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Background: LRRK2-targeting therapeutics that inhibit LRRK2 kinase activity have advanced to clinical trials in idiopathic Parkinson's disease (iPD). LRRK2 phosphorylates Rab10 on endolysosomes in phagocytic cells to promote some types of immunological responses. The identification of factors that regulate LRRK2-mediated Rab10 phosphorylation in iPD, and whether phosphorylated-Rab10 levels change in different disease states, or with disease progression, may provide insights into the role of Rab10 phosphorylation in iPD and help guide therapeutic strategies targeting this pathway. Methods: Capitalizing on past work demonstrating LRRK2 and phosphorylated-Rab10 interact on vesicles that can shed into biofluids, we developed and validated a high-throughput single-molecule array assay to measure extracellular pT73-Rab10. Ratios of pT73-Rab10 to total Rab10 measured in biobanked serum samples were compared between informative groups of transgenic mice, rats, and a deeply phenotyped cohort of iPD cases and controls. Multivariable and weighted correlation network analyses were used to identify genetic, transcriptomic, clinical, and demographic variables that predict the extracellular pT73-Rab10 to total Rab10 ratio. Results: pT73-Rab10 is absent in serum from Lrrk2 knockout mice but elevated by LRRK2 and VPS35 mutations, as well as SNCA expression. Bone-marrow transplantation experiments in mice show that serum pT73-Rab10 levels derive primarily from circulating immune cells. The extracellular ratio of pT73-Rab10 to total Rab10 is dynamic, increasing with inflammation and rapidly decreasing with LRRK2 kinase inhibition. The ratio of pT73-Rab10 to total Rab10 is elevated in iPD patients with greater motor dysfunction, irrespective of disease duration, age, sex, or the usage of PD-related or anti-inflammatory medications. pT73-Rab10 to total Rab10 ratios are associated with neutrophil degranulation, antigenic responses, and suppressed platelet activation. Conclusions: The extracellular serum ratio of pT73-Rab10 to total Rab10 is a novel pharmacodynamic biomarker for LRRK2-linked innate immune activation associated with disease severity in iPD. We propose that those iPD patients with higher serum pT73-Rab10 levels may benefit from LRRK2-targeting therapeutics that mitigate associated deleterious immunological responses. [ABSTRACT FROM AUTHOR]

Akt3 is one of the three members of the serine/threonine protein kinase B (AKT) family, which regulates multiple cellular processes. We have previously demonstrated that global knockout of Akt3 in mice promotes atherogenesis in a macrophage-dependent manner. Whether enhanced Akt3 kinase activity affects atherogenesis is not known. In this study, we crossed atherosclerosis-prone ApoE-/- mice with a mouse strain that has enhanced Akt3 kinase activity (Akt3nmf350) and assessed atherosclerotic lesion formation and the role of macrophages in atherogenesis. Significant reduction in atherosclerotic lesion area and macrophage accumulation in lesions were observed in ApoE-/-/Akt3nmf350 mice fed a Western-type diet. Experiments using chimeric ApoE-/- mice with either ApoE-/-/Akt3nmf350 bone marrow or ApoE-/- bone marrow cells showed that enhanced Akt3 activity specifically in bone marrow-derived cells is atheroprotective. The atheroprotective effect of Akt3nmf350 was more pronounced in male mice. In line with this result, the release of the pro-inflammatory cytokines IL-6, MCP1, TNF-α, and MIP-1α was reduced by macrophages from male but not female ApoE-/-/Akt3nmf350 mice. Levels of IL-6 and TNF-α were also reduced in atherosclerotic lesions of ApoE-/-/Akt3nmf350 male mice compared to ApoE-/- mice. Macrophages from male ApoE-/-/Akt3nmf350 mice were also more resistant to apoptosis in vitro and in vivo and tended to have more pronounced M2 polarization in vitro. These findings demonstrated that enhanced Akt3 kinase activity in macrophages protects mice from atherosclerosis in hyperlipidemic mice in a gender-dependent manner. [ABSTRACT FROM AUTHOR]

Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phosphomimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis. [ABSTRACT FROM AUTHOR]

Background: Neutrophils depend heavily on glycolysis for energy production under normal conditions. In contrast, neutrophils require energy supplied by mitochondrial oxidative phosphorylation (OXPHOS) during chemotaxis. However, the mechanism by which the energy supply changes from glycolysis to OXPHOS remains unknown. Leucine-rich repeat kinase 2 (LRRK2) is partially present in the outer mitochondrial membrane fraction. Lrrk2-deficient cells show mitochondrial fragmentation and reduced OXPHOS activity. We have previously reported that mitofusin (MFN) 2 is involved in chemotaxis and OXPHOS activation upon chemoattractant N-formyl-Met-Leu-Phe (fMLP) stimulation in differentiated HL-60 (dHL-60) cells. It has been previously reported that LRRK2 binds to MFN2 and partially colocalizes with MFN2 at the mitochondrial membranes. This study investigated the involvement of LRRK2 in chemotaxis and MFN2 activation in neutrophils and dHL-60 cells. Methods: Lrrk2 knockout neutrophils and Lrrk2 knockdown dHL-60 cells were used to examine the possible involvement of LRRK2 in chemotaxis. Lrrk2 knockdown dHL-60 cells were used a tetracycline-inducible small hairpin RNA (shRNA) system to minimize the effects of LRRK2 knockdown during cell culture. The relationship between LRRK2 and MFN2 was investigated by measuring the GTP-binding activity of MFN2 in Lrrk2 knockdown dHL-60 cells. The effects of LRRK2 kinase activity on chemotaxis were examined using the LRRK2 kinase inhibitor MLi-2. Results: fMLP-induced chemotactic activity was reduced in Lrrk2 knockout neutrophils in vitro and in vivo. Lrrk2 knockdown in dHL-60 cells expressing Lrrk2 shRNA also reduced fMLP-induced chemotactic activity. Lrrk2 knockdown dHL-60 cells showed reduced OXPHOS activity and suppressed mitochondrial morphological change, similar to Mfn2 knockdown dHL-60 cells. The amount of LRRK2 in the mitochondrial fraction and the GTP-binding activity of MFN2 increased upon fMLP stimulation, and the MFN2 GTP-binding activity was suppressed in Lrrk2 knockdown dHL-60 cells. Furthermore, the kinase activity of LRRK2 and Ser935 phosphorylation of LRRK2 were reduced upon fMLP stimulation, and LRRK2 kinase inhibition by MLi-2 increased the migration to fMLP. Conclusions: LRRK2 is involved in neutrophil chemotaxis and the GTP-binding activity of MFN2 upon fMLP stimulation. On the other hand, the kinase activity of LRRK2 shows a negative regulatory effect on fMLP-induced chemotactic activity in dHL-60 cells. -J661noP-dBkFUp6aS7aWr Video Abstract [ABSTRACT FROM AUTHOR]

Epithelial tissue is at the forefront of innate immunity, playing a crucial role in the recognition and elimination of pathogens. Met is a receptor tyrosine kinase that is necessary for epithelial cell survival, proliferation, and regeneration. Here, we showed that Met is essential for the induction of cytokine production by cytosolic nonself double-stranded RNA through retinoic acid-inducible gene-I-like receptors (RLRs) in epithelial cells. Surprisingly, the tyrosine kinase activity of Met was dispensable for promoting cytokine production. Rather, the intracellular carboxy terminus of Met interacted with mitochondrial antiviral-signaling protein (MAVS) in RLR-mediated signaling to directly promote MAVS signalosome formation. These studies revealed a kinase activity-independent function of Met in the promotion of antiviral innate immune responses, defining dual roles of Met in both regeneration and immune responses in the epithelium. [ABSTRACT FROM AUTHOR]

