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Hypertension is associated with poor outcome and higher mortality in patients with ischemic stroke. The impairment of adaptive vascular mechanisms under hypertensive condition compromises collateral blood flow after arterial occlusion in patients with acute ischemic stroke resulting in hypoperfusion. The increased oxidative stress caused by hypoperfusion is thought to be a trigger for the rapid evolution of ischemic infarct volume under hypertensive condition. However, the cellular factors and pathways that contribute to the exacerbation of ischemic brain injury under hypertensive condition is not yet understood. The current study reveals that predisposition to hypertension leads to basal loss of function of the neuron-specific tyrosine phosphatase STEP, which plays a crucial role in neuroprotection against excitotoxic insult. The findings further show that a mild ischemic insult in hypertensive rats triggers an early onset and sustained activation of the neuronal extracellular signal regulated kinase (ERK MAPK), a member of the mitogen activated protein kinase family and a substrate of STEP. This leads to rapid increase in the activation of neuronal NF-κB, expression of neuronal cyclooxygenase-2 and subsequent biosynthesis of the pro-inflammatory mediator prostaglandin E2, resulting in rapid morphological transformation of microglia to the pro-inflammatory state and subsequent exacerbation of ischemic brain injury. Restoration of STEP signaling with intravenous administration of a STEP-derived peptide mimetic reduces the pro-inflammatory response in neurons, activation of microglia, and ischemic brain injury. The findings suggest that the basal loss of STEP function under hypertensive condition contributes to the exacerbation of ischemic brain injury by enhancing post-ischemic inflammatory response. The study not only presents a novel role of STEP in regulating neuroimmune communication but also highlights the therapeutic potential of a STEP-mimetic in mitigating ischemic brain damage under hypertensive condition. [ABSTRACT FROM AUTHOR]

Rice tocopherols, vitamin E compounds with antioxidant activity, play essential roles in human health. Even though the key genes involved in vitamin E biosynthetic pathways have been identified in plants, the genetic architecture of vitamin E content in rice grain remains unclear. A genome-wide association study (GWAS) on 179 genotypically diverse rice accessions with 34,323 SNP markers was conducted to detect QTLs that define total and a-tocopherol contents in rice grains. Total and a-tocopherol contents had a strong positive correlation and varied greatly across the accessions, ranging from 0.230-31.76 and 0.011-30.83 (mg/g), respectively. A total of 13 QTLs were identified, which were spread across five of the rice chromosomes. Among the 13 QTLs, 11 were considered major with phenotypic variation explained (PVE) greater than 10%. Twelve transcription factor (TF) genes, one microprotein (miP), and a transposon were found to be associated with the QTLs with putative roles in controlling tocopherol contents. Moreover, intracellular transport proteins, ABC transporters, nonaspanins, and SNARE, were identified as associated genes on chromosomes 1 and 8. In the vicinity of seven QTLs, protein kinases were identified as key signaling factors. Haplotype analysis revealed the QTLs qAlph1.1, qTot1.1, qAlph2.1, qAlph6.1, qTot6.1, and qTot8.3 to have significant haplogroups. Quantitative RT-PCR validated the expression direction and magnitude of WRKY39 (Os02g0265200), PIP5Ks (Os08g0450800), and MADS59 (Os06g0347700) in defining the major tocopherol contents. This study provides insights for ongoing biofortification efforts to breed and/or engineer vitamin E and antioxidant levels in rice and other cereals. [ABSTRACT FROM AUTHOR]

Statins are one of the most important classes of drugs. In this analytical review, we elucidate the intricate molecular mechanisms and toxicological rationale regarding both the on‐ (targeting 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase [HMGCR]) and off‐target effects of statins. Statins interact with a number of membrane kinases, such as epidermal growth factor receptor (EGFR), erb‐b2 receptor tyrosine kinase 2 (HER2) and MET proto‐oncogene, receptor tyrosine kinase (MET), as well as cytosolic kinases, such as SRC proto‐oncogene, non‐receptor tyrosine kinase (Src) and show inhibitory activity at nanomolar concentrations. In addition, they interact with calcium ATPases and peroxisome proliferator‐activated receptor α (PPARα/NR1C1) at higher concentrations. Statins interact with mitochondrial complexes III and IV, and their inhibition of coenzyme Q10 synthesis also impairs the functioning of complexes I and II. Statins act as inhibitors of kinases, calcium ATPases and mitochondrial complexes, while activating PPARα. These off‐target effects likely contribute to the side effects observed in patients undergoing statin therapy, including musculoskeletal symptoms and hepatic effects. Interestingly, some off‐target effects of statins could also be the cause of favourable outcomes, relating to repurposing statins in conditions such as inflammatory disorders and cancer. [ABSTRACT FROM AUTHOR]

Plants respond to cold stress at multiple levels, including increasing cytosolic calcium (Ca2+) influx and triggering the expression of cold-responsive genes. In this study, we show that the Ca2+-permeable channel CYCLIC NUCLEOTIDE–GATED CHANNEL20 (CNGC20) positively regulates freezing tolerance in Arabidopsis (Arabidopsis thaliana) by mediating cold-induced Ca2+ influx. Moreover, we demonstrate that the leucine-rich repeat receptor-like kinase PLANT PEPTIDE CONTAINING SULFATED TYROSINE1 RECEPTOR (PSY1R) is activated by cold, phosphorylating and enhancing the activity of CNGC20. The psy1r mutant exhibits decreased cold-evoked Ca2+ influx and freezing tolerance. Conversely, COLD-RESPONSIVE PROTEIN KINASE1 (CRPK1), a protein kinase that negatively regulates cold signaling, phosphorylates and facilitates the degradation of CNGC20 under prolonged periods of cold treatment, thereby attenuating freezing tolerance. This study thus identifies PSY1R and CRPK1 kinases that regulate CNGC20 activity and stability, respectively, thereby antagonistically modulating freezing tolerance in plants. Two kinases phosphorylate distinct sites in a Ca2+-permeable channel to regulate its activity and stability, thereby antagonistically modulating freezing tolerance in plants. [ABSTRACT FROM AUTHOR]

Protein S‐acylation or palmitoylation is a reversible post‐translational modification that influences many proteins encoded in plant genomes. Exciting progress in the past 3 years demonstrates that S‐acylation modulates subcellular localization, interacting profiles, activity, or turnover of substrate proteins in plants, participating in developmental processes and responses to abiotic or biotic stresses. In this review, we summarize and discuss the role of S‐acylation in the targeting of substrate proteins. We highlight complex roles of S‐acylation in receptor signaling. We also point out that feedbacks of protein S‐acyl transferase by signaling initiated from their substrate proteins may be a recurring theme. Finally, the reversibility of S‐acylation makes it a rapid and efficient way to respond to environmental cues. Future efforts on exploring these important aspects of S‐acylation will give a better understanding of how plants enhance their fitness under ever changing and often harsh environments. [ABSTRACT FROM AUTHOR]

Background/Aim • Propofol is a common anesthetic used during surgery. MircoRNA (miRNA), especially miR- 4430, is currently a research hotspot in GBC. This study investigates whether propofol regulates GBC through the miR-4430/SLC19A3 axis. Materials and methods • NOZ and SGC996 cells, human gallbladder cancer cell lines, were obtained from BLUEFBIO (Shanghai, China). The cells were treated with propofol, and the experimental setup included a control group. qRT-PCR was used to measure the content of miR- 4430 and solute carrier family 19 member 3 (SLC19A3). The cell viability, migration, and apoptosis were analyzed by colony formation assay, wound healing assay, and flow cytometry, respectively. Western blot tested the levels of SLC91A3, cyclin D-dependent kinases 6 (CDK6), and Bax proteins. Mechanically, dual-luciferase reporter assay notarized the link of miR-4430 with SLC19A3. Results • Propofol significantly repressed the proliferation of GBC cells, as indicated by a pronounced reduction in colony formation in both NOZ and SGC996 cells (P = .025, 95% CI:1.2, 2.4). The inhibitory effect of propofol was reversed by the enhanced expression of miR-4430 in GBC cells. Furthermore, SLC19A3 was identified as a direct target of miR-4430. Silencing SLC19A3 reversed the altered cell behavior observed in propofol-treated GBC cells (P = .019, 95% CI:1.6, 3.5). Additionally, overexpression of SLC19A3 significantly attenuated both cell proliferation and migration influenced by miR-4430 in propofol-treated GBC cells (P = .034, 95% CI:1.4, 2.8). Conclusion • Propofol changed the cell behaviors of GBC by modulating the miR-4430/SLC19A3 axis. [ABSTRACT FROM AUTHOR]

