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Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a limited number of known driver mutations but considerable cancer cell heterogeneity. Phosphoproteomics provides a direct read‐out of aberrant signaling and the resultant clinically relevant phenotype. Mass spectrometry (MS)‐based proteomics and phosphoproteomics were applied to 42 PDAC tumors. Data encompassed over 19 936 phosphoserine or phosphothreonine (pS/T; in 5412 phosphoproteins) and 1208 phosphotyrosine (pY; in 501 phosphoproteins) sites and a total of 3756 proteins. Proteome data identified three distinct subtypes with tumor intrinsic and stromal features. Subsequently, three phospho‐subtypes were apparent: two tumor intrinsic (Phos1/2) and one stromal (Phos3), resembling known PDAC molecular subtypes. Kinase activity was analyzed by the Integrative iNferred Kinase Activity (INKA) scoring. Phospho‐subtypes displayed differential phosphorylation signals and kinase activity, such as FGR and GSK3 activation in Phos1, SRC kinase family and EPHA2 in Phos2, and EGFR, INSR, MET, ABL1, HIPK1, JAK, and PRKCD in Phos3. Kinase activity analysis of an external PDAC cohort supported our findings and underscored the importance of PI3K/AKT and ERK pathways, among others. Interestingly, unfavorable patient prognosis correlated with higher RTK, PAK2, STK10, and CDK7 activity and high proliferation, whereas long survival was associated with MYLK and PTK6 activity, which was previously unknown. Subtype‐associated activity profiles can guide therapeutic combination approaches in tumor and stroma‐enriched tissues, and emphasize the critical role of parallel signaling pathways. In addition, kinase activity profiling identifies potential disease markers with prognostic significance. [ABSTRACT FROM AUTHOR]

Summary: Leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) comprise the largest class of membrane‐localized receptor‐like kinases in plants. Leucine‐rich repeat receptor‐like kinases are key immune sectors contributing to pattern‐triggered immunity (PTI), but whether LRR‐RLK mediates effector‐triggered immunity (ETI) in plants remains unclear.In this study, we evaluated the function of LRR‐RLKs in regulating ETI by using a virus‐induced gene silencing (VIGS)‐based reverse genetic screening assay, and identified a LRR‐RLK named ETI‐dependent receptor‐like kinase 1 (EDK1) required for ETI triggered by the avirulence effector AVRblb2 secreted by Phytophthora infestans and its cognate receptor Rpi‐blb2. Silencing or knockout of EDK1 compromised immunity mediated by Rpi‐blb2 and the cell death triggered by recognition of AVRblb2.NLR‐required for cell death 4 (NRC4), a signaling component acts downstream of Rpi‐blb2, was identified that interacts with EDK1 using the LC–MS analysis and the interaction was further evaluated by co‐immunoprecipitation. EDK1 promotes protein accumulation of NRC4 in a kinase‐dependent manner and positively regulates resistance to P. infestans in Nicotiana benthamiana.Our study revealed that EDK1 positively regulates plant ETI through modulating accumulation of the NLR signaling component NRC4, representing a new regulatory role of the membrane‐localized LRR‐RLKs in plant immunity. [ABSTRACT FROM AUTHOR]

Severe cardiac remodeling leading to heart failure in individuals harboring pathogenic LMNA variants, known as cardiolaminopathy, poses a significant clinical challenge. Currently, there is no effective treatment for lamin-related diseases. Exploring the intricate molecular landscape underlying this condition, with a specific focus on abnormal mechanotransduction, will propel our understanding of cardiolaminopathy. The LMNA gene undergoes alternative splicing to create A-type lamins, a part of the intermediate filament protein family. A-type lamins are located underneath the nuclear envelope, and given their direct interaction with chromatin, they serve as mechanosensory of the cell by interacting with the cytoskeleton and safeguarding the transcriptional program of cells. Nucleated cells in the cardiovascular system depend on precise mechanical cues for proper function and adaptation to stress. Mechanosensitive signaling pathways are essential in regulating mechanotransduction. They play a pivotal role in various molecular and cellular processes and commence numerous downstream effects, leading to transcriptional activation of target genes involved in proliferation, migration, and (anti-)apoptosis. Most pathways are known to be regulated by kinases, and this area remains largely understudied in cardiomyopathies. Heart failure is linked to disrupted mechanotransduction, where LMNA mutations affect nuclear integrity, impacting the response to extracellular matrix signals and the environment. The Hippo pathway, anchored by YAP1/WWTR1, emerges as a central player by orchestrating cellular responses to mechanical signals. However, the involvement of Hippo and YAP1/WWTR1 in cardiolaminopathy is unclear and likely mutation- and tissue-specific, warranting further investigation. Here, we highlight the involvement of multiple signaling pathways in mechanotransduction in cardiolaminopathy. We delve into (non-)canonical functions of key signaling components, which may hold critical clues for understanding disease pathogenesis. In summary, we comprehensively examine the mechanobiology of A-type lamins, the role of mechanosensitive signaling pathways, and their intricate interplay in the pathogenesis of cardiolaminopathy. A better understanding of these mechanisms is paramount for developing targeted therapies and interventions for individuals afflicted with this debilitating cardiac condition. Prior studies overlooked accurate gene nomenclature in protein and pathway names. Our review addresses this gap, ensuring precision by aligning names with correct gene nomenclature. Plain English Summary: Mutations in the A-type lamin gene (LMNA) can cause a laminopathy. A specific manifestation of this disease leads to cardiolaminopathy, a serious heart condition. The lamin network, located at the inner nuclear membrane, is a central player in transforming forces within cells. As cells move and function, they rely on the ability to sense and respond to these forces, a process named mechanosensing and -response. This review provides an overview of the key molecular pathways involved in the development of heart failure. The molecular mechanisms underlying LMNA cardiomyopathy are poorly understood because the interaction between the signaling pathways is challenging to elucidate. Deciphering these pathways is key to understanding the underlying mechanisms of disease and finding novel targets to alter the pathways and lessen the symptoms of diseases. [ABSTRACT FROM AUTHOR]

Periodontal defects' localization affects wound healing and bone remodeling, with faster healing in the upper jaw compared to the lower jaw. While differences in blood supply, innervation, and odontogenesis contribute, cell-intrinsic variances may exist. Few studies explored cell signaling in periodontal ligament stem cells (PDLSC), overlooking mandible-maxilla disparitiesUsing kinomics technology, we investigated molecular variances in PDLSC. Characterization involved stem cell surface markers, proliferation, and differentiation capacities. Kinase activity was analyzed via multiplex kinase profiling, mapping differential activity in known gene regulatory networks. Upstream kinase analysis identified stronger EphA receptor expression in the mandible, potentially inhibiting osteogenic differentiation. The PI3K-Akt pathway showed higher activity in lower-jaw PDLSC. PDLSC from the upper jaw exhibit superior proliferation and differentiation capabilities. Differential activation of gene regulatory pathways in upper vs. lower-jaw PDLSC suggests implications for regenerative therapies. [ABSTRACT FROM AUTHOR]

