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In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. [ABSTRACT FROM AUTHOR]

Motivation: Phosphoproteomic experiments are increasingly used to study the changes in signaling occurring across different conditions. It has been proposed that changes in phosphorylation of kinase target sites can be used to infer when a kinase activity is under regulation. However, these approaches have not yet been benchmarked due to a lack of appropriate benchmarking strategies. Results: We used curated phosphoproteomic experiments and a gold standard dataset containing a total of 184 kinase-condition pairs where regulation is expected to occur to benchmark and compare different kinase activity inference strategies: Z-test, Kolmogorov Smirnov test, Wilcoxon rank sum test, gene set enrichment analysis (GSEA), and a multiple linear regression model. We also tested weighted variants of the Z-test and GSEA that include information on kinase sequence specificity as proxy for affinity. Finally, we tested how the number of known substrates and the type of evidence (in vivo, in vitro or in silico) supporting these influence the predictions. Conclusions: Most models performed well with the Z-test and the GSEA performing best as determined by the area under the ROC curve (Mean AUC = 0.722). Weighting kinase targets by the kinase target sequence preference improves the results marginally. However, the number of known substrates and the evidence supporting the interactions has a strong effect on the predictions. [ABSTRACT FROM AUTHOR]

Background: Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson's disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Methods: Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients' fibroblasts and in cell-based, biochemical assays. Results: Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Conclusions: Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients' fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development. [ABSTRACT FROM AUTHOR]

[Display omitted] • A simple assay, AUDECY, to measure histidine kinase activity is developed. • AUDECY-based high-throughput screening identified VanS inhibitors. • Identified VanS inhibitors showed efficacy to vancomycin-resistant Enterococci. Bacterial two-component systems (TCSs), which typically consist of a sensor histidine kinase (HK) and a response regulator (RR), have been investigated as attractive antibacterial drug targets. Unfortunately, current HK activity assays based on the quantification of autophosphorylated HKs are hampered by the instability of the phosphohistidine (pHis) product, rendering them ill-suited for high-throughput screenings. To address this challenge, we developed a simple HK activity assay using readily available reagents, which we have termed AUDECY (AUtophosphorylation-DEphosphorylation CYcle assay). Instead of trying to preserve the fragile pHis, we deliberately decomposed it with a pHis-specific phosphatase to constitute an ATPase-like cycle for convenient colorimetric measurements. This kinetic assay was successfully employed for the kinetic characterization of E. coli EnvZ and for high-throughput inhibitor screening of vancomycin-resistant Enterococcus faecium (VRE) VanS, of which histidine kinase activity was hardly detectable with conventional methods. Through the screening, we identified OSU-03012, a potent VanS HK inhibitor, which sensitized VRE toward vancomycin, highlighting the potential of AUDECY in HK inhibitor discovery. [ABSTRACT FROM AUTHOR]

Background: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. Results: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. Conclusions: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements. [ABSTRACT FROM AUTHOR]

Neurite formation, a fundamental process in neuronal maturation, requires the coordinated regulation of cytoskeletal reorganization and membrane transport. Compared to the understanding of cytoskeletal functions, less is known about the supply of membranes to growing neurites. Lemur kinase 1A ( LMTK1A) is an endosomal protein kinase that is highly expressed in neurons. We recently reported that LMTK1A regulates the trafficking of Rab11-positive recycling endosomes in growing axons and dendrites. Here, we used the kinase-negative (kn) mutant to investigate the role of the kinase activity of LMTK1A in its cellular localization and interactions with the cytoskeleton in Neuro2A and PC-12 cells. Kinase activity was required for the localization of LMTK1A in the perinuclear endocytic recycling compartment. Perinuclear accumulation was microtubule dependent, and LMTK1A wild type (wt) localized mainly on microtubules, whereas kn LMTK1A was found in the actin-rich cell periphery. In the neurites of PC-12 cells, LMTK1A showed contrasting distributions depending on the kinase activity, with wt being located in the microtubule-rich shaft and the kn form in the actin-rich tip. Taken together, these results suggest that the kinase activity of LMTK1A regulates the pathway for endosomal vesicles to transfer from microtubules to actin filaments at the tip of growing neurites. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the growth-inhibitory effect of 7-piperazinethylchrysin (PEC) on melanoma cell lines. Methods: Cell viability was analyzed by trypan blue exclusion assays and the cell cycle by flow cytometry using ModFit LT software. Specifically, cells were stained with propidium iodide (0.5 mg/mL) supplemented with RNase A (50 mg/mL), and analyzed using flow cytometry and ModFit LT software. Results: In A375 and B16F10 cell cultures, proliferation was reduced to 79 and 72%, respectively, on treatment with 30 µM PEC. PEC increased the proportion of A375 cells in G1/G0 phase to 71.23%, versus 42.76% in untreated cells. In B16F10 and A375 cells, treatment with PEC caused the inhibition of Mek 1/2 kinase activity and suppressed Erk 1/2 phosphorylation. The level of cAMP-response element binding protein was increased by PEC. The expression of microphthalmia-linked transcription factor was also increased by PEC treatment. Marked enhancement was observed in the level of tyrosinase in melanoma cells on treatment with PEC. Analysis of PBG-D expression showed a marked increase in B16F10 and A375 cells on the addition of PEC to cell cultures at 72 h. The level of PBG D expression was increased by 9-and 8.5-fold in B16F10 and A375 cells, respectively, on incubation with 30 µM PEC. The addition of a Mek 1/2 inhibitor (U0126) to the cultures promoted PEC-mediated growth inhibition. Conclusion: PEC inhibited melanoma cell proliferation, apparently by blocking the cell cycle at G0/G1 and downregulating the Ras/Raf/Mek/Erk pathway. [ABSTRACT FROM AUTHOR]

Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acutemyeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase g. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibitedmtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreasedmtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML. [ABSTRACT FROM AUTHOR]

