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Abstract Karyopherin α 2 (KPNA2, importin α1), a transport factor shuttling between the nuclear and cytoplasmic compartments, is involved in the nuclear import of proteins and participates in cellular processes such as cell cycle regulation, apoptosis, and transcriptional regulation. However, it is still unclear which signaling regulates the nucleocytoplasmic distribution of KPNA2 in response to cellular stress. In this study, we report that oxidative stress increases nuclear retention of KPNA2 through alpha serine/threonine‐protein kinase (AKT1)‐mediated reduction of serine 62 (S62) phosphorylation. We first found that AKT1 activation was required for H2O2‐induced nuclear accumulation of KPNA2. Immunoprecipitation and quantitative proteomic analysis revealed that the phosphorylation of KPNA2 at S62 was decreased under H2O2‐induced oxidative stress. We showed that cyclin‐dependent kinase 1 (CDK1), a kinase responsible for KPNA2 S62 phosphorylation, contributes to the localization of KPNA2 in the cytoplasm. AKT1 knockdown increased KPNA2 S62 phosphorylation and inhibited CDK1 activation. Furthermore, H2O2‐induced AKT1 activation promoted nuclear KPNA2 interaction with nucleophosmin 1 (NPM1), resulting in attenuation of NPM1‐mediated cyclin D1 gene transcription. Thus, we infer that the AKT1‐CDK1 axis regulates the nucleocytoplasmic shuttling and function of KPNA2 through spatiotemporal regulation of KPNA2 S62 phosphorylation under oxidative stress conditions.

Abstract Noninherited diseases and age‐associated vision loss are often associated with retinal degeneration. The retina is a postmitotic neural tissue lacking endogenous regeneration capacity. Therefore, understanding the mechanism of retinal degeneration in diseases is pivotal. Posttranslational modifications (PTMs) determine protein function during physiological and pathological processes, including signal transduction, protein localization, and protein activation. Advanced detection technologies have revealed over 400 different PTMs including acetylation, methylation, phosphorylation, ubiquitination and SUMOylation. Here, we discuss PTMs in retinal degeneration diseases to aid in our understanding of their molecular basis and suggest potential future clinical treatment.

Abstract Interferons (IFNs) activate JAK‐STAT pathways to induce downstream effector genes for host defense against invaded pathogens and tumors. Here both type I (β) and II (γ) IFNs are shown that can activate the transcription factor IRF3 in parallel with STAT1. IRF3‐deficiency impairs transcription of a subset of downstream effector genes induced by IFN‐β and IFN‐γ. Mechanistically, IFN‐induced activation of IRF3 is dependent on the cGAS‐STING‐TBK1 axis. Both IFN‐β and IFN‐γ cause mitochondrial DNA release into the cytosol. In addition, IFNs induce JAK1‐mediated tyrosine phosphorylation of cGAS at Y214/Y215, which is essential for its DNA binding activity and signaling. Furthermore, deficiency of cGAS, STING, or IRF3 impairs IFN‐β‐ or IFN‐γ‐mediated antiviral and antitumor activities. The findings reveal a novel IRF3 activation pathway parallel with the canonical STAT1/2 activation pathways triggered by IFNs and provide an explanation for the pleiotropic roles of the cGAS‐STING‐IRF3 axis in host defense.

A robust body of work has demonstrated that a reduction in cAMP‐specific 3′,5′‐cyclic phosphodiesterase 4D isoform 7 (PDE4D7) is linked with negative prostate cancer outcomes; however, the exact molecular mechanism that underpins this relationship is unknown. Epigenetic profiling has shown that the PDE4D gene can be hyper‐methylated in transmembrane serine protease 2 (TMPRSS2)–ETS transcriptional regulator ERG (ERG) gene‐fusion‐positive prostate cancer (PCa) tumours, and this inhibits messenger RNA (mRNA) expression, leading to a paucity of cellular PDE4D7 protein. In an attempt to understand how the resulting aberrant cAMP signalling drives PCa growth, we immunopurified PDE4D7 and identified binding proteins by mass spectrometry. We used peptide array technology and proximity ligation assay to confirm binding between PDE4D7 and ATP‐dependent RNA helicase A (DHX9), and in the design of a novel cell‐permeable disruptor peptide that mimics the DHX9‐binding region on PDE4D7. We discovered that PDE4D7 forms a signalling complex with the DExD/H‐box RNA helicase DHX9. Importantly, disruption of the PDE4D7–DHX9 complex reduced proliferation of LNCaP cells, suggesting the complex is pro‐tumorigenic. Additionally, we have identified a novel protein kinase A (PKA) phosphorylation site on DHX9 that is regulated by PDE4D7 association. In summary, we report the existence of a newly identified PDE4D7–DHX9 signalling complex that may be crucial in PCa pathogenesis and could represent a potential therapeutic target.

Post‐translational modifications are key in the regulation of activity, structure, localization, and stability of most proteins in eukaryotes. Phosphorylation is potentially the most studied post‐translational modification, also due to its reversibility and thereby the regulatory role this modification often plays. While most research attention was focused on kinases in the past, phosphatases remain understudied, most probably because the addition and presence of the modification is more easily studied than its removal and absence. Here, we report the identification of an uncharacterized protein tyrosine phosphatase PPH‐7 in C. elegans, a member of the evolutionary conserved PTPN family of phosphatases. Lack of PPH‐7 function led to reduction of fertility and embryonic lethality at elevated temperatures. Proteomics revealed changes in the regulation of targets of the von Hippel–Lindau (VHL) E3 ligase, suggesting a potential role for PPH‐7 in the regulation of VHL.

Abstract Mitogen‐activated protein kinase (MAPK) cascade is the center of plant signal transduction system that amplify immune signals into cellular responses by phosphorylating diverse substrates. The MAPK cascade consisting of MAPK kinase kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs is well characterized in plants, in which Raf‐like kinases are generally regarded as MAPKKKs. However, it is rarely reported that Raf‐like MAPKKKs function as middle regulators to link MAPK and its downstream transcription factors in plant immunity. Verticillium wilt, caused by the soil‐borne vascular fungus Verticillium dahliae, is a serious disease in many plants, including cotton. The previous studies showed that GhMPK9 (a MAPK) is involved in the response to Verticillium wilt. Here, the Raf‐like kinase GhRAF39_1 is reported as helper regulates the phosphorylation of WRKY transcription factor GhWRKY40a by GhMPK9. The phosphorylated GhWRKY40a can further activate the transcription of GhERF1b to up‐regulate defense‐related genes while inhibit the transcription of GhABF2 to regulate the stomatal opening, thus improving the resistance to Verticillium wilt in cotton. This study reveals a new signaling module of GhMPK9‐GhRAF39_1‐GhWRKY40a to regulate GhERF1b‐ and GhABF2‐mediated defense responses, which triggers plant defense against Verticillium wilt.