Phosphorylation proteomics is the basis for the study of abnormally activated kinase signaling pathways in breast cancer, which facilitates the discovery of new oncogenic agents and drives the discovery of potential targets for early diagnosis and therapy of breast cancer. In this study, we have explored the aberrantly active kinases in breast cancer development and to elucidate the role of PRKCD_pY313 in triple negative breast cancer (TNBC) progression. We collected 47 pairs of breast cancer and paired far-cancer normal tissues and analyzed phosphorylated tyrosine (pY) peptides by Superbinder resin and further enriched the phosphorylated serine/threonine (pS/pT) peptides using TiO2 columns. We mapped the kinases activity of different subtypes of breast cancer and identified PRKCD_pY313 was upregulated in TNBC cell lines. Gain-of-function assay revealed that PRKCD_pY313 facilitated the proliferation, enhanced invasion, accelerated metastasis, increased the mitochondrial membrane potential and reduced ROS level of TNBC cell lines, while Y313F mutation and low PRKCD_pY313 reversed these effects. Furthermore, PRKCD_pY313 significantly upregulated Src_pY419 and p38_pT180/pY182, while low PRKCD_pY313 and PRKCD_Y313F had opposite effects. Dasatinib significantly inhibited the growth of PRKCD_pY313 overexpression cells, and this effect could be enhanced by Adezmapimod. In nude mice xenograft model, PRKCD_pY313 significantly promoted tumor progression, accompanied by increased levels of Ki-67, Bcl-xl and Vimentin, and decreased levels of Bad, cleaved caspase 3 and ZO1, which was opposite to the trend of Y313F group. Collectively, the heterogeneity of phosphorylation exists in different molecular subtypes of breast cancer. PRKCD_pY313 activates Src and accelerates TNBC progression, which could be inhibited by Dasatinib. [ABSTRACT FROM AUTHOR]

Protein kinase CK2 (formerly known as casein kinase 2 or II), a ubiquitous and constitutively active enzyme, is widely recognized as one of the most pleiotropic serine/threonine kinases. It plays a critical role in numerous signaling pathways, with hundreds of bona fide substrates. However, despite considerable research efforts, our understanding of the entire CK2 substratome and its functional associations with the majority of these substrates is far from being completely deciphered. In this context, we aim to provide an overview of how CK2 recognizes its substrates. We will discuss the pros and cons of the existing methods to manipulate CK2 activity in cells, as well as exploring the dynamic response of substrate phosphorylation to CK2 modulation. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an essential phytohormone for plant responses to complex and variable environmental conditions. The molecular basis of the ABA signaling pathway has been well elucidated. SnRK2.2 and SnRK2.3 are key protein kinases participating in ABA responses, and the regulation of their activity plays an important role in signaling. Previous mass spectroscopy analysis of SnRK2.3 suggested that ubiquitin and homologous proteins may bind directly to the kinase. Ubiquitin typically recruits E3 ubiquitin ligase complexes to target proteins, marking them for degradation by the 26S proteasome. Here, we show that SnRK2.2 and SnRK2.3 interact with ubiquitin but are not covalently attached to the protein, resulting in the suppression of their kinase activity. The binding between SnRK2.2, SnRK2.3, and ubiquitin is weakened under prolonged ABA treatment. Overexpression of ubiquitin positively regulated the growth of seedlings exposed to ABA. Our results thus demonstrate a novel function for ubiquitin, which negatively regulates ABA responses by directly inhibiting SnRK2.2 and SnRK2.3 kinase activity. [ABSTRACT FROM AUTHOR]

Summary: MAPKs are universal eukaryotic signaling factors whose functioning is assumed to depend on the recognition of a common docking motif (CD) by its activators, substrates, and inactivators.We studied the role of the CD domain of Arabidopsis MPK4 by performing interaction studies and determining the ligand‐bound MPK4 crystal structure.We revealed that the CD domain of MPK4 is essential for interaction and activation by its upstream MAPKKs MKK1, MKK2, and MKK6. Cys181 in the CD site of MPK4 was shown to become sulfenylated in response to reactive oxygen species in vitro. To test the function of C181 in vivo, we generated wild‐type (WT) MPK4‐C181, nonsulfenylatable MPK4‐C181S, and potentially sulfenylation mimicking MPK4‐C181D lines in the mpk4 knockout background. We analyzed the phenotypes in growth, development, and stress responses, revealing that MPK4‐C181S has WT activity and complements the mpk4 phenotype. By contrast, MPK4‐C181D cannot be activated by upstream MAPKK and cannot complement the phenotypes of mpk4.Our findings show that the CD motif is essential and is required for activation by upstream MAPKK for MPK4 function. Furthermore, growth, development, or immunity functions require upstream activation of the MPK4 protein kinase. [ABSTRACT FROM AUTHOR]

Summary: Fms‐like tyrosine kinase 3 (FLT3) is frequently mutated in haematological malignancies. Although canonical FLT3 mutations including internal tandem duplications (ITDs) and tyrosine kinase domains (TKDs) have been extensively studied, little is known about the clinical significance of non‐canonical FLT3 mutations. Here, we first profiled the spectrum of FLT3 mutations in 869 consecutively newly diagnosed acute myeloid leukaemia (AML), myelodysplastic syndrome and acute lymphoblastic leukaemia patients. Our results showed four types of non‐canonical FLT3 mutations depending on the affected protein structure: namely non‐canonical point mutations (NCPMs) (19.2%), deletion (0.7%), frameshift (0.8%) and ITD outside the juxtamembrane domain (JMD) and TKD1 regions (0.5%). Furthermore, we found that the survival of patients with high‐frequency (>1%) FLT3‐NCPM in AML was comparable to those with canonical TKD. In vitro studies using seven representative FLT3‐deletion or frameshift mutant constructs showed that the deletion mutants of TKD1 and the FLT3‐ITD mutant of TKD2 had significantly higher kinase activity than wild‐type FLT3, whereas the deletion mutants of JMD had phosphorylation levels comparable with wild‐type FLT3. All tested deletion mutations and ITD were sensitive to AC220 and sorafenib. Collectively, these data enrich our understanding of FLT3 non‐canonical mutations in haematological malignancies. Our results may also facilitate prognostic stratification and targeted therapy of AML with FLT3 non‐canonical mutations. [ABSTRACT FROM AUTHOR]

The article discusses the ALPK3 gene and its potential role in cardiomyopathies. While there are conflicting findings regarding ALPK3's kinase activity, recent studies suggest that ALPK3 functions as a pseudokinase rather than a true kinase. The article presents evidence from in vitro and in vivo experiments, including an in vitro phosphorylation assay and the generation of knock-in mice with a mutated ALPK3 gene. These findings suggest that ALPK3 variants associated with cardiomyopathy may affect other aspects of the protein rather than its kinase activity. [Extracted from the article]