Protein kinases have essential responsibilities in controlling several cellular processes, and their abnormal regulation is strongly related to the development of cancer. The implementation of protein kinase inhibitors has significantly transformed cancer therapy by modifying treatment strategies. These inhibitors have received substantial FDA clearance in recent decades. Protein kinases have emerged as primary objectives for therapeutic interventions, particularly in the context of cancer treatment. At present, 69 therapeutics have been approved by the FDA that target approximately 24 protein kinases, which are specifically prescribed for the treatment of neoplastic illnesses. These novel agents specifically inhibit certain protein kinases, such as receptor protein-tyrosine kinases, protein-serine/threonine kinases, dual-specificity kinases, nonreceptor protein-tyrosine kinases, and receptor protein-tyrosine kinases. This review presents a comprehensive overview of novel targets of kinase inhibitors, with a specific focus on cyclin-dependent kinases (CDKs) and epidermal growth factor receptor (EGFR). The majority of the reviewed studies commenced with an assessment of cancer cell lines and concluded with a comprehensive biological evaluation of individual kinase targets. The reviewed articles provide detailed information on the structural features of potent anticancer agents and their specific activity, which refers to their ability to selectively inhibit cancer-promoting kinases including CDKs and EGFR. Additionally, the latest FDA-approved anticancer agents targeting these enzymes were highlighted accordingly. [ABSTRACT FROM AUTHOR]

Since genetic mutations during brain development play a significant role in the genesis of epilepsy, and such genetically determined epilepsies are the most difficult to treat, there is a need to study the mechanisms of epilepsy development with deletions of various transcription factors. We utilized heterozygous mice (Sip1wt/fl) with a neuronal deletion of the transcription factor Sip1 (Smad interacting protein 1) in the cerebral cortex. These mice are characterized by cognitive impairment and are prone to epilepsy. It is known that the brain-derived neurotrophic factor (BDNF) has a neuroprotective effect in various neurodegenerative diseases. Therefore, we created and applied an adeno-associated construct carrying the BDNF sequence selectively in neurons. Using in vitro and in vivo research models, we were able to identify a key gen, the disruption of whose expression accompanies the deletion of Sip1 and contributes to hyperexcitation of neurons in the cerebral cortex. Overexpression of BDNF in cortical neurons eliminated epileptiform activity in neurons obtained from heterozygous Sip1 mice in a magnesium-free model of epileptiform activity (in vitro). Using PCR analysis, it was possible to identify correlations in the expression profile of genes encoding key proteins responsible for neurotransmission and neuronal survival. The effects of BDNF overexpression on the expression profiles of these genes were also revealed. Using BDNF overexpression in cortical neurons of heterozygous Sip1 mice, it was possible to achieve 100% survival in the pilocarpine model of epilepsy. At the level of gene expression in the cerebral cortex, patterns were established that may be involved in the protection of brain cells from epileptic seizures and the restoration of cognitive functions in mice with Sip1 deletion. [ABSTRACT FROM AUTHOR]

Purpose: Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. Methods and results: USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. Conclusions: While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC. [ABSTRACT FROM AUTHOR]

The LIM domain kinases (LIMKs) are important actin cytoskeleton regulators. These proteins, LIMK1 and LIMK2, are nodes downstream of Rho GTPases and are the key enzymes that phosphorylate cofilin/actin depolymerization factors to regulate filament severing. They therefore perform an essential role in cascades that control actin depolymerization. Signaling of the LIMKs is carefully regulated by numerous inter‐ and intra‐molecular mechanisms. In this review, we discuss recent findings that improve the understanding of LIM domain kinase regulation mechanisms. We also provide an up‐to‐date review of the role of the LIM domain kinases, their architectural features, how activity is impacted by other proteins, and the implications of these findings for human health and disease. [ABSTRACT FROM AUTHOR]

The accuracy of cell division requires precise regulation of the cellular machinery governing DNA/genome duplication, ensuring its equal distribution among the daughter cells. The control of the centrosome cycle is crucial for the formation of a bipolar spindle, ensuring error‐free segregation of the genome. The cell and centrosome cycles operate in close synchrony along similar principles. Both require a single duplication round in every cell cycle, and both are controlled by the activity of key protein kinases. Nevertheless, our comprehension of the precise cellular mechanisms and critical regulators synchronizing these two cycles remains poorly defined. Here, we present our hypothesis that the spatiotemporal regulation of a dynamic equilibrium of mitotic kinases activities forms a molecular clock that governs the synchronous progression of both the cell and the centrosome cycles. [ABSTRACT FROM AUTHOR]

Oxidative stress has long been implicated in Parkinson's disease (PD) pathogenesis, although the sources and regulation of reactive oxygen species (ROS) production are poorly defined. Pathogenic mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are associated with increased kinase activity and a greater risk of PD. The substrates and downstream consequences of elevated LRRK2 kinase activity are still being elucidated, but overexpression of mutant LRRK2 has been associated with oxidative stress, and antioxidants reportedly mitigate LRRK2 toxicity. Here, using CRISPR-Cas9 gene-edited HEK293 cells, RAW264.7 macrophages, rat primary ventral midbrain cultures, and PD patient–derived lymphoblastoid cells, we found that elevated LRRK2 kinase activity was associated with increased ROS production and lipid peroxidation and that this was blocked by inhibitors of either LRRK2 kinase or NADPH oxidase 2 (NOX2). Oxidative stress induced by the pesticide rotenone was ameliorated by LRRK2 kinase inhibition and was absent in cells devoid of LRRK2. In a rat model of PD induced by rotenone, a LRRK2 kinase inhibitor prevented the lipid peroxidation and NOX2 activation normally seen in nigral dopaminergic neurons in this model. Mechanistically, LRRK2 kinase activity was shown to regulate phosphorylation of serine-345 in the p47phox subunit of NOX2. This, in turn, led to translocation of p47phox from the cytosol to the membrane-associated gp91phox (NOX2) subunit, activation of the NOX2 enzyme complex, and production of ROS. Thus, LRRK2 kinase activity may drive cellular ROS production in PD through the regulation of NOX2 activity. Editor's summary: Production of reactive oxygen species (ROS) has been implicated in Parkinson's disease (PD) pathogenesis, but the source and regulation of ROS are unclear. Overexpression of mutant leucine-rich repeat kinase 2 (LRRK2), which is known to increase PD risk, is associated with oxidative stress–induced tissue damage. Keeney and colleagues now report in several different cellular models of PD and a rat model that LRRK2 kinase activity regulates phosphorylation of serine-345 in the p47phox subunit of NADPH oxidase 2 (NOX2), leading to NOX2 activation and ROS production. These findings implicate LRRK2 kinase activity in cellular ROS production mediated by NOX2 activity. —Orla Smith [ABSTRACT FROM AUTHOR]

