type, and TRP channels), dysfunction in intracellular calcium storage and extrusion mechanisms, altered expression of potassium channels, and a change in smooth muscle calcium sensitization may contribute to the age-related dysregulation of myogenic tone. Flow-mediated vasodilation, a hallmark of endothelial function, is compromised in ageing. This endothelial dysfunction is related to increased oxidative stress, lower nitric oxide bioavailability, and a low-grade inflammatory response, further exacerbating vascular dysfunction. Resistance artery remodeling in ageing emerges as a hypertrophic response of the vessel wall that is typically observed in conjunction with outward remodeling (in normotension), or as inward hypertrophic remodeling (in hypertension). The remodeling process involves oxidative stress, inflammation, reorganization of actin cytoskeletal components, and extracellular matrix fiber proteins. Reactive oxygen species (ROS) signaling and chronic low-grade inflammation play substantial roles in age-related vascular dysfunction. Due to its role in the regulation of vascular tone and structural proteins, the RhoA/Rho-kinase pathway is an important target in age-related vascular dysfunction and diseases. Understanding the intricate interplay of these factors is crucial for developing targeted interventions to mitigate the consequences of ageing on resistance arteries and enhance the overall vascular health. [ABSTRACT FROM AUTHOR]

The regulation of protein kinases by dephosphorylation is a key mechanism that defines the activity of immune cells. A balanced process of the phosphorylation/dephosphorylation of key protein kinases by dual-specificity phosphatases is required for the realization of the antitumor immune response. The family of dual-specificity phosphatases is represented by several isoforms found in both resting and activated macrophages. The main substrate of dual-specificity phosphatases are three components of mitogen-activated kinase signaling cascades: the extracellular signal-regulated kinase ERK1/2, p38, and Janus kinase family. The results of the study of model tumor-associated macrophages supported the assumption of the crucial role of dual-specificity phosphatases in the formation and determination of the outcome of the immune response against tumor cells through the selective suppression of mitogen-activated kinase signaling cascades. Since mitogen-activated kinases mostly activate the production of pro-inflammatory mediators and the antitumor function of macrophages, the excess activity of dual-specificity phosphatases suppresses the ability of tumor-associated macrophages to activate the antitumor immune response. Nowadays, the fundamental research in tumor immunology is focused on the search for novel molecular targets to activate the antitumor immune response. However, to date, dual-specificity phosphatases received limited discussion as key targets of the immune system to activate the antitumor immune response. This review discusses the importance of dual-specificity phosphatases as key regulators of the tumor-associated macrophage function. [ABSTRACT FROM AUTHOR]

PhoP-PhoR, one of the 12 two-component systems (TCSs) that empower M. tuberculosis to sense and adapt to diverse environmental conditions, remains essential for virulence, and therefore, represents a major target to develop novel anti-TB therapies. Although both PhoP and PhoR have been structurally characterized, the signal(s) that this TCS responds to remains unknown. Here, we show that PhoR is a sensor of acidic pH/high salt conditions, which subsequently activate PhoP via phosphorylation. In keeping with this, transcriptomic data uncover that acidic pH- inducible expression of PhoP regulon is significantly inhibited in a PhoR-deleted M. tuberculosis. Strikingly, a set of PhoP regulon genes displayed a low pH-dependent activation even in the absence of PhoR, suggesting the presence of non-canonical mechanism(s) of PhoP activation. Using genome-wide interaction-based screening coupled with phosphorylation assays, we identify a non-canonical mechanism of PhoP phosphorylation by the sensor kinase PrrB. To investigate how level of P~PhoP is regulated, we discovered that in addition to its kinase activity PhoR functions as a phosphatase of P~PhoP. Our subsequent results identify the motif/residues responsible for kinase/phosphatase dual functioning of PhoR. Collectively, these results uncover that contrasting kinase and phosphatase functions of PhoR determine the homeostatic mechanism of regulation of intra-mycobacterial P~PhoP which controls the final output of the PhoP regulon. Together, these results connect PhoR to pH-dependent activation of PhoP with downstream functioning of the regulator. Thus, PhoR plays a central role in mycobacterial adaptation to low pH conditions within the host macrophage phagosome, and a PhoR-deleted M. tuberculosis remains significantly attenuated in macrophages and animal models. Author summary: Virulence-associated PhoP-PhoR of M. tuberculosis represents an attractive target to develop anti-tubercular therapy, but to date, the signal(s) that this regulatory system responds to remains unknown. We discovered that acidic pH and high salt conditions activate PhoP using a PhoR-dependent mechanism. Thus, pH inducible PhoP regulon expression is significantly impacted in a PhoR-depleted M. tuberculosis H37Rv. Our subsequent investigations reveal that homeostatic mechanism of regulation of P~PhoP relies on kinase/phosphatase dual functioning of PhoR, which determines mycobacterial pH homeostasis by controlling the final output of the PhoP regulon. Unexpectedly, global regulatory studies uncover that there can be PhoR-independent mechanism(s) of in vivo activation of PhoP. While probing for a non-canonical mechanism, we demonstrate that the sensor kinase (SK) PrrB phosphorylates PhoP. These results connect two SKs with signal-dependent activation of PhoP. [ABSTRACT FROM AUTHOR]

Ribosomal protein S6 kinases belong to a family of highly conserved enzymes in eukaryotes that regulate cell growth, proliferation, survival, and the stress response. It is well established that the activation and downstream signalling of p70S6Ks involve multiple phosphorylation events by key regulators of cell growth, survival, and energy metabolism. Here, we report for the first time the covalent modification of p70S6K1 by coenzyme A (CoA) in response to oxidative stress, which regulates its kinase activity. The site of CoA binding (CoAlation) was mapped by mass spectrometry to cysteine 217 (Cys217), located in the kinase activation loop and only one amino acid away from the tripeptide DFG motif, which facilitates ATP-binding. The CoAlation of recombinant p70S6K1 was demonstrated in vitro and was shown to inhibit its kinase activity. Our molecular docking and dynamics analysis revealed the most likely mode for CoA binding to p70S6K1. This mechanism involves the non-covalent binding of the CoA ADP moiety to the p70S6K1 nucleotide-binding pocket, positioning the CoA thiol group in close proximity to form a covalent bond with the surface-exposed Cys217 residue. These findings support a “dual anchor” mechanism for protein kinase inhibition by CoAlation in cellular response to oxidative stress. Furthermore, the inhibition of S6K1 by CoAlation may open new avenues for developing novel inhibitors.