Aberrant signaling through the AKT kinase mediates oncogenic phenotypes including cell proliferation, survival, and therapeutic resistance. Here, we utilize a bioluminescence reporter for AKT kinase activity (BAR) to noninvasively assess the therapeutic efficacy of the EGFR inhibitor erlotinib in KRAS-mutated lung cancer therapy. A549 non-small cell lung cancer cell line, engineered to express BAR, enabled the evaluation of compounds targeting the EGFR/PI3K/AKT pathway in vitro as well as in mouse models. We found that erlotinib treatment of resistant A549 subcutaneous and orthotopic xenografts resulted in significant AKT inhibition as determined by an 8- to 13-fold (P b .0001) increase in reporter activity 3 hours after erlotinib (100 mg/kg) administration compared to the control. This was confirmed by a 25%(P b .0001) decrease in pAKT ex vivo and a decrease in tumor growth. Treatment of the orthotopic xenograft with varying doses of erlotinib (25, 50, and 100mg/kg) revealed a dose- and time-dependent increase in reporter activity (10-, 12-, and 23-fold). Correspondingly, a decrease in phospho-AKT levels (0%, 16%, and 28%, respectively) and a decrease in the AKT dependent proliferation marker PCNA (0%, 50%, and 50%) were observed. We applied µ-CT imaging for noninvasive longitudinal quantification of lung tumor loadwhich revealed a corresponding decrease in tumor growth in a dose-dependent manner. These findings demonstrate the utility of BAR to noninvasively monitor AKT activity in preclinical studies in response to AKT modulating agents. These results also demonstrate that BAR can be applied to study drug dosing, drug combinations, and treatment efficacy in orthotopic mouse lung tumor models. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular recycling and degradation pathway that depends on membrane trafficking. Rab GTPases are central for autophagy but their regulation especially through the activity of Rab GEFs remains largely elusive. We employed a RNAi screen simultaneously monitoring different populations of autophagosomes and identified 34 out of 186 Rab GTPase, GAP and GEF family members as potential autophagy regulators, amongst them SMCR8. SMCR8 uses overlapping binding regions to associate with C9ORF72 or with a C9ORF72- ULK1 kinase complex holo-assembly, which function in maturation and formation of autophagosomes, respectively. While focusing on the role of SMCR8 during autophagy initiation, we found that kinase activity and gene expression of ULK1 are increased upon SMCR8 depletion. The latter phenotype involved association of SMCR8 with the ULK1 gene locus. Global mRNA expression analysis revealed that SMCR8 regulates transcription of several other autophagy genes including WIPI2. Collectively, we established SMCR8 as multifaceted negative autophagy regulator. [ABSTRACT FROM AUTHOR]

The aquatic spring viremia of carp virus (SVCV) causes significant mortality in common carp ( Cyprinus carpio ), and TBK1 plays a crucial role in the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) system by phosphorylating its substrates to induce type I interferons (IFNs) and cellular antiviral responses. In this study, we report that zebrafish STAT6 is induced during SVCV infection and reduces IFNφ1 expression by suppressing TBK1 phosphorylation. A typical IFN stimulatory response element (ISRE) motif was found in the promoter region of zebrafish STAT6, and zebrafish STAT6 transcription was significantly upregulated in the early stages of virus infection. Overexpression of STAT6 interfered with IFNφ1 promoter activity in response to SVCV infection. Additionally, TBK1-, but not MITA-mediated activation of the IFNφ1 promoter was impaired by STAT6. Co-immunoprecipitation and Western blot experiments indicated that MITA and IRF3 were significantly phosphorylated by TBK1, and that the N-terminal kinase domain of TBK1 was critical in this process. In the final step, STAT6 interacted with the N-terminal kinase domain of TBK1 causing dephosphorylation, which resulted in reductions in the phosphorylation of IRF3 and the production of IFNφ1. These results indicate that fish STAT6 can attenuate the kinase activity of TBK1, leading to suppression of IFNφ1 expression which may in turn facilitate virus replication. [ABSTRACT FROM AUTHOR]

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain. [ABSTRACT FROM AUTHOR]

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted. [ABSTRACT FROM AUTHOR]

Kinase enzymes are an important class of drug targets, particularly in cancer. Cell-based kinase assays are needed to understand how potential kinase inhibitors act on their targets in a physiologically relevant context. Current cell-based kinase assays rely on antibody-based detection of endogenous substrates, inaccurate disease models, or indirect measurements of drug action. Here we expand on previous work from our lab to introduce a 96-well plate compatible approach for measuring cell-based kinase activity in disease-relevant human chronic myeloid leukemia cell lines using an exogenously added, multi-functional peptide substrate. Our cellular models natively express the BcrAbl oncogene and are either sensitive or have acquired resistance to well-characterized BcrAbl tyrosine kinase inhibitors. This approach measures IC50 values comparable to established methods of assessing drug potency, and its robustness indicates that it can be employed in drug discovery applications. This medium-throughput assay could bridge the gap between single target focused, high-throughput in vitro assays and lower-throughput cell-based follow-up experiments. [ABSTRACT FROM AUTHOR]

Summary The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans . Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease. [ABSTRACT FROM AUTHOR]

Kinases play a key role in cellular signaling, and the overactivation or overexpression of these kinases has been linked to a variety of cancers. Tyrosine kinase inhibitors treat the mechanism of these cancers by targeting the specific kinases that are overactive. Some patients, however, do not respond to these inhibitors or develop resistance to these inhibitors during treatment. Additionally, even within cancers of the same tissue type, different kinases may be overactive in different patients. For example, some lung cancers overexpress epidermal growth factor receptor (EGFR) and respond to EGFR inhibitors, whereas other lung cancers do not overexpress EGFR and receive no benefit from this treatment. Even among patients exhibiting EGFR overexpression, some do not respond to EGFR kinase inhibitors because other kinases, such as Met kinase, are also overactivated. Here we describe a quantitative and specific multiplexed microfluidic assay using a hydrogel immobilized substrate for measuring the kinase activity of Met and Abl kinase from cancer cells. We immobilized kinase-specific substrates on macroporous hydrogel micropillars in microchannels. These microchannels were incubated with 6 μl of a kinase reaction solution containing cancer cell lysate, and we measured kinase activity via fluorescence detection of a phosphotyrosine antibody. We showed that the assay can specifically measure the activity of both Met and Abl kinase within one microchannel and has the potential to measure the activity of as many as five kinases within one microchannel. The assay also detected Met kinase inhibition from lysates of cancer cells grown in the Met kinase inhibitor PHA665752. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Recognition of nucleic acids results in the production of type I IFNs, which activate the JAK/STAT pathway and promote the expression of IFN-stimulated genes. In a search for modulators of this pathway, we discovered an unexpected requirement for cyclin-dependent kinases (CDK) in the production of type I IFN following nucleic acid sensing and virus infection. Inhibition of CDK activity or knockdown of CDK levels leads to a striking block in STAT activation and IFN-stimulated gene expression. CDKs are not required for the initial nucleic acid sensing leading to IFN-β mRNA induction, nor for the response to exogenous IFN-α/β, but are critical for IFN-β release into culture supernatants, suggesting a posttranscriptional role for CDKs in type I IFN production. In the absence of CDK activity, we demonstrate a translational block specific for IFN-β, in which IFN-β mRNA is removed from the actively translating polysomes, while the distribution of other cellular mRNAs or global translation rates are unaffected. Our findings reveal a critical role for CDKs in the translation of IFN-β. [ABSTRACT FROM AUTHOR]

