Kinome-Wide Profiling Identifies Human WNK3 as a Target of Cajanin Stilbene Acid from Cajanus cajan (L.) Millsp.

Pigeon Pea (Cajanus cajan (L.) Millsp.) is a common food crop used in many parts of the world for nutritional purposes. One of its chemical constituents is cajanin stilbene acid (CSA), which exerts anticancer activity in vitro and in vivo. In an effort to identify molecular targets of CSA, we performed a kinome-wide approach based on the measurement of the enzymatic activities of 252 human kinases. The serine-threonine kinase WNK3 (also known as protein kinase lysine-deficient 3) was identified as the most promising target of CSA with the strongest enzymatic activity inhibition in vitro and the highest binding affinity in molecular docking in silico. The lowest binding affinity and the predicted binding constant pKi of CSA (-9.65 kcal/mol and 0.084 µM) were comparable or even better than those of the known WNK3 inhibitor PP-121 (-9.42 kcal/mol and 0.123 µM). The statistically significant association between WNK3 mRNA expression and cellular responsiveness to several clinically established anticancer drugs in a panel of 60 tumor cell lines and the prognostic value of WNK3 mRNA expression in sarcoma biopsies for the survival time of 230 patients can be taken as clues that CSA-based inhibition of WNK3 may improve treatment outcomes of cancer patients and that CSA may serve as a valuable supplement to the currently used combination therapy protocols in oncology.

Nanomediator-Effector Cascade Systems for Amplified Protein Kinase Activity Imaging and Phosphorylation-Induced Drug Release In Vivo.

Protein kinases constitute a rich pool of biomarkers and therapeutic targets of tremendous diseases including cancer. However, sensing kinase activity in vivo while implementing treatments according to kinase hyperactivation remains challenging. Herein, we present a nanomediator-effector cascade system that can in situ magnify the subtle events of kinase-catalyzed phosphorylation via DNA amplification machinery to achieve kinase activity imaging and kinase-responsive drug release in vivo. In this cascade, the phosphorylation-mediated disassembly of DNA/peptide complex on the nanomediators initiated the detachment of fluorescent hairpin DNAs from the nanoeffectors via hybridization chain reaction (HCR), leading to fluorescence recovery and therapeutic cargo release. We demonstrated that this nanosystem simultaneously enabled trace protein kinase A (PKA) activity imaging and on-demand drug delivery for inhibition of tumor cell growth both in vitro and in vivo, affording a kinase-specific sense-and-treat paradigm for cancer theranostics.

Acrylamide treatment alters the level of Ca2+ and Ca2+-related protein kinase in spinal cords of rats.

This study aimed to analyze the neurological changes induced by acrylamide (ACR) poisoning and their underlying mechanisms within the spinal cords of male adult Wistar rats. The rats were randomly divided into three groups (n = 9 rats per group). ACR was intraperitoneally injected to produce axonopathy according to the daily dosing schedules of 20 or 40 mg/kg/day of ACR for eight continuous weeks (three times per week). During the exposure period, body weights and gait scores were assessed, and the concentration of Ca2+ was calculated in 27 mice. Protein kinase A (PKA), protein kinase C (PKC), cyclin-dependent protein kinase 5 (CDK5), and P35 were assessed by electrophoretic resolution and Western blotting. The contents of 3'-cyclic adenosine monophosphate (cAMP) and calmodulin (CaM) were determined using ELISA kits, and the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and PKC were determined using the commercial Signa TECTPKAassay kits. Compared with control rats, treatment with 20 and 40 mg/kg of ACR decreased body weight and increased gait scores at 8 weeks. Intracellular Ca2+ levels increased significantly in treated rats; CaM, PKC, CDK5, and P35 levels were significantly decreased; and PKA and cAMP levels remained unchanged. CaMKII, PKA, and PKC activities increased significantly. The results indicated that ACR can damage neurofilaments by affecting the contents and activities of CaM, CaMKII, PKA, cAMP, PKC, CDK5, and P35, which could result in ACR toxic neuropathy.

STOML2 potentiates metastasis of hepatocellular carcinoma by promoting PINK1-mediated mitophagy and regulates sensitivity to lenvatinib.