Background: Ataxia-telangiectasia (A-T) is a progressive multisystemic neurodegenerative disease. The phenotypic spectrum includes conditions (variant A-T) with mild, late-onset, and atypical clinical presentations characterized by the prevalence of dyskinetic rather than ataxic features. Cases: We describe the clinical presentations of 3 siblings with early-onset truncal ataxia without obvious neurological deterioration or biological markers of classic A-T phenotype. We performed functional and genetic evaluation of 3 siblings with very mild neurological phenotype. Genetic evaluation with a next-generation sequencing panel for genes causative of cerebellar ataxia detected 2 known ATM gene variants, missense c.9023G>A p.(Arg3008His), and leaky splicing c.1066-6T>G variants. Functional studies showed mildly reduced ATM expression and residual kinase activity in the probands compared with healthy controls. Conclusions: These results suggest the importance of investigating ATM variants even in the presence of clinical and biological atypical cases to ensure specific therapeutic regimens and oncological surveillance in these patients. [ABSTRACT FROM AUTHOR]

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97–cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97–cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97–cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97–cyclin interaction. High selective pressure on the formation of pUL97–cyclin complexes was identified by the use of clinically relevant mutants. [ABSTRACT FROM AUTHOR]

Simple Summary: Colorectal cancer (CRC) is the most common gastrointestinal tract malignancy. Previous reports have shown that cancerous phenotypes in the intestine are dependent on c-MYC target gene expression. Unfortunately, finding c-MYC inhibitors has proven difficult because c-MYC does not have a deep surface-binding pocket. Considering that c-MYC is maintained upregulated through β-catenin-mediated transcriptional activation and ERK-mediated post-translational stabilization, and since we have previously demonstrated that c-MYC transcriptional activation is affected by p38α as a β-catenin chromatin-associated kinase, here, we investigated p38α's involvement in c-MYC protein stabilization in CRC. Interestingly, we found that p38α sustains c-MYC's stability by preventing its ubiquitination and proteasomal degradation. Moreover, we showed that p38α inhibitors exhibit a synthetic lethality effect when used in combination with MEK inhibitors in CRC cells. Our findings identify p38α as a promising therapeutic target that acts on the pharmacologically "undruggable" c-MYC protein, with implications for countering c-MYC-mediated CRC proliferation, metastasization, and chemoresistance. c-MYC is one of the most important factors involved in colorectal cancer (CRC) initiation and progression; indeed, it is found to be upregulated in up to 80% of sporadic cases. During colorectal carcinogenesis, c-MYC is maintained upregulated through β-catenin-mediated transcriptional activation and ERK-mediated post-translational stabilization. Our data demonstrate that p38α, a kinase involved in CRC metabolism and survival, contributes to c-Myc protein stability. Moreover, we show that p38α, like ERK, stabilizes c-MYC protein levels by preventing its ubiquitination. Of note, we found that p38α phosphorylates c-MYC and interacts with it both in vitro and in cellulo. Extensive molecular analyses in the cellular and in vivo models revealed that the p38α kinase inhibitors, SB202190 and ralimetinib, affect c-MYC protein levels. Ralimetinib also exhibited a synthetic lethality effect when used in combination with the MEK1 inhibitor trametinib. Overall, our findings identify p38α as a promising therapeutic target, acting directly on c-MYC, with potential implications for countering c-MYC-mediated CRC proliferation, metastatic dissemination, and chemoresistance. [ABSTRACT FROM AUTHOR]

The dynamic interface between invading viral pathogens and programmed cell death (PCD) of the host is a finely regulated process. Host cellular demise at the end of the viral life cycle ensures the release of progeny virions to initiate new infection cycles. Rotavirus (RV), a diarrheagenic virus with double-stranded RNA genome, has been reported to trigger different types of PCD such as apoptosis and pyroptosis in a highly regulated way to successfully disseminate progeny virions. Recently our lab also showed that induction of MLKL-driven programmed necroptosis by RV. However, the host cellular machinery involved in RV-induced necroptosis and the upstream viral trigger responsible for it remained unaddressed. In the present study, the signalling upstream of MLKL-driven necroptosis has been delineated where the involvement of Receptor interacting serine/threonine kinase 3 (RIPK3) and 1 (RIPK1) from the host side and RV non-structural protein 4 (NSP4) as the viral trigger for necroptosis has been shown. Interestingly, RV-NSP4 was found to be an integral component of the necrosome complex by interacting with RIPK1, thereby bypassing the requirement of RIPK1 kinase activity. Subsequently, NSP4-driven elevated cytosolic Ca2+ concentration and Ca2+-binding to NSP4 lead further to RHIM domain-dependent RIPK1-RIPK3 interaction, RIPK3-dependent MLKL phosphorylation, and eventual necroptosis. Overall, this study presents the interplay between RV-NSP4 and the host cellular necrosome complex to induce necroptotic death of host cells. [Display omitted] • RV harnesses the kinase-dependent function of RIPK3 and kinase-independent function of RIPK1 to promote necroptosis. • RV non-structural protein 4 is the major viral modulator of necroptosis. • RV-NSP4 associates with RIPK1 which further assembles into the necrosome containing RIPK1-RIPK3-MLKL leading to necroptosis. • RV-NSP4 driven ER-to-cytosol calcium efflux facilitates necroptosis by fostering the integrity of the necrosome complex. [ABSTRACT FROM AUTHOR]

The eukaryotic cell cycle is driven by the activity of cyclin-dependent kinases (CDKs). CDK activity rises over 50-fold during the cell cycle, from a low level in G1 to a high level in mitosis. However, it is not known whether the entire range of CDK activity is necessary for cell cycle progression, or whether cells can tolerate a reduction in CDK activity level. Here, in fission yeast, we show that sublethal CDK inhibition lengthens the time cells spend in mitosis but does not cause misordering of mitotic events. Maximum attainable CDK activity exceeds the amount necessary for mitosis, and thus forms a CDK activity buffer between sufficient and maximal possible CDK activities. This CDK activity buffer is needed for mitotic completion when CDK activity is compromised, and CDK inhibition only becomes lethal to cells when this buffer is exhausted. Finally, we explore what factors influence this CDK activity buffer, and find that it is influenced by CDK-counteracting phosphatases. Therefore, maximum attainable CDK activity is not necessary for mitosis but provides robustness to CDK activity reduction to ensure mitotic completion. [ABSTRACT FROM AUTHOR]

Protein kinases (PKs) are established gameplayers in biological signalling pathways, and a large body of evidence points to their dysregulation in diseases, in particular cancer, where rewiring of PK networks occurs frequently. Fluorescent biosensors constitute attractive tools for probing biomolecules and monitoring dynamic processes in complex samples. A wide variety of genetically encoded and synthetic biosensors have been tailored to report on PK activities over the last decade, enabling interrogation of their function and insight into their behaviour in physiopathological settings. These optical tools can further be used to highlight enzymatic alterations associated with the disease, thereby providing precious functional information which cannot be obtained through conventional genetic, transcriptomic or proteomic approaches. This review focuses on fluorescent peptide biosensors, recent developments and strategies that make them attractive tools to profile PK activities for biomedical and diagnostic purposes, as well as insights into the challenges and opportunities brought by this unique toolbox of chemical probes. [ABSTRACT FROM AUTHOR]

