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Myxoid liposarcoma is a rare mesenchymal malignancy, which is characterized by a FUS-DDIT3 fusion known as chromosomal translocation t(12;16)(q13;p11) and arises in the fat tissue. Although surgery with radiation has been established as a standard treatment, myxoid liposarcoma shows frequent recurrence and poor prognosis, thus requiring new therapeutic approaches. Patient-derived cell lines represent a critical tool for basic and preclinical research. However, only two such myxoid liposarcoma cell lines have been reported, and they are not available in cell banks. The aim of this study was to establish and characterize a novel myxoid liposarcoma cell line. Using surgically resected tumor tissue from a 47-year-old male patient, we established the NCC-MLPS1-C1 cell line. NCC-MLPS1-C1 cells were characterized by FUS-DDIT3 fusion, slow growth, spheroid formation, and invasive capability. We screened the effect of anti-cancer agents on the proliferation of NCC-MLPS1-C1 cells. The cells displayed a remarkable response to multitarget kinase inhibitors of RET, PDGFR-β, VEGFR, or FGFR. NCC-MLPS1-C1 cells and the tumor tissue shared common profiles of kinase activity including identified drug targets, such as RET and PDGFR-β. We believe that NCC-MLPS1-C1 cells will represent a useful tool for basic and preclinical studies of myxoid liposarcoma. [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]

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.

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., (Copyright © 2024 Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

It is important to develop fundamental understanding on the utilization of inorganic nanoparticles (NPs) in health and environment as their use in the agricultural food sector, medical applications, cosmetics, paints are growing rapidly. Agricultural food sector is one of the major fields where these particles of less than 100 nm diameter are applied. This sector uses this revolutionary technology for increased growth of crop; thus, its effect on human body not only need to be studied, but also its effect on human beneficial gut flora that are in symbiosis relationship with human kind needs attention. Here, experiments were conducted on one of the dominant beneficial human gut bacteria (spore-forming lactic acid Bacillus, Bacillus coagulans) in presence of both titanium dioxide (TiO2) anatase nanoparticles (as this nanomaterial used mostly in plants among metal-based nanomaterials) and zinc oxide (ZnO) NPs (as this nanomaterial used mostly in drugs as antimicrobial agent to destroy harmful bacteria, nowadays also used as antiviral agent). ZnO NPs show toxicity at and above 1 mg/L, whereas bacteria show more growth than control in presence of TiO2 anatase NP at 1 mg/L in absence of light. TEM and SEM images and results of enzyme assay revealed that NPs do not aggregate, they act on bacteria, but bacterial cell wall integrity behave differently in presence of different NPs with high level of control on regulation of energy; hence, nanoparticle interaction mechanism with bacteria is studied thoroughly. [ABSTRACT FROM AUTHOR]

PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ.

The many variants of human Ca2+/calmodulin-dependent protein kinase II (CaMKII) differ in the lengths and sequences of disordered linkers connecting the kinase domains to the oligomeric hubs of the holoenzyme. CaMKII activity depends on the balance between activating and inhibitory autophosphorylation (on Thr 286 and Thr 305/306, respectively, in the human α isoform). Variation in the linkers could alter transphosphorylation rates within a holoenzyme and the balance of autophosphorylation outcomes. We show, using mammalian cell expression and a single-molecule assay, that the balance of autophosphorylation is flipped between CaMKII variants with longer and shorter linkers. For the principal isoforms in the brain, CaMKII-α, with a ~30 residue linker, readily acquires activating autophosphorylation, while CaMKII-β, with a ~200 residue linker, is biased towards inhibitory autophosphorylation. Our results show how the responsiveness of CaMKII holoenzymes to calcium signals can be tuned by varying the relative levels of isoforms with long and short linkers.

The molecular mechanism of action of the HER2‐targeted antibody trastuzumab is only partially understood, and the direct effects of trastuzumab on the gastric cancer signaling network are unknown. In this study, we compared the molecular effect of trastuzumab and the HER kinase inhibitor afatinib on the receptor tyrosine kinase (RTK) network and the downstream‐acting intracellular kinases in gastric cancer cell lines. The molecular effects of trastuzumab and afatinib on the phosphorylation of 49 RTKs and 43 intracellular kinase phosphorylation sites were investigated in three gastric cancer cell lines (NCI‐N87, MKN1, and MKN7) using proteome profiling. To evaluate these effects, data were analyzed using mixed models and clustering. Moreover, proliferation assays were performed. Our comprehensive quantitative analysis of kinase activity in gastric cancer cell lines indicates that trastuzumab and afatinib selectively influenced the HER family RTKs. The effects of trastuzumab differed between cell lines, depending on the presence of activated HER2. The effects of trastuzumab monotherapy were not transduced to the intracellular kinase network. Afatinib alone or in combination with trastuzumab influenced HER kinases in all cell lines; that is, the effects of monotherapy and combination therapy were transduced to the intracellular kinase network. These results were confirmed by proliferation analysis. Additionally, the MET‐amplified cell line Hs746T was identified as afatinib nonresponder. The dependence of the effect of trastuzumab on the presence of activated HER2 might explain the clinical nonresponse of some patients who are routinely tested for HER2 expression and gene amplification in the clinic but not for HER2 activation. The consistent effects of afatinib on HER RTKs and downstream kinase activation suggest that afatinib might be an effective candidate in the future treatment of patients with gastric cancer irrespective of the presence of activated HER2. However, MET amplification should be taken into account as potential resistance factor.