Receptor guanylyl cyclases (GCs) are single membrane spanning, multidomain enzymes, that synthesize cGMP in response to natriuretic peptides or other ligands. They are evolutionarily conserved from sea urchins to humans and regulate diverse physiologies. Most family members are phosphorylated on 4 to 7 conserved serines or threonines at the beginning of their kinase homology domains. This review describes studies that demonstrate that phosphorylation and dephosphorylation are required for activation and inactivation of these enzymes, respectively. Phosphorylation sites in GC-A, GC-B, GC-E, and sea urchin receptors are discussed, as are mutant receptors that mimic the dephosphorylated inactive or phosphorylated active forms of GC-A and GC-B, respectively. A salt bridge model is described that explains why phosphorylation is required for enzyme activation. Potential kinases, phosphatases, and ATP regulation of GC receptors are also discussed. Critically, knock-in mice with glutamate substitutions for receptor phosphorylation sites are described. The inability of opposing signaling pathways to inhibit cGMP synthesis in mice where GC-A or GC-B cannot be dephosphorylated demonstrates the necessity of receptor dephosphorylation in vivo. Cardiac hypertrophy, oocyte meiosis, long-bone growth/achondroplasia, and bone density are regulated by GC phosphorylation, but additional processes are likely to be identified in the future. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptors (HER)—also known as EGFR or ErbB receptors—are a subfamily of receptor tyrosine kinases (RTKs) that play crucial roles in cell growth, division, and differentiation. HER4 (ErbB4) is the least studied member of this family, partly because its expression is lower in later stages of development. Recent work has suggested that HER4 can play a role in metastasis by regulating cell migration and invasiveness; however, unlike EGFR and HER2, the precise role that HER4 plays in tumorigenesis is still unresolved. Early work on HER family proteins suggested that there are direct interactions between the four members, but to date, there has been no single study of all four receptors in the same cell line with the same biophysical method. Here, we quantitatively measure the degree of association between HER4 and the other HER family proteins in live cells with a time‐resolved fluorescence technique called pulsed interleaved excitation fluorescence cross‐correlation spectroscopy (PIE‐FCCS). PIE‐FCCS is sensitive to the oligomerization state of membrane proteins in live cells, while simultaneously measuring single‐cell protein expression levels and diffusion coefficients. Our PIE‐FCCS results demonstrate that HER4 interacts directly with all HER family members in the cell plasma membrane. The interaction between HER4 and other HER family members intensified in the presence of a HER4‐specific ligand. Our work suggests that HER4 is a preferred dimerization partner for all HER family proteins, even in the absence of ligands. [ABSTRACT FROM AUTHOR]

Viral encephalitis is characterized by inflammation of the brain parenchyma caused by a variety of viruses, among which the Japanese encephalitis (JE) virus (JEV) is a typical representative arbovirus. Neuronal death, neuroinflammation, and breakdown of the blood brain barrier (BBB) constitute vicious circles of JE progression. Currently, there is no effective therapy to prevent this damage. Growth arrest specific gene 6 (GAS6) is a secreted growth factor that binds to the TYRO3, AXL, and MERTK (TAM) family of receptor tyrosine kinases and has been demonstrated to participate in neuroprotection and suppression of inflammation in many central nervous system (CNS) diseases which has great potential for JE intervention. In this study, we found that GAS6 expression in the brain was decreased and was reversely correlated with viral load and neuronal loss. Mice with GAS6/TAM signalling deficiency showed higher mortality and accelerated neuroinflammation during peripheral JEV infection, accompanied by BBB breakdown. GAS6 directly promoted the expression of tight junction proteins in bEnd.3 cells and strengthened BBB integrity, partly via AXL. Mice administered GAS6 were more resistant to JEV infection due to increased BBB integrity, as well as decreased viral load and neuroinflammation. Thus, targeted GAS6 delivery may represent a strategy for the prevention and treatment of JE especially in patients with impaired BBB. [ABSTRACT FROM AUTHOR]

As one of the factors regulating tumour angiogenesis, angiopoietin-4 (ANGPT4), which plays an important role in promoting tumour proliferation, survival, expansion and angiogenesis, is highly expressed in some tumours, such as lung adenocarcinoma, glioblastoma and ovarian cancer. This may be related to the fact that ANGPT4 affects the blood vessels and lymphatic system of the tumour. Specifically, ANGPT4 could play an effective role in promoting cancer by affecting the tyrosine kinase receptor TIE2, ERK1/2 and PI3K/AKT signalling pathways. Therefore, ANGPT4 may be an important biomarker for the occurrence and development of cancer and poor prognosis. In addition, the inhibition of ANGPT4 may be a useful cancer treatment. This paper reviews the latest preclinical research on ANGPT4, emphasizes its role in tumourigenesis and broadens our understanding of the carcinogenic function of ANGPT4 and the development of ANGPT4 inhibitors. This is the latest version of the revised version of the previous article. [ABSTRACT FROM AUTHOR]

The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed. [ABSTRACT FROM AUTHOR]

Introduction: The combination of BRAF and tyrosine kinase (TK) inhibitors has been demonstrated to be highly effective in inhibiting tumor development and is an approach for overcoming resistance in clinical trials. Accordingly, a novel series of 1,2,4-oxadiazole/quinazoline-4-one hybrids was developed as antiproliferative multitargeted inhibitors. Methods: The structures of the newly synthesized compounds 9a-o were validated using IR, NMR, MS, and elemental techniques. 9a-o were tested as antiproliferative agents. Results and Discussion: The results showed that the majority of the tested compounds showed significant antiproliferative action with 9b, 9c, 9h, 9k, and 9l being the most potent. Compounds 9b, 9c, 9h, 9k, and 9l were tested as EGFR and BRAF[sup V600E] inhibitors. These in vitro tests revealed that compounds 9b, 9c, and 9h are strong antiproliferative agents that may act as dual EGFR/BRAF[sup V600E] inhibitors. 9b, 9c, and 9h were further investigated for their inhibitory effect on mutant EGFR (EGFR[sup T790M]), and the results showed that the tested compounds had considerable inhibitory action. Cell cycle study and apoptosis detection demonstrated that compound 9b exhibits cell cycle arrest at the G2/M transition. Molecular docking simulations reveal the binding mechanism of the most active antiproliferative agents. [ABSTRACT FROM AUTHOR]

Oxadiazoles an important heterocyclic scaffold of medicinal importance in the field of drug discovery. In the study, a library of oxadiazole based compounds was selected for screening against STAT-3 as anti-cancer target. STAT3 is a potential target of interest in cancer therapy. A total of 544 screened library of compounds was subjected to molecular docking against STAT-3 (6NJS and 6NQU). The compounds with good dock score and binding interations were further subjected to in-silico ADME analysis followed by toxicity estimation. A total of 141 hits were selected against 6NJS and 50 hits against 6NQU and further screened for kinetic properties and drug likeliness. The compounds were screened on the basis of physico-chemical properties, solubility, gastrointestinal absorption, BBB permeability, synthetic accessibility, Lipinski and other violations. Best compounds obtained after ADME analysis were further subjected for toxicity analysis. Carcinogenecity, mutagenicity, Ames and other important parameters were considered for toxicity based screening. The best leads thus obtained (compound 114 and 40) were further subjected to molecular dynamics against the respective target proteins. MD simulations were run to access the stability of C-114 and C-40 along with the dynamic behaviour of both complexes for about 100 ns and shows good stability with the proteins. [ABSTRACT FROM AUTHOR]

Central insulin resistance has been linked to the development of neurodegenerative diseases and mood disorders. Various proteins belonging to the enzyme family of protein tyrosine phosphatases (PTPs) act as inhibitors of insulin signaling. Protein tyrosine phosphatase receptor type J (PTPRJ) has been identified as a negative regulator in insulin signaling in the periphery. However, the impact of PTPRJ on insulin signaling and its functional role in neuronal cells is largely unknown. Therefore, we generated a Ptprj knockout (KO) cell model in the murine neuroblast cell line Neuro2a by CRISPR‐Cas9 gene editing. Ptprj KO cells displayed enhanced insulin signaling, as shown by increased phosphorylation of the insulin receptor (INSR), IRS‐1, AKT, and ERK1/2. Further, proximity ligation assays (PLA) revealed both direct interaction of PTPRJ with the INSR and recruitment of this phosphatase to the receptor upon insulin stimulation. By RNA sequencing gene expression analysis, we identified multiple gene clusters responsible for glucose uptake and metabolism, and genes involved in the synthesis of various lipids being mainly upregulated under PTPRJ deficiency. Furthermore, multiple Ca2+ transporters were differentially expressed along with decreased protein biosynthesis. This was accompanied by an increase in endoplasmic reticulum (ER) stress markers. On a functional level, PTPRJ deficiency compromised cell differentiation and neurite outgrowth, suggesting a role in nervous system development. Taken together, PTPRJ emerges as a negative regulator of central insulin signaling, impacting neuronal metabolism and neurite outgrowth. [ABSTRACT FROM AUTHOR]