Introduction: The signalling cascades that contribute to lupus pathogenesis are incompletely understood. We address this by using an unbiased activity‐based kinome screen of murine lupus. Methods: An unbiased activity‐based kinome screen (ABKS) of 196 kinases was applied to two genetically different murine lupus strains. Systemic and renal lupus were evaluated following in vivo PLK1blockade. The upstream regulators and downstream targets of PLK1 were also interrogated. Results: Multiple signalling cascades were noted to be more active in murine lupus spleens, including PLK1. In vivo administration of a PLK1‐specific inhibitor ameliorated splenomegaly, anti‐dsDNA antibody production, proteinuria, BUN and renal pathology in MRL.lpr mice (P < 0.05). Serum IL‐6, IL‐17 and kidney injury molecule 1 (KIM‐1) were significantly decreased after PLK1 inhibition. PLK1 inhibition reduced germinal centre and marginal zone B cells in the spleen, but changes in T cells were not significant. In vitro, splenocytes were treated with anti‐mouse CD40 Ab or F(ab')2 fragment anti‐mouse IgM. After 24‐h stimulation, IL‐6 secretion was significantly reduced upon PLK1 blockade, whereas IL‐10 production was significantly increased. The phosphorylation of mTOR was assessed in splenocyte subsets, which revealed a significant change in myeloid cells. PLK1 blockade reduced phosphorylation associated with mTOR signalling, while Aurora‐A emerged as a potential upstream regulator of PLK1. Conclusion: The Aurora‐A → PLK1 → mTOR signalling axis may be central in lupus pathogenesis, and emerges as a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Despite the successes of immunotherapy in cancer treatment over recent decades, less than <10%–20% cancer cases have demonstrated durable responses from immune checkpoint blockade. To enhance the efficacy of immunotherapies, combination therapies suppressing multiple immune evasion mechanisms are increasingly contemplated. To better understand immune cell surveillance and diverse immune evasion responses in tumor tissues, we comprehensively characterized the immune landscape of more than 1,000 tumors across ten different cancers using CPTAC pan-cancer proteogenomic data. We identified seven distinct immune subtypes based on integrative learning of cell type compositions and pathway activities. We then thoroughly categorized unique genomic, epigenetic, transcriptomic, and proteomic changes associated with each subtype. Further leveraging the deep phosphoproteomic data, we studied kinase activities in different immune subtypes, which revealed potential subtype-specific therapeutic targets. Insights from this work will facilitate the development of future immunotherapy strategies and enhance precision targeting with existing agents. [Display omitted] • Proteogenomics reveals seven immune subtypes spanning 10 cancer types • DNA alterations associate with immune subtypes and affect proteomic profiles • Kinase activation in immune subtypes suggests potential therapeutic targets • Digital pathology reveals infiltrating cells associated with immune subtypes Immunotherapy holds strong promise for cancer treatment but at present benefits only a small proportion of cases. A pan-cancer analysis of the immune landscape in more than 1,000 tumors across ten cancer types reveals immune surveillance and immune evasion mechanisms as well as potential molecular target that could augment future immunotherapy and precision medicine strategies. [ABSTRACT FROM AUTHOR]

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P2 1 2 1 2 1 with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = β = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic R factor and R free values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8–9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies. • Structure elucidation of Vibrio Cholera Nucleoside Diphosphate Kinase (VNDK). • VNDK shows highest affinity for GTP amongst NTPs. • VNDK possesses kinase and DNase activities. • VNDK is maximally stable between pH 8 and 9, and exhibits elevated thermolability. [ABSTRACT FROM AUTHOR]

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca21 imaging and patchclamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons. [ABSTRACT FROM AUTHOR]

CK2 is a constitutively active protein kinase that assuring a constant level of phosphorylation to its numerous substrates supports many of the most important biological functions. Nevertheless, its activity has to be controlled and adjusted in order to cope with the varying needs of a cell, and several examples of a fine-tune regulation of its activity have been described. More importantly, aberrant regulation of this enzyme may have pathological consequences, e.g. in cancer, chronic inflammation, neurodegeneration, and viral infection. Our review aims at summarizing our current knowledge about CK2 regulation. In the first part, we have considered the most important stimuli shown to affect protein kinase CK2 activity/expression. In the second part, we focus on the molecular mechanisms by which CK2 can be regulated, discussing controversial aspects and future perspectives. [ABSTRACT FROM AUTHOR]

Background: Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance.Methods: We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm  with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4-11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors.Results: Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4-11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4-11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks.Conclusions: We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples. [ABSTRACT FROM AUTHOR]

Nucleophosmin (NPM1) mutations are the most frequent genetic alteration in acute myeloid leukemia (AML) and aberrant cytoplasm‐dislocated NPM1 mutant is a distinct biological characterization of this disease. Our group previously reported that NPM1 mutant elevated autophagy activity and autophagy activation contributed to leukemic cell survival. However, the molecular mechanisms by which cytoplasmic NPM1 mutant involving in the autophagy pathway has not been fully elucidated. Here, we showed that Unc‐51‐like kinase 1 (ULK1) as a core autophagy protein was highly expressed in NPM1‐mA positive OCI‐AML3 cells and primary NPM1‐mutated AML blasts. Meanwhile, we found that NPM1‐mA could interact with ULK1 protein and positively regulated ULK1 protein levels. Mechanically, NPM1‐mA promoted TRAF6‐dependent K63 ubiquitination and further maintained ULK1 stability and kinase activity via miR‐146a. In addition, ULK1 high expression‐mediated autophagy activation and facilitated to leukemic cell proliferation. Finally, we demonstrated that restoring ULK1 expression, ULK1 inhibitor SBI‐0206965 treatment and using shULK1 partially rescued the effect of NPM1‐mA on autophagy and cell survival. In conclusion, our findings suggest that NPM1 mutant interacts with ULK1, and thus, maintains its protein stability, which is required for NPM1 mutant‐mediated autophagic cell survival. These data extend our understanding of the functions of NPM1 mutant in the regulation of autophagy activation in NPM1‐mutated AML. [ABSTRACT FROM AUTHOR]