BACKGROUND: Dysregulation of both mitochondrial biogenesis and mitophagy is critical to sustain oncogenic signaling pathways. However, the mechanism of mitophagy in promoting hepatocellular carcinoma (HCC) progression remains poorly understood. In this study, we investigated the clinical significance and biological involvement of mitochondrial inner membrane protein STOML2 in HCC. METHODS: STOML2 was identified by gene expression profiles of HCC tissues and was measured in tissue microarray and cell lines. Gain/loss-of-function experiment was applied to study the biological function of STOML2 in HCC. Flow cytometry, Western blotting, laser confocal microscopy, transmission electron microscopy, and co-immunoprecipitation were used to detect and analyze mitophagy. ChIP and luciferase reporter assay were conducted to evaluate the relationship between STOML2 and HIF-1α. The sensitivity to lenvatinib was assessed in HCC both in vitro and in vivo. RESULTS: Increased expression of STOML2 was found in HCC compared with paired peritumoral tissues. It was more significant in HCC with metastasis and correlated with worse overall survival and higher probability of recurrence after hepatectomy. Upregulation of STOML2 accelerated HCC cells colony formation, migration and invasion. Mechanically, TCGA dataset-based analysis showed enrichment of autophagy-related pathways in STOML2 highly-expressed HCC. Next, STOML2 was demonstrated to interact and stabilize PINK1 under cellular stress, amplify PINK1-Parkin-mediated mitophagy and then promote HCC growth and metastasis. Most interestingly, HIF-1α was upregulated and transcriptionally increased STOML2 expression in HCC cells under the treatment of lenvatinib. Furthermore, higher sensitivity to lenvatinib was found in HCC cells when STOML2 was downregulated. Combination therapy with lenvatinib and mitophagy inhibitor hydroxychloroquine obtained best efficacy. CONCLUSIONS: Our findings suggested that STOML2 could amplify mitophagy through interacting and stabilizing PINK1, which promote HCC metastasis and modulate the response of HCC to lenvatinib. Combinations of pharmacologic inhibitors that concurrently block both angiogenesis and mitophagy may serve as an effective treatment for HCC.

From Beef to Bees: High-Throughput Kinome Analysis to Understand Host Responses of Livestock Species to Infectious Diseases and Industry-Associated Stress.

Within human health research, the remarkable utility of kinase inhibitors as therapeutics has motivated efforts to understand biology at the level of global cellular kinase activity (the kinome). In contrast, the diminished potential for using kinase inhibitors in food animals has dampened efforts to translate this research approach to livestock species. This, in our opinion, was a lost opportunity for livestock researchers given the unique potential of kinome analysis to offer insight into complex biology. To remedy this situation, our lab developed user-friendly, cost-effective approaches for kinome analysis that can be readily incorporated into most research programs but with a specific priority to enable the technology to livestock researchers. These contributions include the development of custom software programs for the creation of species-specific kinome arrays as well as comprehensive deconvolution and analysis of kinome array data. Presented in this review are examples of the application of kinome analysis to highlight the utility of the technology to further our understanding of two key complex biological events of priority to the livestock industry: host immune responses to infectious diseases and animal stress responses. These advances and examples of application aim to provide both mechanisms and motivation for researchers, particularly livestock researchers, to incorporate kinome analysis into their research programs.

Effect of sitagliptin on expression of skeletal muscle peroxisome proliferator-activated receptor γ coactivator-1α and irisin in a rat model of type 2 diabetes mellitus.