An ever-growing number of studies highlight the importance of S-acylation, a reversible protein-lipid modification, for diverse aspects of intracellular signaling. In this review, we summarize the current understanding of how S-acylation regulates perhaps the best-known class of signaling enzymes, protein kinases. We describe how S-acylation acts as a membrane targeting signal that localizes certain kinases to specific membranes, and how such membrane localization in turn facilitates the assembly of signaling hubs consisting of an S-acylated kinase’s upstream activators and/or downstream targets. We further discuss recent findings that S-acylation can control additional aspects of the function of certain kinases, including their interactions and, surprisingly, their activity, and how such regulation might be exploited for potential therapeutic gain. We go on to describe the roles and regulation of de-S-acylases and how extracellular signals drive dynamic (de) S-acylation of certain kinases. We discuss how S-acylation has the potential to lead to “emergent properties” that alter the temporal profile and/or salience of intracellular signaling events. We close by giving examples of other S-acylation– dependent classes of signaling enzymes and by discussing how recent biological and technological advances should facilitate future studies into the functional roles of S-acylation–dependent signaling.An ever-growing number of studies highlight the importance of S-acylation, a reversible protein-lipid modification, for diverse aspects of intracellular signaling. In this review, we summarize the current understanding of how S-acylation regulates perhaps the best-known class of signaling enzymes, protein kinases. We describe how S-acylation acts as a membrane targeting signal that localizes certain kinases to specific membranes, and how such membrane localization in turn facilitates the assembly of signaling hubs consisting of an S-acylated kinase’s upstream activators and/or downstream targets. We further discuss recent findings that S-acylation can control additional aspects of the function of certain kinases, including their interactions and, surprisingly, their activity, and how such regulation might be exploited for potential therapeutic gain. We go on to describe the roles and regulation of de-S-acylases and how extracellular signals drive dynamic (de) S-acylation of certain kinases. We discuss how S-acylation has the potential to lead to “emergent properties” that alter the temporal profile and/or salience of intracellular signaling events. We close by giving examples of other S-acylation– dependent classes of signaling enzymes and by discussing how recent biological and technological advances should facilitate future studies into the functional roles of S-acylation–dependent signaling. [ABSTRACT FROM AUTHOR]

Definition: In recent decades, phosphoinositides (or PIs) have emerged as essential signaling molecules. Despite their low cellular abundance, PIs are found to be involved in various cellular processes, including cell migration, vesicular trafficking, cell cycle regulation, metabolism, cytoskeletal remodeling, autophagy, aging, apoptosis, and cell signaling. Recent studies have shown that aberrant activity of either lipid kinases or phosphatases leads to various medical implications like cancer, diabetes, and microbial infections, suggesting an essential role for these lipid molecules and enzymes in their metabolism. This entry focused on one of the critical enzymes involved in phosphoinositide metabolism: phosphatidyl inositol 4-kinase (PI4-Kinase). [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are produced during cellular metabolism and in response to environmental stress. While low levels of ROS play essential physiological roles, excess ROS can damage cellular components, leading to cell death or transformation. ROS can also regulate protein interactions in cancer cells, thereby affecting processes such as cell growth, migration, and angiogenesis. Dysregulated interactions occur via various mechanisms, including amino acid modifications, conformational changes, and alterations in complex stability. Understanding ROS-mediated changes in protein interactions is crucial for targeted cancer therapies. In this review, we examine the role that ROS mechanisms in regulating pathways through protein–protein interactions. [ABSTRACT FROM AUTHOR]

The host kinase casein kinase 2 (CSNK2) has been proposed to be an antiviral target against β-coronaviral infection. To pharmacologically validate CSNK2 as a drug target in vivo, potent and selective CSNK2 inhibitors with good pharmacokinetic properties are required. Inhibitors based on the pyrazolo[1,5-a]pyrimidine scaffold possess outstanding potency and selectivity for CSNK2, but bioavailability and metabolic stability are often challenging. By strategically installing a fluorine atom on an electron-rich phenyl ring of a previously characterized inhibitor 1, we discovered compound 2 as a promising lead compound with improved in vivo metabolic stability. Compound 2 maintained excellent cellular potency against CSNK2, submicromolar antiviral potency, and favorable solubility, and was remarkably selective for CSNK2 when screened against 192 kinases across the human kinome. We additionally present a co-crystal structure to support its on-target binding mode. In vivo, compound 2 was orally bioavailable, and demonstrated modest and transient inhibition of CSNK2, although antiviral activity was not observed, possibly attributed to its lack of prolonged CSNK2 inhibition. [ABSTRACT FROM AUTHOR]

The purpose of this review is to highlight transformative advances that have been made in the field of biomolecular condensates, with special emphasis on condensate material properties, physiology, and kinases, using the With-No-Lysine (WNK) kinases as a prototypical example. To convey how WNK kinases illustrate important concepts for biomolecular condensates, we start with a brief history, focus on defining features of biomolecular condensates, and delve into some examples of how condensates are implicated in cellular physiology (and pathophysiology). We then highlight how WNK kinases, through the action of "WNK droplets" that ubiquitously regulate intracellular volume and kidney-specific "WNK bodies" that are implicated in distal tubule salt reabsorption and potassium homeostasis, exemplify many of the defining features of condensates. Finally, this review addresses the controversies within this emerging field and questions to address. [ABSTRACT FROM AUTHOR]