The success of Staphylococcus aureus, as both a human and animal pathogen, stems from its ability to rapidly adapt to a wide spectrum of environmental conditions. Two-component systems (TCSs) play a crucial role in this process. Here, we describe a novel staphylococcal virulence factor, SpdC, an Abi-domain protein, involved in signal sensing and/or transduction. We have uncovered a functional link between the WalKR essential TCS and the SpdC Abi membrane protein. Expression of spdC is positively regulated by the WalKR system and, in turn, SpdC negatively controls WalKR regulon genes, effectively constituting a negative feedback loop. The WalKR system is mainly involved in controlling cell wall metabolism through regulation of autolysin production. We have shown that SpdC inhibits the WalKR-dependent synthesis of four peptidoglycan hydrolases, SceD, SsaA, LytM and AtlA, as well as impacting S. aureus resistance towards lysostaphin and cell wall antibiotics such as oxacillin and tunicamycin. We have also shown that SpdC is required for S. aureus biofilm formation and virulence in a murine septicemia model. Using protein-protein interactions in E. coli as well as subcellular localization in S. aureus, we showed that SpdC and the WalK kinase are both localized at the division septum and that the two proteins interact. In addition to WalK, our results indicate that SpdC also interacts with nine other S. aureus histidine kinases, suggesting that this membrane protein may act as a global regulator of TCS activity. Indeed, using RNA-Seq analysis, we showed that SpdC controls the expression of approximately one hundred genes in S. aureus, many of which belong to TCS regulons. [ABSTRACT FROM AUTHOR]

The inositol pyrophosphates 5-InsP7 (diphosphoinositol pentakisphosphate) and 1,5-InsP8 (bis-diphosphoinositol tetrakisphosphate) are highly energetic cellular signals interconverted by the diphosphoinositol pentakisphosphate kinases (PPIP5Ks). Here, we used CRISPR to KO PPIP5Ks in the HCT116 colon cancer cell line. This procedure eliminates 1,5-InsP8 and raises 5-InsP7 levels threefold. Expression of p53 and p21 was up-regulated; proliferation and G1/S cell-cycle transition slowed. Thus, PPIP5Ks are potential targets for tumor therapy. Deletion of the PPIP5Ks elevated [ATP] by 35%; both [ATP] and [5-InsP7] were restored to WT levels by overexpression of PPIP5K1, and a kinase-compromised PPIP5K1 mutant had no effect. This covariance of [ATP] with [5-InsP7] provides direct support for an energy-sensing attribute (i.e., 1 mM Km for ATP) of the 5-InsP7-generating inositol hexakisphosphate kinases (IP6Ks). We consolidate this conclusion by showing that 5-InsP7 levels are elevated on direct delivery of ATP into HCT116 cells using liposomes. Elevated [ATP] in PPIP5K-/- HCT116 cells is underpinned by increased mitochondrial oxidative phosphorylation and enhanced glycolysis. To distinguish between 1,5-InsP8 and 5-InsP7 as drivers of the hypermetabolic and p53-elevated phenotypes, we used IP6K2 RNAi and the pan-IP6K inhibitor, N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl) purine (TNP), to return 5-InsP7 levels in PPIP5K-/- cells to those of WT cells without rescuing 1,5-InsP8 levels. Attenuation of IP6K restored p53 expression but did not affect the hypermetabolic phenotype. Thus, we conclude that 5-InsP7 regulates p53 expression, whereas 1,5-InsP8 regulates ATP levels. These findings attribute hitherto unsuspected functionality for 1,5-InsP8 to bioenergetic homeostasis. [ABSTRACT FROM AUTHOR]

Guanylate kinase is an essential and conserved enzyme in nucleotide biosynthetic pathway that transfers phosphoryl group of ATP to GMP for yielding GDP. Here, we report the phosphorylation of guanylate kinase from Mycobacterium tuberculosis (mGmk) by eukaryotic-type Ser/Thr kinase, PknA. Mass spectrometric studies identified Thr101 and Thr169 as phosphorylatable residues in mGmk. To evaluate the significance of phosphorylation in these threonines, two point (T101A and T169A) and one double (T101A-T169A) mutants were generated. The kinase assay with these mutant proteins revealed the major contribution of Thr169 compared with Thr101 in the phosphorylation of mGmk. Kinetic analysis indicated that p-mGmk was deficient in its enzymatic activity compared with that of its un-phosphorylated counterpart. Surprisingly, its phosphoablated (T169A) as well as phosphomimic (T169E) variants exhibited decreased activity as was observed with p-mGmk. Structural analysis suggested that phosphorylation of Thr169 might affect its interaction with Arg166, which is crucial for the functioning of mGmk. In fact, the R166A and R166K mutant proteins displayed a drastic decrease in enzymatic activity compared with that of the wild-type mGmk. Molecular dynamics (MD) studies of mGmk revealed that upon phosphorylation of Thr169, the interactions of Arg165/Arg166 with Glu158, Asp121 and residues of the loop in GMP-binding domain are perturbed. Taken together, our results illuminate the mechanistic insights into phosphorylation-mediated modulation of the catalytic activity of mGmk. [ABSTRACT FROM AUTHOR]

Pyruvate kinase (PK) deficiency is a rare genetic disease that causes chronic hemolytic anemia. There are currently no targeted therapies for PKdeficiency. Here,we describe the identification and characterization of AG-348, an allosteric activator ofPKthat is currently in clinical trials for the treatment of PK deficiency. We demonstrate that AG-348 can increase the activity of wild-type and mutant PK enzymes in biochemical assays and in patient red blood cells treated ex vivo. These data illustrate the potential for AG-348 to restore the glycolytic pathway activity in patients withPKdeficiencyand ultimately lead to clinical benefit. [ABSTRACT FROM AUTHOR]