Inherited retinal degenerative diseases (IRDs) are a group of rare diseases that lead to a progressive loss of photoreceptor cells and, ultimately, blindness. The overactivation of cGMP-dependent protein kinase G (PKG), one of the key effectors of cGMP-signaling, was previously found to be involved in photoreceptor cell death and was studied in murine IRD models to elucidate the pathophysiology of retinal degeneration. However, PKG is a serine/threonine kinase (STK) with several hundred potential phosphorylation targets and, so far, little is known about the specificity of the target interaction and downstream effects of PKG activation. Here, we carried out both the kinome activity and phosphoproteomic profiling of organotypic retinal explant cultures derived from the rd10 mouse model for IRD. After treating the explants with the PKG inhibitor CN03, an overall decrease in peptide phosphorylation was observed, with the most significant decrease occurring in seven peptides, including those from the known PKG substrate cyclic-AMP-response-element-binding CREB, but also Ca2+/calmodulin-dependent kinase (CaMK) peptides and TOP2A. The phosphoproteomic data, in turn, revealed proteins with decreased phosphorylation, as well as proteins with increased phosphorylation. The integration of both datasets identified common biological networks altered by PKG inhibition, which included kinases predominantly from the so-called AGC and CaMK families of kinases (e.g., PKG1, PKG2, PKA, CaMKs, RSKs, and AKTs). A pathway analysis confirmed the role of CREB, Calmodulin, mitogen-activated protein kinase (MAPK) and CREB modulation. Among the peptides and pathways that showed reduced phosphorylation activity, the substrates CREB, CaMK2, and CaMK4 were validated for their retinal localization and activity, using immunostaining and immunoblotting in the rd10 retina. In summary, the integrative analysis of the kinome activity and phosphoproteomic data revealed both known and novel PKG substrates in a murine IRD model. This data establishes a basis for an improved understanding of the biological pathways involved in cGMP-mediated photoreceptor degeneration. Moreover, validated PKG targets like CREB and CaMKs merit exploration as novel (surrogate) biomarkers to determine the effects of a clinical PKG-targeted treatment for IRDs. [ABSTRACT FROM AUTHOR]

Simple Summary: Recurrent non-medullary thyroid cancer (NMTC) is difficult to treat and therapy options are limited. Of the available compounds, serine/threonine kinase (STK) inhibitors are currently widely used. However, this form of targeted therapy is not always effective and additional response-related biological information may improve both accuracy and efficacy. Using in-depth STK activity profiling, in this study, we aimed to determine whether benign and malignant thyroid tumors can be differentiated based on these profiles. In addition, we analyzed the impact of the BRAF V600E-specific inhibitor dabrafenib, as well as the generic RAF inhibitors sorafenib and regorafenib, in a subgroup of BRAF V600E and non-BRAF V600E papillary thyroid carcinomas. We demonstrate that STK activity profiling can differentiate benign from malignant thyroid tumors. Furthermore, the BRAF V600E-specific inhibitor dabrafenib can distinguish BRAF V600E from non-BRAF V600E papillary thyroid carcinomas. We conclude that STK activity profiling is beneficial when the goal is to differentiate benign from malignant thyroid tumors. In addition, this approach aids in the selection of likely effective (novel) kinase inhibitors for treatment of recurrent thyroid and other cancers. Differentiated non-medullary thyroid cancer (NMTC) can be effectively treated by surgery followed by radioactive iodide therapy. However, a small subset of patients shows recurrence due to a loss of iodide transport, a phenotype frequently associated with BRAF V600E mutations. In theory, this should enable the use of existing targeted therapies specifically designed for BRAF V600E mutations. However, in practice, generic or specific drugs aimed at molecular targets identified by next generation sequencing (NGS) are not always beneficial. Detailed kinase profiling may provide additional information to help improve therapy success rates. In this study, we therefore investigated whether serine/threonine kinase (STK) activity profiling can accurately classify benign thyroid lesions and NMTC. We also determined whether dabrafenib (BRAF V600E-specific inhibitor), as well as sorafenib and regorafenib (RAF inhibitors), can differentiate BRAF V600E from non-BRAF V600E thyroid tumors. Using 21 benign and 34 malignant frozen thyroid tumor samples, we analyzed serine/threonine kinase activity using PamChip®peptide microarrays. An STK kinase activity classifier successfully differentiated malignant (26/34; 76%) from benign tumors (16/21; 76%). Of the kinases analyzed, PKC (theta) and PKD1 in particular, showed differential activity in benign and malignant tumors, while oncocytic neoplasia or Graves' disease contributed to erroneous classifications. Ex vivo BRAF V600E-specific dabrafenib kinase inhibition identified 6/92 analyzed peptides, capable of differentiating BRAF V600E-mutant from non-BRAF V600E papillary thyroid cancers (PTCs), an effect not seen with the generic inhibitors sorafenib and regorafenib. In conclusion, STK activity profiling differentiates benign from malignant thyroid tumors and generates unbiased hypotheses regarding differentially active kinases. This approach can serve as a model to select novel kinase inhibitors based on tissue analysis of recurrent thyroid and other cancers. [ABSTRACT FROM AUTHOR]

Cellular inhibitor of apoptosis proteins (cIAPs) are RING‐containing E3 ubiquitin ligases that ubiquitylate receptor‐interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2MutR). cIap1/2MutR/MutR mice died during embryonic development due to RIPK1‐mediated apoptosis. While expression of kinase‐inactive RIPK1D138N rescued embryonic development, Ripk1D138N/D138N/cIap1/2MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2MutR and RIPK1D138N were still susceptible to TNF‐induced apoptosis and necroptosis, implying additional kinase‐independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock‐out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1‐mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF‐signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1. Synopsis: cIAPs control RIPK1‐dependent and ‐independent TNF toxicity in mouse. This study finds ubiquitylation and inhibition of RIPK1 by cIAPs to be required for mouse embryonic development, while RIPK1‐independent cIAP inhibition of TNF‐induced cell death regulates tissue inflammation in adult mice. cIAP1/2 E3 ubiquitin ligase activity is required for mouse embryonic development.Catalytic activity of cIAPs regulates RIPK1 kinase activity to promote embryonic survival.TNF induces apoptosis and necroptosis in cells lacking the cIAPs‐RIPK1 regulatory axis.cIAPs control non‐overlapping signalling processes downstream of RIPK1 and TNFR1. [ABSTRACT FROM AUTHOR]

Protein kinases are key regulators of cell signaling and have been important therapeutic targets for three decades. ATP-competitive drugs directly inhibit the activity of kinases but these enzymes work as part of complex protein networks in which protein–protein interactions (often referred to as kinase docking) may govern a more complex activation pattern. Kinase docking is indispensable for many signaling disease-relevant Ser/Thr kinases and it is mediated by a dedicated surface groove on the kinase domain which is distinct from the substrate-binding pocket. Thus, interfering with kinase docking provides an alternative strategy to control kinases. We describe activity sensors developed for p90 ribosomal S6 kinase (RSK) and mitogen-activated protein kinases (MAPKs: ERK, p38, and JNK) whose substrate phosphorylation is known to depend on kinase-docking-groove-mediated protein–protein binding. The in vitro assays were based on fragment complementation of the NanoBit luciferase, which is facilitated upon substrate motif phosphorylation. The new phosphorylation-assisted luciferase complementation (PhALC) sensors are highly selective and the PhALC assay is a useful tool for the quantitative analysis of kinase activity or kinase docking, and even for high-throughput screening of academic compound collections. [ABSTRACT FROM AUTHOR]

Motile bacteria have a chemotaxis system that enables them to sense their environment and direct their swimming toward favorable conditions. Chemotaxis involves a signaling process in which ligand binding to the extracellular domain of the chemoreceptor alters the activity of the histidine kinase, CheA, bound ~300 Å away to the distal cytoplasmic tip of the receptor, to initiate a phosphorylation cascade that controls flagellar rotation. The cytoplasmic domain of the receptor is thought to propagate this signal via changes in dynamics and/or stability, but it is unclear how these changes modulate the kinase activity of CheA. To address this question, we have used hydrogen deuterium exchange mass spectrometry to probe the structure and dynamics of CheA within functional signaling complexes of the Escherichia coli aspartate receptor cytoplasmic fragment, CheA, and CheW. Our results reveal that stabilization of the P4 catalytic domain of CheA correlates with kinase activation. Furthermore, differences in activation of the kinase that occur during sensory adaptation depend on receptor destabilization of the P3 dimerization domain of CheA. Finally, hydrogen exchange properties of the P1 domain that bears the phosphorylated histidine identify the dimer interface of P1/P1' in the CheA dimer and support an ordered sequential binding mechanism of catalysis, in which dimeric P1/P1' has productive interactions with P4 only upon nucleotide binding. Thus stabilization/destabilization of domains is a key element of the mechanism of modulating CheA kinase activity in chemotaxis, and may play a role in the control of other kinases. [ABSTRACT FROM AUTHOR]