Recent studies have revealed that B-subgroup rapidly accelerated fibrosarcoma (RAF) kinases have pivotal roles in hormone signaling and stress responses across a wide range of organisms. In this forum, I explore their evolution and diverse signaling pathways, highlighting the significance of B-RAF kinases in plant growth and plant–environment interactions while discussing open questions for future research. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to explore the expression, clinical significance, and functional mechanism of FYN in lower-grade gliomas (LGG). Methods: The mRNA and protein expression of FYN in LGG tissues were detected using databases including OncoLnc, GEPIA, and Human protein atlas (HPA). The UCSC Xena browser, TIMER, STRING and Metascape databases were used to investigate Kaplan–Meier survival curves, correlations between FYN expression and various types of immune cell infiltration, protein interaction network and possible functional mechanism. Results: FYN expression in LGG, IDH mutation or 1p19q co-deletion subgroup was significantly higher than in corresponding control groups (p < 0.05). Patients with higher FYN expression had longer overall survival (p < 0.05). Male or no 1p19q co-deletion groups with higher FYN expression also had longer overall survival (p < 0.05). FYN expression had close correlation with infiltrating levels of cell purity, CD4+T cells, macrophages, and CD8+T cells (p < 0.05). Protein interaction network result showed correlation among FYN, SH2D1A, LCK, CAV1, SRC, CBL and PTK2. Functional enrichment analysis revealed that FYN and its related genes mainly participated in bacterial invasion of epithelial cells and natural killer cell mediated cytotoxicity. Peptidyl-tyrosine phosphorylation, negative regulation of anoikis, immune effector process, transmembrane receptor protein tyrosine kinase signaling pathway, epidermal growth factor receptor signaling pathway, and negative regulation of protein modification process may be the critical biological process. Conclusions: FYN is up-expressed in LGG and related to its good prognosis. It participated in tumor pathophysiological processes and may be a therapeutic target for LGG. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is the most common type of cancer in the world. In lung adenocarcinoma (LUAD), studies on receptor tyrosine kinase ROS proto-oncogene 1 (ROS1) have mainly focused on the oncogenic effects of its fusion mutations, whereas ROS1 has been reported to be aberrantly expressed in a variety of cancers and can extensively regulate the growth, survival, and proliferation of tumor cells through multiple signaling pathways. The comprehensive analysis of ROS1 expression has not been fully investigated regarding its predictive value for LUAD patients. Methods: Gene expression profiles collected from The Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) databases were used to build and validate prognostic risk models. The association of ROS1 with overall survival and the immune landscape was obtained from the Tumor Immune Estimation Resource (TIMER) database. The following analyses were performed using the R package to determine the model's validity: pathway dysregulation analysis, gene set enrichment analysis, Gene Oncology analysis, immune invasion analysis, chemotherapy, radiotherapy, and immunotherapy sensitivity analysis. Finally, we conducted a pan-cancer analysis and performed in vitro experiments to explore the regulatory role of intercellular adhesion molecule 5 (ICAM5) in the progression of LUAD. Results: We constructed a 17-gene model that categorized patients into two risk groups. The model had predictive accuracy for tumor prognosis and was specific for patients with high ROS1 expression. Comprehensive analysis showed that patients in the high-risk group were characterized by marked dysregulation of multiple pathways (eg, unfolded protein response), immune suppression of the tumor microenvironment, and poor benefit from immunotherapy and radiotherapy compared with patients in the low-risk group. PLX4720 may be a suitable treatment for the high-risk patient population. The ICAM5 gene has been demonstrated to inhibit the proliferation, cell cycle, invasion, and migration of LUAD cells. Conclusion: We constructed a 17-gene prognostic risk model and found differences in immune-related cells, biological processes, and prognosis among patients in different risk groups based on the correlation between ROS1 and immunity. Personalized therapy may play an essential role in treatment. We further investigated the role of ICAM5 in inhibiting the malignant bioactivity of LUAD cells. [ABSTRACT FROM AUTHOR]

Inflammation-induced osteoclast proliferation is a crucial contributor to impaired bone metabolism. Kurarinone (KR), a flavonoid extracted from the Radix Sophorae Flavescentis, exhibits notable anti-inflammatory properties. Nevertheless, the precise influence of KR on osteoclast formation remains unclear. This study's objective was to assess the impact of KR on osteoclast activity in vitro and unravel its underlying mechanism. Initially, a target network for KR-osteoclastogenesis-osteoporosis was constructed using network pharmacology. Subsequently, the intersecting targets were identified through the Venny platform and a PPI network was created using Cytoscape 3.9.1. Key targets within the network were identified employing topological algorithms. GO enrichment and KEGG pathway analysis were then performed on these targets to explore their specific functions and pathways. Additionally, molecular docking of potential core targets of KR was conducted, and the results were validated through cell experiments. A total of 83 target genes overlapped between KR and osteoclastogenesis-osteoporosis targets. Enrichment analysis revealed their role in inflammatory response, protein tyrosine kinase activity, osteoclast differentiation, and MAPK and NF-κB signaling pathways. PPI analysis and molecular docking demonstrate that key targets MAPK14 and MAPK8 exhibit more stable binding with KR compared to other proteins. In vitro experiments demonstrate that KR effectively inhibits osteoclast differentiation and bone resorption without cellular toxicity. It suppresses key osteoclast genes (NFATc1, c-Fos, TRAP, MMP9, Ctsk, Atp6v2), hinders IκB-α degradation, and inhibits ERK and JNK phosphorylation, while not affecting p38 phosphorylation. The results indicate that KR may inhibit osteoclast maturation and bone resorption by blocking NF-κB and MAPK signaling pathways, suggesting its potential as a natural therapeutic agent for osteoporosis. [ABSTRACT FROM AUTHOR]

Glioblastoma, the most aggressive form of brain tumor, poses significant challenges in terms of treatment success and patient survival. Current treatment modalities for glioblastoma include radiation therapy, surgical intervention, and chemotherapy. Unfortunately, the median survival rate remains dishearteningly low at 12-15 months. One of the major obstacles in treating glioblastoma is the recurrence of tumors, making chemotherapy the primary approach for secondary glioma patients. However, the efficacy of drugs is hampered by the presence of the blood-brain barrier and multidrug resistance mechanisms. Consequently, considerable research efforts have been directed toward understanding the underlying signaling pathways involved in glioma and developing targeted drugs. To tackle glioma, numerous studies have examined kinase-downstream signaling pathways such as RAS-RAF-MEKERK-MPAK. By targeting specific signaling pathways, heterocyclic compounds have demonstrated efficacy in glioma therapeutics. Additionally, key kinases including phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase, cytoplasmic tyrosine kinase (CTK), receptor tyrosine kinase (RTK) and lipid kinase (LK) have been considered for investigation. These pathways play crucial roles in drug effectiveness in glioma treatment. Heterocyclic compounds, encompassing pyrimidine, thiazole, quinazoline, imidazole, indole, acridone, triazine, and other derivatives, have shown promising results in targeting these pathways. As part of this review, we propose exploring novel structures with low toxicity and high potency for glioma treatment. The development of these compounds should strive to overcome multidrug resistance mechanisms and efficiently penetrate the blood-brain barrier. By optimizing the chemical properties and designing compounds with enhanced drug-like characteristics, we can maximize their therapeutic value and minimize adverse effects. Considering the complex nature of glioblastoma, these novel structures should be rigorously tested and evaluated for their efficacy and safety profiles. [ABSTRACT FROM AUTHOR]

As cell signaling research has advanced, it has become clearer that signal transduction has complex spatiotemporal regulation that goes beyond foundational linear transduction models. Several technologies have enabled these discoveries, including fluorescent biosensors designed to report live biochemical signaling events. As genetically encoded and live-cell compatible tools, fluorescent biosensors are well suited to address diverse cell signaling questions across different spatial scales of regulation. In this review, methods of examining spatial signaling regulation and the design of fluorescent biosensors are introduced. Then, recent biosensor developments that illuminate the importance of spatial regulation in cell signaling are highlighted at several scales, including membranes and organelles, molecular assemblies, and cell/tissue heterogeneity. In closing, perspectives on how fluorescent biosensors will continue enhancing cell signaling research are discussed.

We computationally predicted all phosphorylation sites in the sequence of the human laminin γ1-chain (LAMC1), and computationally identified, for the first time, all kinases for experimentally observed phosphorylated residues of the LAMC1 and all missense deleterious LAMC1 mutations found in different cancer types that interfere with LAMC1 phosphorylation. Also, we mapped the above data to all the biologically functional interaction sequences of the LAMC1. Five kinases (CKII, GPCRK1, PKA, PKC, and CKI) are most enriched for LAMC1 phosphorylation, and the significance of ecto-kinases in this process was emphasized. PKA and PKC targeted more residues inside and close to functional interaction sequences compared with other kinases and in the functional interaction sequence RPESFAIYKRTR. Most phosphorylation-interfering mutations were found in cutaneous melanoma and uterine endometrioid carcinoma. The mutation R255H interfered with the experimentally observed phosphorylation of LAMC1 inside the functional interaction sequence TDIRVTLNRLNTF, while the mutations S181Y and S213Y interfered with the experimentally observed phosphorylation of LAMC1 outside the functional interaction sequences. Mutations R359C,H, R589H, R657C,H, R663I,G, and T1207 interfered with the predicted phosphorylation inside or close to the functional interaction sequences, whereas other mutations interfered outside. PKA- and PKC-predicted phosphorylation was mostly interfered with by mutations inside functional interaction sequences. Phosphorylation-interfering mutations and functional interaction sequences were suggested to promote specific cancer types or cancer progression in general. [ABSTRACT FROM AUTHOR]

In recent decades, phosphoinositides (or PIs) have emerged as essential signaling molecules. Despite their low cellular abundance, PIs are found to be involved in various cellular processes, including cell migration, vesicular trafficking, cell cycle regulation, metabolism, cytoskeletal remodeling, autophagy, aging, apoptosis, and cell signaling. Recent studies have shown that aberrant activity of either lipid kinases or phosphatases leads to various medical implications like cancer, diabetes, and microbial infections, suggesting an essential role for these lipid molecules and enzymes in their metabolism. This entry focused on one of the critical enzymes involved in phosphoinositide metabolism: phosphatidyl inositol 4-kinase (PI4-Kinase).