Abstract The dystrophin protein has well‐characterized roles in force transmission and maintaining membrane integrity during muscle contraction. Studies have reported decreased expression of dystrophin in atrophying muscles during wasting conditions, and that restoration of dystrophin can attenuate atrophy, suggesting a role in maintaining muscle mass. Phosphorylation of S3059 within the cysteine‐rich region of dystrophin enhances binding between dystrophin and β‐dystroglycan, and mimicking phosphorylation at this site by site‐directed mutagenesis attenuates myotube atrophy in vitro. To determine whether dystrophin phosphorylation can attenuate muscle wasting in vivo, CRISPR‐Cas9 was used to generate mice with whole body mutations of S3059 to either alanine (DmdS3059A) or glutamate (DmdS3059E), to mimic a loss of, or constitutive phosphorylation of S3059, on all endogenous dystrophin isoforms, respectively. Sciatic nerve transection was performed on these mice to determine whether phosphorylation of dystrophin S3059 could attenuate denervation atrophy. At 14 days post denervation, atrophy of tibialis anterior (TA) but not gastrocnemius or soleus muscles, was partially attenuated in DmdS3059E mice relative to WT mice. Attenuation of atrophy was associated with increased expression of β‐dystroglycan in TA muscles of DmdS3059E mice. Dystrophin S3059 phosphorylation can partially attenuate denervation‐induced atrophy, but may have more significant impact in less severe modes of muscle wasting.

Abstract Cancer metastasis is the leading cause of mortality in patients with hepatocellular carcinoma (HCC). To meet the rapid malignant growth and transformation, tumor cells dramatically increase the consumption of nutrients, such as amino acids. Peptide transporter 1 (PEPT1), a key transporter for small peptides, has been found to be an effective and energy‐saving intracellular source of amino acids that are required for the growth of tumor cells. Here, the role of PEPT1 in HCC metastasis and its underlying mechanisms is explored. PEPT1 is upregulated in HCC cells and tissues, and high PEPT1 expression is associated with poor prognosis in patients with HCC. PEPT1 overexpression dramatically promoted HCC cell migration, invasion, and lung metastasis, whereas its knockdown abolished these effects both in vitro and in vivo. Mechanistic analysis revealed that high PEPT1 expression increased cellular dipeptides in HCC cells that are responsible for activating the MAP4K4/G3BP2 signaling pathway, ultimately facilitating the phosphorylation of G3BP2 at Thr227 and enhancing HCC metastasis. Taken together, these findings suggest that PEPT1 acts as an oncogene in promoting HCC metastasis through dipeptide‐induced MAP4K4/G3BP2 signaling and that the PEPT1/MAP4K4/G3BP2 axis can serve as a promising therapeutic target for metastatic HCC.

The deposition of highly phosphorylated and aggregated tau is a characteristic of tauopathies, including Alzheimer's disease. It has long been known that different isoforms of tau are aggregated in different cell types and brain regions in each tauopathy. Recent advances in analytical techniques revealed the details of the biochemical and structural biological differences of tau specific to each tauopathy. In this review, we explain recent advances in the analysis of post‐translational modifications of tau, particularly phosphorylation, brought about by the development of mass‐spectrometry and Phos‐tag technology. We then discuss the structure of tau filaments in each tauopathy revealed by the advent of cryo‐EM. Finally, we describe the progress in biofluid and imaging biomarkers for tauopathy. This review summarizes current efforts to elucidate the characteristics of pathological tau and the landscape of the use of tau as a biomarker to diagnose and determine the pathological stage of tauopathy.

Abstract Targeting cancer‐specific metabolic processes is a promising therapeutic strategy. Here, this work uses a compound library that directly inhibits metabolic enzymes to screen the potential metabolic targets in lung adenocarcinoma (LUAD). SHIN1, the specific inhibitor of serine hydroxymethyltransferase 1/2 (SHMT1/2), has a highly specific inhibitory effect on LUAD cells, and this effect depends mainly on the overexpression of SHMT2. This work clarifies that mitogen‐activated protein kinase 1 (MAPK1)‐mediated phosphorylation at Ser90 is the key mechanism underlying SHMT2 upregulation in LUAD and that this phosphorylation stabilizes SHMT2 by reducing STIP1 homology and U‐box containing protein 1 (STUB1)‐mediated ubiquitination and degradation. SHMT2‐Ser90 dephosphorylation decreases S‐adenosylmethionine levels in LUAD cells, resulting in reduced N6‐methyladenosine (m6A) levels in global RNAs without affecting total protein or DNA methylation. Methylated RNA immunoprecipitation sequencing (MeRIP‐Seq) and RNA sequencing (RNA‐Seq) analyses further demonstrate that SHMT2‐Ser90 dephosphorylation accelerates the RNA degradation of oncogenic genes by reducing m6A modification, leading to the inhibition of tumorigenesis. Overall, this study elucidates a new regulatory mechanism of SHMT2 during oncogenesis and provides a theoretical basis for targeting SHMT2 as a therapeutic target in LUAD.

Background Myosin phosphatase targeting subunit 2 (MYPT2) is an important subunit of cardiac MLC (myosin light chain) phosphatase, which plays a crucial role in regulating the phosphorylation of MLC to phospho‐MLC (p‐MLC). A recent study demonstrated mineralocorticoid receptor‐related hypertension is associated with RhoA/Rho‐associated kinase/MYPT1 signaling upregulation in smooth muscle cells. Our purpose is to investigate the effect of MYPT2 on cardiac function and fibrosis in mineralocorticoid receptor‐related hypertension. Methods and Results HL‐1 murine cardiomyocytes were incubated with different concentrations or durations of aldosterone. After 24‐hour stimulation, aldosterone increased CTGF (connective tissue growth factor) and MYPT2 and decreased p‐MLC in a dose‐dependent manner. MYPT2 knockdown decreased CTGF. Cardiac‐specific MYPT2‐knockout (c‐MYPT2−/−) mice exhibited decreased type 1 phosphatase catalytic subunit β and increased p‐MLC. A disease model of mouse was induced by subcutaneous aldosterone and 8% NaCl food for 4 weeks after uninephrectomy. Blood pressure elevation and left ventricular hypertrophy were observed in both c‐MYPT2−/− and MYPT2+/+ mice, with no difference in heart weights or nuclear localization of mineralocorticoid receptor in cardiomyocytes. However, c‐MYPT2−/− mice had higher ejection fraction and fractional shortening on echocardiography after aldosterone treatment. Histopathology revealed less fibrosis, reduced CTGF, and increased p‐MLC in c‐MYPT2−/− mice. Basal global radial strain and global longitudinal strain were higher in c‐MYPT2−/− than in MYPT2+/+ mice. After aldosterone treatment, both global radial strain and global longitudinal strain remained higher in c‐MYPT2−/− mice compared with MYPT2+/+ mice. Conclusions Cardiac‐specific MYPT2 knockout leads to decreased myosin light chain phosphatase and increased p‐MLC. MYPT2 deletion prevented cardiac fibrosis and dysfunction in a model of mineralocorticoid receptor‐associated hypertension.