The creatine kinase (CK) phosphagen system is fundamental to cellular energy homeostasis. Cardiomyocytes express three CK isoforms, namely the mitochondrial sarcomeric CKMT2 and the cytoplasmic CKM and CKB. We hypothesized that augmenting CK in vitro would preserve cell viability and function and sought to determine efficacy of the various isoforms. The open reading frame of each isoform was cloned into pcDNA3.1, followed by transfection and stable selection in human embryonic kidney cells (HEK293). CKMT2- CKM- and CKB-HEK293 cells had increased protein and total CK activity compared to non-transfected cells. Overexpressing any of the three CK isoforms reduced cell death in response to 18h hypoxia at 1% O2 followed by 2h re-oxygenation as assayed using propidium iodide: by 33% in CKMT2, 47% in CKM and 58% in CKB compared to non-transfected cells (P<0.05). Loading cells with creatine did not modify cell survival. Transient expression of CK isoforms in HL-1 cardiac cells elevated isoenzyme activity, but only CKMT2 over-expression protected against hypoxia (0.1% for 24h) and reoxygenation demonstrating 25% less cell death compared to non-transfected control (P<0.01). The same cells were not protected from doxorubicin toxicity (250nM for 48h), in contrast to the positive control. These findings support increased CK activity as protection against ischaemia-reperfusion injury, in particular, protection via CKMT2 in a cardiac-relevant cell line, which merits further investigation in vivo. [ABSTRACT FROM AUTHOR]

Protein kinases play crucial regulatory roles in the physiological activities in the human body. Understanding protein kinase activity and its inhibition is essential for the management of human diseases. Considering the limitations of the existing protein kinase-related analysis methods, the aim of the present study was to develop a fluorescent biosensor based on Eu(BTC) (H 2 O) 6 (BTC = 1,3,5-Benzenetricarboxylic acid) for evaluating protein kinase activity and the relevant inhibitors. A fluorophore-labelled substrate polypeptide was phosphorylated under the catalysis of protein kinase. This phosphorylated peptide can be coordinated explicitly with the europium site of Eu(BTC) (H 2 O) 6 to detect the protein kinase. The developed biosensor performed well, with a detection limit of 0.00003 U μL−1, and it showed good selectivity and universality. Protein kinase activity could also be detected in MCF-7 cells using this method. Furthermore, in terms of inhibitor screening using the Eu(BTC) (H 2 O) 6 -based sensor, both H-89 and ellagic acid were found to inhibit protein kinase activity with IC 50 values of 1.09 and 19.88 nmol L−1, respectively. Overall, this biosensor has broad application prospects in monitoring and controlling protein kinase activity. • A new biosensing platform for detecting PKA activity was established using Eu(BTC) (H 2 O) 6. • Akt1 was selected to prove the universality of this biosensor for protein kinases. • This biosensor screened two common PKA inhibitors, H-89 and ellagic acid. • This method is helpful for monitoring and treating protein kinase-related diseases. [ABSTRACT FROM AUTHOR]

Key points The Mg2+ and Ca2+ conducting transient receptor potential melastatin 7 (TRPM7) channel-enzyme (chanzyme) has been implicated in immune cell function., Mice heterozygous for a TRPM7 kinase deletion are hyperallergic, while mice with a single point mutation at amino acid 1648, silencing kinase activity, are not., As mast cell mediators trigger allergic reactions, we here determine the function of TRPM7 in mast cell degranulation and histamine release., Our data establish that TRPM7 kinase activity regulates mast cell degranulation and release of histamine independently of TRPM7 channel function., Our findings suggest a regulatory role of TRPM7 kinase activity on intracellular Ca2+ and extracellular Mg2+ sensitivity of mast cell degranulation., Abstract Transient receptor potential melastatin 7 (TRPM7) is a divalent ion channel with a C-terminally located α-kinase. Mice heterozygous for a TRPM7 kinase deletion (TRPM7+/∆K) are hypomagnesaemic and hyperallergic. In contrast, mice carrying a single point mutation at amino acid 1648, which silences TRPM7 kinase activity (TRPM7KR), are not hyperallergic and are resistant to systemic magnesium (Mg2+) deprivation. Since allergic reactions are triggered by mast cell-mediated histamine release, we investigated the function of TRPM7 on mast cell degranulation and histamine release using wild-type (TRPM7+/+), TRPM7+/∆K and TRPM7KR mice. We found that degranulation and histamine release proceeded independently of TRPM7 channel function. Furthermore, extracellular Mg2+ assured unperturbed IgE-DNP-dependent exocytosis, independently of TRPM7. However, impairment of TRPM7 kinase function suppressed IgE-DNP-dependent exocytosis, slowed the cellular degranulation rate, and diminished the sensitivity to intracellular calcium (Ca2+) in G protein-induced exocytosis. In addition, G protein-coupled receptor (GPCR) stimulation revealed strong suppression of histamine release, whereas removal of extracellular Mg2+ caused the phenotype to revert. We conclude that the TRPM7 kinase activity regulates murine mast cell degranulation by changing its sensitivity to intracellular Ca2+ and affecting granular mobility and/or histamine contents. [ABSTRACT FROM AUTHOR]

DAPK3 belongs to family of DAPK (death-associated protein kinases) and is involved in the regulation of progression of the cell cycle, cell proliferation, apoptosis and autophagy. It is considered as a tumor suppressor kinase, suggesting the loss of its function in case of certain specific mutations. The T112M, D161N and P216S mutations in DAPK3 have been observed in cancer patients. These DAPK3 mutants have been associated with very low kinase activity, which results in the cellular progression towards cancer. However, a clear understanding of the structural and biophysical variations that occur in DAPK3 with these mutations, resulting in the decreased kinase activity has yet not been deciphered. We performed a molecular dynamic simulation study to investigate such structural variations. Our results revealed that mutations caused a significant structural variation in DAPK3, majorly concentrated in the flexible loops that form part of the ATP binding pocket. Interestingly, D161N and P216S mutations collapsed the ATP binding pocket through flexible loops invasion, hindering ATP binding which resulted in very low kinase activity. On the contrary, T112M mutant DAPK3 reduces ATP binding potential through outward distortion of flexible loops. In addition, the mutant lacked characteristic features of the active protein kinase including proper interaction between HR/FD and DFG motifs, well structured hydrophobic spine and Lys42–Glu64 salt bridge interaction. These observations could possibly explain the underlying mechanism associated with the loss of kinase activity with T112M, D161N and P216S mutation in DAPK3. [ABSTRACT FROM AUTHOR]

