Allosteric regulation of kinase activity.

The articles in this special issue highlight how modern cellular, biochemical, biophysical and computational techniques are allowing deeper and more detailed studies of allosteric kinase regulation.

Oxidative and Nitrous Stress Underlies Vascular Malfunction Induced by Ionizing Radiation and Diabetes.

Oxidative stress results from the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in quantities exceeding the potential activity of the body's antioxidant system and is one of the risk factors for the development of vascular dysfunction in diabetes and exposure to ionizing radiation. Being the secondary products of normal aerobic metabolism in living organisms, ROS and RNS act as signaling molecules that play an important role in the regulation of vital organism functions. Meanwhile, in high concentrations, these compounds are toxic and disrupt various metabolic pathways. The various stress factors (hyperglycemia, gamma-irradiation, etc.) trigger free oxygen and nitrogen radicals accumulation in cells that are capable to damage almost all cellular components including ion channels and transporters such as Na+/K+-ATPase, BKCa, and TRP channels. Vascular dysfunctions are governed by interaction of ROS and RNS. For example, the reaction of ROS with NO produces peroxynitrite (ONOO-), which not only oxidizes DNA, cellular proteins, and lipids, but also disrupts important signaling pathways that regulate the cation channel functions in the vascular endothelium. Further increasing in ROS levels and formation of ONOO- leads to reduced NO bioavailability and causes endothelial dysfunction. Thus, imbalance of ROS and RNS and their affect on membrane ion channels plays an important role in the pathogenesis of vascular dysfunction associated with various disorders.

Multiplexed Profiling of CDK Kinase Activities in Tumor Biopsies with Fluorescent Peptide Biosensors.

Detection of disease biomarkers constitutes a major challenge for the development of personalized and predictive diagnostics as well as companion assays. Protein kinases (PKs) involved in the coordination of cell cycle progression and proliferation that are hyperactivated in human cancers constitute attractive pharmacological targets and relevant biomarkers. Although it is relatively straightforward to assess the relative abundance of PKs in a biological sample, there is not always a direct correlation with enzymatic activity, which is regulated by several posttranslational mechanisms. Studies of relative abundance therefore convey limited information, and the lack of selective, sensitive, and standardized tools together with the inherent complexity of biological samples makes it difficult to quantify PK activities in physio-pathological tissues. To address this challenge, we have developed a toolbox of fluorescent biosensors that report on CDK activities in a sensitive, selective, dose-dependent, and quantitative fashion, which we have implemented to profile CDK activity signatures in cancer cell lines and biopsies from human tumors. In this study, we report on a standardized and calibrated biosensing approach to quantify CDK1,2,4, and 6 activities simultaneously through a combination of four different biosensors in a panel of 40 lung adenocarcinoma and 40 follicular lymphoma samples. CDK activity profiling highlighted two major patterns which were further correlated with age, sex of patients, tumor size, grade, and genetic and immunohistochemical features of the biopsies. Multiplex CDKACT biosensing technology provides new and complementary information relative to current genetic and immunohistochemical characterization of tumor biopsies, which will be useful for diagnostic purposes, potentially guiding therapeutic decision. These fluorescent peptide biosensors offer promise for personalized diagnostics based on kinase activity profiling.

CaMKII autophosphorylation is the only enzymatic event required for synaptic memory.

Ca2+/calmodulin (CaM)-dependent kinase II (CaMKII) plays a critical role in long-term potentiation (LTP), a well-established model for learning and memory through the enhancement of synaptic transmission. Biochemical studies indicate that CaMKII catalyzes a phosphotransferase (kinase) reaction of both itself (autophosphorylation) and of multiple downstream target proteins. However, whether either type of phosphorylation plays any role in the synaptic enhancing action of CaMKII remains hotly contested. We have designed a series of experiments to define the minimal requirements for the synaptic enhancement by CaMKII. We find that autophosphorylation of T286 and further binding of CaMKII to the GluN2B subunit are required both for initiating LTP and for its maintenance (synaptic memory). Once bound to the NMDA receptor, the synaptic action of CaMKII occurs in the absence of target protein phosphorylation. Thus, autophosphorylation and binding to the GluN2B subunit are the only two requirements for CaMKII in synaptic memory.