Abstract The eukaryote‐specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho‐deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild‐type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl‐terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho‐enabled version of eS6z, the phospho‐deficient seedlings retained a mild pointed‐leaf phenotype, root growth was reduced, and certain cell cycle‐related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho‐deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain‐of‐function defects. As previously published, the phospho‐deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho‐deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

Abstract Here, a sucrose non‐fermenting‐1‐related protein kinase alpha subunit (TaSnRK1α‐1A) is identified as associated with cold stress through integration of genome‐wide association study, bulked segregant RNA sequencing, and virus‐induced gene silencing. It is confirmed that TaSnRK1α positively regulates cold tolerance by transgenes and ethyl methanesulfonate (EMS) mutants. A plastid‐lipid‐associated protein 6, chloroplastic‐like (TaPAP6L‐2B) strongly interacting with TaSnRK1α‐1A is screened. Molecular chaperone DJ‐1 family protein (TaDJ‐1‐7B) possibly bridged the interaction of TaSnRK1α‐1A and TaPAP6L‐2B. It is further revealed that TaSnRK1α‐1A phosphorylated TaPAP6L‐2B. Subsequently, a superior haplotype TaPAP6L‐2B30S/38S is identified and confirmed that both R30S and G38S are important phosphorylation sites that influence TaPAP6L‐2B in cold tolerance. Overexpression (OE) and EMS‐mutant lines verified TaPAP6L positively modulating cold tolerance. Furthermore, transcriptome sequencing revealed that TaPAP6L‐2B‐OE lines significantly increased jasmonic acid (JA) content, possibly by improving precursor α‐linolenic acid contributing to JA synthesis and by repressing JAR1 degrading JA. Exogenous JA significantly improved the cold tolerance of wheat plants. In summary, TaSnRK1α profoundly regulated cold stress, possibly through phosphorylating TaPAP6L to increase endogenous JA content of wheat plants.

Abstract In tauopathy conditions, such as Alzheimer's disease (AD), highly soluble and natively unfolded tau polymerizes into an insoluble filament; however, the mechanistic details of this process remain unclear. In the brains of AD patients, only a minor segment of tau forms β‐helix‐stacked protofilaments, while its flanking regions form disordered fuzzy coats. Here, it is demonstrated that the tau AD nucleation core (tau‐AC) sufficiently induced self‐aggregation and recruited full‐length tau to filaments. Unexpectedly, phospho‐mimetic forms of tau‐AC (at Ser324 or Ser356) show markedly reduced oligomerization and seeding propensities. Biophysical analysis reveal that the N‐terminus of tau‐AC facilitates the fibrillization kinetics as a nucleation motif, which becomes sterically shielded through phosphorylation‐induced conformational changes in tau‐AC. Tau‐AC oligomers are efficiently internalized into cells via endocytosis and induced endogenous tau aggregation. In primary hippocampal neurons, tau‐AC impaired axon initial segment plasticity upon chronic depolarization and is mislocalized to the somatodendritic compartments. Furthermore, it is observed significantly impaired memory retrieval in mice intrahippocampally injected with tau‐AC fibrils, which corresponds to the neuropathological staining and neuronal loss in the brain. These findings identify tau‐AC species as a key neuropathological driver in AD, suggesting novel strategies for therapeutic intervention.

Abstract Background Adolescent idiopathic scoliosis (AIS) is a scoliotic deformity of unknown etiology that occurs during adolescent development. Abnormal bone metabolism is closely related to AIS, but the cause is uncertain. Recent studies have shown that heat shock protein 27 (HSP27) and its phosphorylation (pHSP27) play important roles in bone metabolism. However, whether HSP27 and pHSP27 are involved in abnormal bone metabolism in AIS is unclear. Methods Osteoblasts (OBs) and bone marrow stem cells (BMSCs) were extracted from the facet joints and bone marrow of AIS patients and controls who underwent posterior spinal fusion surgery. The expression levels of HSP27 and pHSP27, as well as the expression levels of bone formation markers in OBs from AIS patients and controls, were examined by quantitative real‐time PCR (qRT–PCR) and Western blotting. The mineralization ability of OBs from AIS patients and controls was analyzed by alizarin red staining after osteogenic differentiation. Heat shock and thiolutin were used to increase the levels of pHSP27 in OBs, and the levels of bone formation markers were also investigated. In addition, the levels of pHSP27 and the bone formation ability of BMSCs from AIS patients and controls were investigated after heat shock treatment. Results Lower pHSP27 levels and impaired osteogenic differentiation abilities were observed in the OBs of AIS patients than in those of controls. Thiolutin increased HSP27 phosphorylation and increased the mRNA levels of SPP1 and ALPL in OBs from AIS patients. Heat shock treatment increased SPP1 and HSP27 mRNA expression, pHSP27 levels, OCN expression, and mineralization ability of both OBs and BMSCs from AIS patients. Conclusion Heat shock treatment and thiolutin can increase the levels of pHSP27 and further promote the bone formation of OBs and BMSCs from AIS patients. Therefore, decreased pHSP27 levels may be associated with abnormal bone metabolism in AIS patients.

TP53 gene abnormalities represent the most important biomarker in chronic lymphocytic leukemia (CLL). Altered protein modifications could also influence p53 function, even in the wild‐type protein. We assessed the impact of p53 protein phosphorylations on p53 functions as an alternative inactivation mechanism. We studied p53 phospho‐profiles induced by DNA‐damaging agents (fludarabine, doxorubicin) in 71 TP53‐intact primary CLL samples. Doxorubicin induced two distinct phospho‐profiles: profile I (heavily phosphorylated) and profile II (hypophosphorylated). Profile II samples were less capable of activating p53 target genes upon doxorubicin exposure, resembling TP53‐mutant samples at the transcriptomic level, whereas standard p53 signaling was triggered in profile I. ATM locus defects were more common in profile II. The samples also differed in the basal activity of the hypoxia pathway: the highest level was detected in TP53‐mutant samples, followed by profile II and profile I. Our study suggests that wild‐type TP53 CLL cells with less phosphorylated p53 show TP53‐mutant‐like behavior after DNA damage. p53 hypophosphorylation and the related lower ability to respond to DNA damage are linked to ATM locus defects and the higher basal activity of the hypoxia pathway.