Abstract Esophageal cancer is one of the most common types of cancer, which is a leading cause of cancer-related death worldwide. Based on histological behavior, it is mainly of two types (i) Esophageal squamous cell carcinoma (ESCC), and (ii) esophageal adenocarcinoma (EAD or EAC). In astronomically immense majority of malignancies, receptor tyrosine kinases (RTKs) have been kenned to play a consequential role in cellular proliferation, migration, and metastasis of the cells. The post-translational modifications (PTMs) including phosphorylation of tyrosine (pY) residue of the tyrosine kinase (TK) domain have been exploited for treatment in different malignancies. Lung cancer where pY residues of EGFR have been exploited for treatment purpose in lung adenocarcinoma patients, but we do not have such kind of felicitously studied and catalogued data in ESCC patients. Thus, the goal of this review is to summarize the studies carried out on ESCC to explore the role of RTKs, tyrosine kinase inhibitors, and their pertinence and consequentiality for the treatment of ESCC patients.

PKZ is a novel and unique eIF2α protein kinase identified in fish. Although PKZ is most homologous to PKR, particularly in the C-terminal catalytic domain, it contains two N-terminal Z-DNA-binding domains (Zα1 and Zα2) instead of the dsRNA binding domains (dsRBDs) in PKR. As a novel member of eIF2α kinase family, the available data suggest that PKZ has some distinct mechanisms for recognition, binding, and B-Z DNA transition. Functionally, PKZ seems to be activated by the binding of Zα to Z-DNA and participates in innate immune responses. In this review, we summarize the recent progress on fish PKZ. [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]

Abstract 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, and autoradiography 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.

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]

• 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]

Abstract 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.

RIPK3 mediates cell death and regulates inflammatory responses. Although genetic studies have suggested that RIPK3-MLKL-mediated necroptosis leads to embryonic lethality in Fadd or Caspase-8-deficient mice, the exact mechanisms are not fully understood. Here, we generated Ripk3 mutant mice by altering the RIPK3 kinase domain (Ripk3Δ/Δ mice), thus abolishing its kinase activity. Ripk3Δ/Δ cells were resistant to necroptosis stimulation in vitro, and Ripk3Δ/Δ mice were protected from necroptotic diseases. Although the Ripk3Δ/Δ mutation rescued embryonic lethality in Fadd−/− embryos, Fadd−/− Ripk3Δ/Δ mice died within 1 day after birth due to massive inflammation. These results indicate that Ripk3 ablation rescues embryonic lethality in Fadd-deficient mice by suppressing two RIPK3-mediating processes: necroptosis during embryogenesis and inflammation during postnatal development in Fadd−/− mice.

Tyrosine autophosphorylation plays a crucial regulatory role in the kinase activities of fibroblast growth factor receptors (FGFRs), and in the recruitment and activation of downstream intracellular signaling pathways. Biophysical and biochemical investigations of FGFR kinase domains in membrane environments offer key insights into phosphorylation mechanisms. Hence, we constructed nickel chelating nanodiscs based on a 22-residue peptide. The spontaneous anchoring of N-terminal His6-tagged FGFR1c kinase domain (FGFR1K) onto peptide nanodiscs grants FGFR1K orientations occurring on native plasma membranes. Following membrane incorporation, the autophosphorylation of FGFR1K, as exemplified by Y653 and Y654 in the A-loop and the total tyrosine phosphorylation, increase significantly. This in vitro reconstitution system may be applicable to studies of other membrane associated phenomena.