Full-length Bruton’s tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3–SH2–kinase core. Precisely how the BTK N-terminal domains (the Pleckstrin homology/Tec homology [PHTH] domain and proline-rich regions [PRR] contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3–SH2–kinase core with no electron density visible for the PHTH–PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH–PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3–SH2–kinase core. On the way to activation, disassembly of the SH3–SH2–kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.

Simple Summary: Understanding the changes that occur in proteins as they perform their cellular function is critical to understanding our biology. Here, we investigated PKA, a protein partially responsible for processing signals that allows cells to respond to their environment, as well as how the shape of this protein changes as it performs its role by chemically altering other proteins. We analyzed a repertoire of publicly available crystal structures of PKA and identified regions of PKA that change shape based on its physical interactions with its ligands, or other signaling proteins. Studies of these shape changes allow researchers to better explore PKA for making advances in improving existing pharmacological therapies and gaining a deeper understanding of disease biology. Cyclic-AMP-dependent protein kinase A (PKA) is a critical enzyme involved in various signaling pathways that plays a crucial role in regulating cellular processes including metabolism, gene transcription, cell proliferation, and differentiation. In this study, the mechanisms of allostery in PKA were investigated by analyzing the vast repertoire of crystal structures available in the RCSB database. From existing structures of murine and human PKA, we elucidated the conformational ensembles and protein dynamics that are altered in a ligand-dependent manner. Distance metrics to analyze conformations of the G-loop were proposed to delineate different states of PKA and were compared to existing structural metrics. Furthermore, ligand-dependent flexibility was investigated through normalized B′-factors to better understand the inherent dynamics in PKA. The presented study provides a contemporary approach to traditional methods in engaging the use of crystal structures for understanding protein dynamics. Importantly, our studies provide a deeper understanding into the conformational ensemble of PKA as the enzyme progresses through its catalytic cycle. These studies provide insights into kinase regulation that can be applied to both PKA individually and protein kinases as a class. [ABSTRACT FROM AUTHOR]

Background: The therapeutic properties of phytochemicals found in medicinal plants, as well as in dietary plants and herbs, have had a significant impact on the development of several herbal remedies for a range of ailments. Amalaki churna, a well-known ayurvedic composition, has been traditionally used to treat a number of disorders and has been shown to have anti-inflammatory, anti-diabetic, and anti-cancer properties, among others. The presence of large number of phytochemicals in this ayurvedic formulation seems to be responsible for its diverse actions. However, little is known about the precise molecular interaction between phytochemicals and their protein targets. This study puts forward a methodical and structured approach to determine the targets for the phytochemicals recovered from the aqueous extract of Amalaki churna and to establish the phytochemical-protein cross talk occurring at the molecular level. Materials and Methods: Various phytochemicals were identified from AMCAE using spectroscopic techniques such as HPLC, NMR (¹H, 13C, and 2D NMR), and UPLC-MS analysis. The phytochemical-protein cross talk was discovered utilizing network-based pharmacology, with phytochemicals serving as core nodes. Seven hub proteins were considered most important from the string network of the protein targets using variable parameters such as Degree Centrality (DCY), Closeness Centrality (CCY) and Betweenness Centrality (BCY). Molecular docking and molecular dynamic simulations were carried out to ascertain the stability of the best docked phytochemical-protein complexes. The in silico findings were further validated using c-Src kinase protein as a model. Results: In this study, we have fractionated and identified 8 phytochemicals from Amalaki churna. We were able to find a total of 387 protein targets from the Drug bank and Binding DB by using structural similarity search. A network with 273 nodes and 417 edges that was acquired from Drug Bank demonstrated the substantial cross talk between phytochemicals and their protein targets. In a similar manner, phytochemical similarity search from Binding data bank resulted in a protein-phytochemical network with 143 nodes and 275 edges. Further, seven hub proteins with most interconnectedness were selected as the top most protein targets of the phytochemicals. Molecular modelling and docking experiments show that phytochemicals fit well into the target proteins' active sites. Molecular dynamic studies were used to further demonstrate the durability and stability of the protein phytochemical complexes. Phytochemicals were also shown to alter cell cycle regulation, disrupt cell survival pathways, limit cell migratory capacity, and induce apoptosis. The disruption of many cellular pathways was demonstrated using c-Src kinase as a model, in which down-regulation of the tyrosine kinase by phytochemicals led in the disruption of its downstream proteins such as Akt1, cyclin D1 and vimentin. Conclusion: Network analysis, followed by molecular docking, molecular dynamics modelling, and in vitro studies, clearly underline the importance of protein-phytochemical cross-talk. [ABSTRACT FROM AUTHOR]

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions. [ABSTRACT FROM AUTHOR]