Rotavirus non-structural protein 4 usurps host cellular RIPK1-RIPK3 complex to induce MLKL-dependent necroptotic cell death.

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.

The diverging roles of insulin-like growth factor binding proteins in pulmonary arterial hypertension.

Pulmonary hypertension (PH) is a progressive, severe and to date not curable disease of the pulmonary vasculature. Alterations of the insulin-like growth factor 1 (IGF-1) system are known to play a role in vascular pathologies and IGF-binding proteins (IGFBPs) are important regulators of the bioavailability and function of IGFs. In this study, we show that circulating plasma levels of IGFBP-1, IGFBP-2 and IGFBP-3 are increased in idiopathic pulmonary arterial hypertension (IPAH) patients compared to healthy individuals. These binding proteins inhibit the IGF-1 induced IGF-1 receptor (IGF1R) phosphorylation and exhibit diverging effects on the IGF-1 induced signaling pathways in human pulmonary arterial cells (i.e. healthy as well as IPAH-hPASMCs, and healthy hPAECs). Furthermore, IGFBPs are differentially expressed in an experimental mouse model of PH. In hypoxic mouse lungs, IGFBP-1 mRNA expression is decreased whereas the mRNA for IGFBP-2 is increased. In contrast to IGFBP-1, IGFBP-2 shows vaso-constrictive properties in the murine pulmonary vasculature. Our analyses show that IGFBP-1 and IGFBP-2 exhibit diverging effects on IGF-1 signaling and display a unique IGF1R-independent kinase activation pattern in human pulmonary arterial smooth muscle cells (hPASMCs), which represent a major contributor of PAH pathobiology. Furthermore, we could show that IGFBP-2, in contrast to IGFBP-1, induces epidermal growth factor receptor (EGFR) signaling, Stat-3 activation and expression of Stat-3 target genes. Based on our results, we conclude that the IGFBP family, especially IGFBP-1, IGFBP-2 and IGFBP-3, are deregulated in PAH, that they affect IGF signaling and thereby regulate the cellular phenotype in PH.

Kinase Inhibitors and Kinase-Targeted Cancer Therapies: Recent Advances and Future Perspectives.

Over 120 small-molecule kinase inhibitors (SMKIs) have been approved worldwide for treating various diseases, with nearly 70 FDA approvals specifically for cancer treatment, focusing on targets like the epidermal growth factor receptor (EGFR) family. Kinase-targeted strategies encompass monoclonal antibodies and their derivatives, such as nanobodies and peptides, along with innovative approaches like the use of kinase degraders and protein kinase interaction inhibitors, which have recently demonstrated clinical progress and potential in overcoming resistance. Nevertheless, kinase-targeted strategies encounter significant hurdles, including drug resistance, which greatly impacts the clinical benefits for cancer patients, as well as concerning toxicity when combined with immunotherapy, which restricts the full utilization of current treatment modalities. Despite these challenges, the development of kinase inhibitors remains highly promising. The extensively studied tyrosine kinase family has 70% of its targets in various stages of development, while 30% of the kinase family remains inadequately explored. Computational technologies play a vital role in accelerating the development of novel kinase inhibitors and repurposing existing drugs. Recent FDA-approved SMKIs underscore the importance of blood-brain barrier permeability for long-term patient benefits. This review provides a comprehensive summary of recent FDA-approved SMKIs based on their mechanisms of action and targets. We summarize the latest developments in potential new targets and explore emerging kinase inhibition strategies from a clinical perspective. Lastly, we outline current obstacles and future prospects in kinase inhibition.

Targeting FGFR1 by ß,ß-dimethylacrylalkannin suppresses the proliferation of colorectal cancer in cellular and xenograft models.