Genetic information is transcribed from genomic DNA to mRNA, which is then translated into three-dimensional proteins. mRNAs can undergo various post-transcriptional modifications, including RNA editing that alters mRNA sequences, ultimately affecting protein function. In this study, RNA editing was identified at the 499th base (c.499) of human vaccinia-related kinase 2 (VRK2). This RNA editing changes the amino acid in the catalytic domain of VRK2 from isoleucine (with adenine base) to valine (with guanine base). Isoleucine-containing VRK2 has higher kinase activity than the valine-containing VRK2, which leads to an increase in tumor cell proliferation. Earlier we reported that VRK2 directly interacts with dystrobrevin-binding protein (dysbindin) and results in reducing its stability. Herein, we demonstrate that isoleucine-containing VRK2 decreases the level of dysbindin than valine-containing VRK2. Dysbindin interacts with cyclin D and thereby regulates its expression and function. The reduction in the level of dysbindin by isoleucine-containing VRK2 further enhances the cyclin D expression, resulting in increased tumor growth and reduction in survival rates. It has also been observed that in patient samples, VRK2 level was elevated in breast cancer tissue compared to normal breast tissue. Additionally, the isoleucine form of VRK2 exhibited a greater increase in breast cancer tissue. Therefore, it is concluded that VRK2, especially dependent on the 167th variant amino acid, can be one of the indexes of tumor progression and proliferation., Competing Interests: The authors declare that they have no conflicts of interest to report regarding the present study., (© 2024 Gwak et al.)

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

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]

FMS‐like tyrosine kinase 3 (FLT3) is one of the most frequently mutated genes in hematological malignancies. FLT3 internal tandem duplication (FLT3‐ITD) mutations located in juxtamembrane domain (JMD) and tyrosine kinase domain 1 (TKD1) regions account for two‐thirds of all FLT3 mutations. The outcome of patients remains unsatisfactory, with low survival rates. It is not yet known whether the different mutations within the FLT3 gene are all associated with patient outcome. In addition, the cause of FLT3‐ITD in‐frame duplication events remains unknown. Although there are some published studies investigating the FLT3‐ITD mutation and its clinical implications in Chinese acute myeloid leukemia (AML) patients, sample sizes tend to be small and detailed molecular profiles of FLT3 mutations are lacking in these studies. In our study, 227 FLT3‐ITD sequences were analyzed from 227 Chinese de novo AML patients. ITD were next classified into 3 types based on molecular profiles of insertion DNA sequences: DNA complete duplication (type I), DNA partial duplication (type II) and complete random sequence (type III). From the 154 patients, we confirmed that high ITD allelic ratio (≥.5) and allogeneic stem cell transplant treatment under CR1 are independent prognostic factors. We also presented evidence that ITD integration sites in the hinge region or beta1‐sheet region are an unfavorable prognostic factor in adult AML patients with FLT3‐ITD mutations. These findings may help to decipher the mechanisms of FLT3‐ITD in‐frame duplication events and stratify patients when considering different therapeutic combinations. A total of 227 FLT3‐ITD sequences were analyzed from 227 Chinese adult de novo acute myeloid leukemia (AML) patients. ITD could be classified into 3 types based on molecular profiles. ITD integration sites in the hinge region or beta1‐sheet region are an unfavorable prognostic factor in adult AML patients with FLT3‐ITD mutations. [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 flowering plants, the developmental switch to the reproductive phase is tightly regulated and involves the integration of internal and external signals. FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) integrate signals from multiple pathways. FT and TSF function as florigenic substances, and share high sequence similarity with mammalian Raf kinase inhibitor protein (RKIP). Despite their strong similarity to RKIP, the kinase inhibitory activity of FT and TSF remains to be investigated. We performed a yeast two-hybrid screen and found that TSF interacted with FRUCTOKINASE6 (FRK6), which phosphorylates fructose for various metabolic pathways. Among the seven Arabidopsis FRKs, FRK6 and FRK7 have high sequence similarity; therefore, we investigated whether TSF interacts with FRK6 and FRK7. In vitro pull-down assays and bimolecular fluorescence complementation assays revealed that TSF interacts with FRK6 in the nucleus, but not with FRK7. Kinase activity assays suggested that TSF inhibits the kinase activity of FRK6, whereas FT does not. By contrast, neither TSF nor FT inhibits the kinase activity of FRK7. The frk6 and frk7 mutants show slightly delayed flowering, but only under short-day (SD) conditions. Plastochron length is also affected in both frk6 and frk7 mutants under SD conditions. FT expression levels decreased in frk6 mutants, but not in frk7 mutants. Taken together, our findings suggest that TSF physically interacts with FRK6 and affects its kinase activity, whereas FT does not, although these proteins share high sequence similarity.