Selective macroautophagy/autophagy of the endoplasmic reticulum, known as reticulophagy/ER-phagy, is essential to maintain ER homeostasis. We recently showed that members of the autophagy receptor family RETREG/FAM134 are regulated by phosphorylation-dependent ubiquitination. In an unbiased screen we had identified several kinases downstream of MTOR with profound impact on reticulophagy flux, including ATR and CSNK2/CK2. Inhibition of CSNK2 by SGC-CK2-1 prevented regulatory ubiquitination of RETREG1/FAM134B and RETREG3/FAM134C upon autophagy activation as well as the formation of high-density RETREG1- and RETREG3-clusters. Here we report on additional resource data of global proteomics upon CSNK2 and ATR inhibition, respectively. Our data suggests that the function of CSNK2 is mainly limited to the ER/reticulophagy and Golgi/Golgiphagy, while ATR inhibition by VE-822 affects the vast majority of organelles/selective autophagy pathways.Abbreviation: ATRi: ATR inhibitor VE-822; CSNK2i: CSNK2 inhibitor SGC-CK2-1; ER: endoplasmic reticulum [ABSTRACT FROM AUTHOR]

Introduction: The Janus kinase (JAK) family includes four cytoplasmic tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) constitutively bound to several cytokine receptors. JAKs phosphorylate downstream signal transducers and activators of transcription (STAT). JAK-STAT5 pathways play a critical role in basophil and mast cell activation. Previous studies have demonstrated that inhibitors of JAK-STAT pathway blocked the activation of mast cells and basophils. Methods: In this study, we investigated the in vitro effects of ruxolitinib, a JAK1/2 inhibitor, on IgE- and IL-3-mediated release of mediators from human basophils, as well as substance P-induced mediator release from skin mast cells (HSMCs). Results: Ruxolitinib concentration-dependently inhibited IgE-mediated release of preformed (histamine) and de novo synthesized mediators (leukotriene C4) from human basophils. Ruxolitinib also inhibited anti-IgE- and IL-3-mediated cytokine (IL-4 and IL-13) release from basophils, as well as the secretion of preformed mediators (histamine, tryptase, and chymase) from substance Pactivated HSMCs. Discussion: These results indicate that ruxolitinib, inhibiting the release of several mediators from human basophils and mast cells, is a potential candidate for the treatment of inflammatory disorders. [ABSTRACT FROM AUTHOR]

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings. Targeted protein degradation (TPD) is a key modality for drug discovery. Here the authors present the discovery and analysis of reversible DCAF1-PROTACs, which show efficacy in cellular environments resistant to VHL-PROTACs or with acquired resistance to CRBN-PROTACs. [ABSTRACT FROM AUTHOR]

Tissue fibrosis remains unamenable to meaningful therapeutic interventions and is the primary cause of chronic graft failure after organ transplantation. Eukaryotic translation initiation factor (eIF4E), a key translational regulator, serves as convergent target of multiple upstream profibrotic signaling pathways that contribute to mesenchymal cell (MC) activation. Here, we investigate the role of MAP kinase--interacting serine/threonine kinase--induced (MNK-induced) direct phosphorylation of eIF4E at serine 209 (Ser209) in maintaining fibrotic transformation of MCs and determine the contribution of the MNK/ eIF4E pathway to the pathogenesis of chronic lung allograft dysfunction (CLAD). MCs from patients with CLAD demonstrated constitutively higher eIF4E phosphorylation at Ser209, and eIF4E phospho-Ser209 was found to be critical in regulating key fibrogenic protein autotaxin, leading to sustained β-catenin activation and profibrotic functions of CLAD MCs. MNK1 signaling was upregulated in CLAD MCs, and genetic or pharmacologic targeting of MNK1 activity inhibited eIF4E phospho-Ser209 and profibrotic functions of CLAD MCs in vitro. Treatment with an MNK1/2 inhibitor (eFT-508) abrogated allograft fibrosis in an orthotopic murine lung-transplant model. Together these studies identify what we believe is a previously unrecognized MNK/eIF4E/ATX/β-catenin signaling pathway of fibrotic transformation of MCs and present the first evidence, to our knowledge, for the utility of MNK inhibitors in fibrosis. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites balance proliferation, persistence, and spread in their metazoan hosts. AGC kinases, such as PKG, PKA, and the PDK1 ortholog SPARK, integrate environmental signals to toggle parasites between replicative and motile life stages. Recent studies have cataloged pathways downstream of apicomplexan PKG and PKA; however, less is known about the global integration of AGC kinase signaling cascades. Here, conditional genetics coupled to unbiased proteomics demonstrates that SPARK complexes with an elongin-like protein to regulate the stability of PKA and PKG in the model apicomplexan Toxoplasma gondii. Defects attributed to SPARK depletion develop after PKG and PKA are down-regulated. Parasites lacking SPARK differentiate into the chronic form of infection, which may arise from reduced activity of a coccidian-specific PKA ortholog. This work delineates the signaling topology of AGC kinases that together control transitions within the asexual cycle of this important family of parasites. [ABSTRACT FROM AUTHOR]

Pulmonary hypertension (PH) is a chronic progressive disease with high mortality. There has been more and more research focusing on the role of AMPK in PH. AMPK consists of three subunits—α, β, and γ. The crosstalk among these subunits ultimately leads to a delicate balance to affect PH, which results in conflicting conclusions about the role of AMPK in PH. It is still unclear how these subunits interfere with each other and achieve balance to improve or deteriorate PH. Several signaling pathways are related to AMPK in the treatment of PH, including AMPK/eNOS/NO pathway, Nox4/mTORC2/AMPK pathway, AMPK/BMP/Smad pathway, and SIRT3-AMPK pathway. Among these pathways, the role and mechanism of AMPK/eNOS/NO and Nox4/mTORC2/AMPK pathways are clearer than others, while the SIRT3-AMPK pathway remains still unclear in the treatment of PH. There are drugs targeting AMPK to improve PH, such as metformin (MET), MET combination, and rhodiola extract. In addition, several novel factors target AMPK for improving PH, such as ADAMTS8, TUFM, and Salt-inducible kinases. However, more researches are needed to explore the specific AMPK signaling pathways involved in these novel factors in the future. In conclusion, AMPK plays an important role in PH. [ABSTRACT FROM AUTHOR]

The polar auxin transport is required for proper plant growth and development. D6 PROTEIN KINASE (D6PK) is required for the phosphorylation of PIN‐FORMED (PIN) auxin efflux carriers to regulate auxin transport, while the regulation of D6PK stabilization is still poorly understood. Here, we found that Cytosolic ABA Receptor Kinases (CARKs) redundantly interact with D6PK, and the interactions are dependent on CARKs' kinase activities. Similarly, CARK3 also could interact with paralogs of D6PK, including D6PKL1, D6PKL2, and D6PKL3. The genetic analysis shows that D6PK acts the downstream of CARKs to regulate Arabidopsis growth, including hypocotyl, leaf area, vein formation, and the length of silique. Loss‐of‐function of CARK3 in overexpressing GFP‐D6PK plants leads to reduce the level of D6PK protein, thereby rescues plant growth. In addition, the cell‐free degradation assays indicate that D6PK is degraded through 26 S proteasome pathway, while the phosphorylation by CARK3 represses this process in cells. In summary, D6PK stabilization by the CARK family is required for auxin‐mediated plant growth and development. Summary statement: The AGC VIII kinase D6 PROTEIN KINASE (D6PK) phosphorylates PIN‐FORMED (PIN) auxin efflux carriers, which is essential for auxin transport in plant cells. This study uncovers a new mechanism for D6PK stability through Cytosolic ABA Receptor Kinases‐mediated phosphorylation to regulate auxin‐controlled Arabidopsis growth and development. [ABSTRACT FROM AUTHOR]