Tumor progression locus 2 (TPL2) (MAP3K8) is a central signaling node in the inflammatory response of peripheral immune cells. We find that TPL2 kinase activity modulates microglial cytokine release and is required for microglia-mediated neuron death in vitro. In acute in vivo neuroinflammation settings, TPL2 kinase activity regulates microglia activation states and brain cytokine levels. In a tauopathy model of chronic neurodegeneration, loss of TPL2 kinase activity reduces neuroinflammation and rescues synapse loss, brain volume loss, and behavioral deficits. Single-cell RNA sequencing analysis indicates that protection in the tauopathy model was associated with reductions in activated microglia subpopulations as well as infiltrating peripheral immune cells. Overall, using various models, we find that TPL2 kinase activity can promote multiple harmful consequences of microglial activation in the brain including cytokine release, iNOS (inducible nitric oxide synthase) induction, astrocyte activation, and immune cell infiltration. Consequently, inhibiting TPL2 kinase activity could represent a potential therapeutic strategy in neurodegenerative conditions. [ABSTRACT FROM AUTHOR]

Summary: Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf‐like kinase HT1 as a required step for stomatal movements caused by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2‐induced stomatal responses warrants in‐depth investigation.We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1.We show that CO2/HCO3−‐enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase‐dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2‐dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2/HCO3− and present structural modeling that explains the MPK12:HT1 interaction interface.These data add to the model that MPK12 kinase‐activity‐independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO2 concentration. [ABSTRACT FROM AUTHOR]

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death. [ABSTRACT FROM AUTHOR]

The aim of the study was to evaluate the role of kisspeptin-10 (KiSS-10) in the regulation of collagen content in cardiac fibroblasts. An attempt was also made to describe the mechanism of the effect of KiSS-10 on collagen metabolism. The studies indicate that kisspeptin-10 significantly increases the content of intracellular collagen in the myocardium. KiSS-10 also elevates the level of phosphorylated focal adhesion kinase (FAK) in human cardiac fibroblasts. The inhibition of FAK negates the stimulatory effect of KiSS-10 on collagen deposition in vitro. These changes correlate with an increase in the level of propeptides of procollagen type I (PICP) and III (PIIICP) in fibroblast culture medium and mouse PIIICP in serum. Moreover, this hormone inhibits the release of metalloproteinases (MMP-1,-2,-9) and elevates the secretion of their tissue inhibitors (TIMP-1,-2,-4). KiSS-10 also enhances the expression of α1 chains of procollagen type I and III in vitro. Thus, KiSS-10 is involved in the regulation of collagen metabolism and cardiac fibrosis. Augmentation of collagen deposition by KiSS-10 is dependent on the protein synthesis elevation, inhibition of MMPs activity (increase of TIMPs release) or decrease of MMPs concentration. The profibrotic activity of KiSS-10 is mediated by FAK and is not dependent on TGF-β1. [ABSTRACT FROM AUTHOR]

The dysregulation of protein kinases is associated with multiple diseases due to the kinases' involvement in a variety of cell signaling pathways. Manipulating protein kinase function, by controlling the active site, is a promising therapeutic and investigative strategy to mitigate and study diseases. Kinase active sites share structural similarities, making it difficult to specifically target one kinase, and allosteric control allows specific regulation and study of kinase function without directly targeting the active site. Allosteric sites are distal to the active site but coupled via a dynamic network of inter-atomic interactions between residues in the protein. Establishing an allosteric control over a kinase requires understanding the allosteric wiring of the protein. Computational techniques offer effective and inexpensive mapping of the allosteric sites on a protein. Here, we discuss the methods to map and regulate allosteric communications in proteins, and strategies to establish control over kinase functions in live cells and organisms. Protein molecules, or 'sensors,' are engineered to function as tools to control allosteric activity of the protein as these sensors have high spatiotemporal resolution and help in understanding cell phenotypes after immediate activation or inactivation of a kinase. Traditional methods used to study protein functions, such as knockout, knockdown, or mutation, cannot offer a sufficiently high spatiotemporal resolution. We discuss the modern repertoire of tools to regulate protein kinases as we enter a new era in deciphering cellular signaling and developing novel approaches to treat diseases associated with signal dysregulation. [ABSTRACT FROM AUTHOR]

Hirschsprung disease (HSCR) is a congenital disorder caused by the failure of enteric neural crest cells (ENCCs) to colonize the distal bowel, resulting in absence of enteric nervous system. While a range of molecules and signaling pathways have been found to contribute to HSCR development, the risk factors and pathogenesis of this disease in many patients remain unknown. We previously demonstrated that increased activity of the prostaglandin E2 (PGE2)/PGE2 receptor subtype EP2 pathway can be a risk factor for HSCR. In this study, an Ednrb-deficient mouse model of HSCR was generated and used to investigate if PGE2/EP2 pathway could be a potential therapeutic target for HSCR. We found that downregulation of PGE2/EP2 signaling by siRNA-mediated ablation of a PGE2 synthase or pharmacologic blockage of EP2 enhanced ENCC colonization in the distal bowel of Ednrb−/− mice and alleviated their HSCR-like symptoms. Furthermore, blockage of EP2 was shown to promote ENCC migration through upregulating p38 mitogen-activated protein kinase activity, which was downregulated in the colon of Ednrb−/− mice and in the distal aganglionic bowel of HSCR patients. These data provide evidence that maternal exposure during embryonic development to an environment with dysregulated activation of the PGE2/EP2 pathway may predispose genetically susceptible offspring to HSCR, and avoidance or early disruption of maternal events (e.g. inflammation) that possibly enhance PGE2/EP2 signaling during pregnancy would reduce the occurrence and severity of this disease. Key messages: Knockdown of PTGES alleviates HSCR severity in Ednrb−/− mice. Blockage of EP2-mediated PGE2 signaling alleviates HSCR severity in Ednrb−/− mice. Blockage of EP2-mediated PGE2 signaling promotes ENCC migration via enhancing p38 activity. [ABSTRACT FROM AUTHOR]

Although structurally similar to type II counterparts, type I or activin receptor-like kinases (ALKs) are set apart by a metastable helix–loop–helix (HLH) element preceding the protein kinase domain that, according to a longstanding paradigm, serves passive albeit critical roles as an inhibitor-to-substrate-binding switch. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder activation of ACVR1/ALK2 leading to sporadic heterotopic bone deposition in patients presenting with fibrodysplasia ossificans progressiva, or FOP. To determine the protein structural–functional basis for the gain of function, R206H mutant, Q207D (aspartate-substituted caALK2) and HLH subdomain-truncated (208 Ntrunc) forms were compared to one another and the wild-type enzyme through in vitro kinase and protein–protein interaction analyses that were complemented by signaling read-out (p-Smad) in primary mouse embryonic fibroblasts and Drosophila S2 cells. Contrary to the paradigm, the HLH subdomain actively suppressed the phosphotransferase activity of the enzyme, even in the absence of FKBP12. Unexpectedly, perturbation of the HLH subdomain elevated kinase activity at a distance, i.e., allosterically, at the ATP-binding and polypeptide-interacting active site cleft. Accessibility to polypeptide substrate (BMP Smad C-terminal tails) due to allosterically altered conformations of type I active sites within heterohexameric cytoplasmic signaling complexes—assembled noncanonically by activin-type II receptors extracellularly—is hypothesized to produce a gain of function of the R206H mutant protein responsible for episodic heterotopic ossification in FOP. [ABSTRACT FROM AUTHOR]