OBJECTIVE: To evaluate the effect of sitagliptin on skeletal muscle expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), irisin, and phosphoadenylated adenylate activated protein kinase (p-AMPK) in a rat model of type 2 diabetes mellitus (T2DM). METHODS: A high-fat diet/streptozotocin T2DM rat model was established. Rats were divided into T2DM, low-dose sitagliptin (ST1), high-dose sitagliptin (ST2), and normal control groups (NC). PGC-1α, irisin, and p-AMPK protein levels in skeletal muscle were measured by western blot, and PCG-1α and Fndc5 mRNA levels were assessed by reverse transcription-polymerase chain reaction. RESULTS: Fasting plasma glucose (FPG), fasting insulin (FIns), homeostatic model assessment-insulin resistance (HOMA-IR), and tumor necrosis factor-α (TNF-α) were significantly up-regulated in the T2DM compared with the other groups, and FPG, FIns, total cholesterol, triglycerides, TNF-α, and HOMA-IR were significantly down-regulated in the ST2 compared with the ST1 group. PGC-1α, irisin, and p-AMPK expression levels decreased successively in the ST2, ST1, and DM groups compared with the NC, and were all significantly up-regulated in the ST2 compared with the ST1 group. CONCLUSION: Down-regulation of PGC-1α and irisin in skeletal muscle may be involved in T2DM. Sitagliptin can dose-dependently up-regulate PCG-1α and irisin, potentially improving insulin resistance and glycolipid metabolism and inhibiting inflammation.

The Trend of ripk1/ripk3 and mlkl Mediated Necroptosis Pathway in Patients with Different Stages of Prostate Cancer as Promising Progression Biomarkers.

BACKGROUND: Programmed cell death is critical to maintain tissue homeostasis. Necroptosis, as well as apoptosis, has been considered as another form of regulated cell death which can be used as an effective way to overcome apoptosis-resistant tumor tissue growth. The aim of present study was to test whether or not ripk1, ripk3, or mlkl expression levels, as the key necroptotic modulators in different stages of prostate tumor growth. METHODS: Sixty-seven prostate tissues representing histologically confirmed cancer were selected. The cancer samples were categorized into 4 different stages based on cellular differentiation, tumor growth rate, and extra tissue expansion to regional lymph nodes, average PSA levels, and tumor volume. RNA extraction, cDNA synthesis and quantitative real time PCR were done based on standard guidelines. RESULTS: No statistically significant changes in ripk1 expression showed in all three stages (stage II to IV). The expression pattern of ripk3 represented a remarkable elevation in early stage, while, predominantly repressed in final cancer stage (IV). Also, there has been a significant negative correlation between ripk3 gene expression and tumor size and PSA levels. CONCLUSIONS: We cannot exclude the importance of the key regulator proteins in development and progression of prevalent lethal disease like prostate cancer. The ripk1/ripk3 mediated necroptosis pathway is more activated in early stages of prostate cancer via induced ripk3 expression, while repressed during prostate cancer final stages. Also, the repression of ripk3 is related to elevation of both PSA levels and tumor volume which represented the tumor progression in final stages.

Ultrasensitive peptide-based electrochemical detection of protein kinase activity amplified by RAFT polymerization.

Since the oversecretion of protein kinases is indicative of multiple human diseases, the screening of their activities is quite important to clinical diagnosis and targeted therapy. In this work, an ultrasensitive peptide-based electrochemical biosensor was presented for the detection of protein kinase activity by using the reversible addition-fragmentation chain transfer (RAFT) polymerization technique as a signal amplification strategy. First, the substrate peptides were tethered to a gold electrode surface via the thiol terminals. After the phosphorylation of substrate peptides by protein kinases, the carboxyl group-containing dithiobenzoates were labeled to the phosphorylated sites via the robust phosphate-Zr4+-carboxylate linkages. Finally, the RAFT polymerization was initiated using ferrocenylmethyl methacrylates (FcMMAs) and dithiobenzoates as the monomers and the RAFT agents, respectively. The grafting of ferrocenyl polymer chains efficiently recruits a great number of electro-active Fc probes to each phosphorylated site, leading to a drastic amplification of the electrochemical signal. With PKA (protein kinase A) as the target, the detection limit of the peptide-based biosensor can be as low as 1.05 mU mL-1. Moreover, it can selectively differentiate the target from other interferents and is applicable for the screening of potential inhibitors as well as the detection of protein kinase activity in complex cell lysates. Therefore, the peptide-based biosensor shows great promise as a universal tool for protein kinase activity detection and inhibitor screening.

Electrochemically Controlled RAFT Polymerization for Highly Sensitive Electrochemical Biosensing of Protein Kinase Activity.