Positive transcription elongation factor b (P-TEFb) is a RNA polymerase II carboxyl-terminal domain (Pol II CTD) kinase that phosphorylates Ser2 of the CTD and promotes the elongation phase of transcription. Despite the fact that P-TEFb has role in many cellular processes, the role of this kinase complex remains to be understood in mammalian early developmental events. In this study, using immunocytochemical analyses, we found that the P-TEFb components, CDK9, Cyclin T1 and Cyclin T2 were localized to nuclear speckles, as well as in nucleolar-like bodies in pig germinal vesicle oocytes. Using nascent RNA labeling and small molecule inhibitors, we showed that inhibition of CDK9 activity abolished the transcription of GV oocytes globally. Moreover, using fluorescence in situ hybridization, in absence of CDK9 kinase activity the production of ribosomal RNAs was impaired. We also presented the evidences indicating that P-TEFb kinase activity is essential for resumption of oocyte meiosis and embryo development. Treatment with CDK9 inhibitors resulted in germinal vesicle arrest in maturing oocytes in vitro. Inhibition of CDK9 kinase activity did not interfere with in vitro fertilization and pronuclear formation. However, when in vitro produced zygotes were treated with CDK9 inhibitors, their development beyond the 4-cell stage was impaired. In these embryos, inhibition of CDK9 abrogated global transcriptional activity and rRNA production. Collectively, our data suggested that P-TEFb kinase activity is crucial for oocyte maturation, embryo development and regulation of RNA transcription in pig. [ABSTRACT FROM AUTHOR]

Background: Recent studies have linked certain single nucleotide polymorphisms in the leucine-rich repeat kinase 2 (LRRK2) gene with Parkinson’s disease (PD). Among the mutations, LRRK2 c.4883G>C (R1628P) variant was identified to have a significant association with the risk of PD in ethnic Han-Chinese populations. But the molecular pathological mechanisms of R1628P mutation in PD is still unknown. Principle Findings: Unlike other LRRK2 mutants in the Roc-COR-Kinase domain, the R1628P mutation didn’t alter the LRRK2 kinase activity and promote neuronal death directly. LRRK2 R1628P mutation increased the binding affinity of LRRK2 with Cyclin-dependent kinase 5 (Cdk5). Interestingly, R1628P mutation turned its adjacent amino acid residue S1627 on LRRK2 protein to a novel phosphorylation site of Cdk5, which could be defined as a typical type II (+) phosphorylation-related single nucleotide polymorphism. Importantly, we showed that the phosphorylation of S1627 by Cdk5 could activate the LRRK2 kinase, and neurons ectopically expressing R1628P displayed a higher sensitivity to 1-methyl-4-phenylpyridinium, a bioactive metabolite of environmental toxin MPTP, in a Cdk5-dependent manner. Conclusion: Our data indicate that Parkinson-related LRRK2 mutation R1628P leads to Cdk5 phosphorylation of LRRK2 at S1627, which would upregulate the kinase activity of LRRK2 and consequently cause neuronal death. [ABSTRACT FROM AUTHOR]

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS. In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity. [ABSTRACT FROM AUTHOR]

Most patients with allergic asthma are sensitized to house dust mite (HDM). The allergenicity of HDM largely depends on disruption of the integrity and proinflammatory activation of the airway epithelium. In this study, we hypothesized that Pim1 kinase activity attenuates HDM-induced asthma by preserving airway epithelial integrity. The effects of Pim1 kinase activity on barrier function and release of the proinflammatory mediators IL-1 and CCL20 were studied in vitro in 16HBE and primary bronchial epithelial cells (PBECs). Pim1-proficient and -deficient mice were exposed to a HDM-driven model of allergic asthma, and airway hyperresponsiveness (AHR) was measured upon methacholine challenge. Airway inflammation and proinflammatory mediators in lung tissue and BAL fluid were determined. We observed that inhibition of Pim1 kinase prolongs the HDMinduced loss of barrier function in 16HBE cells and sensitizes PBECs to HDM-induced barrier dysfunction. Additionally, inhibition of Pim1 kinase increased the HDM-induced proinflammatory activity of 16HBE cells as measured by IL-1 secretion. In line herewith, HDM exposure induced an enhanced production of the proinflammatory chemokines CCL17 and CCL20 in Pim1-deficient mice compared with wild-type controls. While we observed a marked increase in eosinophilic and neutrophilic granulocytes as well as mucus cell metaplasia and AHR to methacholine in mice exposed to HDM, these parameters were independent of Pim1 kinase activity. In contrast, levels of the Th2-cytokines IL-5 and IL-10 were significantly augmented in HDM-treated Pim1-deficient mice. Taken together, our study shows that Pim1 kinase activity maintains airway epithelial integrity and protects against HDM-induced proinflammatory activation of the airway epithelium. [ABSTRACT FROM AUTHOR]

The Drosophila bone morphogenetic protein encoded by decapentaplegic (dpp) controls ventral head morphogenesis by expression in the head primordia, eye-antennal imaginal discs. These are epithelial sacs made of two layers: columnar disc proper cells and squamous cells of the peripodial epithelium. dpp expression related to head formation occurs in the peripodial epithelium; cisregulatory mutations disrupting this expression display defects in sensory vibrissae, rostral membrane, gena, and maxillary palps. Here we document that disruption of this dpp expression causes apoptosis in peripodial cells and underlying disc proper cells. We further show that peripodial Dpp acts directly on the disc proper, indicating that Dpp must cross the disc lumen to act. We demonstrate that palp defects are mechanistically separable from the other mutant phenotypes; both are affected by the c-Jun N-terminal kinase pathway but in opposite ways. Slight reduction of both Jun N-terminal kinase and Dpp activity in peripodial cells causes stronger vibrissae, rostral membrane, and gena defects than Dpp alone; additionally, strong reduction of Jun N-terminal kinase activity alone causes identical defects. A more severe reduction of dpp results in similar vibrissae, rostral membrane, and gena defects, but also causes mutant maxillary palps. This latter defect is correlated with increased peripodial Jun N-terminal kinase activity and can be caused solely by ectopic activation of Jun N-terminal kinase. We conclude that formation of sensory vibrissae, rostral membrane, and gena tissue in head morphogenesis requires the action of Jun N-terminal kinase in peripodial cells, while excessive Jun N-terminal kinase signaling in these same cells inhibits the formation of maxillary palps. [ABSTRACT FROM AUTHOR]