The inhibitory-kappaB kinases (IKKs) IKKα and IKKβ play central roles in regulating the non-canonical and canonical NF-κB signalling pathways. Whilst the proteins that transduce the signals of each pathway have been extensively characterised, the clear dissection of the functional roles of IKKα-mediated non-canonical NF-κB signalling versus IKKβ-driven canonical signalling remains to be fully elucidated. Progress has relied upon complementary molecular and pharmacological tools; however, the lack of highly potent and selective IKKα inhibitors has limited advances. Herein, we report the development of an aminoindazole-pyrrolo[2,3-b]pyridine scaffold into a novel series of IKKα inhibitors. We demonstrate high potency and selectivity against IKKα over IKKβ in vitro and explain the structure–activity relationships using structure-based molecular modelling. We show selective target engagement with IKKα in the non-canonical NF-κB pathway for both U2OS osteosarcoma and PC-3M prostate cancer cells by employing isoform-related pharmacodynamic markers from both pathways. Two compounds (SU1261 [IKKα Ki = 10 nM; IKKβ Ki = 680 nM] and SU1349 [IKKα Ki = 16 nM; IKKβ Ki = 3352 nM]) represent the first selective and potent pharmacological tools that can be used to interrogate the different signalling functions of IKKα and IKKβ in cells. Our understanding of the regulatory role of IKKα in various inflammatory-based conditions will be advanced using these pharmacological agents. [ABSTRACT FROM AUTHOR]

Simple Summary: The Wnt signaling pathway plays a pivotal role in governing developmental processes and maintaining stem cell characteristics, while also exhibiting significant implications in cancer pathogenesis. While initially characterized in colorectal cancer, aberrations in Wnt signaling are pervasive across various cancer types. Both intrinsic and extrinsic factors modulate Wnt signaling, mainly influencing the strictly coordinated kinase cascade within Wnt signaling. Understanding the complex interplay of canonical and non-canonical Wnt pathways and their potential dysregulation in disorders holds promise for the development of innovative therapeutic strategies in cancer treatment. Since the initial identification of oncogenic Wnt in mice and Drosophila, the Wnt signaling pathway has been subjected to thorough and extensive investigation. Persistent activation of Wnt signaling exerts diverse cancer characteristics, encompassing tumor initiation, tumor growth, cell senescence, cell death, differentiation, and metastasis. Here we review the principal signaling mechanisms and the regulatory influence of pathway-intrinsic and extrinsic kinases on cancer progression. Additionally, we underscore the divergences and intricate interplays of the canonical and non-canonical Wnt signaling pathways and their critical influence in cancer pathophysiology, exhibiting both growth-promoting and growth-suppressing roles across diverse cancer types. [ABSTRACT FROM AUTHOR]

Bacteria possess diverse classes of signaling systems that they use to sense and respond to their environments and execute properly timed developmental transitions. One widespread and evolutionarily ancient class of signaling systems are the Hanks‐type Ser/Thr kinases, also sometimes termed "eukaryotic‐like" due to their homology with eukaryotic kinases. In diverse bacterial species, these signaling systems function as critical regulators of general cellular processes such as metabolism, growth and division, developmental transitions such as sporulation, biofilm formation, and virulence, as well as antibiotic tolerance. This multifaceted regulation is due to the ability of a single Hanks‐type Ser/Thr kinase to post‐translationally modify the activity of multiple proteins, resulting in the coordinated regulation of diverse cellular pathways. However, in part due to their deep integration with cellular physiology, to date, we have a relatively limited understanding of the timing, regulatory hierarchy, the complete list of targets of a given kinase, as well as the potential regulatory overlap between the often multiple kinases present in a single organism. In this review, we discuss experimental methods and curated datasets aimed at elucidating the targets of these signaling pathways and approaches for using these datasets to develop computational models for quantitative predictions of target motifs. We emphasize novel approaches and opportunities for collecting data suitable for the creation of new predictive computational models applicable to diverse species. [ABSTRACT FROM AUTHOR]

Several blinding diseases affecting the retina and optic nerve are exacerbated by or caused by dysregulated inflammation and oxidative stress. These diseases include uveitis, age related macular degeneration, diabetic retinopathy and glaucoma. Consequently, despite their divergent symptoms, treatments that reduce oxidative stress and suppress inflammation may be therapeutic. The production of inflammatory cytokines and their activities are regulated by a class of proteins termed Suppressors of Cytokine Signaling (SOCS). SOCS1 and SOCS3 are known to dampen signaling via pathways employing Janus kinases and signal transducer and activatorof transcription proteins (JAK/STAT), Toll-like Receptors (TLR), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), mitogen activated kinase (MAPK) andNLR family pyrin domain containing 3 (NLRP3). We have developed cellpenetrating peptides from the kinase inhibitory region of the SOCS1 and SOCS3 (denoted as R9-SOCS1-KIR and R9-SOCS3-KIR) and tested them in retinal pigment epithelium (RPE) cellsand inmacrophage cell lines.SOCS-KIRpeptides exhibited antiinflammatory, anti-oxidant and anti-angiogenic properties. In cell culture, both Th1 and Th17 cells were suppressed together with the inhibition of other inflammatory markers. We also observed a decrease in oxidants and a simultaneous rise in neuroprotective and anti-oxidant effectors. In addition, treatment prevented the loss of gap junction proteins and the ensuing drop in transepithelial electrical resistance in RPE cells. When tested in mouse models by eye drop instillation, they showed protection against autoimmune uveitis, as a prophylactic as well as a therapeutic. Mice with endotoxin-induced uveitis were protected by eye drop administration as well. R9-SOCS3-KIR was particularly effective against the pathways acting through STAT3, e.g. IL-6 and VEGF-A mediated responses that lead to macular degeneration. Eye drop administration of R9-SOCS3-KIR stimulated production of antioxidant effectorsandreducedclinicalsymptomsinmousemodel of oxidative stress that replicates the RPE injury occurring in AMD. Because these peptides suppress multiple pathogenic stimuli and because they can be delivered topically to the cornea, they are attractive candidates for therapeutics for uveitis, macular degeneration, diabetic retinopathy and glaucoma. [ABSTRACT FROM AUTHOR]

Signal processing by intracellular kinases controls near all biological processes but how signal pathway functions evolve with changed cellular context is poorly understood. Functional specificity of c-Jun N-terminal Kinases (JNK) are partly encoded by signal strength. Here we reveal that intracellular pH (pHi) is a significant component of the JNK network and defines signal response to specific stimuli. We show pHi regulates JNK activity in response to cell stress, with the relationship between pHi and JNK activity dependent on specific stimuli and upstream kinases activated. Using the optogenetic clustering tag CRY2, we show that an increase in pHi promotes the light-induced phase transition of ASK1 to augment JNK activation. While increased pHi similarly promoted CRY2-tagged JNK2 to form light-induced condensates, this attenuated JNK activity. Mathematical modelling of feedback signalling incorporating pHi and differential contributions by ASK1 and JNK2 condensates was sufficient to delineate signal responses to specific stimuli. Taking pHi and ASK1/JNK2 signal contributions into consideration may delineate oncogenic versus tumour suppressive JNK functions and cancer cell drug responses. The transduction of signals by c-Jun N-terminal kinases (JNK) is highly contextual and has diverse cell functions. Here, the authors reveal tiered kinases in the JNK pathway are regulated by intracellular pH with changed signaling outcomes. [ABSTRACT FROM AUTHOR]