Phosphorylation of proteins catalyzed by protein kinases (PKs) is essential to many biological processes; the sensitive detection of PK activity and the screening of PK inhibitors are thus integral to disease diagnosis and drug discovery. Herein, a highly sensitive biosensor has been fabricated for the electrochemical detection of PK activity by exploiting the electrochemically controlled reversible addition-fragmentation chain transfer (eRAFT) polymerization as a novel amplification strategy. The fabrication of the eRAFT-polymerization-based electrochemical biosensor involves (1) the immobilization of substrate peptides onto a gold electrode by way of gold-sulfur self-assembly, (2) the site-specific phosphorylation of substrate peptides by PKs, (3) the anchoring of carboxyl-group-containing chain transfer agents (CTAs) to the phosphorylated sites, and (4) the eRAFT polymerization under a potentiostatic condition, using ferrocenylmethyl methacrylate (FcMMA) as the monomer. Through the eRAFT polymerization, long polymer chains containing numerous electroactive Fc tags can be de novo grafted from each phosphorylated site, resulting in significant amplification of the electrochemical detection signal. The as-fabricated biosensor is highly selective and features a very low detection limit of 1.02 mU mL-1, in the presence of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent PK (PKA) as the model target. Results also demonstrate that it can be applied to the screening of PK inhibitors and the detection of PK activity in complex serum samples and cell lysates. Moreover, it holds the merits of easy fabrication, high efficiency, and low cost, which make it a promising tool for the detection of PK activity and the screening of potential PK inhibitors.

Prognostic and clinicopathological significance of MLKL expression in cancer patients: a meta-analysis.

BACKGROUND: MLKL is the most important executor of necroptosis pathway. Recent studies have demonstrated that MLKL could serve as a potential prognostic biomarker for cancer patients. However, most studies reported so far are limited in discrete outcome and sample size. METHODS: We systematically searched PubMed, Embase, Web of Science and CNKI to obtain all relevant articles about the prognostic value of abnormally expressed MLKL in patients with any type of tumor. Odds ratios or hazards ratios (HRs) with corresponding 95% confidence intervals (CIs) were pooled to estimate the association between MLKL expression and clinicopathological characteristics or survival of cancer patients. RESULTS: A total of 6 eligible studies with 613 cancer patients were enrolled in our meta-analysis. Our results demonstrated that decreased expression level of MLKL was significantly associated with poor overall survival (OS) (pooled HR 0.26, 95%CI 0.17-0.40, high/low) and event-free survival (EFS) (pooled HR 0.45, 95%CI 0.23-0.87, high/low) in cancer patients. Furthermore, subgroup analysis divided by type of cancer, sample size, follow-up time and Newcastle-Ottawa Scale (NOS) score showed consistent prognostic value. In addition, our analysis revealed that decreased expression level of MLKL was significantly associated with advanced tumor stage, more lymph node metastasis and older age. CONCLUSIONS: In conclusion, our meta-analysis suggested that decreased MLKL expression might be a convinced unfavorable prognostic factor that could help the clinical decision-making process.

The kinase activity of the channel-kinase protein TRPM7 regulates stability and localization of the TRPM7 channel in polarized epithelial cells.

The channel-kinase transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein with ion channel and kinase domains. The kinase activity of TRPM7 has been linked to the regulation of a broad range of cellular activities, but little is understood as to how the channel itself is regulated by its own kinase activity. Here, using several mammalian cell lines expressing WT TRPM7 or kinase-inactive variants, we discovered that compared with the cells expressing WT TRPM7, cells in which TRPM7's kinase activity was inactivated had faster degradation, elevated ubiquitination, and increased intracellular retention of the channel. Mutational analysis of TRPM7 autophosphorylation sites further revealed a role for Ser-1360 of TRPM7 as a key residue mediating both TRPM7 stability and intracellular trafficking. Additional trafficking roles were uncovered for Ser-1403 and Ser-1567, whose phosphorylation by TRPM7's kinase activity mediated the interaction of the channel with the signaling protein 14-3-3θ. In summary, our results point to a critical role for TRPM7's kinase activity in regulating proteasome-mediated turnover of the TRPM7 channel and controlling its cellular localization in polarized epithelial cells. Overall, these findings improve our understanding of the significance of TRPM7's kinase activity for functional regulation of its channel activity.