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

• LIPUS can directly impact the intracellular dynamics of macromolecules. • The diffusivities of cytoplasmic macromolecules increase during LIPUS exposure. • LIPUS stimulation accelerates passive nucleocytoplasmic transport. • The ERK activity can be amplified by its increased diffusivity under LIPUIS exposure. Whilst a number of studies have demonstrated that low-intensity pulsed ultrasound (LIPUS) is a promising therapeutic ultrasound technique that can be used for delivering mild mechanical stimuli to target tissue non-invasively, the underlying biophysical mechanisms still remain unclear. Most mechanism studies have focused explicitly on the effects of LIPUS on the cell membrane and mechanosensitive receptors. In the present study, we propose an additional mechanism by which LIPUS propagation through living cells may directly impact intracellular dynamics, particularly the diffusion transport of biomolecules. To support our hypothesis, human epithelial-like cells (SaOS-2 and HeLa) seeded on a confocal dish placed on a microscope stage were exposed to LIPUS with various exposure conditions (ultrasound frequencies of 0.5, 1 and 3 MHz, peak acoustic pressure of 200 and 400 kPa, a pulse repetition frequency of 1 kHz and a 20 % duty cycle), and the diffusivities of various sizes of biomolecules in the cytoplasm area were measured using fluorescence recovery after photobleaching (FRAP). Furthermore, giant unilamellar vesicles (GUVs) filled with macromolecules were used to examine the physical causal relationship between LIPUS and molecular diffusion changes. Nucleocytoplasmic transport coefficients were also measured by modified FRAP that bleaches the whole cell nuclear region. Extracellular signal-regulated kinases (ERK) activity (the phosphorylation dynamics) was monitored using fluorescence resonance energy transfer (FRET) microscopy. All the measurements were taken during, before and after the LIPUS exposure. Our experimental results clearly showed that the diffusion coefficients of macromolecules within the cell increased with acoustic pressure by 12.1 to 33.5 % during the sonication, and the increments were proportional to their molecular sizes regardless of the ultrasound frequency used. This observation in living cells was consistent with the GUVs exposed to the LIPUS, which indicated that the diffusivity increase was a passive physical response to the acoustic energy of LIPUS. Under the 1 MHz LIPUS exposure with 400 kPa, the passive nucleocytoplasmic transport of enhanced green fluorescent protein (EGFP) was accelerated by 21.4 %. With the same LIPUS exposure condition, both the diffusivity and phosphorylation of ERK induced by EGF treatment were significantly elevated simultaneously, which implied that LIPUS could also modify the kinase kinetics in the signal transduction process. Taken together, this study is the first attempt to uncover the physical link between LIPUS and the dynamics of intracellular macromolecules and related biological processes that LIPUS can possibly increase the diffusivity of intracellular macromolecules, leading to the changes in the basic cellular processes: passive nucleocytoplasmic transport and ERK. Our findings can provide a novel perspective that the mechanotransduction process that the intracellular region, in addition to the cell membrane, can convert the acoustic stimuli of LIPUS to biochemical signals. [ABSTRACT FROM AUTHOR]

[Display omitted] • SASH1 is identified as a novel binding partner for Eph receptors through SAM-SAM interactions, with the highest affinity for EphA8. • The high-resolution crystal structure of the EphA8/SASH1 SAM-SAM complex reveals molecular basis governing the specific inter-molecular interactions. • Cancer mutations (EphA8 R942H or G978D) impair the interaction between EphA8 and SASH1, highlighting potential implications in tumorigenesis. • SASH1 enhances EphA8 receptor kinase activation through their SAM-SAM interaction. The Eph (erythropoietin-producing human hepatocellular) receptor family, the largest subclass of receptor tyrosine kinases (RTKs), plays essential roles in embryonic development and neurogenesis. The intracellular Sterile Alpha Motif (SAM) domain presents a critical structural feature that distinguishes Eph receptors from other RTKs and participates in recruiting and binding downstream molecules. This study identified SASH1 (SAM and SH3 domain containing 1) as a novel Eph receptor-binding partner through SAM-SAM domain interactions. Our comprehensive biochemical analyses revealed that SASH1 selectively interacts with Eph receptors via its SAM1 domain, displaying the highest affinity for EphA8. The high-resolution crystal structure of the EphA8-SASH1 complex provided insights into the specific intermolecular interactions between these proteins. Cellular assays confirmed that EphA8 and SASH1 co-localize and co-precipitate in mammalian cells, with cancer mutations (EphA8 R942H or G978D) impairing this interaction. We demonstrated that SAM-SAM interaction is critical for SASH1-mediated regulation of EphA8 kinase activity, shedding new light on the Eph signaling pathway and expanding our understanding of the molecular basis of the tumor suppressor gene SASH1. [ABSTRACT FROM AUTHOR]

Src family kinases (SFKs) are non-receptor tyrosine kinases and are involved in various cellular functions (proliferation, differentiation, migration, survival and invasion) by regulating downstream pathways. Considerable evidence suggests that co- and post-translational modifications are highly related to the activation of SFKs and their downstream signals. How SFKs are activated and how their subsequent cascades were regulated has been reviewed in previous reports. However, the contribution of co- and post-translational modification to SFKs activation has not been fully elucidated. This review focuses on the effect of these modifications on SFKs activity according to structural and biochemical studies and uncovers the significance of co-and post-translational modifications in the regulation of SFKs activity. [ABSTRACT FROM AUTHOR]

Protein phosphorylation is a key mechanism that regulates cellular physiological functions such as proliferation, migration, cell cycle progression, apoptosis, and differentiation. Aberrations in kinase activity and subsequent dysregulation of protein phosphorylation occur in the process of carcinogenesis and cancer progression and are considered to be therapeutic targets and biomarkers in oncology. Gel electrophoresis has versatile utility in the study of protein phosphorylation. Phosphorylated proteins can be enriched prior to gel electrophoresis, and the proteins separated by gel electrophoresis are visualized by colorimetric methods or western blotting with antibodies, which specifically detect phosphorylation. Phosphorylated proteins migrate differently from non-phosphorylated proteins in gels containing substrates with affinity for phosphorylation. All these methods can be combined in multiple ways, generating unique data from different viewpoints. The identification of separated proteins can be achieved by mass spectrometry, making it possible to integrate protein and genetic data. Peptide array allow the evaluation of kinase activity in heterogeneous samples. As protein phosphorylation and kinase activity are under the regulation of multiple mechanisms and their statuses influence the structures and functions of the other proteins, a multidisciplinary approach is required, and gel electrophoresis will play an important role in the study of protein phosphorylation. [ABSTRACT FROM AUTHOR]

Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε. [ABSTRACT FROM AUTHOR]

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

A fluorometric strategy for protein kinase A (PKA) activity detection was proposed based on the recognition of phosphate and guanidine groups by applying gold nanoclusters (AuNCs) functionalized by peptide as the fluorescent probe. • A fluorometric strategy for PKA activity sensing was constructed based on specific phosphate-guanidine interaction. • Specially designed peptides were applied to modify AuNPs and AuNCs, respectively. • The recognition of phosphate and guanidine groups triggered the FRET process. • The inhibition effect of H-89 on PKA activity was also investigated. • The analysis of PKA activity in complicated biologic sample was demonstrated in HepG2 cell lysates. In this paper, a fluorometric strategy for protein kinase A (PKA) activity detection was proposed based on the recognition of phosphate and guanidine groups. Two artificial substrate peptides (T1 and T2) were specially designed, T1, containing PKA specific recognition domain, was self-assembled on gold nanoclusters (T1-AuNCs). T2, possessing guanidine groups at arginine residues for the interaction with phosphorylation site, was used for modifying gold nanoparticles (T2-AuNPs). In the presence of adenosine triphosphate (ATP) and PKA, T1 was phosphorylated and then the phosphorylated T1 specially combined with T2 via phosphate-guanidine recognition. So, T1-AuNCs and T2-AuNPs were brought in close distance and fluorescence resonance energy transfer (FRET) process from T1-AuNCs to T2-AuNPs was triggered to result in the fluorescence quenching of T1-AuNCs. Accordingly, PKA activity was efficiently determined depending on the fluorescent signal changes within a good linearity from 0.04 to 8.0 U mL−1 with the detection limit down to 0.013 U mL−1. Moreover, this sensing platform was used to investigate the inhibition effect of PKA inhibitor (H-89) with good performance. Also, the practical application of this strategy for analysis of PKA activity in complicated biologic sample was demonstrated in HepG2 cell lysates. [ABSTRACT FROM AUTHOR]

Mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene have been implicated in the pathogenesis of Parkinson's disease (PD). Identification of PD-associated LRRK2 mutations has led to the development of novel animal models, primarily in mice. However, the characteristics of human LRRK2 and mouse Lrrk2 protein have not previously been directly compared. Here we show that proteins from different species have different biochemical properties, with the mouse protein being more stable but having significantly lower kinase activity compared to the human orthologue. In examining the effects of PD-associated mutations and risk factors on protein function, we found that conserved substitutions such as G2019S affect human and mouse LRRK2 proteins similarly, but variation around position 2385, which is not fully conserved between humans and mice, induces divergent in vitro behavior. Overall our results indicate that structural differences between human and mouse LRRK2 are likely responsible for the different properties we have observed for these two species of LRRK2 protein. These results have implications for disease modelling of LRRK2 mutations in mice and on the testing of pharmacological therapies in animals. [ABSTRACT FROM AUTHOR]

Cyclin-dependent kinase 1 (CDK1) is essential for cell-cycle progression. While dependence of CDK activity on cyclin levels is well established, molecular mechanisms that regulate their binding are less understood. Here, we report for the first time that CDK1:cyclin-B binding is not default but rather determined by the evolutionarily conserved catalytic residue, lysine-33 in CDK1. We demonstrate that the charge state of this lysine allosterically remodels the CDK1:cyclin-B interface. Cell cycle-dependent acetylation of lysine-33 or its mutation to glutamine, which mimics acetylation, abrogates cyclin-B binding. Using biochemical approaches and atomistic molecular dynamics simulations, we have uncovered both short-range and long-range effects of perturbing the charged state of the catalytic lysine, which lead to inhibition of kinase activity. Specifically, although loss of the charge state of catalytic lysine did not impact ATP binding significantly, it altered its orientation in the active site. In addition, the catalytic lysine also acts as an intra-molecular electrostatic tether at the active site to orient structural elements interfacing with cyclin-B. Physiologically, opposing activities of SIRT1 and P300 regulate acetylation and thus control the charge state of lysine-33. Importantly, cells expressing acetylation mimic mutant of Cdc2/CDK1 in yeast are arrested in G2 and fail to divide, indicating the requirement of the deacetylated state of the catalytic lysine for cell division. Thus, by illustrating the molecular role of the catalytic lysine and cell cycle-dependent deacetylation as a determinant of CDK1:cyclin-B interaction, our results redefine the current model of CDK1 activation and cell-cycle progression. Unlabelled Image • If and how CDK1:Cyclin-B interaction is regulated is still unknown. • CDK1 acetylation at its catalytic lysine-33 is regulated by SIRT1 and P300. • Deacetylated lysine-33 allosterically remodels the CDK1:cyclin-B interface. • Charge state of CDK1 catalytic lysine-33 is essential for CDK1:cyclin-B binding. • Acetyl-mimic mutation of Cdc2/CDK1 inhibits G2-M transition and affects viability. [ABSTRACT FROM AUTHOR]

Summary: In Arabidopsis, embryonic development follows a stereotypical pattern of cell division. Although many factors have been reported to participate in establishment of the proper embryonic pattern, the molecular mechanisms underlying pattern formation are unclear. In this study we showed that RAF22 and RAF28, two RAF‐like mitogen‐activated protein kinase kinase kinases (MAPKKKs) in Arabidopsis, are involved in the regulation of embryogenesis. The double knockout mutant of RAF22 and RAF28 was embryo lethal. A large proportion of the raf22−/−raf28+/− mutant embryos exhibited various defects, including disordered proembryo cell divisions, disruption of the bilaterally symmetrical structure, abnormally formative divisions of hypophysis and exaggerated suspensor growth. Whereas the kinase active form of RAF22 could complement these embryonic aberrant phenotypes, the kinase inactive form could not. The restrictive expression of the basal cell fate marker WOX8 in the abnormally dividing suspensor cells and the apical cell linage marker WOX2 in the abnormal proembryos indicated that apical and basal cell fates were unchanged in the abnormal embryos. The polar distribution of the auxin maxima and the PIN1 and PIN7 auxin transporters was markedly altered in the abnormal embryos. Our results suggest that RAF22 and RAF28 are important components of embryogenesis and that auxin polar transport may be involved in this regulation. Significance Statement: Embryogenesis in Arabidopsis is strictly regulated by several factors, including polar auxin transport; however, not all underlying mechanisms have been fully described. In this study, we report that the kinase activity of two RAF‐like mitogen‐activated protein kinase kinase kinases, i.e. RAF22 and RAF28, is required for normal auxin distribution through the polar localization of the PIN1 and PIN7 auxin transporters, and that RAF22 and RAF28 are essential regulators of auxin transport involved in plant embryogenesis. [ABSTRACT FROM AUTHOR]

In this work, a novel and sensitive electrogenerated chemiluminescence (ECL) biosensor for protein kinase A (PKA) activity analysis and relevant inhibitor screening was proposed based on bimetallic catalysis signal amplification and recognition of Au and Pt nanoparticles loaded metal-organic frameworks (Au&Pt@UiO-66) nanocomposite. After being phosphorylated by PKA in the presence of ATP, Au&Pt@UiO-66 probes were specifically chelated to the modified electrode by forming Zr-O-P bonds between the surface defects of UiO-66 and the phosphorylated kemptide. Due to the high synergistic catalysis of Au&Pt@UiO-66 nanocomposites to the luminol-H 2 O 2 reaction, the ECL signal of luminol was greatly enhanced. Moreover, UiO-66 afford numerous Zr defect sites for high efficient phosphate group recognition, and can also prevent the nanoparticles from aggregating during catalytic reactions. Thus, the excellent performance of the ECL biosensor with high sensitivity and superior stability was obtained. Under the optimized conditions, the detection limit for PKA activity was 0.009 UmL −1 (S/N = 3). Meanwhile, the ECL biosensor was successfully applied in inhibitor screening and cell lysates PKA activity analysis, showing great promise in kinase related research. [ABSTRACT FROM AUTHOR]