Protein kinases are molecular machines with rich sequence variation that distinguishes the two main evolutionary branches – tyrosine kinases (TKs) from serine/threonine kinases (STKs). Using a sequence co-variation Potts statistical energy model we previously concluded that TK catalytic domains are more likely than STKs to adopt an inactive conformation with the activation loop in an autoinhibitory folded conformation, due to intrinsic sequence effects. Here we investigate the structural basis for this phenomenon by integrating the sequence-based model with structure-based molecular dynamics (MD) to determine the effects of mutations on the free energy difference between active and inactive conformations, using a thermodynamic cycle involving many (n = 108) protein-mutation free energy perturbation (FEP) simulations in the active and inactive conformations. The sequence and structure-based results are consistent and support the hypothesis that the inactive conformation DFG-out Activation Loop Folded, is a functional regulatory state that has been stabilized in TKs relative to STKs over the course of their evolution via the accumulation of residue substitutions in the activation loop and catalytic loop that facilitate distinct substrate binding modes in trans and additional modes of regulation in cis for TKs. In this study, the authors identify a mechanism for the distinct conformational preferences of tyrosine kinases vs serine/threonine kinases and suggest that the evolution of tyrosine kinase function can explain these conformational differences. [ABSTRACT FROM AUTHOR]

Neuroectoderm-derived tumors characteristically express gangliosides such as GD3 and GD2. Many studies have reported that gangliosides GD3/GD2 enhance malignant phenotypes of cancers. Recently, we reported that human gliomas expressing GD3/GD2 exhibited enhanced malignant phenotypes. Here, we investigated the function of GD3/GD2 in glioma cells and GD3/GD2-expressing glioma-derived exosomes. As reported previously, transfectant cells of human glioma U251 MG expressing GD3/GD2 showed enhanced cancer phenotypes compared with GD3/GD2-negative controls. When GD3/GD2-negative cells were treated with exosomes secreted from GD3/GD2-positive cells, clearly increased malignant properties were observed. Furthermore, increased phosphorylation of signaling molecules was detected after 5-15 min of exosome treatment, ie, higher tyrosine phosphorylation of platelet-derived growth factor receptor, focal adhesion kinase, and paxillin was found in treated cells than in controls. Phosphorylation of extracellular signal-regulated kinase-1/2 was also enhanced. Consequently, it is suggested that exosomes secreted from GD3/GD2-positive gliomas play important roles in enhancement of the malignant properties of glioma cells, leading to total aggravation of heterogenous cancer tissues, and also in the regulation of tumor microenvironments. [ABSTRACT FROM AUTHOR]

Salt-inducible kinases (SIKs) are serine/threonine kinases of the adenosine monophosphate-activated protein kinase family. Acting as mediators of a broad array of neuronal and hormonal signaling pathways, SIKs play diverse roles in many physiological and pathological processes. Phosphorylation by the upstream kinase liver kinase B1 is required for SIK activation, while phosphorylation by protein kinase A induces the binding of 14-3-3 protein and leads to SIK inhibition. SIKs are subjected to auto-phosphorylation regulation and their activity can also be modulated by Ca2+/calmodulindependent protein kinase in response to cellular calcium influx. SIKs regulate the physiological processes through direct phosphorylation on various substrates, which include class IIa histone deacetylases, cAMP-regulated transcriptional coactivators, phosphatase methylesterase-1, among others. Accumulative body of studies have demonstrated that SIKs are important regulators of the cardiovascular system, including early works establishing their roles in sodium sensing and vascular homeostasis and recent progress in pulmonary arterial hypertension and pathological cardiac remodeling. SIKs also regulate inflammation, fibrosis, and metabolic homeostasis, which are essential pathological underpinnings of cardiovascular disease. The development of small molecule SIK inhibitors provides the translational opportunity to explore their potential as therapeutic targets for treating cardiometabolic disease in the future. [ABSTRACT FROM AUTHOR]

During nervous system development, Sonic hedgehog (Shh) guides developing commissural axons toward the floor plate of the spinal cord. To guide axons, Shh binds to its receptor Boc and activates downstream effectors such as Smoothened (Smo) and Src family kinases (SFKs). SFK activation requires Smo activity and is also required for Shh-mediated axon guidance. Here we report that β-arrestin1 and β-arrestin2 (β-arrestins) serve as scaffolding proteins that link Smo and SFKs in Shh-mediated axon guidance. We found that β-arrestins are expressed in rat commissural neurons. We also found that Smo, β-arrestins, and SFKs form a tripartite complex, with the complex formation dependent on β-arrestins. β-arrestin knockdown blocked the Shh-mediated increase in Src phosphorylation, demonstrating that β-arrestins are required to activate Src kinase downstream of Shh. β-arrestin knockdown also led to the loss of Shh-mediated attraction of rat commissural axons in axon turning assays. Expression of two different dominant-negative β-arrestins, β-arrestin1 V53D which blocks the internalization of Smo and β-arrestin1 P91G-P121E which blocks its interaction with SFKs, also led to the loss of Shh-mediated attraction of commissural axons. In vivo, the expression of these dominant-negative β-arrestins caused defects in commissural axon guidance in the spinal cord of chick embryos of mixed sexes. Thus we show that β-arrestins are essential scaffolding proteins that connect Smo to SFKs and are required for Shh-mediated axon guidance. [ABSTRACT FROM AUTHOR]

Protein kinase function and interactions with drugs are controlled in part by the movement of the DFG and ɑC-Helix motifs that are related to the catalytic activity of the kinase. Small molecule ligands elicit therapeutic effects with distinct selectivity profiles and residence times that often depend on the active or inactive kinase conformation(s) they bind. Modern AI-based structural modeling methods have the potential to expand upon the limited availability of experimentally determined kinase structures in inactive states. Here, we first explored the conformational space of kinases in the PDB and models generated by AlphaFold2 (AF2) and ESMFold, two prominent AI-based protein structure prediction methods. Our investigation of AF2's ability to explore the conformational diversity of the kinome at various multiple sequence alignment (MSA) depths showed a bias within the predicted structures of kinases in DFG-in conformations, particularly those controlled by the DFG motif, based on their overabundance in the PDB. We demonstrate that predicting kinase structures using AF2 at lower MSA depths explored these alternative conformations more extensively, including identifying previously unobserved conformations for 398 kinases. Ligand enrichment analyses for 23 kinases showed that, on average, docked models distinguished between active molecules and decoys better than random (average AUC (avgAUC) of 64.58), but select models perform well (e.g., avgAUCs for PTK2 and JAK2 were 79.28 and 80.16, respectively). Further analysis explained the ligand enrichment discrepancy between low- and high-performing kinase models as binding site occlusions that would preclude docking. The overall results of our analyses suggested that, although AF2 explored previously uncharted regions of the kinase conformational space and select models exhibited enrichment scores suitable for rational drug discovery, rigorous refinement of AF2 models is likely still necessary for drug discovery campaigns. Author summary: Greater abundance of kinase structural data in inactive conformations, currently lacking in structural databases, would improve our understanding of how protein kinases function, and expand drug discovery and development for this important family of therapeutic targets. Modern approaches utilizing artificial intelligence and machine learning, like AlphaFold2 and ESMFold, have potential for efficiently capturing novel protein conformations. We provide evidence for a bias within AlphaFold2 and ESMFold to predict structures of kinases in their active states, similar to their overrepresentation in the PDB. We show that lowering the multiple sequence alignment depth used in the AlphaFold2 algorithm can help explore the kinase conformational space more broadly. Through a series of quantitative and visual analyses of the models, we also offer a critique of AlphaFold2-generated models, highlighting their potential utility in drug discovery, but also underscoring the need for further refinement to enhance their suitability for rational drug design. Furthermore, many of the high-quality and high-enriching models we make available may represent starting points for novel drug discovery campaigns. [ABSTRACT FROM AUTHOR]