Background. Aberrant protein kinase regulation leading to abnormal substrate phosphorylation is associated with several human diseases. Despite the promise of therapies targeting kinases, many human kinases remain understudied. Most existing computational tools predicting phosphorylation cover less than 50% of known human kinases. They utilize local feature selection based on protein sequences, motifs, domains, structures, and/or functions, and do not consider the heterogeneous relationships of the proteins. In this work, we present KSFinder, a tool that predicts kinase-substrate links by capturing the inherent association of proteins in a network comprising 85% of the known human kinases. We also postulate the potential role of two understudied kinases based on their substrate predictions from KSFinder. Methods. KSFinder learns the semantic relationships in a phosphoproteome knowledge graph using a knowledge graph embedding algorithm and represents the nodes in low- dimensional vectors. A multilayer perceptron (MLP) classifier is trained to discern kinase-substrate links using the embedded vectors. KSFinder uses a strategic negative generation approach that eliminates biases in entity representation and combines data from experimentally validated non-interacting protein pairs, proteins from different subcellular locations, and random sampling. We assess KSFinder's generalization capability on four different datasets and compare its performance with other state- of-the-art prediction models. We employ KSFinder to predict substrates of 68 "dark'' kinases considered understudied by the Illuminating the Druggable Genome program and use our text-mining tool, RLIMS-P along with manual curation, to search for literature evidence for the predictions. In a case study, we performed functional enrichment analysis for two dark kinases - HIPK3 and CAMKK1 using their predicted substrates. Results. KSFinder shows improved performance over other kinase-substrate prediction models and generalized prediction ability on different datasets. We identified literature evidence for 17 novel predictions involving an understudied kinase. All of these 17 predictions had a probability score ≥ 0:7 (nine at > 0.9, six at 0.8-0.9, and two at 0.7-0.8). The evaluation of 93,593 negative predictions (probability ≤0.3) identified four false negatives. The top enriched biological processes of HIPK3 substrates relate to the regulation of extracellular matrix and epigenetic gene expression, while CAMKK1 substrates include lipid storage regulation and glucose homeostasis. Conclusions. KSFinder outperforms the current kinase-substrate prediction tools with higher kinase coverage. The strategically developed negatives provide a superior generalization ability for KSFinder. We predicted substrates of 432 kinases, 68 of which are understudied, and hypothesized the potential functions of two dark kinases using their predicted substrates. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid (LPA) signalling is essential for maintaining germ cell viability during mouse spermatogenesis; however, its role in human spermatozoa is unknown. We previously demonstrated that peroxiredoxin 6 (PRDX6) calcium-independent phospholipase A2 (iPLA2) releases lysophospholipids such as LPA or arachidonic acid (AA) and that inhibiting PRDX6 iPLA2 activity impairs sperm cell viability. The exogenous addition of LPA bypassed the inhibition of PRDX6 iPLA2 activity and maintained the active phosphoinositide 3-kinase (PI3K)/AKT pathway. Here, we aimed to study PI3K/AKT pathway regulation via LPA signalling and protein kinases in maintaining sperm viability. The localization of LPARs in human spermatozoa was determined using immunocytochemistry, and P-PI3K and P-AKT substrate phosphorylations via immunoblotting. Sperm viability was determined using the hypo-osmotic swelling test. LPAR1, 3, 5 and 6 were located on the sperm plasma membrane. The inhibition of LPAR1-3 with Ki16425 promoted the impairment of sperm viability and decreased the phosphorylation of PI3K AKT substrates. Inhibitors of PKC, receptor-type PTK and PLC impaired sperm viability and the PI3K/AKT pathway. Adding 1-oleoyl-2-acetyl-snglycerol (OAG), a cell-permeable analog of diacylglycerol (DAG), prevented the loss of sperm viability and maintained the phosphorylation of PI3K. In conclusion, human sperm viability is supported by LPAR signalling and regulated by PLC, PKC and RT-PTK by maintaining phosphorylation levels of PI3K and AKT substrates. [ABSTRACT FROM AUTHOR]

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions.

Phase-separated condensates can act as activation platforms for kinases that can sense the molecular features of condensates (e.g., polyubiquitin). Liquid–liquid phase separation (LLPS) compartmentalizes kinases, substrates, and cofactors to promote substrate selectivity and spatiotemporal control of signaling events. Some kinases can act as scaffolds for phase separation, whereas others act as clients that depend on the LLPS properties of scaffolds. Tyrosine phosphorylation can initiate LLPS by recruiting SH2 domain-containing proteins, which in turn recruit other proteins into the forming condensate. Oncogenic kinase fusions often include oligomerization domains and intrinsically disordered regions of the fusion partner, which may promote LLPS-mediated activation of the kinase fusion. Liquid–liquid phase separation (LLPS) is emerging as a mechanism of spatiotemporal regulation that could answer long-standing questions about how order is achieved in biochemical signaling. In this review we discuss how LLPS orchestrates kinase signaling, either by creating condensate structures that are sensed by kinases or by direct LLPS of kinases, cofactors, and substrates – thereby acting as a mechanism to compartmentalize kinase–substrate relationships, and in some cases also sequestering the kinase away from inhibitory factors. We also examine the possibility that selective pressure promotes genomic rearrangements that fuse pro-growth kinases to LLPS-prone protein sequences, which in turn drives aberrant kinase activation through LLPS. [ABSTRACT FROM AUTHOR]

Excessive hepatic gluconeogenesis partially accounts for the occurrence of type 2 diabetes mellitus. Serum- and glucocorticoid inducible-kinase 1 (SGK1) is linked to the development of metabolic syndrome, such as obesity, hypertension, and hyperglycemia. However, the regulatory role of SGK1 in glucose metabolism of liver remains uncertain. Our microarray analysis showed that SGK1 expression was strongly induced by 8-Br-cAMP and suppressed by metformin in primary mouse hepatocytes. Hepatic SGK1 expression was markedly increased in obese and diabetic mice. Metformin treatment decreased hepatic SGK1 expression levels in db/db mice. Inhibition or knockdown of SGK1 suppressed gluconeogenesis in primary mouse hepatocytes, with decreased expressions of key gluconeogenic genes. Furthermore, SGK1 silencing in liver decreased hepatic glucose production in C57BL/6 mice. Knockdown of SGK1 had no impact on CREB phosphorylation level but increased AKT and FoxO1 phosphorylation levels with decreased expressions of transcription factors including FoxO1 and hepatocyte nuclear factors. Adenovirus-mediated expression of dominant-negative AMPK antagonized metformin-suppressed SGK1 expression induced by 8-Br-cAMP. These findings demonstrate that hepatic specific silence of SGK1 might be a potential therapeutic strategy for type 2 diabetes. [ABSTRACT FROM AUTHOR]

Phosphorylation of proteins is a ubiquitous mechanism of regulating their function, localization, or activity. Protein kinases, enzymes that use ATP to phosphorylate protein substrates are, therefore, powerful signal transducers in eukaryotic cells. The mechanism of phosphoryl-transfer is universally conserved among protein kinases, which necessitates the tight regulation of kinase activity for the orchestration of cellular processes with high spatial and temporal fidelity. In response to a stimulus, many kinases enhance their own activity by autophosphorylating a conserved amino acid in their activation loop, but precisely how this reaction is performed is controversial. Classically, kinases that autophosphorylate their activation loop are thought to perform the reaction in trans, mediated by transient dimerization of their kinase domains. However, motivated by the recently discovered regulation mechanism of activation loop cis-autophosphorylation by a kinase that is autoinhibited in trans, we here review the various mechanisms of autoregulation that have been proposed. We provide a framework for critically evaluating biochemical, kinetic, and structural evidence for protein kinase dimerization and autophosphorylation, and share some thoughts on the implications of these mechanisms within physiological signaling networks. [ABSTRACT FROM AUTHOR]