BACKGROUND: Colorectal cancer (CRC) continues to be a major global health challenge, ranking as a top cause of cancer-related mortality. Alarmingly, the five-year survival rate for CRC patients hovers around a mere 10-30 %. The disruption of fibroblast growth factor receptor (FGFRs) signaling pathways is significantly implicated in the onset and advancement of CRC, presenting a promising target for therapeutic intervention in CRC management. Further investigation is essential to comprehensively elucidate FGFR1's function in CRC and to create potent therapies that specifically target FGFR1. PURPOSE: This study aims to demonstrate the oncogenic role of FGFR1 in colorectal cancer and to explore the potential of ß,ß-dimethylacrylalkannin (ß,ß-DMAA) as a therapeutic option to inhibit FGFR1. METHODS: In this research, we employed a comprehensive suite of techniques including tissue array, kinase profiling, computational docking, knockdown assay to predict and explore the inhibitor of FGFR1. Furthermore, we utilized kinase assay, pull-down, cell proliferation tests, and Patient derived xenograft (PDX) mouse models to further investigate a novel FGFR1 inhibitor and its impact on the growth of CRC. RESULTS: In our research, we discovered that FGFR1 protein is markedly upregulated in colorectal cancer tissues, suggesting a significant role in regulating cellular proliferation, particularly in patients with colorectal cancer. Furthermore, we conducted a computational docking, kinase profiling analysis, simulation and identified that ß,ß-DMAA could directly bind with FGFR1 within ATP binding pocket domain. Cell-based assays confirmed that ß,ß-DMAA effectively inhibited the proliferation of colon cancer cells and also triggered cell cycle arrest, apoptosis, and altered FGFR1-mediated signaling pathways. Moreover, ß,ß-DMAA effectively attenuated the development of PDX tumors in mice that were FGFR1-positive, with no notable toxicity observed. In summary, our study highlights the pivotal role of FGFR1 in colorectal cancer, suggesting that inhibiting FGFR1 activity could be a promising strategy for therapeutic intervention. We present strong evidence that targeting FGFR1 with ß,ß-DMAA is a viable approach for the management of colorectal cancer. Given its low toxicity and high efficacy, ß,ß-DMAA, as an FGFR1 inhibitor, warrants further investigation in clinical settings for the treatment of FGFR1-positive tumors.

Kinase activities in pancreatic ductal adenocarcinoma with prognostic and therapeutic avenues.

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.

PhosMap: An ensemble bioinformatic platform to empower interactive analysis of quantitative phosphoproteomics.

BACKGROUND: Liquid chromatography-mass spectrometry (LC-MS)-based quantitative phosphoproteomics has been widely used to detect thousands of protein phosphorylation modifications simultaneously from the biological specimens. However, the complicated procedures for analyzing phosphoproteomics data has become a bottleneck to widening its application. METHODS: Here, we develop PhosMap, a versatile and scalable tool to accomplish phosphoproteomics data analysis. A standardized phosphorylation data format was created for data analyses, from data preprocessing to downstream bioinformatic analyses such as dimension reduction, differential phosphorylation analysis, kinase activity, survival analysis, and so on. For better usability, we distribute PhosMap as a Docker image for easy local deployment upon any of Windows, Linux, and Mac system. RESULTS: The source code is deposited at https://github.com/BADD-XMU/PhosMap. A free PhosMap webserver (https://huggingface.co/spaces/Bio-Add/PhosMap), with easy-to-follow fashion of dashboards, is curated for interactive data analysis. CONCLUSIONS: PhosMap fills the technical gap of large-scale phosphorylation research by empowering researchers to process their own phosphoproteomics data expediently and efficiently, and facilitates better data interpretation.

Structural insights into the regulation of protein-arginine kinase McsB by McsA.

In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.

From gene to mechanics: a comprehensive insight into the mechanobiology of LMNA mutations in cardiomyopathy.

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.

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.

Novel Roles of the Greatwall Kinase Rim15 in Yeast Oxidative Stress Tolerance through Mediating Antioxidant Systems and Transcriptional Regulation.