Alpha-protein kinase 1 (ALPK1) is a pathogen recognition receptor that detects ADP-heptose (ADPH), a lipopolysaccharide biosynthesis intermediate, recently described as a pathogen-associated molecular pattern in Gram-negative bacteria. ADPH binding to ALPK1 activates its kinase domain and triggers TIFA phosphorylation on threonine 9. This leads to the assembly of large TIFA oligomers called TIFAsomes, activation of NF-κB and pro-inflammatory gene expression. Furthermore, mutations in ALPK1 are associated with inflammatory syndromes and cancers. While this kinase is of increasing medical interest, its activity in infectious or non-infectious diseases remains poorly characterized. Here, we use a non-radioactive ALPK1 in vitro kinase assay based on the use of ATPγS and protein thiophosphorylation. We confirm that ALPK1 phosphorylates TIFA T9 and show that T2, T12 and T19 are also weakly phosphorylated by ALPK1. Interestingly, we find that ALPK1 itself is phosphorylated in response to ADPH recognition during Shigella flexneri and Helicobacter pylori infection and that disease-associated ALPK1 mutants exhibit altered kinase activity. In particular, T237M and V1092A mutations associated with ROSAH syndrome and spiradenoma/spiradenocarcinoma respectively, exhibit enhanced ADPH-induced kinase activity and constitutive assembly of TIFAsomes. Altogether, this study provides new insights into the ADPH sensing pathway and disease-associated ALPK1 mutants. [ABSTRACT FROM AUTHOR]

Glucose metabolism and DNA repair are fundamental cellular processes frequently dysregulated in cancer. In this study, we define a direct role for the glycolytic Aldolase A (ALDOA) protein in DNA double-strand break (DSB) repair. ALDOA is a fructose biphosphate Aldolase that catalyses fructose-1,6-bisphosphate to glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP), during glycolysis. Here, we show that upon DNA damage induced by ionising radiation (IR), ALDOA translocates from the cytoplasm into the nucleus, where it partially co-localises with the DNA DSB marker γ-H2AX. DNA damage was shown to be elevated in ALDOA-depleted cells prior to IR and following IR the damage was repaired more slowly. Consistent with this, cells depleted of ALDOA exhibited decreased DNA DSB repair via non-homologous end-joining and homologous recombination. In support of the defective repair observed in its absence, ALDOA was found to associate with the major DSB repair effector kinases, DNA-dependent Protein Kinase (DNA-PK) and Ataxia Telangiectasia Mutated (ATM) and their autophosphorylation was decreased when ALDOA was depleted. Together, these data establish a role for an essential metabolic protein, ALDOA in DNA DSB repair and suggests that targeting ALDOA may enable the concurrent targeting of cancer metabolism and DNA repair to induce tumour cell death. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinases (RTKs) are typically activated through a precise sequence of intracellular phosphorylation events starting with a tyrosine residue on the activation loop (A-loop) of the kinase domain (KD). From this point the mono-phosphorylated enzyme is active, but subject to stringent regulatory mechanisms which can vary dramatically across the different RTKs. In the absence of extracellular stimulation, fibroblast growth factor receptor 2 (FGFR2) exists in the mono-phosphorylated state in which catalytic activity is regulated to allow rapid response upon ligand binding, whilst restricting ligand-independent activation. Failure of this regulation is responsible for pathologic outcomes including cancer. Here we reveal the molecular mechanistic detail of KD control based on combinatorial interactions of the juxtamembrane (JM) and the C-terminal tail (CT) regions of the receptor. JM stabilizes the asymmetric dimeric KD required for substrate phosphorylation, whilst CT binding opposes dimerization, and down-regulates activity. Direct binding between JM and CT delays the recruitment of downstream effector proteins adding a further control step as the receptor proceeds to full activation. Our findings underscore the diversity in mechanisms of RTK oligomerisation and activation. Interactions between the peripheral juxtamembrane and C-terminal tail regions and the phosphorylation state of the kinase domain of the receptor tyrosine kinase FGFR2 fine tunes its signaling outputs. [ABSTRACT FROM AUTHOR]

Src is a protein tyrosine kinase commonly activated downstream of transmembrane receptors and plays key roles in cell growth, migration, and survival signaling pathways. In conventional dendritic cells (cDCs), Src is involved in the activation of the non-enzymatic functions of indoleamine 2,3-dioxygenase 1 (IDO1), an immunoregulatory molecule endowed with both catalytic activity and signal transducing properties. Prompted by the discovery that the metabolite spermidine confers a tolerogenic phenotype on cDCs that is dependent on both the expression of IDO1 and the activity of Src kinase, we here investigated the spermidine mode of action. We found that spermidine directly binds Src in a previously unknown allosteric site located on the backside of the SH2 domain and thus acts as a positive allosteric modulator of the enzyme. Besides confirming that Src phosphorylates IDO1, here we showed that spermidine promotes the protein-protein interaction of Src with IDO1. Overall, this study may pave the way toward the design of allosteric modulators able to switch on/off the Src-mediated pathways, including those involving the immunoregulatory protein IDO1. [ABSTRACT FROM AUTHOR]

The article focuses on the interplay between autophagy and glycolysis and reveals that ULK1 activation leads to the phosphorylation of lactate dehydrogenase A (LDHA), promoting lactate production. It mentions the Lactate then mediates the lactylation of Vps34, enhancing its association with key autophagy proteins and increasing its lipid kinase activity, thereby promoting autophagy and endolysosomal trafficking.

Extensive research has been conducted for decades to elucidate the molecular and regulatory mechanisms for phytochrome-mediated light signaling in plants. As a result, tens of downstream signaling components that physically interact with phytochromes are identified, among which negative transcription factors for photomorphogenesis, PHYTOCHROME-INTERACTING FACTORs (PIFs), are well known to be regulated by phytochromes. In addition, phytochromes are also shown to inactivate an important E3 ligase complex consisting of CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) and SUPPRESSORs OF phyA-105 (SPAs). This inactivation induces the accumulation of positive transcription factors for plant photomorphogenesis, such as ELONGATED HYPOCOTYL 5 (HY5). Although many downstream components of phytochrome signaling have been studied thus far, it is not fully elucidated which intrinsic activity of phytochromes is necessary for the regulation of these components. It should be noted that phytochromes are autophosphorylating protein kinases. Recently, the protein kinase activity of phytochrome A (phyA) has shown to be important for its function in plant light signaling using Avena sativa phyA mutants with reduced or increased kinase activity. In this review, we highlight the function of phyA as a protein kinase to explain the regulation of plant photoresponses by phyA. [ABSTRACT FROM AUTHOR]

The obligate biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) employs virulence effectors to disturb host immunity and causes devastating stripe rust disease. However, our understanding of how Pst effectors regulate host defense responses remains limited. In this study, we determined that the Pst effector Hasp98, which is highly expressed in Pst haustoria, inhibits plant immune responses triggered by flg22 or nonpathogenic bacteria. Overexpression of Hasp98 in wheat (Triticum aestivum) suppressed avirulent Pst‐triggered immunity, leading to decreased H2O2 accumulation and promoting P. striiformis infection, whereas stable silencing of Hasp98 impaired P. striiformis pathogenicity. Hasp98 interacts with the wheat mitogen‐activated protein kinase TaMAPK4, a positive regulator of plant resistance to stripe rust. The conserved TEY motif of TaMAPK4 is important for its kinase activity, which is required for the resistance function. We demonstrate that Hasp98 inhibits the kinase activity of TaMAPK4 and that the stable silencing of TaMAPK4 compromises wheat resistance against P. striiformis. These results suggest that Hasp98 acts as a virulence effector to interfere with the MAPK signaling pathway in wheat, thereby promoting P. striiformis infection. [ABSTRACT FROM AUTHOR]