Hibernation consists of alternating torpor–arousal phases, during which animals cope with repetitive hypothermia and ischaemia-reperfusion. Due to limited transcriptomic and methylomic information for facultative hibernators, we here conducted RNA and whole-genome bisulfide sequencing in liver of hibernating Syrian hamster (Mesocricetus auratus). Gene ontology analysis was performed on 844 differentially expressed genes and confirmed the shift in metabolic fuel utilization, inhibition of RNA transcription and cell cycle regulation as found in seasonal hibernators. Additionally, we showed a so far unreported suppression of mitogen-activated protein kinase (MAPK) and protein phosphatase 1 pathways during torpor. Notably, hibernating hamsters showed upregulation of MAPK inhibitors (dual-specificity phosphatases and sproutys) and reduced levels of MAPK-induced transcription factors (TFs). Promoter methylation was found to modulate the expression of genes targeted by these TFs. In conclusion, we document gene regulation between hibernation phases, which may aid the identification of pathways and targets to prevent organ damage in transplantation or ischaemia-reperfusion. [ABSTRACT FROM AUTHOR]

Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named crk10-A397T in which alanine 397 has been replaced by a threonine in the αC helix of the kinase domain of CRK10, known to be a crucial regulatory module in mammalian kinases. The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10 , the first such mutant to have been identified for a CRK in Arabidopsis. [ABSTRACT FROM AUTHOR]

Maintaining normal cellular behavior is essential for the survival of organisms. One of the main mechanisms to control cellular behavior is protein phosphorylation. The process of protein phosphorylation is reversible under the regulation of protein kinases and protein phosphatases. The importance of kinases in numerous cellular processes has been well recognized. In recent years, protein phosphatases have also been demonstrated to function actively and specifically in various cellular processes and thus have gained more and more attention from researchers. In the animal kingdom, regeneration frequently occurs to replace or repair damaged or missing tissues. Emerging evidence has revealed that protein phosphatases are crucial for organ regeneration. In this review, after providing a brief overview of the classification of protein phosphatases and their functions in several representative developmental processes, we highlight the critical roles that protein phosphatases play in organ regeneration by summarizing the most recent research on the function and underlying mechanism of protein phosphatase in the regeneration of the liver, bone, neuron, and heart in vertebrates. [ABSTRACT FROM AUTHOR]

Heart failure is associated with atrioventricular (AV) node dysfunction, and AV node dysfunction in the setting of heart failure is associated with an increased risk of mortality and heart failure hospitalisation. This study aims to understand the causes of AV node dysfunction in heart failure by studying changes in the whole nodal transcriptome. The mouse transverse aortic constriction model of pressure overload-induced heart failure was studied; functional changes were assessed using electrocardiography and echocardiography and the transcriptome of the AV node was quantified using RNAseq. Heart failure was associated with a significant increase in the PR interval, indicating a slowing of AV node conduction and AV node dysfunction, and significant changes in 3,077 transcripts (5.6% of the transcriptome). Many systems were affected: transcripts supporting AV node conduction were downregulated and there were changes in transcripts identified by GWAS as determinants of the PR interval. In addition, there was evidence of remodelling of the sarcomere, a shift from fatty acid to glucose metabolism, remodelling of the extracellular matrix, and remodelling of the transcription and translation machinery. There was evidence of the causes of this widespread remodelling of the AV node: evidence of dysregulation of multiple intracellular signalling pathways, dysregulation of 109 protein kinases and 148 transcription factors, and an immune response with a proliferation of neutrophils, monocytes, macrophages and B lymphocytes and a dysregulation of 40 cytokines. In conclusion, inflammation and a widespread transcriptional remodelling of the AV node underlies AV node dysfunction in heart failure. [ABSTRACT FROM AUTHOR]

Protein tyrosine kinases (PTKs) are a large enzyme family that regulates many cellular processes. The key to their broad role in signaling is their tunable substrate specificity and regulatory mechanisms that allow each to respond to appropriate regulatory signals and phosphorylate the correct physiological protein substrates. Thus, in addition to the general PTK catalytic platform, each PTK acquires unique structural motifs that confer a unique combination of catalytic and regulatory properties. Understanding the structural basis for these properties is essential for understanding and manipulating the PTK-based signaling networks in normal and cancer cells. C-terminal Src kinase (Csk) and its homolog, Csk-homologous kinase (Chk), phosphorylate Src family kinases on a C-terminal Tyr residue and negatively regulate their kinase activity. While this regulatory function is biologically essential, Csk and Chk have also been excellent model PTKs for dissecting the structural basis of PTK catalysis and regulation. In this article, we review the structure-function studies of Csk and Chk that shed light on the regulatory and catalytic mechanisms of protein tyrosine kinases in general.

Accompanying the process of growth and development, plants are exposed to ever-changing environments, which consequently trigger abiotic or biotic stress responses. The large protein family known as receptor-like protein kinases (RLKs) is involved in the regulation of plant growth and development, as well as in the response to various stresses. Understanding the biological function and molecular mechanism of RLKs is helpful for crop breeding. Research on the role and mechanism of RLKs has recently received considerable attention regarding the balance between plant growth and environmental adaptability. In this paper, we systematically review the classification of RLKs, the regulatory roles of RLKs in plant development (meristem activity, leaf morphology and reproduction) and in stress responses (disease resistance and environmental adaptation). This review focuses on recent findings revealing that RLKs simultaneously regulate plant growth and stress adaptation, which may pave the way for the better understanding of their function in crop improvement. Although the exact crosstalk between growth constraint and plant adaptation remains elusive, a profound study on the adaptive mechanisms for decoupling the developmental processes would be a promising direction for the future research. [ABSTRACT FROM AUTHOR]