Abstract Background Muscle atrophy, leading to muscular dysfunction and weakness, is an adverse outcome of sustained period of glucocorticoids usage. However, the molecular mechanism underlying this detrimental condition is currently unclear. Pyruvate dehydrogenase kinase 4 (PDK4), a central regulator of cellular energy metabolism, is highly expressed in skeletal muscle and has been implicated in the pathogenesis of several diseases. The current study was designed to investigated and delineate the role of PDK4 in the context of muscle atrophy, which could be identified as a potential therapeutic avenue to protect against dexamethasone‐induced muscle wasting. Methods The dexamethasone‐induced muscle atrophy in C2C12 myotubes was evaluated at the molecular level by expression of key genes and proteins involved in myogenesis, using immunoblotting and qPCR analyses. Muscle dysfunction was studied in vivo in wild‐type and PDK4 knockout mice treated with dexamethasone (25 mg/kg body weight, i.p., 10 days). Body weight, grip strength, muscle weight and muscle histology were assessed. The expression of myogenesis markers were analysed using qPCR, immunoblotting and immunoprecipitation. The study was extended to in vitro human skeletal muscle atrophy analysis. Results Knockdown of PDK4 was found to prevent glucocorticoid‐induced muscle atrophy and dysfunction in C2C12 myotubes, which was indicated by induction of myogenin (0.3271 ± 0.102 vs 2.163 ± 0.192, ****P

It is well known that the Kaiso protein (encoded by the ZBTB33 gene) is a transcription factor, and Kaiso–P120ctn [P120 catenin (CTNND1)] interaction increases the translocation of Kaiso from the nucleus into the cytoplasm. However, the regulatory mechanisms of Kaiso compartmentalisation are far from clear. Here, we reported that RAC‐alpha serine/threonine‐protein kinase (AKT1) could phosphorylate threonine residue 606 (T606) within the RSSTIP motif of Kaiso in the cytoplasm. The T606‐phosphorylated Kaiso (pT606‐Kaiso) could directly bind to 14‐3‐3 family proteins, and depletion of T606 phosphorylation by T606A mutation abolished most of the Kaiso–14‐3‐3 binding. In addition, the Kaiso–P120ctn interaction was essential for pT606‐Kaiso accumulation in the cytoplasm. Notably, enforced stratifin (14‐3‐3σ; SFN) overexpression could increase pT606‐Kaiso accumulation in the cytoplasm and de‐repress the transcription of Kaiso target gene cadherin 1 (CDH1), which is a tumour suppressor. Decreased amounts of both pT606‐Kaiso and CDH1 proteins were frequently observed in human gastric cancer tissues compared to paired normal controls. The mRNA levels of 14‐3‐3σ and Kaiso target gene CDH1 showed highly significant positive correlations in both human normal tissues and cancer cell lines by bioinformatics analyses. Furthermore, Kaiso T606A mutant (unable to be phosphorylated) significantly increased the migration and invasion of cancer cells in vitro and promoted the growth of these cells in vivo. In conclusion, Kaiso could be phosphorylated at T606 by AKT1 and pT606‐Kaiso accumulates in the cytoplasm through binding to 14‐3‐3/P120ctn, which de‐represses the Kaiso target gene CDH1 in normal tissues. Decreased Kaiso phosphorylation might contribute to the development of gastrointestinal cancer. The status of Kaiso phosphorylation is a determinant factor for the role of Kaiso in the development of cancer.

Abstract Histones are DNA‐binding basic proteins found in chromosomes. After the histone translation, its amino tail undergoes various modifications, such as methylation, acetylation, phosphorylation, ubiquitination, malonylation, propionylation, butyrylation, crotonylation, and lactylation, which together constitute the “histone code.” The relationship between their combination and biological function can be used as an important epigenetic marker. Methylation and demethylation of the same histone residue, acetylation and deacetylation, phosphorylation and dephosphorylation, and even methylation and acetylation between different histone residues cooperate or antagonize with each other, forming a complex network. Histone‐modifying enzymes, which cause numerous histone codes, have become a hot topic in the research on cancer therapeutic targets. Therefore, a thorough understanding of the role of histone post‐translational modifications (PTMs) in cell life activities is very important for preventing and treating human diseases. In this review, several most thoroughly studied and newly discovered histone PTMs are introduced. Furthermore, we focus on the histone‐modifying enzymes with carcinogenic potential, their abnormal modification sites in various tumors, and multiple essential molecular regulation mechanism. Finally, we summarize the missing areas of the current research and point out the direction of future research. We hope to provide a comprehensive understanding and promote further research in this field.

Abstract The ankle‐link complex (ALC) consists of USH2A, WHRN, PDZD7, and ADGRV1 and plays an important role in hair cell development. At present, its architectural organization and signaling role remain unclear. By establishing Adgrv1 Y6236fsX1 mutant mice as a model of the deafness‐associated human Y6244fsX1 mutation, the authors show here that the Y6236fsX1 mutation disrupts the interaction between adhesion G protein‐coupled receptor V subfamily member 1 (ADGRV1) and other ALC components, resulting in stereocilia disorganization and mechanoelectrical transduction (MET) deficits. Importantly, ADGRV1 inhibits WHRN phosphorylation through regional cAMP‐PKA signaling, which in turn regulates the ubiquitination and stability of USH2A via local signaling compartmentalization, whereas ADGRV1 Y6236fsX1 does not. Yeast two‐hybrid screening identified the E3 ligase WDSUB1 that binds to WHRN and regulates the ubiquitination of USH2A in a WHRN phosphorylation‐dependent manner. Further FlAsH‐BRET assay, NMR spectrometry, and mutagenesis analysis provided insights into the architectural organization of ALC and interaction motifs at single‐residue resolution. In conclusion, the present data suggest that ALC organization and accompanying local signal transduction play important roles in regulating the stability of the ALC.

Salt‐inducible kinase 2 (SIK2; also known as serine/threonine‐protein kinase SIK2) is overexpressed in several cancers and has been implicated in cancer progression. However, the mechanisms by which SIK2 regulates cancer cell motility, migration and metastasis in ovarian cancer have not been fully discovered. Here, we identify that SIK2 promotes ovarian cancer cell motility, migration and metastasis in vitro and in vivo. Mechanistically, SIK2 regulated cancer cell motility and migration by myosin light chain kinase, smooth muscle (MYLK)‐meditated phosphorylation of myosin light chain 2 (MYL2). SIK2 directly phosphorylated MYLK at Ser343 and activated its downstream effector MYL2, promoting ovarian cancer cell motility and metastasis. In addition, we found that adipocytes induced SIK2 phosphorylation at Ser358 and MYLK phosphorylation at Ser343, enhancing ovarian cancer cell motility. Moreover, SIK2 protein expression was positively correlated with the expression of MYLK‐pS343 in ovarian cancer cell lines and tissues. The co‐expression of SIK2 and MYLK‐pS343 was associated with reduced median overall survival in human ovarian cancer samples. Taken together, SIK2 positively regulates ovarian cancer motility, migration and metastasis, suggesting that SIK2 is a potential candidate for ovarian cancer treatment.