Background: PINK1 (PTEN-induced putative kinase 1) gene is the causal gene for recessive familial type 6 of Parkinson's disease (PARK6), which is an early-onset autosomal recessive inherited neurodegenerative disease. PINK1 has been reported to exert both autophosphorylation and phosphorylation activity, affecting cell damage under stress and other physiological responses. However, there has been no report on the identification of PINK1 autophosphorylation sites and their physiological functions. Methods: (1) We adopted mass spectrometry assay to identify the autophosphorylation site of PINK1, andautoradiography assay was further conducted to confirm this result. (2) Kinase activity assay was used to compare the kinase activity of both Ser465 mutant PINK1 and disease-causing mutant PINK1. (3) We use Pulse-chase analysis to measure whether Ser465 may affect PINK1 degradation. (4) Immunocytochemistry staining was used to study the PINK1 subcellular localization and Parkin transition in subcellular level. Result: In our study, we identified the 465th serine residue (Ser465) as one of the autophosphorylation sites in PINK1 protein. The inactivation of Ser465 can decrease the kinase activity of PINK1. Either dissipated or excessive Ser465 site phosphorylation of PINK1 can slow down its degradation. PINK1 autophosphorylation contributes to the transit of Parkin to mitochondria, and has no effect on its subcellular localization. PARK6 causal mutations, T313 M and R492X, display the same characteristics as Ser465A mutation PINK1 protein, such as decreasing PINK1 kinase activity and affecting its interaction with Parkin. Conclusion: Ser465 was identified as one of the autophosphorylation sites of PINK1, which affected PINK1 kinase activity. In addition, Ser465 is involved in the degradation of PINK1 and the transit of Parkin to mitochondria. T313 M and R492X, two novel PARK6 mutations on Thr313 and Arg492, were similar to Ser465 mutation, including decreasing PINK1 phosphorylation activity and Parkin subcellular localization. [ABSTRACT FROM AUTHOR]

Background: Circadian clocks are found in organisms of almost all domains including photosynthetic Cyanobacteria, whereby large diversity exists within the protein components involved. In the model cyanobacterium Synechococcus elongatus PCC 7942 circadian rhythms are driven by a unique KaiABC protein clock, which is embedded in a network of input and output factors. Homologous proteins to the KaiABC clock have been observed in Bacteria and Archaea, where evidence for circadian behavior in these domains is accumulating. However, interaction and function of non-cyanobacterial Kai-proteins as well as homologous input and output components remain mainly unclear. Results: Using a universal BLAST analyses, we identified putative KaiC-based timing systems in organisms outside as well as variations within Cyanobacteria. A systematic analyses of publicly available microarray data elucidated interesting variations in circadian gene expression between different cyanobacterial strains, which might be correlated to the diversity of genome encoded clock components. Based on statistical analyses of co-occurrences of the clock components homologous to Synechococcus elongatus PCC 7942, we propose putative networks of reduced and fully functional clock systems. Further, we studied KaiC sequence conservation to determine functionally important regions of diverged KaiC homologs. Biochemical characterization of exemplary cyanobacterial KaiC proteins as well as homologs from two thermophilic Archaea demonstrated that kinase activity is always present. However, a KaiA-mediated phosphorylation is only detectable in KaiC1 orthologs. Conclusion: Our analysis of 11,264 genomes clearly demonstrates that components of the Synechococcus elongatus PCC 7942 circadian clock are present in Bacteria and Archaea. However, all components are less abundant in other organisms than Cyanobacteria and KaiA, Pex, LdpA, and CdpA are only present in the latter. Thus, only reduced KaiBC-based or even simpler, solely KaiC-based timing systems might exist outside of the cyanobacterial phylum, which might be capable of driving diurnal oscillations. [ABSTRACT FROM AUTHOR]

We prove an averaging principle which asserts convergence of diffusion processes on domains separated by semi-permeable membranes, when diffusion coefficients tend to infinity while the flux through the membranes remains constant. In the limit, points in each domain are lumped into a single state of a limit Markov chain. The limit chain's intensities are proportional to the membranes' permeability and inversely proportional to the domains' sizes. Analytically, the limit is an example of a singular perturbation in which boundary and transmission conditions play a crucial role. This averaging principle is strongly motivated by recent signaling pathways models of mathematical biology, which are discussed toward the end of the paper. [ABSTRACT FROM AUTHOR]

The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinasedriven decision-making. [ABSTRACT FROM AUTHOR]

Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16 INK4a -Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16 INK4a leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma. [ABSTRACT FROM AUTHOR]

Conformational changes associated to sensing mechanisms of heme-based protein sensors are a key molecular event that seems to modulate not only the protein activity but also the potential of the Fe III/II redox couple of the heme domain. In this work, midpoint potentials ( E m ) assigned to the Fe III/II redox couple of the heme domain of FixL from Rhizobium etli ( Re FixL) in the unliganded and liganded states were determined by spectroelectrochemistry in the presence of inorganic mediators. In comparison to the unliganded Re FixL protein (+ 19 mV), the binding to ligands that switch off the kinase activity induces a negative shift, i. e. E m = − 51, − 57 and − 156 mV for O 2 , imidazole and CN − , respectively. Upon binding to CO, which does not affect the kinase active, E m was observed at + 21 mV. The potential values observed for Fe III/II of the heme domain of Re FixL upon binding to CO and O 2 do not follow the expected trend based on thermodynamics, assuming that positive potential shift would be expected for ligands that bind to and therefore stabilize the Fe II state. Our results suggest that the conformational changes that switch off kinase activity upon O 2 binding have knock-on effects to the local environment of the heme, such as solvent rearrangement, destabilize the Fe II state and counterbalances the Fe II -stabilizing influence of the O 2 ligand. [ABSTRACT FROM AUTHOR]

Background: The chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined. Methods: Primary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca2+ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK. Results: Here, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca2+ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca2+ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12. Conclusions: Our findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca2+ channels upstream of p38 MAPK activation. [ABSTRACT FROM AUTHOR]

Background: The use of targeted agents to impel dual inhibition of anti-apoptotic mechanisms and mTOR-mediated pro-survival signaling in colorectal carcinoma (CRC) cell lines with KRAS or BRAF mutation has been shown to induce apoptosis, a timely result given CRC entities harboring such mutations are in need of new therapies. Since CRC comprises heterogeneous tumors with predominant hypoxic components, we investigated effects of an inhibitor of anti-apoptotic Bcl-2 family proteins (ABT-737) in combination with an mTOR inhibitor (AZD8055)-collectively referred to as combo-Rx, in hypoxic CRC cell lines.Methods: Cell viability measures, expression of proteins implicated in apoptosis and MAPK/PI3K-AKT/mTOR pathway signaling, and profiling of composite kinase activities were undertaken in a panel of 14 cell lines.Results: In hypoxic conditions, combo-Rx suppressed viability of 13 of the cell lines, albeit ABT-737 did not significantly potentiate the inhibitory effect of single-agent AZD8055 in six of the models. Hypoxic KRAS/PIK3CA-mutant HCT-116 and HCT-15 cell lines (both with low endogenous expression of the anti-apoptotic Mcl-1 protein and showing augmented inhibition of viability following the addition of ABT-737 to AZD8055) responded to combo-Rx by induction of apoptosis but with the simultaneous strong Mcl-1 up-regulation and activation of MAPK/PI3K-conducted signaling. In contrast, in hypoxic KRAS-mutant LoVo (devoid of PIK3CA mutation), BRAF/PIK3CA-mutant RKO, and wild-type Colo320DM cell lines (all with high endogenous Mcl-1 expression and being resistant to the additional effect of ABT-737 to AZD8055), combo-Rx did not elicit apoptotic or pro-survival responses.Conclusions: The concurrent inhibition of anti-apoptotic proteins and mTOR-mediated signaling in hypoxic KRAS/PIK3CA-mutant CRC cell lines resulted in pro-survival responses in parallel with the intended anti-proliferative effects, a finding that should be of note if considering combinatory targeting of multiple pathways in this CRC entity. [ABSTRACT FROM AUTHOR]