Chronic kidney disease (CKD) is a common clinical disease with an increasing incidence, affecting 10 to 15% of the world's population. Hypertension is the most common and modifiable risk factor for preventing adverse cardiovascular outcomes in patients with CKD. A survey from developed countries shows that 47% of hypertensive patients over the age of 20 have uncontrolled blood pressure (BP), and the control rate is even lower in developing countries. CKD is both a common cause of uncontrolled hypertension and a risk factor for altered sequelae. In particular, studies have demonstrated that abnormal blood-pressure patterns in CKD patients, such as non-dipping-blood-pressure patterns, are associated with a significantly increased risk of cardiovascular (CV) disease. The distal convoluted tubule (DCT) is a region of the kidney, and although only 5–10% of the sodium (Na+) filtered by the glomerulus is reabsorbed by DCT, most studies agree that Na-Cl cotransporter (NCC) in human, rabbit, mouse, and rat kidneys is the most important route of sodium reabsorption across the DCT for maintaining the homeostasis of sodium. The regulation of NCC involves a large and complex network structure, including certain physiological factors, kinases, scaffold proteins, transporter phosphorylation, and other aspects. This regulation network includes various levels. Naturally, cross-talk between the components of this system must occur in order to relay the important signals to the transporter to play its role. Knowledge of the mechanisms regulating NCC activation is critical for understanding and treating hypertension and CKD. Previous studies from our laboratory have investigated the mechanisms through which NCC is activated in several different models. In the following sections, we review the literature on the mechanisms of NCC in relation to hypertension in CKD. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is a WHO grade 4 tumor and is the most malignant form of glioma. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a mitochondrial enzyme involved in folate metabolism, has been reported to be highly expressed in several human tumors. However, little is known about the role of MTHFD2 in GBM. In this study, we aimed to explore the biological functions of MTHFD2 in GBM and identify the associated mechanisms. We performed experiments such as immunohistochemistry, Western blot, and transwell assays and found that MTHFD2 expression was lower in high-grade glioma than in low-grade glioma. Furthermore, a high expression of MTHFD2 was associated with a favorable prognosis, and MTHFD2 levels showed good prognostic accuracy for glioma patients. The overexpression of MTHFD2 could inhibit the migration, invasion, and proliferation of GBM cells, whereas its knockdown induced the opposite effect. Mechanistically, our findings revealed that MTHFD2 suppressed GBM progression independent of its enzymatic activity, likely by inducing cytoskeletal remodeling through the regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, thereby influencing GBM malignance. Collectively, these findings uncover a potential tumor-suppressor role of MTHFD2 in GBM cells. MTHFD2 may act as a promising diagnostic and therapeutic target for GBM treatment. [ABSTRACT FROM AUTHOR]

As a widely prescribed anti-diabetic drug, metformin has been receiving novel attention for its analgesic potential. In the study of the complex etiology of neuropathic pain (NeP), male and female individuals exhibit quite different responses characterized by higher pain sensitivity and greater NeP incidence in women. This "gender gap" in our knowledge of sex differences in pain processing strongly limits the sex-oriented treatment of patients suffering from NeP. Besides, the current investigation of the analgesic potential of metformin has not addressed the "gender gap" problem. Hence, this study focuses on metformin and sex-dependent analgesia in a murine model of NeP induced by chronic constriction injury of the sciatic nerve. We investigated sexual dimorphism in signaling pathways involved by 7 days of metformin administration, such as changes in AMP-activated protein kinase and the positive regulation of autophagy machinery, discovering that metformin affected in a sexually dimorphic manner the immunological and inflammatory response to nerve lesion. These effects were complemented by morphological and adaptive changes occurring after peripheral nerve injury. Altogether these data can contribute to explaining a number of potential mechanisms responsible for the complete recovery from NeP found in male mice, as opposed to the failure of long-lasting recovery in female animals. [ABSTRACT FROM AUTHOR]

Protein kinases are dynamic enzymes that display complex regulatory mechanisms. Although they possess a structurally conserved catalytic domain, significant conformational dynamics are evident both within a single kinase and across different kinases in the kinome. Here, we highlight methods for exploring this conformational space and its dynamics using kinase domains from ABL1 (Abelson kinase), PKA (protein kinase A), AurA (Aurora A), and PYK2 (proline-rich tyrosine kinase 2) as examples. Such experimental approaches combined with AI-driven methods, such as AlphaFold, will yield discoveries about kinase regulation, the catalytic process, substrate specificity, the effect of disease-associated mutations, as well as new opportunities for structure-based drug design. [Display omitted] • Protein kinases are dynamic with many functionally relevant states. • Multiple methods provide insights into kinase dynamics and conformations. • NMR is a powerful approach for determining the conformational landscape of kinases. • Computational and AI methods provide testable predictions about kinase conformations. • A complete kinase landscape is a powerful tool for understanding kinase function. [ABSTRACT FROM AUTHOR]

Background: Hypoxic-ischemic encephalopathy (HIE) is a major cause of neonatal morbidity and mortality worldwide. While the application of therapeutic hypothermia has improved neurodevelopmental outcomes for some survivors of HIE, this lone treatment option is only available to a subset of affected neonates. Src kinase, an enzyme central to the apoptotic cascade, is a potential pharmacologic target to preserve typical brain development after HIE. Here, we present evidence of the neuroprotective effects of targeting Src kinase in preclinical models of HIE. Methods: We performed a comprehensive literature search using the National Library of Medicine's MEDLINE database to compile studies examining the impact of Src kinase regulation on neurodevelopment in animal models. Each eligible study was assessed for bias. Results: Twenty studies met the inclusion criteria, and most studies had an intermediate risk for bias. Together, these studies showed that targeting Src kinase resulted in a neuroprotective effect as assessed by neuropathology, enzymatic activity, and neurobehavioral outcomes. Conclusion: Src kinase is an effective neuroprotective target in the setting of acute hypoxic injury. Src kinase inhibition triggers multiple signaling pathways of the sub-membranous focal adhesions and the nucleus, resulting in modulation of calcium signaling and prevention of cell death. Despite the significant heterogeneity of the research studies that we examined, the available evidence can serve as proof-of-concept for further studies on this promising therapeutic strategy. [ABSTRACT FROM AUTHOR]