Abstract Status epilepticus (SE) remains a significant cause of morbidity and mortality and often is refractory to standard first‐line treatments. A rapid loss of synaptic inhibition and development of pharmacoresistance to benzodiazepines (BZDs) occurs early during SE, while NMDA and AMPA receptor antagonists remain effective treatments after BZDs have failed. Multimodal and subunit‐selective receptor trafficking within minutes to an hour of SE involves GABA‐A, NMDA, and AMPA receptors and contributes to shifts in the number and subunit composition of surface receptors with differential impacts on the physiology, pharmacology, and strength of GABAergic and glutamatergic currents at synaptic and extrasynaptic sites. During the first hour of SE, synaptic GABA‐A receptors containing γ2 subunits move to the cell interior while extrasynaptic GABA‐A receptors with δ subunits are preserved. Conversely, NMDA receptors containing N2B subunits are increased at synaptic and extrasynaptic sites, and homomeric GluA1 (“GluA2‐lacking”) calcium permeant AMPA receptor surface expression also is increased. Molecular mechanisms, largely driven by NMDA receptor or calcium permeant AMPA receptor activation early during circuit hyperactivity, regulate subunit‐specific interactions with proteins involved with synaptic scaffolding, adaptin‐AP2/clathrin‐dependent endocytosis, endoplasmic reticulum (ER) retention, and endosomal recycling. Reviewed here is how SE‐induced shifts in receptor subunit composition and surface representation increase the excitatory to inhibitory imbalance that sustains seizures and fuels excitotoxicity contributing to chronic sequela such as “spontaneous recurrent seizures” (SRS). A role for early multimodal therapy is suggested both for treatment of SE and for prevention of long‐term comorbidities.

Abstract Background The MYC oncoprotein, also known as the master regulator of genes, is a transcription factor that regulates numerous physiological processes, including cell cycle control, apoptosis, protein synthesis and cell adhesion, among others. MYC is overexpressed in approximately 70% of human cancers. Given its pervasive role in cancer biology, MYC down‐regulation has become an attractive cancer treatment strategy. Methods The CRISPR/Cas9 method was used to produce KO cell models. Western blot was used to analyzed the expressions of MYC and TATA‐binding proteinassociated factors 10 (TAF10) in cancer cells (MCF7, A549, HepG2 cells) Cell culture studies were performed to determine the mechanisms by which small molecules (Z363119456, Z363) affects MYC and TAF10 expressions and functions. Mouse studies were carried out to investigate the impact of Z363 regulation on tumor growth. Results Z363 activate Thyroid hormone Receptor‐interacting Protein 12 (TRIP12), which phosphorylates MYC at Thr58, resulting in MYC ubiquitination and degradation and thereby regulating MYC target genes. Importantly, TRIP12 also induces TAF10 degradation, which reduces MYC protein levels. TRIP12, an E3 ligase, controls MYC levels both directly and indirectly by inhibiting MYC or TAF10 activity. Conclusions In summary,these results demonstrate the anti‐cancer properties of Z363, a small molecule that is co‐regulated by TAF10 and MYC.

Objective: To assess the impact of the IFIT3/TBK1 signalling pathway in activating plasmacytoid dendritic cells (pDCs) and its role in the development of SSc., Methods: Utilized single-cell RNA sequencing (scRNA-seq) and high-throughput transcriptome RNA sequencing to reveal the differential abundance of pDCs and the role of the key gene IFIT3 in SSc. Conducted in vitro cell experiments to evaluate the effect of IFIT3/TBK1 signalling pathway intervention on pDC activation cytokine release and fibroblast function. Constructed an IFIT3 -/- mouse model using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing to assess the potential benefits of intervening in the IFIT3/TBK1 signalling pathway on skin and lung fibrosis in the SSc mouse model., Results: The IFIT3/TBK1 signalling pathway plays a crucial role in activating pDCs, with IFIT3 acting as an upstream regulator of TBK1. Intervention in the IFIT3/TBK1 signalling pathway can inhibit pDC activation cytokine release and impact fibroblast function. The IFIT3 -/- mouse model shows potential benefits of targeting the IFIT3/TBK1 signalling pathway in reducing skin and lung fibrosis in the SSc mouse model., Conclusion: This study provides new insights into potential therapeutic targets for SSc, highlighting the critical role of the IFIT3/TBK1 signalling pathway in SSc development., Highlights: This study elucidates the pivotal role of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc). This study identified the key regulatory gene involved in systemic sclerosis (SSc) as IFIT3. This study has found that IFIT3 functions as an upstream regulatory factor, activating TBK1. This study provides Evidence of the regulatory effects of the IFIT3/TBK1 pathway on plasmacytoid dendritic cells (pDCs). This study validated the therapeutic potential using the IFIT3 -/- mouse model., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Receptor tyrosine kinases exhibit ligand-induced activity and uptake into cells via endocytosis. In the case of epidermal growth factor (EGF) receptor (EGFR), the resulting endosomes are trafficked to the perinuclear region, where dephosphorylation of receptors occurs, which are subsequently directed to degradation. Traveling endosomes bearing phosphorylated EGFRs are subjected to the activity of cytoplasmic phosphatases as well as interactions with the endoplasmic reticulum (ER). The peri-nuclear region harbors ER-embedded phosphatases, a component of the EGFR-bearing endosome-ER contact site. The ER is also emerging as a central player in spatiotemporal control of endosomal motility, positioning, tubulation, and fission. Past studies strongly suggest that the physical interaction between the ER and endosomes forms a reaction "unit" for EGFR dephosphorylation. Independently, endosomes have been implicated to enable quantization of EGFR signals by modulation of the phosphorylation levels. Here, we review the distinct mechanisms by which endosomes form the logistical means for signal quantization and speculate on the role of the ER., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Abstract Background There is increasing evidence that circular RNAs (circRNAs) play a significant role in pathological processes including tumorigenesis. In contrast to exonic circRNAs, which are the most frequently reported circRNAs in cancer so far, the studies of intronic circRNAs have been greatly lagged behind. Here, we aimed to investigate the regulatory role of intronic circRNAs in head and neck squamous cell carcinoma (HNSCC). Methods We conducted whole‐transcriptome sequencing with four pairs of primary tumor tissues and adjacent normal tissues from HNSCC patients. Then, we characterized circGNG7 expression in HNSCC tissues and cell lines and explored its association with the prognosis of HNSCC patients. We also identified interactions between circGNG7 and functional proteins, which alter downstream signaling that regulate HNSCC progression. Results In this study, we identified a new intronic circRNA, circGNG7, and validated its functional roles in HNSCC progression. CircGNG7 was predominately localized to the cytoplasm, and its expression was downregulated in both HNSCC tissues andCAL27, CAL33, SCC4, SCC9, HN6, and HN30 cells. Low expression of circGNG7 was significantly correlated with poor prognosis in HNSCC patients. Consistent with this finding, overexpression of circGNG7 strongly inhibited tumor cell proliferation, colony formation, in vitro migration, and in vivo tumor growth. Mechanistically, the expression of circGNG7 in HNSCC cells was regulated by the transcription factor SMAD family member 4 (SMAD4). Importantly, we discovered that circGNG7 could bind to serine residues 78 and 82 of the functional heat shock protein 27 (HSP27), occupying its phosphorylation sites and hindering its phosphorylation, which reduced HSP27‐JNK/P38 mitogen‐activated protein kinase (MAPK) oncogenic signaling. Downregulation of circGNG7 expression in HNSCC increased HSP27‐JNK/P38 MAPK signaling and promoted tumor progression. Conclusions Our results revealed that a new intronic circRNA, circGNG7, functions as a strong tumor suppressor and that circGNG7/HSP27‐JNK/P38 MAPK signaling is a novel mechanism by which HNSCC progression can be controlled.