The accessibility of DNA to different cellular functions requires a dynamic regulation of chromatin organization that is mediated by different epigenetic modifications, which regulate chromatin accessibility and degree of compaction. These epigenetic modifications, particularly the acetylation of histone H4 in lysine 14 (H4K16ac), determine the degree of chromatin accessibility to different nuclear functions, as well as to DNA damage drugs. H4K16ac is regulated by the balance between two alternative histone modifications, acetylation and deacetylation, which are mediated by acetylases and deacetylases. Tip60/KAT5 acetylates, and SIRT2 deacetylates histone H4K16. However, the balance between these two epigenetic enzymes is unknown. VRK1 regulates the level of H4K16 acetylation by activating Tip60. We have shown that the VRK1 and SIRT2 are able to form a stable protein complex. For this work, we used in vitro interaction, pull-down and in vitro kinase assays. In cells, their interaction and colocalization were detected by immunoprecipitation and immunofluorescence. The kinase activity of VRK1 is inhibited by a direct interaction of its N-terminal kinase domain with SIRT2 in vitro. This interaction causes a loss of H4K16ac similarly to the effect of a novel VRK1 inhibitor (VRK-IN-1) or VRK1 depletion. The use of specific SIRT2 inhibitors in lung adenocarcinoma cells induces H4K16ac, contrary to the novel VRK-IN-1 inhibitor, which prevents H4K16ac and a correct DNA damage response. Therefore, the inhibition of SIRT2 can cooperate with VRK1 in the accessibility of drugs to chromatin in response to DNA damage caused by doxorubicin. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is an established risk factor for neurodegenerative diseases. In this study, we used the Closed Head Injury Model of Engineered Rotational Acceleration (CHIMERA) to investigate the effects of a single high-energy TBI in rTg4510 mice, a mouse model of tauopathy. Fifteen male rTg4510 mice (4 mo) were impacted at 4.0 J using interfaced CHIMERA and were compared to sham controls. Immediately after injury, the TBI mice showed significant mortality (7/15; 47%) and a prolonged duration of loss of the righting reflex. At 2 mo post-injury, surviving mice displayed significant microgliosis (Iba1) and axonal injury (Neurosilver). Western blotting indicated a reduced p-GSK-3β (S9):GSK-3β ratio in TBI mice, suggesting chronic activation of tau kinase. Although longitudinal analysis of plasma total tau suggested that TBI accelerates the appearance of tau in the circulation, there were no significant differences in brain total or p-tau levels, nor did we observe evidence of enhanced neurodegeneration in TBI mice compared to sham mice. In summary, we showed that a single high-energy head impact induces chronic white matter injury and altered GSK-3β activity without an apparent change in post-injury tauopathy in rTg4510 mice. [ABSTRACT FROM AUTHOR]

Sucrose-nonfermenting 1 (SNF1)-related kinase 1 (SnRK1) is a central hub in carbon and energy signaling in plants, and is orthologous with SNF1 in yeast and the AMP-activated protein kinase (AMPK) in animals. Previous studies of SnRK1 relied on in vitro activity assays or monitoring of putative marker gene expression. Neither approach gives unambiguous information about in vivo SnRK1 activity. We have monitored in vivo SnRK1 activity using Arabidopsis (Arabidopsis thaliana) reporter lines that express a chimeric polypeptide with an SNF1/SnRK1/AMPK-specific phosphorylation site. We investigated responses during an equinoctial diel cycle and after perturbing this cycle. As expected, in vivo SnRK1 activity rose toward the end of the night and rose even further when the night was extended. Unexpectedly, although sugars rose after dawn, SnRK1 activity did not decline until about 12 h into the light period. The sucrose signal metabolite, trehalose 6-phosphate (Tre6P), has been shown to inhibit SnRK1 in vitro. We introduced the SnRK1 reporter into lines that harbored an inducible trehalose-6-phosphate synthase construct. Elevated Tre6P decreased in vivo SnRK1 activity in the light period, but not at the end of the night. Reporter polypeptide phosphorylation was sometimes negatively correlated with Tre6P, but a stronger and more widespread negative correlation was observed with glucose-6-phosphate. We propose that SnRK1 operates within a network that controls carbon utilization and maintains diel sugar homeostasis, that SnRK1 activity is regulated in a context-dependent manner by Tre6P, probably interacting with further inputs including hexose phosphates and the circadian clock, and that SnRK1 signaling is modulated by factors that act downstream of SnRK1. [ABSTRACT FROM AUTHOR]

Ste20‐like kinase (SLK) is involved in cell proliferation and migration in somatic cells. This study aims to explore SLK expression and function in mouse oocyte meiosis. Western blot, immunofluorescence, Co‐immunoprecipitation, drug treatment, cRNA construct and in vitro transcription, microinjection of morpholino oilgo (MO) and cRNA were performed in oocytes. High and stable protein expression of SLK was detected in mouse oocyte meiosis, with dynamic distribution in the nucleus, chromosomes and spindle apparatus. SLK phosphorylation emerges around meiotic resumption and reaches a peak during metaphase I (MI) and metaphase II. SLK knockdown with MO or expression of kinase‐dead SLK K63R dramatically delays meiotic resumption due to sequentially suppressed phosphorylation of Polo‐like kinase 1 (Plk1) and cell division cycle 25C (CDC25C) and dephosphorylation of cyclin‐dependent kinase 1 (CDK1). SLK depletion promotes ubiquitination‐mediated degradation of paxillin, an antagonist to α‐tubulin deacetylation, and thus destroys spindle assembly and chromosome alignment; these phenotypes can be substantially rescued by exogenous expression of SLK kinase active fragment. Additionally, exogenous SLK effectively promotes meiotic progression and spindle assembly in aging oocytes with reduced SLK. Collectively, this study reveals SLK is required for meiotic resumption and spindle assembly in mouse oocyte meiosis. [ABSTRACT FROM AUTHOR]

Inhibitors targeting Bruton's tyrosine kinase (BTK) have revolutionized the treatment for various B-cell malignancies but are limited by acquired resistance after prolonged treatment as a result of mutations in BTK. Here, by a combination of structural modeling, in vitro assays, and deep phosphotyrosine proteomics, we demonstrated that four clinically observed BTK mutations-C481F, C481Y, C481R, and L528W -inactivated BTK kinase activity both in vitro and in diffused large B-cell lymphoma (DLBCL) cells. Paradoxically, we found that DLBCL cells harboring kinase-inactive BTK exhibited intact B cell receptor (BCR) signaling, unperturbed transcription, and optimal cellular growth. Moreover, we determined that DLBCL cells with kinase-inactive BTK remained addicted to BCR signaling and were thus sensitive to targeted BTK degradation by the proteolysis-targeting chimera. By performing parallel genome-wide CRISPR-Cas9 screening in DLBCL cells with WT or kinase-inactive BTK, we discovered that DLBCL cells with kinase-inactive BTK displayed increased dependence on Toll-like receptor 9 (TLR9) for their growth and/or survival. Our study demonstrates that the kinase activity of BTK is not essential for oncogenic BCR signaling and suggests that BTK's noncatalytic function is sufficient to sustain the survival of DLBCL. [ABSTRACT FROM AUTHOR]

Protein kinases and their substrates form signaling networks partitioned across subcellular compartments to facilitate critical biological processes. While the subcellular roles of many individual kinases have been elucidated, a comprehensive assessment of the synaptic subkinome is lacking. Further, most studies of kinases focus on transcript, protein, and/or phospho-protein expression levels, providing an indirect measure of protein kinase activity. Prior work suggests that gene expression levels are not a good predictor of protein function. Thus, we assessed global serine/threonine protein kinase activity profiles in synaptosomal, nuclear, and cytosolic fractions from rat frontal cortex homogenate using peptide arrays. Comparisons made between fractions demonstrated differences in overall protein kinase activity. Upstream kinase analysis revealed a list of cognate kinases that were enriched in the synaptosomal fraction compared to the nuclear fraction. We identified many kinases in the synaptic fraction previously implicated in this compartment, while also identifying other kinases with little or no evidence for synaptic localization. Our results show the feasibility of assessing subcellular fractions with peptide activity arrays, as well as suggesting compartment specific activity profiles associated with established and novel kinases. [ABSTRACT FROM AUTHOR]

The article discusses the elevated carbon dioxide (CO2) triggers interaction of the Mitogen-activated protein (MAP) kinases MPK4/MPK12 with the HT1 protein kinase, thus inhibiting HT1 kinase activity. It discusses about identifying dominant active HT1 mutants that cause insensitivity to elevated CO2; and the presented findings reveal that MPK4/12 and HT1 together constitute the long-sought primary stomatal CO2/bicarbonate sensor upstream of the CBC1 kinase in plants.