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. Cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported an interaction between pUL97 kinase with a subset of host cyclins, in particular with cyclin T1. Here, we describe an interaction of pUL97 at an even higher affinity with cyclin B1. As a striking feature, the interaction between pUL97 and cyclin B1 proved to be strictly dependent on pUL97 activity, as interaction could be abrogated by treatment with pUL97 inhibitors or by inserting mutations into the conserved kinase domain or the nonconserved C-terminus of pUL97, both producing loss of activity. Thus, we postulate that the mechanism of pUL97-cyclin B1 interaction is determined by an active pUL97 kinase domain.

Background/Aims: The antileukemic potential of isoindigos make them desired candidates for understanding their mechanism of action. We have recently synthesized a novel group of pyridone-annelated isoindigos and identified the derivative 5'-Cl that is cytotoxic to various cancer cell lines. In the present study, we analyzed the effect of this compound on cell cycle of the promyelocytic leukemia cell line HL-60. Methods: HL-60 cells were treated with 5'-Cl and its effect on cell cycle stages were determined by flow cytometry. Expression of cyclins, cyclin dependent kinases (CDKs) and cyclin kinase inhibitors (CKIs) were determined by Western blotting, and activation of CDKs was studied using kinase assays. Results: 5'-Cl remarkably arrested cell cycle in HL-60 cells at the G0/G1 phase in a dose and time-dependent manner. Furthermore, 5'-Cl treatment significantly inhibited expression of D-cyclins, CDK2 and CDK4 and suppressed phosphorylation of the retinoblastoma protein Rb, whereas it increased the level of CKI p21. Molecular modelling experiments show that 5'-Cl may compete with ATP for binding to the catalytic subunit of CDK2 and CDK4 that could lead to inhibition of these enzymes. Indeed, 5'-Cl inhibited the kinase activity of CDK2 and CDK4 both in cell free systems and in treated cells. 5'-Cl also inhibited cell cycle progression in several other tumor cell lines. Conclusion: We demonstrate the potent inhibitory effects of 5'-Cl on HL-60 cells could be mediated by arresting cells in the G0/G1 phase.

Accumulating evidence indicates that early and essential events for receptor-like kinase (RLK) function involve both autophosphorylation and substrate phosphorylation. However, the structural and biochemical basis for these events is largely unclear. Here, we used RLK FERONIA (FER) as a model and crystallized its core kinase domain (FER-KD) and two FER-KD mutants (K565R, S525A) in complexes with ATP/ADP and Mg 2+ in the unphosphorylated state. Unphosphorylated FER-KD was found to adopt an unexpected active conformation in its crystal structure. Moreover, unphosphorylated FER-KD mutants with reduced (S525A) or no catalytic activity (K565R) also adopt similar active conformations. Biochemical studies revealed that FER-KD is a dual-specificity kinase, and its autophosphorylation is accomplished via an intermolecular mechanism. Further investigations confirmed that initiating substrate phosphorylation requires autophosphorylation of the activation segment on T696, S701, and Y704. This study reveals the structural and biochemical basis for the activation and regulatory mechanism of FER, providing a paradigm for the early steps in RLK signaling initiation., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

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]

The molecular mechanism of action of the HER2‐targeted antibody trastuzumab is only partially understood, and the direct effects of trastuzumab on the gastric cancer signaling network are unknown. In this study, we compared the molecular effect of trastuzumab and the HER kinase inhibitor afatinib on the receptor tyrosine kinase (RTK) network and the downstream‐acting intracellular kinases in gastric cancer cell lines. The molecular effects of trastuzumab and afatinib on the phosphorylation of 49 RTKs and 43 intracellular kinase phosphorylation sites were investigated in three gastric cancer cell lines (NCI‐N87, MKN1, and MKN7) using proteome profiling. To evaluate these effects, data were analyzed using mixed models and clustering. Moreover, proliferation assays were performed. Our comprehensive quantitative analysis of kinase activity in gastric cancer cell lines indicates that trastuzumab and afatinib selectively influenced the HER family RTKs. The effects of trastuzumab differed between cell lines, depending on the presence of activated HER2. The effects of trastuzumab monotherapy were not transduced to the intracellular kinase network. Afatinib alone or in combination with trastuzumab influenced HER kinases in all cell lines; that is, the effects of monotherapy and combination therapy were transduced to the intracellular kinase network. These results were confirmed by proliferation analysis. Additionally, the MET‐amplified cell line Hs746T was identified as afatinib nonresponder. The dependence of the effect of trastuzumab on the presence of activated HER2 might explain the clinical nonresponse of some patients who are routinely tested for HER2 expression and gene amplification in the clinic but not for HER2 activation. The consistent effects of afatinib on HER RTKs and downstream kinase activation suggest that afatinib might be an effective candidate in the future treatment of patients with gastric cancer irrespective of the presence of activated HER2. However, MET amplification should be taken into account as potential resistance factor. [ABSTRACT FROM AUTHOR]