Histone modification is aberrantly regulated in cancer and generates an unbalanced state of gene transcription. VprBP, a recently identified kinase, phosphorylates histone H2A on threonine 120 (T120) and is involved in oncogenic transcriptional dysregulation; however, its specific role in colon cancer is undefined. Here, we show that VprBP is overexpressed in colon cancer and directly contributes to epigenetic gene silencing and cancer pathogenesis. Mechanistically, the observed function of VprBP is mediated through H2AT120 phosphorylation (H2AT120p)‐driven transcriptional repression of growth regulatory genes, resulting in a significantly higher proliferative capacity of colon cancer cells. Our preclinical studies using organoid and xenograft models demonstrate that treatment with the VprBP inhibitor B32B3 impairs colonic tumor growth by blocking H2AT120p and reactivating a transcriptional program resembling that of normal cells. Collectively, our work describes VprBP as a master kinase contributing to the development and progression of colon cancer, making it a new molecular target for novel therapeutic strategies.

NDR/LATS‐family protein kinases are conserved among eukaryotes. These protein kinases in yeast and animals phosphorylate specific targets and regulate the cell cycle. Arabidopsis thaliana has eight NDR/LATS‐family protein kinase genes (NDR1‐8), of which NDR2, NDR4, and NDR5 are involved in regulating pollen development. However, the functions of the other NDR/LATS‐family protein kinase genes in plants are unclear. Here, we show that three putative phosphorylation sites of an Arabidopsis basic leucine zipper transcription factor, VIP1, correspond to NDR/LATS‐family protein kinase phosphorylation motifs and that two of these three sites are phosphorylated by NDR2, NDR3, or NDR8 in vitro. Expression of NDR1‐8 was detected in various tissues. An NDR4 NDR6 NDR7 NDR8 quadruple mutation caused embryonic lethality These results suggest that different NDR/LATS‐family protein kinases in plants have distinct physiological roles.

Abstract Background Dysregulation of alternative splicing (AS) induced by serine/arginine‐rich proteins has recently been linked to cancer metastasis. Nonetheless, as a member of the serine/arginine‐rich protein family, the involvement of SRSF11 in colorectal cancer (CRC) is unknown. Methods The TCGA dataset and clinical samples were used to assess SRSF11 expression levels in CRC. For SRSF11, functional experiments were conducted both in vitro and in vivo. RNA‐seq technology was used to analyze and screen SRSF11‐triggered AS events, which were then confirmed by in vivo UV crosslinking and immunoprecipitation (CLIP) and mini‐gene reporter assays. Jalview software was used to determine the preferential binding motif with relation to exon skipping (ES) events. Furthermore, coimmunoprecipitation (Co‐IP) and Phospho‐tag SDS‐PAGE experiments were used to investigate PAK5‐mediated phosphorylation regulation on SRSF11, and in vitro kinase experiments validated the interaction. Results In CRC, SRSF11 was discovered to be overexpressed and associated with a poor prognosis. And SRSF11 played a pro‐metastatic role in vitro and in vivo. By screening SRSF11‐regulated AS events, we identified the binding motif of SRSF11‐triggered splicing‐switching of HSPA12A AS, which specifically regulated HSPA12A AS by directly binding to a motif in exon 2. Mechanistically, the HSPA12A transcript with exon 2 retention increased N‐cadherin expression by promoting RNA stability. Furthermore, the oncogenic kinase PAK5 phosphorylated SRSF11 at serine 287, protecting it from ubiquitination degradation. Conclusions SRSF11 exerts pro‐metastatic effects in CRC by inhibiting the AS of HSPA12A pre‐RNA. Our findings point to SRSF11‐regulated HSPA12A splicing as a novel relationship between SRSF11‐regulated splicing and CRC metastasis and suggest a PAK5/SRSF11/HSPA12A axis as a potential therapeutic target and prognostic biomarker in CRC.

Abstract Angiotensin II (Ang II)‐dependent stimulation of the AT1 receptor in proximal tubules increases sodium reabsorption and blood pressure. Reabsorption is driven by the Na,K‐pump that is acutely stimulated by Ang II, which requires phosphorylation of serine‐938 (S938). This site is present in humans and only known to phosphorylated by PKA. Yet, activation of AT1 decreases cAMP required to activate PKA and inhibiting PKA does not block Ang II‐dependent phosphorylation of S938. We tested the hypothesis that Ang II‐dependent activation is mediated via increased phosphorylation at S938 through a PI3K/AKT‐dependent pathway. Experiments were conducted using opossum kidney cells, a proximal tubule cell line, stably co‐expressing the AT1 receptor and either the wild‐type (α‐1.wild‐type) or an alanine substituted (α‐1.S938A) form of rat kidney Na,K‐pump. A 5‐min exposure to 10 pM Ang II significantly activated Na,K‐pump activity (56%) measured as short‐circuit current across polarized α‐1.wild‐type cells. Wortmannin, at a concentration that selectively inhibits PI3K, blocked that Ang II‐dependent activation. Ang II did not stimulate Na,K‐pump activity in α‐1.S938A cells. Ang II at 10 and 100 pM increased phosphorylation at S938 in α‐1.wild‐type cells measured in whole cell lysates. The increase was inhibited by wortmannin plus H‐89, an inhibitor of PKA, not by either alone. Ang II activated AKT inhibited by wortmannin, not H‐89. These data support our hypothesis and show that Ang II‐dependent phosphorylation at S938 stimulates Na,K‐pump activity and transcellular sodium transport.