Mutations in LRRK2 and GBA1 are key contributors to genetic risk of developing Parkinson's disease (PD). To investigate how LRRK2 kinase activity interacts with GBA and contributes to lysosomal dysfunctions associated with the pathology of PD. The activity of the lysosomal enzyme β-Glucocerebrosidase (GCase) was assessed in a human neuroglioma cell model treated with two selective inhibitors of LRKK2 kinase activity (LRRK2-in-1 and MLi-2) and a GCase irreversible inhibitor, condutirol-beta-epoxide (CBE), under 24 and 72 h experimental conditions. We observed levels of GCase activity comparable to controls in response to 24 and 72 h treatments with LRRK2-in-1 and MLi-2. However, GBA protein levels increased upon 72 h treatment with LRRK2-in-1. Moreover, LC3-II protein levels were increased after both 24 and 72 h treatments with LRRK2-in-1, suggesting an activation of the autophagic pathway. These results highlight a possible regulation of lysosomal function through the LRRK2 kinase domain and suggest an interplay between LRRK2 kinase activity and GBA. Although further investigations are needed, the enhancement of GCase activity might restore the defective protein metabolism seen in PD. [ABSTRACT FROM AUTHOR]

Creatine kinase (CK) is an enzyme that produces and uses phosphocreatine to transfer energy to maintain tissue and cellular energy homeostasis, being considered the main controller of cellular energy homeostasis. Its activity in plasma/serum has been commonly used to evaluate tissue damage, since CK is released into the bloodstream during damage. This review summarizes the current knowledge regarding the use of CK activity in fish, focusing on its potential as an indicator of the impairment of energetic homeostasis and tissue damage during stressful situations, such as exposure to contaminants (metals, pesticides, microplastic), hypoxia, thermal stress, and diseases (fungal, parasitic, and bacterial). Based on the data, we can conclude that tissue CK activity can be used as a suitable indicator of the impairment of energetic homeostasis in fish exposed to different aquaculture challenge conditions, while serum/plasma CK activity can be used as the first evidence of possible tissue damage, due to its release into the bloodstream. [ABSTRACT FROM AUTHOR]

The enantioselective preparation of the two isomers of 4-hydroxy-2-cyclohexanone derivatives 1a,b was achieved, starting from a common cyclohexenone, through asymmetric transfer hydrogenation (ATH) reactions using bifunctional ruthenium catalysts. From these versatile intermediates, a stereoselective route to a cytosine analogue built on a bicyclo [4.1.0]heptane scaffold is described. Nucleoside kinase activity assays with this cyclopropyl-fused cyclohexane nucleoside, together with other related nucleosides (2a–e), were performed, showing that thymine- and guanine- containing compounds have affinity for herpes simplex virus Type 1 (HSV-1) thymidine kinase (TK) but not for human cytosolic TK-1, thus pointing to their selectivity for herpetic TKs but not cellular TKs. [ABSTRACT FROM AUTHOR]

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell–cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, the p21-activated kinase Cla4p regulates polarized morphogenesis and cytokinesis. However, it remains unknown how Cla4p kinase activity is regulated. After pheromone exposure, yeast cells temporally separate the mitotic and mating programs by sequestering Fus2p in the nucleus until cell cycle completion, after which Fus2p exits to facilitate cell fusion. Previously, we showed that sequestration is regulated by two opposing protein kinases, Cla4p and Fus3p. Phosphorylation of Fus2p-S67 by Cla4p promotes nuclear localization by both activating nuclear import and blocking export. During mating, phosphorylation of Fus2p-S85 and Fus2p-S100 by Fus3p promotes nuclear export and blocks import. Here, we find that Cla4p kinase activity is itself down-regulated during mating. Pheromone exposure causes Cla4p hyper-phosphorylation and reduced Fus2p-S67 phosphorylation, dependent on Fus3p. Multiple phosphorylation sites in Cla4p are mating- and/or Fus3p-specific. Of these, Cla4p-S186 phosphorylation reduced the kinase activity of Cla4p, in vitro. A phosphomimetic cla4-S186E mutation caused a strong reduction in Fus2p-S67 phosphorylation and nuclear localization, in vivo. More generally, a non-phosphorylatable mutation, cla4-S186A, caused failure to maintain pheromone arrest and delayed formation of the mating-specific septin morphology. Thus, as cells enter the mating pathway, Fus3p counteracts Cla4p kinase activity to allow proper mating differentiation. [ABSTRACT FROM AUTHOR]

Simple Summary: A review of the molecular and cellular features of the major cancer syndromes associated with radiosensitivity revealed the importance of the ATM protein, either as an impaired kinase in the nucleus or as a complex in the cytoplasm, with the mutated protein responsible for the syndrome. There are a number of genetic syndromes associated with both high cancer risk and clinical radiosensitivity. However, the link between these two notions remains unknown. Particularly, some cancer syndromes are caused by mutations in genes involved in DNA damage signaling and repair. How are the DNA sequence errors propagated and amplified to cause cell transformation? Conversely, some cancer syndromes are caused by mutations in genes involved in cell cycle checkpoint control. How is misrepaired DNA damage produced? Lastly, certain genes, considered as tumor suppressors, are not involved in DNA damage signaling and repair or in cell cycle checkpoint control. The mechanistic model based on radiation-induced nucleoshuttling of the ATM kinase (RIANS), a major actor of the response to ionizing radiation, may help in providing a unified explanation of the link between cancer proneness and radiosensitivity. In the frame of this model, a given protein may ensure its own specific function but may also play additional biological role(s) as an ATM phosphorylation substrate in cytoplasm. It appears that the mutated proteins that cause the major cancer and radiosensitivity syndromes are all ATM phosphorylation substrates, and they generally localize in the cytoplasm when mutated. The relevance of the RIANS model is discussed by considering different categories of the cancer syndromes. [ABSTRACT FROM AUTHOR]

Simple Summary: Acquired resistance of cancer cells to targeted therapy poses a major clinical problem. Hyperactivation of the PI3K/Akt pathway is common in human malignancies, and numerous Akt inhibitors are undergoing clinical evaluation. However, mechanisms of acquired resistance to Akt inhibitors have not been studied extensively. By using phospho-RTK arrays, we demonstrated hyper-phosphorylation of multiple RTKs in Akt-inhibitor-resistant breast cancer cells. We further showed that the EGFR inhibitor could overcome Akt inhibitor resistance. In addition, our study revealed enhanced cancer stemness in resistant cells, and RNA sequencing identified several stem cell regulators that may contribute to acquired resistance. The PI3K/Akt pathway is frequently deregulated in human cancers, and multiple Akt inhibitors are currently under clinical evaluation. Based on the experience from other molecular targeted therapies, however, it is likely that acquired resistance will be developed in patients treated with Akt inhibitors. We established breast cancer models of acquired resistance by prolonged treatment of cells with allosteric or ATP-competitive Akt inhibitors. Phospho-Receptor tyrosine kinase (Phospho-RTK) arrays revealed hyper-phosphorylation of multiple RTKS, including EGFR, Her2, HFGR, EhpB3 and ROR1, in Akt-inhibitor-resistant cells. Importantly, resistance can be overcome by treatment with an EGFR inhibitor. We further showed that cancer stem cells (CSCs) are enriched in breast tumor cells that have developed resistance to Akt inhibitors. Several candidates of CSC regulators, such as ID4, are identified by RNA sequencing. Cosmic analysis indicated that sensitivity of tumor cells to Akt inhibitors can be predicted by ID4 and stem cell/epithelial–mesenchymal transition pathway targets. These findings indicate the potential of targeting the EGFR pathway and CSC program to circumvent Akt inhibitor resistance in breast cancer. [ABSTRACT FROM AUTHOR]