Summary The ability to monitor kinase activity dynamics in live cells greatly aids the study of how signaling events are spatiotemporally regulated. Here, we report on the adaptability of bimolecular kinase activity reporters (bimKARs) as molecular tools to enhance the real-time visualization of kinase activity. We demonstrate that the bimKAR design is truly versatile and can be used to monitor a variety of kinases, including JNK, ERK, and AMPK. Furthermore, bimKARs can have significantly enhanced dynamic ranges over their unimolecular counterparts, allowing the elucidation of previously undetectable kinase activity dynamics. Using these newly designed bimKARs, we investigate the regulation of AMPK by protein kinase A (PKA) in the plasma membrane, and demonstrate that PKA can both negatively and positively regulate AMPK activity in the same cell. [ABSTRACT FROM AUTHOR]

Despite the widespread use of kinase-targeted agents in clear cell renal cell carcinoma (CC-RCC), comprehensive kinase activity evaluation (kinomic profiling) of these tumors is lacking. Thus, kinomic profiling of CC-RCC may assist in devising a classification system associated with clinical outcomes, and help identify potential therapeutic targets. Fresh frozen CC-RCC tumor lysates from 41 clinically annotated patients who had localized disease at diagnosis were kinomically profiled using the PamStation®12 high-content phospho-peptide substrate microarray system (PamGene International). Twelve of these patients also had matched normal kidneys available that were also profiled. Unsupervised hierarchical clustering and supervised comparisons based on tumor vs. normal kidney and clinical outcome (tumor recurrence) were performed and coupled with advanced network modeling and upstream kinase prediction methods. Unsupervised clustering analysis of localized CC-RCC tumors identified 3 major kinomic groups associated with inflammation (A), translation initiation (B), and immune response and cell adhesions (C) processes. Potential driver kinases implicated include PFTAIRE (PFTK1), PKG1, and SRC, which were identified in groups A, B, and C, respectively. Of the 9 patients who had tumor recurrence, only one was found in Group B. Supervised analysis showed decreased kinase activity of CDK1 and RSK1-4 substrates in those which progressed compared to others. Twelve tumors with matching normal renal tissue implicated increased PIM’s and MAPKAPK’s in tumors compared to adjacent normal renal tissue. As such, comprehensive kinase profiling of CC-RCC tumors could provide a functional classification strategy for patients with localized disease and identify potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Flavonoids are plant pigments that have been demonstrated to exert various pharmacological effects including anti-cancer, anti-diabetic, anti-atherosclerotic, anti-bacterial, and anti-inflammatory activities. However, the molecular mechanisms in terms of exact target proteins of flavonoids are not fully elucidated yet. In this study, we aimed to evaluate the anti-inflammatory mechanism of scutellarein (SCT), a flavonoid isolated from Erigeron breviscapus, Clerodendrum phlomidis and Oroxylum indicum Vent that have been traditionally used to treat various inflammatory diseases in China and Brazil. For this purpose, a nitric oxide (NO) assay, polymerase chain reaction (PCR), nuclear fractionation, immunoblot analysis, a kinase assay, and an overexpression strategy were employed. Scutellarein significantly inhibited NO production in a dose-dependent manner and reduced the mRNA expression levels of inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, SCT also dampened nuclear factor (NF)-κB-driven expression of a luciferase reporter gene upon transfection of a TIR-domain-containing adapter-inducing interferon-β (TRIF) construct into Human embryonic kidney 293 (HEK 293) cells; similarly, NF-κ B nuclear translocation was inhibited by SCT. Moreover, the phosphorylation levels of various upstream signaling enzymes involved in NF-κB activation were decreased by SCT treatment in LPS-treated RAW264.7 cells. Finally, SCT strongly inhibited Src kinase activity and also inhibited the autophosphorylation of overexpressed Src. Therefore, our data suggest that SCT can block the inflammatory response by directly inhibiting Src kinase activity linked to NF-κB activation. [ABSTRACT FROM AUTHOR]

Human epidermal growth factor receptor 2 (HER2) is amplified in ~15-20% of human breast cancer and is important for tumor etiology and therapeutic options of breast cancer. Up-regulation of HER2 oncogene initiates cascades of events cumulating to the stimulation of transforming PI3K/AKT signaling, which also plays a dominant role in supporting cell survival and efficacy of HER2-directed therapies. Although investigating the underlying mechanisms by which HER2 promotes cell survival, we noticed a profound reduction in the kinase activity of a pro-apoptotic mixed lineage kinase 3 (MLK3) in HER2-positive (HER2+) but not in HER2-negative (HER2-) breast cancer tissues, whereas both HER2+ and HER2- tumors expressed a comparable level of MLK3 protein. Furthermore, the kinase activity of MLK3 was inversely correlated with HER2+ tumor grades. Moreover, HER2-directed drugs such as trastuzumab and lapatinib as well as depletion of HER2 or HER3 stimulated MLK3 kinase activity in HER2+ breast cancer cell lines. In addition, the noted inhibitory effect of HER2 on MLK3 kinase activity was mediated via its phosphorylation on Ser674 by AKT and that pharmacological inhibitors of PI3K/AKT prevented trastuzumab- and lapatinib-induced stimulation of MLK3 activity. Consistent with the pro-apoptotic function of MLK3, stable knockdown of MLK3 in the HER2+ cell line blunted the pro-apoptotic effects of trastuzumab and lapatinib. These findings suggest that HER2 activation inhibits the pro-apoptotic function of MLK3, which plays a mechanistic role in mediating anti-tumor activities of HER2-directed therapies. In brief, MLK3 represents a newly recognized integral component of HER2 biology in HER2+ breast tumors. [ABSTRACT FROM AUTHOR]

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. [ABSTRACT FROM AUTHOR]