Cytokinesis, the conclusive act of cell division, allows cytoplasmic organelles and chromosomes to be faithfully partitioned between two daughter cells. In animal organisms, its accurate regulation is a fundamental task for normal development and for preventing aneuploidy. Cytokinesis failures produce genetically unstable tetraploid cells and ultimately result in chromosome instability, a hallmark of cancer cells. In animal cells, the assembly and constriction of an actomyosin ring drive cleavage furrow ingression, resulting in the formation of a cytoplasmic intercellular bridge, which is severed during abscission, the final event of cytokinesis. Kinase-mediated phosphorylation is a crucial process to orchestrate the spatio-temporal regulation of the different stages of cytokinesis. Several kinases have been described in the literature, such as cyclin-dependent kinase, polo-like kinase 1, and Aurora B, regulating both furrow ingression and/or abscission. However, others exist, with well-established roles in cell-cycle progression but whose specific role in cytokinesis has been poorly investigated, leading to considering these kinases as "minor" actors in this process. Yet, they deserve additional attention, as they might disclose unexpected routes of cell division regulation. Here, we summarize the role of multifunctional kinases in cytokinesis with a special focus on those with a still scarcely defined function during cell cleavage. Moreover, we discuss their implication in cancer. [ABSTRACT FROM AUTHOR]

In mammals, ovarian function is dependent on the primordial follicle pool and the rate of primordial follicle activation determines a female's reproductive lifespan. Ovarian ageing is characterised by chronic low‐grade inflammation with accelerated depletion of primordial follicles and deterioration of oocyte quality. Macrophages (Mφs) play critical roles in multiple aspects of ovarian functions; however, it remains unclear whether Mφs modulate the primordial follicle pool and what is their role in ovarian ageing. Here, by using super‐ or naturally ovulated mouse models, we demonstrated for the first time that ovulation‐induced local inflammation acted as the driver for selective activation of surrounding primordial follicles in each estrous cycle. This finding was related to infiltrating Mφs in ovulatory follicles and the dynamic changes of the two polarised Mφs, M1 and M2 Mφs, during the process. Further studies on newborn ovaries cocultured with different subtypes of Mφs demonstrated the stimulatory effect of M1 Mφs on primordial follicles, whereas M2 Mφs maintained follicles in a dormant state. The underlying mechanism was associated with the differential regulation of the Phosphatidylinositol 3‐kinase/Mechanistic target of rapamycin (PI3K/mTOR) signaling pathway through secreted extracellular vesicles (EVs) and the containing specific miRNAs miR‐107 (M1 Mφs) and miR‐99a‐5p (M2 Mφs). In aged mice, the intravenous injection of M2‐EVs improved ovarian function and ameliorated the inflammatory microenvironment within the ovary. Thus, based on the anti‐ageing effects of M2 Mφs in old mice, M2‐EVs may represent a new approach to improve inflammation‐related infertility in women. [ABSTRACT FROM AUTHOR]

The Nef protein produced by the viruses HIV-1 and SIV drives efficient viral replication partially by inducing constitutive activation of host cell tyrosine kinases, including members of the Src and Tec families. Here, we uncovered the mechanism by which both HIV-1 and SIV Nef enhanced the activity of the Tec family kinase Btk in vitro and in cells. A Nef mutant that could not bind to the SH3 domain of Src family kinases activated Btk to the same extent as did wild-type Nef, demonstrating that Nef activated Src and Tec family kinases by distinct mechanisms. The Btk SH3-SH2 region formed a homodimer requiring the CD loop in the SH2 domain, which was stabilized by the binding of Nef homodimers. Alanine substitution of Pro327 in the CD loop of the Btk SH2 domain destabilized SH3-SH2 dimers, abolished the interaction with Nef, and prevented activation by Nef in vitro. In cells, Nef stabilized and activated wild-type but not P327A Btk homodimers at the plasma membrane. These data reveal that the interaction with Nef stabilizes Btk dimers through the SH3-SH2 interface to promote kinase activity and show that the HIV-1 Nef protein evolved distinct mechanisms to activate Src and Tec family tyrosine kinases to enhance viral replication. How HIV hijacks Btk: Viruses ensure successful replication, in part, by subverting host signaling pathways. Using biochemical and cellular assays, Aryal et al. uncovered how the HIV-1 protein Nef activates the Tec family kinase Btk, which is critical for HIV-1 replication in myeloid cells. Nef homodimers enabled autophosphorylation of Btk by stabilizing Btk homodimers formed through intermolecular interactions of the SH3 and SH2 domains mediated by Pro327 in the SH2 domain of Btk. This mechanism was distinct from how Btk is normally activated in cells and from how Nef activates the Src kinase family, suggesting a previously unrecognized mode of Tec family kinase regulation and indicating that Nef can induce tyrosine kinase activation through independent mechanisms. [ABSTRACT FROM AUTHOR]

The neurodegenerative disease spinocerebellar ataxia type 1 (SCA1) is known to lead to the progressive degeneration of specific neuronal populations, including cerebellar Purkinje cells (PCs), brainstem cranial nerve nuclei and inferior olive nuclei, and spinocerebellar tracts. The disease-causing protein ataxin-1 is fairly ubiquitously expressed throughout the brain and spinal cord, but most studies have primarily focused on the role of ataxin-1 in the cerebellum and brainstem. Therefore, the functions of ataxin-1 and the effects of SCA1 mutations in other brain regions including the cortex are not well-known. Here, we characterized pathology in the motor cortex of a SCA1 mouse model and performed RNA sequencing in this brain region to investigate the impact of mutant ataxin-1 towards transcriptomic alterations. We identified progressive cortical pathology and significant transcriptomic changes in the motor cortex of a SCA1 mouse model. We also identified progressive, region-specific, colocalization of p62 protein with mutant ataxin-1 aggregates in broad brain regions, but not the cerebellum or brainstem. A cross-regional comparison of the SCA1 cortical and cerebellar transcriptomic changes identified both common and unique gene expression changes between the two regions, including shared synaptic dysfunction and region-specific kinase regulation. These findings suggest that the cortex is progressively impacted via both shared and region-specific mechanisms in SCA1. [ABSTRACT FROM AUTHOR]