Endothelial nitric oxide synthase (eNOS)‐derived nitric oxide (NO) plays a crucial role in maintaining vascular homeostasis. As a hallmark of eNOS activation, phosphorylation of eNOS at Ser1177 induced by activated protein kinase B (PKB/Akt) is pivotal for NO production. The complete activation of Akt requires its phosphorylation of both Thr308 and Ser473. However, which site plays the main role in regulating phosphorylation of eNOS Ser1177 is still controversial. The purpose of the present study is to explore the specific regulatory mechanism of phosphorylated Akt in eNOS activation. Inhibition of Akt Thr308 phosphorylation by a specific inhibitor or by siRNA in vitro led to a decrease in eNOS phosphorylation at Ser1177 and to lower NO concentration in the cell culture medium of HUVECs. However, inhibiting p‐Akt Ser473 had no effect on eNOS phosphorylation at Ser1177. Next, we administered mice with inhibitors to downregulate p‐Akt Ser473 or Thr308 activity. Along with the inhibition of p‐Akt Thr308, vascular p‐eNOS Ser1177 protein was simultaneously downregulated in parallel with a decrease in plasma NO concentration. Additionally, we cultured HUVECs at various temperature conditions (37, 22, and 4 °C). The results showed that p‐Akt Ser473 was gradually decreased in line with the reduction in temperature, accompanied by increased levels of p‐Akt Thr308 and p‐eNOS Ser1177. Taken together, our study indicates that the phosphorylation of Akt at Thr308, but not at Ser473, plays a more significant role in regulating p‐eNOS Ser1177 levels under physiological conditions.

Nonreceptor tyrosine kinase c‐Abl participates in several cellular processes by phosphorylating transcription factors or cofactors. c‐Abl binds and phosphorylates four‐and‐a‐half‐LIM‐only protein 2 (FHL2), but the identity of the phosphorylation sites and their contribution to cell cycle regulation is unclear. In this study, we demonstrate that c‐Abl highly phosphorylates FHL2 at Y97, Y176, Y217, and Y236 through mass spectrometry and tyrosine‐to‐phenylalanine (Y → F) mutant analysis. Proliferation was inhibited in cells expressing wild‐type (WT) FHL2 but not cells expressing the phosphorylation‐defective mutant FHL2(4YF). Moreover, FHL2 contributed to cell cycle arrest at G2/M induced by ionizing radiation (IR). FHL2 WT but not FHL2(4YF) rescued FHL2 function in FHL2‐depleted cells by causing IR‐induced G2/M arrest. These results demonstrate that c‐Abl regulates cell cycle progression by phosphorylating FHL2.

Abstract The marked overexpression of cyclin‐dependent kinase 5 (CDK5) or Notch1 receptor, which plays critical roles in pancreatic ductal adenocarcinoma (PDAC) development, has been detected in numerous PDAC cell lines and tissues. Although, a previous study has demonstrated that CDK5 inhibition disrupts Notch1 functions in human umbilical vein endothelial cells, the mechanism underlying Notch1 activation regulated by CDK5 remains unclear. Herein, we identified a physical interaction between CDK5 and Notch1 in PDAC cells, with the Notch1 peptide phosphorylated by CDK5/p25 kinase. CDK5 blockade resulted in the profound inhibition of Notch signaling. Accordingly, CDK5 inhibition sensitized PDAC cell proliferation and migration following Notch inhibition. In conclusion, CDK5 positively regulates Notch1 function via phosphorylation, which in turn promotes cell proliferation and migration. The combinational inhibition of CDK5 and Notch signaling may be an effective strategy in the treatment of PDAC.

Abstract Background Charcot–Marie–Tooth disease (CMT) is a hereditary monogenic peripheral nerve disease. Variants in the gene encoding myelin protein zero (MPZ) lead to CMT, and different variants have different clinical phenotypes. A variant site, namely, c.389A > G (p.Lys130Arg), in the MPZ gene has been found in Chinese people. The pathogenicity of this variant has been clarified through pedigrees, and peripheral blood‐related functional studies have been conducted. Method Whole‐exome sequencing and Sanger sequencing were used to detect the c.389A > G (p.Lys130Arg) variant in the MPZ gene in family members of the proband. Physical examination was performed in the case group to assess the clinical characteristics of MPZ site variants. The expression of MPZ and phosphorylated MPZ in the blood of 12 cases and 12 randomly selected controls was compared by RT–qPCR, Western blotting, and ELISA. Results The proband and 12 of her family members presented the AG genotype with different clinical manifestations. The expression of MPZ mRNA in the case group was increased compared with that in the control group, and the levels of MPZ and phosphorylated MPZ in peripheral blood were higher than those in normal controls. Conclusion The heterozygous genotype of the c.389A > G (p.Lys130Arg) variant in the MPZ gene mediated the increase in MPZ and phosphorylated MPZ levels in peripheral blood and was found to be involved with CMT.

Background and Purpose: This study was undertaken to compare the performance of plasma p-tau181 with that of [ 18 F]fluorodeoxyglucose (FDG) positron emission tomography (PET) in the identification of early biological Alzheimer disease (AD)., Methods: We included 533 cognitively impaired participants from the Alzheimer's Disease Neuroimaging Initiative. Participants underwent PET scans, biofluid collection, and cognitive tests. Receiver operating characteristic analyses were used to determine the diagnostic accuracy of plasma p-tau181 and [ 18 F]FDG-PET using clinical diagnosis and core AD biomarkers ([ 18 F]florbetapir-PET and cerebrospinal fluid [CSF] p-tau181) as reference standards. Differences in the diagnostic accuracy between plasma p-tau181 and [ 18 F]FDG-PET were determined by bootstrap-based tests. Correlations of [ 18 F]FDG-PET and plasma p-tau181 with CSF p-tau181, amyloid β (Aβ) PET, and cognitive performance were evaluated to compare associations between measurements., Results: We observed that both plasma p-tau181 and [ 18 F]FDG-PET identified individuals with positive AD biomarkers in CSF or on Aβ-PET. In the MCI group, plasma p-tau181 outperformed [ 18 F]FDG-PET in identifying AD measured by CSF (p = 0.0007) and by Aβ-PET (p = 0.001). We also observed that both plasma p-tau181 and [ 18 F]FDG-PET metabolism were associated with core AD biomarkers. However, [ 18 F]FDG-PET uptake was more closely associated with cognitive outcomes (Montreal Cognitive Assessment, Mini-Mental State Examination, Clinical Dementia Rating Sum of Boxes, and logical memory delayed recall, p < 0.001) than plasma p-tau181., Conclusions: Overall, although both plasma p-tau181 and [ 18 F]FDG-PET were associated with core AD biomarkers, plasma p-tau181 outperformed [ 18 F]FDG-PET in identifying individuals with early AD pathophysiology. Taken together, our study suggests that plasma p-tau181 may aid in detecting individuals with underlying early AD., (© 2024 The Authors. European Journal of Neurology published by John Wiley & Sons Ltd on behalf of European Academy of Neurology.)