The main purpose of this study was to investigate the effect of acute inflammation on total creatine kinase (CK) activity in dogs. In these animals, CK is an enzyme found predominantly in skeletal muscle and significantly elevated serum activity is largely associated with muscle damage. Plasma increases in dogs are associated with cell membrane leakage and will therefore be seen in any condition associated with muscular inflammation. The study was induced in 15 mongrel male dogs (n=9 in experimental group and n=6 in control group) at the age of two years and body weight 12-15 kg. The inflammation was reproduced by inoculation of 2 ml turpentine oil subcutaneously in lumbar region. The plasma activity of creatine kinase was evaluated at 0, 6, 24, 48, 72 hours after inoculation and on days 7, 14 and 21 by a kit from Hospitex Diagnostics. In the experimental group, the plasma concentrations of the CK-activity were increased at the 48th hour (97.48±6.92 U/L) and remained significantly higher (p<0.05) at the 72 hour (97.43±2.93 U/L) compared to the control group (77.08±5.27 U/L). The results of this study suggest that the evaluation of creatine kinase in dogs with experimentally induced acute inflammation has a limited diagnostic value. It was observed that the creatine kinase activity is slightly affected by the experimentally induced acute inflammation in dogs. [ABSTRACT FROM AUTHOR]

Computational characterization of differential kinase activity from phosphoproteomics datasets is critical for correctly inferring cellular circuitry and how signaling cascades are altered in drug treatment and/or disease. Kinase-Substrate Enrichment Analysis (KSEA) offers a powerful approach to estimating changes in a kinase's activity based on the collective phosphorylation changes of its identified substrates. However, KSEA has been limited to programmers who are able to implement the algorithms. Thus, to make it accessible to the larger scientific community, we present a web-based application of this method: the KSEA App. Overall, we expect that this tool will offer a quick and user-friendly way of generating kinase activity estimates from highthroughput phosphoproteomics datasets. [ABSTRACT FROM AUTHOR]

Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched domains is a transmembrane adaptor protein primarily involved in negative regulation of T-cell activation by recruitment of C-terminal Src kinase (Csk), a protein tyrosine kinase which represses Src kinase activity through C-terminal phosphorylation. Recruitment of Csk occurs via SH2-domain binding to PAG pTyr317, thus, the interaction is highly dependent on phosphorylation performed by the Src family kinase Fyn, which docks onto PAG using a dual-domain binding mode involving both SH3- and SH2-domains of Fyn. In this study, we investigated Fyn SH3-domain binding to 14-mer peptide ligands derived from Cbp/PAG-enriched microdomains sequence using biochemical, biophysical and computational techniques. Interaction kinetics and dissociation constants for the various ligands were determined by SPR. The local structural impact of ligand association has been evaluated using CD, and molecular modelling has been employed to investigate details of the interactions. We show that data from these investigations correlate with functional effects of ligand binding, assessed experimentally by kinase assays using full-length PAG proteins as substrates. The presented data demonstrate a potential method for modulation of Src family kinase tyrosine phosphorylation through minor changes of the substrate SH3-interacting motif. [ABSTRACT FROM AUTHOR]

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fonds voor Wetenschappelijk Onderzoek (FWO) Introduction Necroptosis is a form of regulated necrosis mediated by the kinase activity of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and executed by RIPK3 and mixed lineage kinase domain like pseudokinase (MLKL). Because necroptosis is involved in necrotic core development in atherosclerotic plaques, we investigated the effect of a RIPK1 S25D/S25D mutation, which prevents the activation of RIPK1 kinase, on atherogenesis in ApoE-/- mice. Furthermore, we pharmacologically inhibited RIPK1 kinase activity by administering GSK'547 to ApoE-/- Fbn1 C1039G+/- mice, a mouse model of advanced atherosclerosis. Purpose We aimed to study the effect of RIPK1 kinase inhibition on atherogenesis through a genetic and a pharmacological approach in ApoE-/- and ApoE-/- Fbn1 C1039G+/- mice, respectively. Methods ApoE-/- and ApoE-/- Fbn1 C1039G+/- mice were fed a Western-type diet (WD) for up to 24 weeks and plaque necroptosis was confirmed. ApoE-/- Ripk1+/+ (n=16) and ApoE-/- Ripk1 S25D/S25D (n=12) mice were fed a WD for 16 weeks to induce plaque formation. ApoE-/- Fbn1 C1039G+/- mice received WD supplemented with GSK'547 (10 mg/kg BW/day, n=12-13/group) for 20 weeks to evaluate the effect of pharmacological RIPK1 kinase inhibition on atherogenesis. Results After 16 weeks WD, atherosclerotic plaques of ApoE-/- Ripk1 S25D/S25D mice were significantly larger as compared to ApoE-/- Ripk1+/+ mice (167±34 vs. 69±18 103 µm2, P=0.01). Absolute cell numbers (350±34 vs. 154±33 nuclei) and deposition of glycosaminoglycans (Alcian blue: 31±6 vs. 14±4%, P=0.023) were increased in plaques from ApoE-/- Ripk1 S25D/S25D mice while macrophage content (Mac3: 2.3±0.4 vs. 9.8±2.4%, P=0.012) was significantly decreased. Increased deposition of glycosaminoglycans was also observed in the vessel wall of ApoE-/- Ripk1 S25D/S25D mice (Alcian blue: 4.3±1.5 vs. 1.4±0.4 %, P=0.04). Together with increased internal elastic lamina area and increased vessel wall thickness, this points towards hypertrophic positive vascular remodeling in ApoE-/- Ripk1 S25D/S25D mice. Plaque apoptosis was not different between both groups. In contrast, pharmacological inhibition of RIPK1 kinase with GSK'547 in ApoE-/- Fbn1 C1039G+/- mice did not significantly alter plaque size and vessel properties after 20 weeks WD, but induced plaque apoptosis (TUNEL: 124±17 vs. 68±8 cells/mm2, P=0.018; cleaved caspase 3: 0.49±0.13 vs. 0.14±0.04 %; p=0.021). Conclusion Inhibition of RIPK1 kinase activity in ApoE-/- Ripk1 S25D/S25D mice accelerated plaque progression and induced positive vascular remodeling. These effects were not observed after pharmacological inhibition with GSK'547 in ApoE-/- Fbn1 C1039G+/- mice. However, a switch to apoptosis was observed after GSK'547 treatment, stressing the complex involvement of RIPK1 in cell survival and death. [ABSTRACT FROM AUTHOR]