JAK2 is cytokine-activated non-receptor tyrosine kinase. Although JAK2 is mainly localized at the plasma membrane, it is also present on the centrosome. In this study, we demonstrated that JAK2 localization to the centrosome depends on the SH2 domain and intact kinase activity. We created JAK2 mutants deficient in centrosomal localization ΔSH2, K882E and (ΔSH2, K882E). We showed that JAK2 WT clone strongly enhances cell proliferation as compared to control cells while JAK2 clones ΔSH2, K882E and (ΔSH2, K882E) proliferate slower than JAK2 WT cells. These mutant clones also progress much slower through the cell cycle as compared to JAK2 WT clone and the enhanced proliferation of JAK2 WT cells is accompanied by increased S −> G2 progression. Both the SH2 domain and the kinase activity of JAK2 play a role in prolactin-dependent activation of JAK2 substrate STAT5. We showed that JAK2 is an important regulator of centrosome function as the SH2 domain of JAK2 regulates centrosome amplification. The cells overexpressing ΔSH2 and (ΔSH2, K-E) JAK2 have almost three-fold the amplified centrosomes of WT cells. In contrast, the kinase activity of JAK2 is dispensable for centrosome amplification. Our observations provide novel insight into the role of SH2 domain and kinase activity of JAK2 in centrosome localization of JAK2 and in the regulation of cell growth and centrosome biogenesis. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol 4-phosphate 5-kinase (PIP5K) family of lipid-modifying enzymes generate the majority of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids found at the plasma membrane in eukaryotic cells. PI(4,5)P2 lipids serve a critical role in regulating receptor activation, ion channel gating, endocytosis, and actin nucleation. Here, we describe how PIP5K activity is regulated by cooperative binding to PI(4,5)P2 lipids and membrane-mediated dimerization of the kinase domain. In contrast to constitutively dimeric phosphatidylinositol 5-phosphate 4-kinase (PIP4K, type II PIPK), solution PIP5K exists in a weak monomer-dimer equilibrium. PIP5K monomers can associate with PI(4,5)P2-containing membranes and dimerize in a protein density-dependent manner. Although dispensable for cooperative PI(4,5)P2 binding, dimerization enhances the catalytic efficiency of PIP5K through a mechanism consistent with allosteric regulation. Additionally, dimerization amplifies stochastic variation in the kinase reaction velocity and strengthens effects such as the recently described stochastic geometry sensing. Overall, the mechanism of PIP5K membrane binding creates a broad dynamic range of lipid kinase activities that are coupled to the density of PI(4,5)P2 and membrane-bound kinase. [ABSTRACT FROM AUTHOR]

Inducing senescence in cancer cells is emerging as a new therapeutic strategy. In order to find ways to enhance senescence induction by palbociclib, a CDK4/6 inhibitor approved for treatment of metastatic breast cancer, we performed functional genetic screens in palbociclib-resistant cells. Using this approach, we found that loss of CDK2 results in strong senescence induction in palbociclib-treated cells. Treatment with the CDK2 inhibitor indisulam, which phenocopies genetic CDK2 inactivation, led to sustained senescence induction when combined with palbociclib in various cell lines and lung cancer xenografts. Treating cells with indisulam led to downregulation of cyclin H, which prevented CDK2 activation. Combined treatment with palbociclib and indisulam induced a senescence program and sensitized cells to senolytic therapy. Our data indicate that inhibition of CDK2 through indisulam treatment can enhance senescence induction by CDK4/6 inhibition. [ABSTRACT FROM AUTHOR]

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease (ILD). Pulmonary fibroblasts play an important role in the development of IPF. Emerging evidence indicates that pulmonary endothelial cells could be the source of pulmonary fibroblasts through endothelial mesenchymal transition (EndoMT), which contributes to pulmonary fibrosis. EndoMT is a complex process in which endothelial cells lose their expression of endothelial markers and give rise to the characteristics of mesenchymal cells, including morphological fibroblast-like change and the expression of mesenchymal markers, which result in cardiac, renal, and dermal fibroses. Furthermore, EndoMT inhibition attenuates pulmonary fibrosis. Herein, we demonstrate that nintedanib, a tyrosine kinase receptor inhibitor, ameliorated murine bleomycin (BLM)-induced pulmonary fibrosis and suppressed the in vivo and in vitro models of EndoMT. We demonstrated that the activity of focal adhesion kinase (FAK), a key EndoMT regulator, increased in murine lung tissues and human pulmonary microvascular endothelial cells after BLM stimulation. Nintedanib treatment inhibited BLM-induced FAK activation and thus suppressed both in vivo and in vitro BLM-induced EndoMT. Importantly, we found that the VEGF/FAK signaling pathway was involved in nintedanib regulating EndoMT. These novel findings help us understand the mechanism and signaling pathway of EndoMT to further develop more efficacious drugs for IPF treatment. [ABSTRACT FROM AUTHOR]

Background: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro. Methods: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients. [ABSTRACT FROM AUTHOR]

Background: The ketogenic diet (KD)has been considered an effective treatment for epilepsy, whereas its underlying mechanisms remain obscure. We have previously reported that the KD feeding increased Neuregulin 1 (NRG1) expression in the hippocampus; disruption of NRG1 signaling by genetically deleting its receptor-ErbB4 abolished KD's effects on inhibitory synaptic activity and seizures. However, it is still unclear about the mechanisms underlying the effect of KD on NRG1 expression and whether the effects of KD require ErbB4 kinase activity. Methods: The effects of the KD on NRG1 expression were assessed via western blotting and real-time PCR. Acetylation level at the Nrg1 promoter locus was examined using the chromatin immunoprecipitation technique. Kainic acid (KA)-induced acute seizure model was utilized to examine the effects of KD and histone deacetylase inhibitor-TSA on seizures. Synaptic activities in the hippocampus were recorded with the technique of electrophysiology. The obligatory role of ErbB4 kinase activity in KD's effects on seizures and inhibitory synaptic activity was evaluated by using ErbB kinase antagonist and transgenic mouse-T796G. Results: We report that KD specifically increases Type I NRG1 expression in the hippocampus. Using the chromatin immunoprecipitation technique, we observe increased acetylated-histone occupancy at the Nrg1 promoter locus of KD-fed mice. Treatment of TSA dramatically elevates NRG1 expression and diminishes the difference between the effects of the control diet (CD) and KD. These data indicate that KD increases NRG1 expression via up-regulating histone acetylation. Moreover, both pharmacological and genetic inhibitions of ErbB4 kinase activity significantly block the KD's effects on inhibitory synaptic activity and seizure, suggesting an essential role of ErbB4 kinase activity. Conclusion: These results strengthen our understanding of the role of NRG1/ErbB4 signaling in KD and shed light on novel therapeutic interventions for epilepsy. [ABSTRACT FROM AUTHOR]