Human calcium/calmodulin-dependent protein kinase IV (CAMKIV) is a member of Ser/Thr protein kinase family. It is regulated by the calcium-calmodulin dependent signal through a secondary messenger, Ca2+, which leads to the activation of its autoinhibited form. The over-expression and mutation in CAMKIV as well as change in Ca2+ concentration is often associated with numerous neurodegenerative diseases and cancers. We have successfully cloned, expressed, and purified a functionally active kinase domain of human CAMKIV. To observe the effect of different pH conditions on the structural and functional properties of CAMKIV, we have used spectroscopic techniques such as circular diachroism (CD) absorbance and fluorescence. We have observed that within the pH range 5.0-11.5, CAMKIV maintained both its secondary and tertiary structures, along with its function, whereas significant aggregation was observed at acidic pH (2.0-4.5). We have also performed ATPase activity assays under different pH conditions and found a significant correlation between the structure and enzymatic activities of CAMKIV. In-silico validations were further carried out by modeling the 3-dimensional structure of CAMKIV and then subjecting it to molecular dynamics (MD) simulations to understand its conformational behavior in explicit water conditions. A strong correlation between spectroscopic observations and the output of molecular dynamics simulation was observed for CAMKIV. [ABSTRACT FROM AUTHOR]

The Escherichia coli chemosensory system consists of large arrays of transmembrane chemoreceptors associated with a dedicated histidine kinase, CheA, and a linker protein, CheW, that couples CheA activity to receptor control. The kinase activity responses to receptor ligand occupancy changes can be highly cooperative, reflecting allosteric coupling of multiple CheA and receptor molecules. Recent structural and functional studies have led to a working model in which receptor core complexes, the minimal units of signaling, are linked into hexagonal arrays through a unique interface 2 interaction between CheW and the P5 domain of CheA. To test this array model, we constructed and characterized CheA and CheW mutants with amino acid replacements at key interface 2 residues. The mutant proteins proved defective in interface 2-specific in vivo cross-linking assays, and formed signaling complexes that were dispersed around the cell membrane rather than clustered at the cell poles as in wild type chemosensory arrays. Interface 2 mutants down-regulated CheA activity in response to attractant stimuli in vivo, but with much less cooperativity than the wild type. Moreover, mutant cells containing fluorophore-tagged receptors exhibited greater basal anisotropy that changed rapidly in response to attractant stimuli, consistent with facile changes in loosely packed receptors. We conclude that interface 2 lesions disrupt important network connections between core complexes, preventing receptors from operating in large, allosteric teams. This work confirms the critical role of interface 2 in organizing the chemosensory array, in directing the clustered array to the cell poles, and in producing its highly cooperative signaling properties. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ 1, α1β2γ 1, α1β2γ 3, α2β1γ 1, α2β2γ1 and α2β2γ 3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lowerKm values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ~25- fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1- complexes. Compared with β1-containing heterotrimers, β2- containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.. [ABSTRACT FROM AUTHOR]

Pseudokinases are classified by the lack of one or several of the highly conserved motifs involved in nucleotide (nt) binding or catalytic activity of protein kinases (PKs). Pseudokinases represent ∼10% of the human kinome and they are found in all evolutionary classes of kinases. It has become evident that pseudokinases, which were initially considered somewhat peculiar dead kinases, are important components in several signalling cascades. Furthermore, several pseudokinases have been linked to human diseases, particularly cancer, which is raising interest for therapeutic approaches towards these proteins. The ATP-binding pocket is a well-established drug target and elucidation of the mechanism and properties of nt binding in pseudokinases is of significant interest and importance. Recent studies have demonstrated that members of the pseudokinase family are very diverse in structure as well as in their ability and mechanism to bind nts or perform phosphoryl transfer reactions. This diversity also precludes prediction of pseudokinase function, or the importance of nt binding for said function, based on primary sequence alone. Currently available data indicate that ∼40% of pseudokinases are able to bind nts, whereas only few are able to catalyse occasional phosphoryl transfer. Pseudokinases employ diverse mechanisms to bind nts, which usually occurs at low, but physiological, affinity. ATP binding serves often a structural role but in most cases the functional roles are not precisely known. In the present review, we discuss the various mechanisms that pseudokinases employ for nt binding and how this often low-affinity binding can be accurately analysed. [ABSTRACT FROM AUTHOR]

AgrC is an integral membrane receptor protein with histidine kinase activity in the accessory gene regulator (agr) quorum-sensing system of Staphylococcus aureus . In this study, proteoliposomes were used as a model to investigate AgrC orientation. Many approaches have been described to determine membrane protein orientation, but they are often complicated and time consuming. In this study, AgrC orientation in liposomes was determined by thiol-reactive reagent labeling and a kinase activity assay. Our results suggest use of a kinase activity assay could get an accurate percentage of functional protein orientation and only cost nearly one-sixth of the time compared with the method based on thiol-reactive reagent labeling. We present an effective and rapid method for determining the orientation of membrane protein kinases like AgrC. [ABSTRACT FROM AUTHOR]

Background: Selective BRAF inhibitors, vemurafenib and dabrafenib, and the MEK inhibitor, trametinib, have been approved for treatment of metastatic melanomas with a BRAF p.V600E mutation. The clinical significance of non-codon 600 mutations remains unclear, in part, due to variation of kinase activity for different mutants.Methods: In this study, we categorized BRAF mutations according to the reported mutant kinase activity. A total of 1027 lung cancer, colorectal cancer or melanoma specimens were submitted for clinical mutation detection by next generation sequencing.Results: Non-codon 600 mutations were observed in 37% of BRAF-mutated tumors. Of all BRAF mutants, 75% were kinase-activated, 15% kinase-impaired and 10% kinase-unknown. The most common kinase-impaired mutant involves codon 594, specifically, p.D594G (c.1781A > G) and p.D594N (c.1780G > A). Lung cancers showed significantly higher incidences of kinase-impaired or kinase-unknown mutants. Kinase-impaired BRAF mutants showed a significant association with concomitant activating KRAS or NRAS mutations, but not PIK3CA mutations, supporting the reported interaction of these mutations.Conclusions: BRAF mutants with impaired or unknown kinase activity as well as concomitant kinase-impaired BRAF mutations and RAS mutations were detected in lung cancers, colorectal cancers and melanomas. Different therapeutic strategies based on the BRAF mutant kinase activity and the concomitant mutations may be worthwhile. [ABSTRACT FROM AUTHOR]