Summary The regulation of many protein kinases by binding to calcium/calmodulin connects two principal mechanisms in signaling processes: protein phosphorylation and responses to dose- and time-dependent calcium signals. We used the calcium/calmodulin-dependent members of the death-associated protein kinase (DAPK) family to investigate the role of a basic DAPK signature loop near the kinase active site. In DAPK2, this loop comprises a novel dimerization-regulated calcium/calmodulin-binding site, in addition to a well-established calcium/calmodulin site in the C-terminal autoregulatory domain. Unexpectedly, impairment of the basic loop interaction site completely abolishes calcium/calmodulin binding and DAPK2 activity is reduced to a residual level, indicative of coupled binding to the two sites. This contrasts with the generally accepted view that kinase calcium/calmodulin interactions are autonomous of the kinase catalytic domain. Our data establish an intricate model of multi-step kinase activation and expand our understanding of how calcium binding connects with other mechanisms involved in kinase activity regulation. [ABSTRACT FROM AUTHOR]

Background: Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Methods: Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. Results: The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM-/- cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. Conclusion: H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection. [ABSTRACT FROM AUTHOR]

The Maf1 protein is a general negative repressor of RNA polymerase III, which is conserved in eukaryotes from yeast to humans. Herein, we show the yeast maf1Δ mutant increases pyruvate kinase activity, the key enzyme in glycolysis and an important player in switching between fermentative and oxidative metabolism. We observed enhanced ethanol production and elevated lipid content in the maf1Δ strain grown on glucose. However, after shifting to a non-fermentable carbon source, the opposite effect was observed, and the mutant cells accumulated smaller lipid droplets. Thus, it has been concluded that the Maf1 protein is essential for regulation of glucose metabolism and lipid homeostasis. [ABSTRACT FROM AUTHOR]

Summary IκB kinase/necrosis factor κB (IKK/NF-κB) signaling exhibits important yet opposing functions in hepatocarcinogenesis. Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer. Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMO LPC-KO mice. Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMO LPC-KO mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1. Collectively, these results show that NEMO prevents hepatocarcinogenesis by inhibiting RIPK1 kinase activity-driven hepatocyte apoptosis through NF-κB-dependent and -independent functions. [ABSTRACT FROM AUTHOR]

Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2-/-) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2-/- mice and mice expressing a kinase-inactive version of TYK2 (Tyk2K923E), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2-/- and Tyk2K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2-/- NK cells (and to a lesser extent of Tyk2K923E NK cells) is impaired. In contrast, the production of interferon g (IFNg) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2K923E NK cells against a range of target cells in vitro is higher than that of Tyk2-/- NK cells. Consistently, Tyk2K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2's kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy accompanies many forms of heart disease, including ischemic disease, hypertension, heart failure, and valvular disease, and it is a strong predictor of increased cardiovascular morbidity and mortality. Deletion of bone marrow kinase in chromosome X (Bmx), an arterial nonreceptor tyrosine kinase, has been shown to inhibit cardiac hypertrophy in mice. This finding raised the possibility of therapeutic use of Bmx tyrosine kinase inhibitors, which we have addressed here by analyzing cardiac hypertrophy in gene-targeted mice deficient in Bmx tyrosine kinase activity. We found that angiotensin II (Ang II)-induced cardiac hypertrophy is significantly reduced in mice deficient in Bmx and in mice with inactivated Bmx tyrosine kinase compared with WT mice. Genome-wide transcriptomic profiling showed that Bmx inactivation suppresses myocardial expression of genes related to Ang II-induced inflammatory and extracellular matrix responses whereas expression of RNAs encoding mitochondrial proteins after Ang II administration was maintained in Bmx-inactivated hearts. Very little or no Bmx mRNA was expressed in human cardiomyocytes whereas human cardiac endothelial cells expressed abundant amounts. Ang II stimulation of endothelial cells increased Bmx phosphorylation, and Bmx gene silencing inhibited downstream STAT3 signaling, which has been implicated in cardiac hypertrophy. Furthermore, activation of the mechanistic target of rapamycin complex 1 pathway by Ang II treatment was decreased in the Bmx-deficient hearts. Our results demonstrate that inhibition of the cross-talk between endothelial cells and cardiomyocytes by Bmx inactivation suppresses Ang II-induced signals for cardiac hypertrophy. These results suggest that the endothelial Bmx tyrosine kinase could provide a target to attenuate the development of cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Raf kinases are important components of the Ras-Raf-Mek-Erk pathway and also cross-talk with other signaling pathways. Araf kinase has been demonstrated to inhibit TGF-β/Smad2 signaling by directly phosphorylating and accelerating degradation of activated Smad2. In this study, we show that the araf gene expresses in zebrafish embryos to produce a shorter transcript variant, araf-tv2, in addition to the full-length variant araf-tv1. araf-tv2 is predicted to encode a C-terminally truncated peptide without the kinase activity domain. Araf-tv2 can physically associate with Araf-tv1 but does not antagonize the inhibitory effect of Araf-tv1 on TGF-β/Smad2 signaling. Instead, Araf-tv2 interacts strongly with Kras and Nras, ultimately blocking MAPK activation by these Ras proteins. In zebrafish embryos, overexpression of araf-tv2 is sufficient to inhibit Fgf/Ras-promoted Erk activation, mesodermal induction, dorsal development, and neuroectodermal posteriorization. Therefore, different isoforms of Araf may participate in similar developmental processes but by regulating different signaling pathways. [ABSTRACT FROM AUTHOR]

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 104/cm2 showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 104/cm2 largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) catalytic subunit p110α is the most frequently mutated kinase in human cancer, and the hot spot mutations E542K, E545K, and H1047R are the most common mutations in p110α. Very little is known about the metabolic consequences of the hot spot mutations of p110α in vivo. In this study, we used adenoviral gene transfer in mice to investigate the effects of the E545K and H1047R mutations on hepatic and whole-body glucose metabolism. We show that hepatic expression of these hot spot mutations results in rapid hepatic steatosis, paradoxically accompanied by increased glucose tolerance, and marked glycogen accumulation. In contrast, wild-type p110α expression does not lead to hepatic accumulation of lipids or glycogen despite similar degrees of upregulated glycolysis and expression of lipogenic genes. The reprogrammed metabolism of the E545K and H1047R p110α mutants was surprisingly not dependent on altered p110α lipid kinase activity. [ABSTRACT FROM AUTHOR]