The Greatwall-family protein kinase Rim15 is associated with the nutrient starvation response, whereas its role in oxidative stress responses remains unclear. Here, acetic acid and peroxide were used as two oxidative stress elicitors. The antioxidant indicator assay under acetic acid stress revealed the impaired growth in rim15Δ related to the regulation of antioxidant systems. Comparative transcriptome analysis revealed that differentially expressed genes (DEGs) are predicted to be mostly regulated by oxidative stress-responsive transcriptional factor Yap1. Among the DEGs, acetic acid stress-induced genes were found, and YAP1 disruption also inhibited their induction. The deletion of Rim15 or the Rim15 kinase domain in yap1Δ did not further decrease the gene expression, suggesting that Rim15 functions together with Yap1 in regulating acetic acid stress-induced genes, which requires Rim15 kinase activity. Additionally, Rim15 regulated H2O2 stress tolerance through partially similar but special mechanisms in that Rim15 kinase activity impacted acetic acid and H2O2 stress tolerance in different degrees, indicating the different mechanisms underlying Rim15-mediated redox regulation against different stressors. These results benefit the better understanding of stress signaling pathways related to Rim15. Given that Rim15 and some of its target genes are conserved across eukaryotes, these results also provide a basis for studies of oxidative stress-related processes in other organisms.

Identifying Differences in Molecular Characteristics Relevant for Remodeling of Periodontal Ligament Stem Cells from the Upper and Lower Jaw.

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.

A versatile kinase mobility shift assay (KiMSA) for PKA analysis and cyclic AMP detection in sperm physiology (and beyond).

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-32P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

Pan-cancer proteogenomics characterization of tumor immunity.

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.

Monitoring Chk1 kinase activity dynamics in live single cell imaging assays.

The ATR/Chk1 pathway is an important regulator of cell cycle progression, notably upon genotoxic stress where it can detect a large variety of DNA alterations and induce a transient cell cycle arrest that promotes DNA repair. In addition to its role in DNA damage response (DDR), Chk1 is also active during a non-perturbed S phase and contributes to prevent a premature entry into mitosis with an incompletely replicated genome, meaning the ATR/Chk1 pathway is an integral part of the cell cycle machinery that preserves genome integrity during cell growth. We recently developed a FRET-based Chk1 kinase activity reporter to directly monitor and quantify the kinetics of Chk1 activation in live single cell imaging assays with unprecedented sensitivity and time resolution. This tool allowed us to monitor Chk1 activity dynamics over time during a normal S phase and following genotoxic stress, and to elucidate the underlying mechanisms leading to its activation. Here, we review available fluorescent tools to study the interplay of cell cycle progression, DNA damage and DDR in individual live cells, and present the full protocol and image analysis pipeline to monitor Chk1 activity in two imaging assays.

Superbinder based phosphoproteomic landscape revealed PRKCD_pY313 mediates the activation of Src and p38 MAPK to promote TNBC progression.

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.

PHOTOTROPIN1 lysine 526 functions to enhance phototropism in Arabidopsis.

MAIN CONCLUSION: After blue-light exposure, ubiquitination of PHOTOTROPIN1 lysine 526 enhances phototropic responses. Arabidopsis blue-light photoreceptor, PHOTOTROPIN1 (PHOT1) mediates a series of blue-light responses that function to optimize photosynthesis efficiency. Blue-light sensing through the N-terminal sensory domain activates the C-terminal kinase activity of PHOT1, resulting in autophosphorylation. In addition to phosphorylation, PHOT1 lysine residue 526 (Lys526), after blue-light exposure, was found to carry a double glycine attachment, indicative of a possible ubiquitination modification. The functionality of PHOT1 Lys526 was investigated by reverse genetic approaches. Arginine replacements of PHOT1 Lys526, together with Lys527, complemented phot1-5 phot2-1 double mutant with attenuated phototropic bending, while blue-light responses: leaf expansion and stomatal opening, were restored to wild type levels. Transgenic seedlings were not different in protein levels of phot1 Lys526 527Arg than the wild type control, suggesting the reduced phototropic responses was not caused by reduction in protein levels. Treating the transformants with proteosome inhibitor, MG132, did not restore phototropic sensitivity. Both transgenic protein and wild type PHOT1 also had similar dark recovery of kinase activity, suggesting that phot1 Lys526 527Arg replacement did not affect the protein stability to cause the phenotype. Together, our results indicate that blocking Lys526 ubiquitination by arginine substitution may have caused the reduced phototropic phenotype. Therefore, the putative ubiquitination on Lys526 functions to enhance PHOT1-mediated phototropism, rather than targeting PHOT1 for proteolysis.

The ETI-dependent receptor-like kinase 1 positively regulates effector-triggered immunity by stabilizing NLR-required for cell death 4 in Nicotiana benthamiana.

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.