Calcium and Nuclear Signaling in Prostate Cancer.

Recently, there have been a number of developments in the fields of calcium and nuclear signaling that point to new avenues for a more effective diagnosis and treatment of prostate cancer. An example is the discovery of new classes of molecules involved in calcium-regulated nuclear import and nuclear calcium signaling, from the G protein-coupled receptor (GPCR) and myosin families. This review surveys the new state of the calcium and nuclear signaling fields with the aim of identifying the unifying themes that hold out promise in the context of the problems presented by prostate cancer. Genomic perturbations, kinase cascades, developmental pathways, and channels and transporters are covered, with an emphasis on nuclear transport and functions. Special attention is paid to the molecular mechanisms behind prostate cancer progression to the malignant forms and the unfavorable response to anti-androgen treatment. The survey leads to some new hypotheses that connect heretofore disparate results and may present a translational interest.

A label-free fluorescent biosensor for the detection of protein kinase activity based on gold nanoclusters/graphene oxide hybrid materials.

Protein kinase (PKA) can regulate many cellular biological processes by phosphorylation substrate peptide or protein. A new fluorescent biosensing method for the detection of PKA activity was developed by using 11-mercaptoundecanoic acid-capped gold nanoclusters (MUA-Au NCs) and graphene oxide (GO) with low background noise. In this strategy, the special designed peptide could be anchored on the surface of MUA-Au NCs by the Au-S bond and also adsorbed on the surface of GO owing to the electrostatic interaction. As a result, the fluorescence of MUA-Au NCs was quenched leading to low background fluorescence due to the forster resonance energy transfer (FRET) between MUA-Au NCs and GO via peptide as a bridge. However, when the substrate peptide was phosphorylated by PKA, the FRET between GO and MUA-Au NCs was disrupted because of the weakened interaction between the phosphorylated peptide and the GO, resulting in recovery of the fluorescence intensity. The developed label-free fluorescence "turn-off-on" method can detect protein kinase activity in the range of 0.6-2.0 U mL-1 with a detection limit of 0.17 U mL-1 (3σ). The feasibility of this present method for kinase inhibitor screening was also studied by assessment of H-89 kinase inhibition with an IC50 value of 0.049 µmol L-1.

Electrochemically mediated polymerization for highly sensitive detection of protein kinase activity.

Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr4+-phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL-1. Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening.

Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children.

BACKGROUND: Fusions involving one of three tropomyosin receptor kinases (TRK) occur in diverse cancers in children and adults. We evaluated the efficacy and safety of larotrectinib, a highly selective TRK inhibitor, in adults and children who had tumors with these fusions. METHODS: We enrolled patients with consecutively and prospectively identified TRK fusion-positive cancers, detected by molecular profiling as routinely performed at each site, into one of three protocols: a phase 1 study involving adults, a phase 1-2 study involving children, or a phase 2 study involving adolescents and adults. The primary end point for the combined analysis was the overall response rate according to independent review. Secondary end points included duration of response, progression-free survival, and safety. RESULTS: A total of 55 patients, ranging in age from 4 months to 76 years, were enrolled and treated. Patients had 17 unique TRK fusion-positive tumor types. The overall response rate was 75% (95% confidence interval [CI], 61 to 85) according to independent review and 80% (95% CI, 67 to 90) according to investigator assessment. At 1 year, 71% of the responses were ongoing and 55% of the patients remained progression-free. The median duration of response and progression-free survival had not been reached. At a median follow-up of 9.4 months, 86% of the patients with a response (38 of 44 patients) were continuing treatment or had undergone surgery that was intended to be curative. Adverse events were predominantly of grade 1, and no adverse event of grade 3 or 4 that was considered by the investigators to be related to larotrectinib occurred in more than 5% of patients. No patient discontinued larotrectinib owing to drug-related adverse events. CONCLUSIONS: Larotrectinib had marked and durable antitumor activity in patients with TRK fusion-positive cancer, regardless of the age of the patient or of the tumor type. (Funded by Loxo Oncology and others; ClinicalTrials.gov numbers, NCT02122913 , NCT02637687 , and NCT02576431 .).