Pathologic inclusions composed of α-synuclein called Lewy pathology are hallmarks of Parkinson's Disease (PD). Dominant inherited mutations in leucine rich repeat kinase 2 (LRRK2) are the most common genetic cause of PD. Lewy pathology is found in the majority of individuals with LRRK2-PD, particularly those with the G2019S-LRRK2 mutation. Lewy pathology in LRRK2-PD associates with increased non-motor symptoms such as cognitive deficits, anxiety, and orthostatic hypotension. Thus, understanding the relationship between LRRK2 and α-synuclein could be important for determining the mechanisms of non-motor symptoms. In PD models, expression of mutant LRRK2 reduces membrane localization of α-synuclein, and enhances formation of pathologic α-synuclein, particularly when synaptic activity is increased. α-Synuclein and LRRK2 both localize to the presynaptic terminal. LRRK2 plays a role in membrane traffic, including axonal transport, and therefore may influence α-synuclein synaptic localization. This study shows that LRRK2 kinase activity influences α-synuclein targeting to the presynaptic terminal. We used the selective LRRK2 kinase inhibitors, MLi-2 and PF-06685360 (PF-360) to determine the impact of reduced LRRK2 kinase activity on presynaptic localization of α-synuclein. Expansion microscopy (ExM) in primary hippocampal cultures and the mouse striatum, in vivo, was used to more precisely resolve the presynaptic localization of α-synuclein. Live imaging of axonal transport of α-synuclein-GFP was used to investigate the impact of LRRK2 kinase inhibition on α-synuclein axonal transport towards the presynaptic terminal. Reduced LRRK2 kinase activity increases α-synuclein overlap with presynaptic markers in primary neurons, and increases anterograde axonal transport of α-synuclein-GFP. In vivo, LRRK2 inhibition increases α-synuclein overlap with glutamatergic, cortico-striatal terminals, and dopaminergic nigral-striatal presynaptic terminals. The findings suggest that LRRK2 kinase activity plays a role in axonal transport, and presynaptic targeting of α-synuclein. These data provide potential mechanisms by which LRRK2-mediated perturbations of α-synuclein localization could cause pathology in both LRRK2-PD, and idiopathic PD. [ABSTRACT FROM AUTHOR]

Bromodomain protein 4 (BRD4) is a transcriptional and epigenetic regulator that is a therapeutic target in many cancers and inflammatory diseases. BRD4 plays important roles in transcription as an active kinase, which phosphorylates the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II), the proto-oncogene c-MYC, and transcription factors TAF7 and CDK9. BRD4 is also a passive scaffold that recruits transcription factors. Despite these well-established functions, there has been little characterization of BRD4's biophysical properties or its kinase activity. We report here that the 156 kD mouse BRD4 exists in an extended dimeric conformation with a sedimentation coefficient of ~6.7 S and a high frictional ratio. Deletion of the conserved B motif (aa 503-548) disrupts BRD4's dimerization. BRD4 kinase activity maps to amino acids 351 to 598, which span bromodomain-2, the B motif, and the BID domain (BD2-B-BID) and contributes to the in vivo phosphorylation of its substrates. As further assessed by analytical ultracentrifugation, BRD4 directly binds purified Pol II CTD. Importantly, the conserved A motif of BRD4 is essential for phosphorylation of Pol II CTD, but not for phosphorylation of TAF7, mapping its binding site to the A motif. Peptides of the viral MLV integrase (MLVIN) protein and cellular histone lysine methyltransferase, NSD3, which have been shown by NMR to bind to the extra-terminal (ET) domain, also are phosphorylated by BRD4. Thus, BRD4 has multiple distinct substrate-binding sites and a common kinase domain. These results provide new insights into the structure and kinase function of BRD4. [ABSTRACT FROM AUTHOR]

CcrZ is a recently discovered cell cycle regulator that connects DNA replication initiation with cell division in pneumococci and may have a similar function in related bacteria. CcrZ is also annotated as a putative kinase, suggesting that CcrZ homologs could represent a novel family of bacterial kinase-dependent cell cycle regulators. Here, we investigate the CcrZ homolog in Bacillus subtilis and show that cells lacking ccrZ are sensitive to a broad range of DNA damage. We demonstrate that increased expression of ccrZ results in over initiation of DNA replication. In addition, increased expression of CcrZ activates the DNA damage response. Using sensitivity to DNA damage as a proxy, we show that the negative regulator for replication initiation (yabA) and ccrZ function in the same pathway. We show that CcrZ interacts with replication initiation proteins DnaA and DnaB, further suggesting that CcrZ is important for replication timing. To understand how CcrZ functions, we solved the crystal structure bound to AMP-PNP to 2.6 Å resolution. The CcrZ structure most closely resembles choline kinases, consisting of a bilobal structure with a cleft between the two lobes for binding ATP and substrate. Inspection of the structure reveals a major restructuring of the substrate-binding site of CcrZ relative to the choline-binding pocket of choline kinases, consistent with our inability to detect activity with choline for this protein. Instead, CcrZ shows activity on D-ribose and 2-deoxy-D-ribose, indicating adaptation of the choline kinase fold in CcrZ to phosphorylate a novel substrate. We show that integrity of the kinase active site is required for ATPase activity in vitro and for function in vivo. This work provides structural, biochemical, and functional insight into a newly identified, and conserved group of bacterial kinases that regulate DNA replication initiation. Author summary: Some bacteria enlist kinases to regulate cell cycle progression and developmental checkpoints. CcrZ is a recently identified cell cycle regulator and putative kinase showing similarity to choline kinases. We identified the ccrZ gene in Bacillus subtilis using a forward genetic approach with the ccrZ deletion conferring sensitivity to a broad range of DNA damage. We show that induced expression of CcrZ causes over initiation of DNA replication, and we provide evidence for an interaction between CcrZ and replication initiation proteins DnaA and DnaB. We find that integrity of the kinase active site is required for function in vivo. To gain insight into kinase function, we solved the crystal structure of B. subtilis CcrZ bound to AMP-PNP. The structure demonstrates that CcrZ evolved from a choline kinase with a bilobed structure and a larger inter-lobe cleft relative to choline kinases. The larger inter-lobe cleft could help accommodate larger substrates than choline. As expected by the structure CcrZ does not have activity on choline, but instead shows activity on ribose and 2 deoxy-ribose. With our results, we propose that CcrZ regulates replication initiation through phosphorylation of a small molecule and interaction with replication initiation proteins. [ABSTRACT FROM AUTHOR]

Objective To explore the kinase activity of novel receptor interacting protein kinase 3 (RIPK3) mutants. Methods 瞬 The four amino acids (Q84WDF87) of RIPK3 were mutated respectively and these mutants were co-transfected with mixed lineage kinase domain like pseudokiiiase (MLKL) into HEK293T cells. The auto-phosphoiylation of these mutants at S232 and phosphorylation of MLKL at S345 were detected by Western blotting. The interaction between RIPK3 and MLKL was tested by co-immimoprecipitatiotL The oligomerization of MLKL was detected by non-reducing gel. Results The kinase activities of REPK3AQ84, RIPK3AW85 and RIPK3AD86 were effectively decreased. Nevertheless, the kinase activities of RIPK3Q84A/RIPK3Q84E, RIPK3W85Y and RIPK3D86A/RIPK3D86Y did not change markedly. The auto-phosphorylatioii of RIPK3W85A at S232 was decreased without affecting phosphorylatioii and oligomerization of MLKL. Conclusion • The amino acid site Q84, W85 or D86 plays a critical role in RIPK3 kinase activity. The kinase activity ofRIPK3W85Ais decreased, but it does not affect MLKL. [ABSTRACT FROM AUTHOR]

Clear cell carcinoma (CCC) is a rare subtype of ovarian cancer resistant to standard platinum chemotherapy, which leads to a poor prognosis for patients with CCC. Kinases are targets for anticancer drugs; few studies have profiled kinase activity to identify kinase inhibitors as novel anticancer drugs. In this study, we aimed to identify novel anticancer drugs for the treatment of CCC with comprehensive kinase activity assay and drug screening. Using ascites from a 51-year old patient, we established and characterized the NCC-cOV1-C1 cell line. We screened the antiproliferative effects of 152 small anticancer compounds and conducted comprehensive kinase activity assays with the PamStation12 platform. The NCC-cOV1-C1 cells harbor copy number variation of HFN1ß amplification, and exhibit constant growth, spheroid formation, and invasion capability. NCC-cOV1-C1 cells responded remarkably to idarubicin HCl and vorinostat. The kinase activity assay revealed that SRC and EGFR were highly activated in NCC-cOV1-C1 cells; the SRC inhibitor dasatinib and the EGFR inhibitor lapatinib exhibited antiproliferative effects and downregulation of downstream signaling. The NCC-cOV1-C1 cell line will be a useful tool for basic and preclinical study of CCC, and the clinical utility of idarubicin HCl, vorinostat, dasatinib, lapatinib is worthy of further investigation. [ABSTRACT FROM AUTHOR]