Background: Empagliflozin has been reported to protect endothelial cell function, regardless of diabetes status. However, the role of empagliflozin in microvascular protection during myocardial ischemia reperfusion injury (I/R) has not been fully understood. Methods: Electron microscopy, western blots, immunofluorescence, qPCR, mutant plasmid transfection, co-immunoprecipitation were employed to explore whether empagliflozin could alleviate microvascular damage and endothelial injury during cardiac I/R injury. Results: In mice, empagliflozin attenuated I/R injury-induced microvascular occlusion and microthrombus formation. In human coronary artery endothelial cells, I/R injury led to adhesive factor upregulation, endothelial nitric oxide synthase inactivation, focal adhesion kinase downregulation, barrier dysfunction, cytoskeletal degradation and cellular apoptosis; however, empagliflozin treatment diminished these effects. Empagliflozin improved mitochondrial oxidative stress, mitochondrial respiration and adenosine triphosphate metabolism in I/R-treated human coronary artery endothelial cells by preventing the phosphorylation of dynamin-related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1), thus repressing mitochondrial fission. The protective effects of empagliflozin on mitochondrial homeostasis and endothelial function were abrogated by the re-introduction of phosphorylated Fis1, but not phosphorylated Drp1, suggesting that Fis1 dephosphorylation is the predominant mechanism whereby empagliflozin inhibits mitochondrial fission during I/R injury. Besides, I/R injury induced Fis1 phosphorylation primarily by activating the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) pathway, while empagliflozin inactivated this pathway by exerting anti-oxidative effects. Conclusions: These results demonstrated that empagliflozin can protect the microvasculature by inhibiting the DNA-PKcs/Fis1/mitochondrial fission pathway during myocardial I/R injury. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) isozymes are maintained in a 'ready-to-go' but 'safe' autoinhibited conformation until second messenger binding unleashes an autoinhibitory pseudosubstrate to allow substrate phosphorylation. However, to gain this 'ready-to-go' conformation, PKC must be processed by a series of complex priming phosphorylations, the mechanism of which was enigmatic until now. Recent findings snapped the pieces of the phosphorylation puzzle into place to unveil a process that involves a newly described motif (TOR interaction motif, TIM), a well-described kinase [mechanistic target of rapamycin complex 2 (mTORC2)], and an often-used mechanism (autophosphorylation) to prime PKC to signal. This review highlights new insights into how phosphorylation controls PKC and discusses them in the context of common mechanisms for AGC kinase regulation by phosphorylation and autophosphorylation. Newly synthesized protein kinase C (PKC) becomes phosphorylated at multiple sites necessary to produce a mature kinase in a signaling-competent and 'ready-to-go' conformation that can rapidly respond to second messengers. The upstream kinase phosphoinositide-dependent kinase 1 (PDK1) has long been known to catalyze phosphorylation of the activation loop to stabilize the active site for catalysis, in turn triggering autophosphorylation at a C-terminal site known as the hydrophobic motif. Why the kinase mechanistic target of rapamycin complex 2 (mTORC2) is also necessary for some PKC isozymes to 'mature' into this signaling-competent species has remained elusive. The identification of an evolutionarily conserved phosphorylation motif (F-X 3 -F-pT) that is present in all mTORC2-regulated AGC kinases – the TOR interaction motif (TIM) – provided the key to unlock the puzzle. A new model has emerged in which nascent PKC is a stable homodimer mediated by the TIM; phosphorylation by mTORC2 dissociates the dimer to facilitate PDK1 binding, activation loop phosphorylation, and ultimately hydrophobic motif autophosphorylation. [ABSTRACT FROM AUTHOR]

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs.

Casein kinase 2 (CK2) is an evolutionarily conserved serine/threonine kinase implicated in a wide range of cellular functions and known to be dysregulated in various diseases such as cancer. Compared to most other kinases, CK2 exhibits several unusual properties, including dual co-substrate specificity and a high degree of promiscuity with hundreds of substrates described to date. Most paradoxical, however, is its apparent constitutive activity: no definitive mode of catalytic regulation has thus far been identified. Here we demonstrate that copper enhances the enzymatic activity of CK2 both in vitro and in vivo. We show that copper binds directly to CK2, and we identify specific residues in the catalytic subunit of the enzyme that are critical for copper-binding. We further demonstrate that increased levels of intracellular copper result in enhanced CK2 kinase activity, while decreased copper import results in reduced CK2 activity. Taken together, these findings establish CK2 as a copper-regulated kinase and indicate that copper is a key modulator of CK2-dependent signaling pathways.

Memory consolidation is associated with the regulation of protein kinases, which impact synaptic functions and promote synaptogenesis. The administration of spermidine (SPD) has been shown to modulate major protein kinases associated with memory improvement, including the Ca2+-dependent protein kinase (PKC) and cAMP-dependent protein kinase (PKA), key players in the cAMP response element-binding protein (CREB) activation. Nevertheless, the initial mechanism underlying SPD-mediated memory consolidation remains unknown, as we hypothesize a potential involvement of the memory consolidation precursor, Ca2+/calmodulin-dependent protein kinase II-α (CaMKIIα), in this process. Based on this, our study aimed to investigate potential interactions among PKC, PKA, and CREB activation, mediated by CaMKIIα activation, in order to elucidate the SPD memory consolidation pathway. Our findings suggest that the post-training administration of the CaMKII inhibitor, KN-62 (0.25 nmol, intrahippocampal), prevented the memory enhancement induced by SPD (0.2 nmol, intrahippocampal) in the inhibitory avoidance task. Through western immunoblotting, we observed that phosphorylation of CaMKIIα in the hippocampus was facilitated 15 min after intrahippocampal SPD administration, resulting in the activation of PKA and CREB, 180 min after infusion, suggesting a possible sequential mechanism, since SPD with KN-62 infusion leads to a downregulation in CaMKIIα/PKA/CREB pathway. However, KN-62 does not alter the memory-facilitating effect of SPD on PKC, possibly demonstrating a parallel cascade in memory acquisition via PKA, without modulating CAMKIIα. These results suggest that memory enhancement induced by SPD administration involves crosstalk between CaMKIIα and PKA/CREB, with no PKC interaction. [Display omitted] • Memory improvement is involved in CAMKIIα and PKA/CREB crosstalk. • KN-62 prevent the memory facilitatory effect of spermidine. • KN-62 downregulates CAMKIIα and PKA/CREB. • PKC is not involved in CAMKIIα signaling cascade induced by spermidine. [ABSTRACT FROM AUTHOR]