DNA-hosted copper nanoclusters/graphene oxide based fluorescent biosensor for protein kinase activity detection.

A novel fluorescent biosensor for protein kinase activity (PKA) detection was designed by applying double-strands DNA-hosted copper nanoclusters (dsDNA-CuNCs) and graphene oxide (GO). One DNA strand of the dsDNA consisted of two domains, one domain can hybridize with another complementary DNA strand to stabilize the fluorescent CuNCs and another domain was adenosine 5'-triphosphate (ATP) aptamer. ATP aptamer of the dsDNA-CuNCs would be spontaneously absorbed onto the GO surface through π-π stacking interactions. Thus GO can efficiently quench the fluorescence (FL) of dsDNA-CuNCs through fluorescence resonance energy transfer (FRET). In the present of ATP, ATP specifically combined with ATP aptamer to form ATP-ATP aptamer binding complexes, which had much less affinity to GO, resulting in the fluorescence recovery of the system. Nevertheless, in the presence of PKA, ATP could be translated into ADP and ADP could not combine with ATP aptamer resulting in the fluorescence quenching of dsDNA-CuNCs again. According to the change of the fluorescence signal, PKA activity could be successfully monitored in the range of 0.1-5.0 U mL-1 with a detection limit (LOD) of 0.039 U mL-1. Besides, the inhibitory effect of H-89 on PKA activity was studied. The sensor was performed for PKA activity detection in cell lysates with satisfactory results.

Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death.

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase.

BACKGROUND: Luciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP. METHODS: Here, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP. RESULTS: In this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors  to validate this new luminescence method. CONCLUSION: With this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

Measuring Kinase Activity-A Global Challenge.

The kinase enzymes within a cell, known collectively as the kinome, play crucial roles in many signaling pathways, including survival, motility, differentiation, stress response, and many more. Aberrant signaling through kinase pathways is often linked to cancer, among other diseases. A major area of scientific research involves understanding the relationships between kinases, their targets, and how the kinome adapts to perturbations of the cellular system. This review will discuss many of the current and developing methods for studying kinase activity, and evaluate their applications, advantages, and disadvantages. J. Cell. Biochem. 118: 3595-3606, 2017. © 2017 Wiley Periodicals, Inc.

Kinase analysis of penile squamous cell carcinoma on multiple platforms to identify potential therapeutic targets.

Penile squamous cell carcinoma (PSCC) is an orphan malignancy with poorly understood biology and suboptimal systemic therapy. Given that kinases may be drivers and readily actionable, we performed comprehensive multiplatform analysis of kinases in PSCC tumor and normal tissue. Fresh frozen tumors were collected from 11 patients with PSCC. After macrodissection to demarcate tumor from normal tissue, the samples underwent multiplatform analysis of kinases. Next Generation Sequencing (NGS) of 517 kinase genes was performed using Agilent Kinome capture and run on the Illumina MiSeq at PE150bp. The NanoString nCounter® platform analyzed the expression of 519 kinase genes. Kinase activity of tissue lysates was measured using PamStation®12 high-content phospho-peptide substrate microarray system. Network mapping was done with GeneGo MetaCore™ and upstream kinase prediction was performed with BioNavigator and the Kinexus database. Ingenuity pathway analysis was performed to integrate elevated kinase activity and gene over-expression with coexisting missense mutations at DNA level. Top pathways upregulated in both the kinase activity and gene expression platforms were PTEN, STAT3, GNRH, IL-8 and B cell receptor signaling. Potentially relevant missense mutations were seen in 176 kinase genes, with the top altered pathways overlapping with gene overexpression being GNRH, NF-kB and STAT3 signaling. ERBB2, ERBB3 and SYK were altered on NGS and also exhibited elevated kinase activity. To summarize, multiplatform comprehensive analysis of kinases discovered potential drivers of PSCC and actionable therapeutic targets. Translational studies are necessary to validate the functional relevance of our data to make advances in this rare malignancy.