Metabolic syndrome (MetS) and its four clinical entities, central obesity, insulin resistance, hypertension and dyslipidemia, are implicated in increasing the risk and mortality of cancer in several organs. However, it is unclear how they are associated with increased risk of prostate cancer. To elucidate the mechanistic link between MetS and prostate carcinogenesis, we characterized the development of MetS and prostate adenocarcinoma in prostate-specific Pten−/− (Ptenp−/−) mice fed a high-fat (HF) diet. We found that male Ptenp−/− mice on an HF diet gained excess body weight and elevated blood glucose, insulin and insulin-like growth factor 1 (IGF1) levels at 20 weeks of age and were obese at 40 weeks. Prostate adenocarcinoma multiplicity at 40 weeks was significantly higher in the mice on an HF diet, suggesting that the HF diet promotes the development of prostate adenocarcinoma. Increased cell proliferation and enhanced AKT activation were found in the prostates of mice on an HF diet. Further transcriptome study revealed that receptor tyrosine kinase regulation, which mediates insulin/IGF1 signaling, was one of the top enriched pathways by HF diet-induced transcriptome changes. Together, our results suggest that HF diet-induced hyperinsulinemia leads to increased activation of insulin/IGF1/AKT signaling in lesioned prostates, promoting the development of adenocarcinoma. [ABSTRACT FROM AUTHOR]

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global epidemic with a high mortality rate. In this study, our goal was to identify the function and associated targets of SARS-CoV-2 from circulating monocytes in the blood and peripheral blood mononuclear cell (PBMC) dataset of patients with COVID-19. METHODS: The Gene Expression Omnibus database (GSE164805 and GSE180594) was used to identify differentially expressed genes (DEGs). Gene ontology function analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of the DEGs were performed using the DAVID database. RESULTS: Gene ontology analysis of DEG revealed that GSE164805 and GSE180594 were involved in the regulation of cell migration, upregulation of cell proliferation, and in the activation of the mitogen-activated protein kinase signaling pathway. Kyoto Encyclopedia of Genes and Genomes analysis of GSE164805 revealed that the DEGs were enriched in peroxisome, melanogenesis, and actin regulation. Peroxisome genes were highly expressed in patients with mild and severe COVID-19. Bioinformatics analysis to compare GSE180594 and public data for the single-cell atlas of the peripheral immune response in patients with COVID-19 showed that interferon-associated genes were highly increased in acute COVID-19 PBMC and in CD14+ and CD16+ monocytes from patients with COVID-19. CONCLUSIONS: We comprehensively analyzed the blood cell gene expression profile data of patients with COVID-19 using bioinformatics methods to preliminary understand the functions and associated targets of DEGs in the blood cells of these patients. Thus, our data provide targets for potential therapies against COVID-19. [ABSTRACT FROM AUTHOR]

Despite the established view of protein kinases as dynamic and versatile allosteric regulatory machines, our knowledge of allosteric functional states, allosteric interaction networks, and the intrinsic folding energy landscapes is surprisingly limited. We discuss the latest developments in structural characterization of allosteric molecular events underlying protein kinase dynamics and functions using structural, biophysical, and computational biology approaches. The recent studies highlighted progress in making the invisible aspects of protein kinase 'life' visible, including the determination of hidden allosteric states and mapping of allosteric energy landscapes, discovery of new mechanisms underlying ligand-induced modulation of allosteric activity, evolutionary adaptation of kinase allostery, and characterization of allosteric interaction networks as the intrinsic driver of kinase adaptability and signal transmission in the regulatory assemblies. • Structural biology captures hidden allosteric states in protein kinases. • Allosteric kinase inhibitors and activators can target the same allosteric site. • Engineering of protein kinase regulation using the allosteric pluripotency mechanism. • Naturally evolved allosteric interaction networks are exploited for kinase regulation. • Deep mutational scanning detects allosteric drug resistance hotspots. [ABSTRACT FROM AUTHOR]

Background and Purpose: Translational controls pervade neurobiology. Nociceptors play an integral role in the detection and propagation of pain signals. Nociceptors can undergo persistent changes in their intrinsic excitability. Pharmacological disruption of nascent protein synthesis diminishes acute and chronic forms of pain‐associated behaviours. However, the targets of translational controls that facilitate plasticity in nociceptors are unclear. Experimental Approach We used ribosome profiling to probe the translational landscape in dorsal root ganglion (DRG) neurons from male Swiss‐Webster mice, after treatment with nerve growth factor and IL‐6. Expression dynamics of c‐Fos were followed with immunoblotting and immunohistochemistry. The involvement of ribosomal protein S6 kinase 1 (S6K1), a downstream component of mTOR signalling, in the control of c‐Fos levels was assessed with low MW inhibitors of S6K1 (DG2) or c‐Fos (T‐5224), studying their effects on nociceptor activity in vitro using multielectrode arrays (MEAs) and pain behaviour in vivo in Swiss‐Webster mice using the hyperalgesic priming model. Key Results: c‐Fos was expressed in sensory neurons. Inflammatory mediators that promote pain in both humans and rodents promote c‐Fos translation. The mTOR effector S6K1 is essential for c‐Fos biosynthesis. Inhibition of S6K1 or c‐Fos with low MW compounds diminished mechanical and thermal hypersensitivity in response to inflammatory cues. Additionally, both inhibitors reduced evoked nociceptor activity. Conclusion and implications: Our data show a novel role of S6K1 in modulating the rapid response to inflammatory mediators, with c‐Fos being one key downstream target. Targeting the S6 kinase pathway or c‐Fos is an exciting new avenue for pain‐modulating compounds. [ABSTRACT FROM AUTHOR]