Endothelin‐1 is a mitogenic peptide that activates several proliferation, survival, and invasiveness pathways. The effects of endothelin‐1 rely on its activation by endothelin‐converting enzyme‐1 (ECE1), which is expressed as four isoforms with different cytoplasmic N termini. Recently, isoform ECE1c has been suggested to have a role in cancer aggressiveness. The N terminus of ECE1c is phosphorylated by protein kinase CK2 (also known as casein kinase 2), and this enhances its stability and promotes invasiveness in colorectal cancer cells. However, it is not known how phosphorylation improves stability and why this is correlated with increased aggressiveness. We hypothesized that CK2 phosphorylation protects ECE1c from N‐terminal ubiquitination and, consequently, from proteasomal degradation. Here, we show that lysine 6 is the bona fide residue involved in ubiquitination of ECE1c and its mutation to arginine (ECE1cK6R) significantly impairs proteasomal degradation, thereby augmenting ECE1c stability, even in the presence of the CK2 inhibitor silmitasertib. Furthermore, colorectal cancer cells overexpressing ECE1cK6R displayed enhanced cancer stem cell (CSC) traits, including increased stemness gene expression, chemoresistance, self‐renewal, and colony formation and spheroid formation in vitro, as well as enhanced tumor growth and metastasis in vivo. These findings suggest that CK2‐dependent phosphorylation enhances ECE1c stability, promoting an increase in CSC‐like traits. Therefore, phospho‐ECE1c may be a biomarker of poor prognosis and a potential therapeutic target for colorectal cancer.

Abstract Signal transducer and activator of transcription 3 (STAT3) is a ubiquitous and pleiotropic transcription factor that plays essential roles in normal development, immunity, response to tissue damage and cancer. We have developed a Venus‐STAT3 bimolecular fluorescence complementation assay that allows the visualization and study of STAT3 dimerization and protein‐protein interactions in living cells. Inactivating mutations on residues susceptible to post‐translational modifications (PTMs) (K49R, K140R, K685R, Y705F and S727A) changed significantly the intracellular distribution of unstimulated STAT3 dimers when the dimers were formed by STAT3 molecules that carried different mutations (ie they were “asymmetric”). Some of these asymmetric dimers changed the proliferation rate of HeLa cells. Our results indicate that asymmetric PTMs on STAT3 dimers could constitute a new level of regulation of STAT3 signaling. We put forward these observations as a working hypothesis, since confirming the existence of asymmetric STAT3 homodimers in nature is extremely difficult, and our own experimental setup has technical limitations that we discuss. However, if our hypothesis is confirmed, its conceptual implications go far beyond STAT3, and could advance our understanding and control of signaling pathways.

Abstract Protein–protein interaction (PPI) plays a crucial role in most biological processes, including signal transduction and cell apoptosis. Importantly, the knowledge of PPIs can be useful for identification of multimeric protein complexes and elucidation of uncharacterized protein functions. Arabidopsis thaliana, the best‐characterized dicotyledonous plant, the steadily increasing amount of information on the levels of its proteome and signaling pathways is progressively enabling more researchers to construct models for cellular processes for the plant, which in turn encourages more experimental data to be generated. In this study, we performed an overview analysis of the 10 major organelles and their associated proteins of the dicotyledonous model plant Arabidopsis thaliana via PPI network, and found that PPI may play an important role in organelle communication. Further, multilocation proteins, especially phosphorylation‐related multilocation proteins, can function as a “needle and thread” via PPIs and play an important role in organelle communication. Similar results were obtained in a monocotyledonous model crop, rice. Furthermore, we provide a research strategy for multilocation proteins by LOPIT technique, proteomics, and bioinformatics analysis and also describe their potential role in the field of plant science. The results provide a new view that the phosphorylation‐related multilocation proteins play an important role in organelle communication and provide new insight into PPIs and novel directions for proteomic research. The research of phosphorylation‐related multilocation proteins may promote the development of organelle communication and provide an important theoretical basis for plant responses to external stress.

Sodium and potassium channels, especially Nav1.5 and Kir2.1, play key roles in the formation of action potentials in cardiomyocytes. These channels interact with, and are regulated by, synapse-associated protein 97 (SAP97). However, the regulatory role of SAP97 in myocyte remains incompletely understood. Here, we investigate the function of SAP97 phosphorylation in the regulation of Nav1.5 and Kir2.1 channel complexes and the upstream regulation of SAP97. We found that SAP97 is phosphorylated by casein kinase II (CK2) in vitro. In addition, transfection of casein kinase 2 interacting protein-1 (CKIP-1) into cardiomyocytes to drive CK2 from the nucleus to the cytoplasm, increased SAP97 phosphorylation and Nav1.5 and Kir2.1 current activity. These findings demonstrated that CKIP-1 modulates the subcellular translocation of CK2, which regulates Nav1.5 and Kir2.1 channel complex formation and activity in cardiomyocytes., (© 2024 Wiley Periodicals LLC.)

Background: The induction of mitochondrial quality control (MQC) mechanisms is essential for the re-establishment of mitochondrial homeostasis and cellular bioenergetics during periods of stress. Although MQC activation has cardioprotective effects in various cardiovascular diseases, its precise role and regulatory mechanisms in alcoholic cardiomyopathy (ACM) remain incompletely understood., Methods: We explored whether two mitochondria-related proteins, phosphoglycerate mutase 5 (Pgam5) and prohibitin 2 (Phb2), influence MQC in male mice during ACM., Results: Myocardial Pgam5 expression was upregulated in a male mouse model of ACM. Notably, following ACM induction, heart dysfunction was markedly reversed in male cardiomyocyte-specific Pgam5 knockout (Pgam5 cKO ) mice. Meanwhile, in alcohol-treated male mouse-derived neonatal cardiomyocytes, Pgam5 depletion preserved cell survival and restored mitochondrial dynamics, mitophagy, mitochondrial biogenesis and the mitochondrial unfolded protein response (mtUPR). We further found that in alcohol-treated cardiomyocyte, Pgam5 binds Phb2 and induces its dephosphorylation at Ser91. Alternative transduction of phospho-mimetic (Phb2 S91D ) and phospho-defective (Phb2 S9A ) Phb2 mutants attenuated and enhanced, respectively, alcohol-related mitochondrial dysfunction in cardiomyocytes. Moreover, transgenic male mice expressing Phb2 S91D were resistant to alcohol-induced heart dysfunction., Conclusions: We conclude that ACM-induced Pgam5 upregulation results in Pgam5-dependent Phb2 S91 dephosphorylation, leading to MQC destabilisation and mitochondrial dysfunction in heart. Therefore, modulating the Pgam5/Phb2 interaction could potentially offer a novel therapeutic strategy for ACM in male mice., Highlights: Pgam5 knockout attenuates alcohol-induced cardiac histopathology and heart dysfunction in male mice. Pgam5 KO reduces alcohol-induced myocardial inflammation, lipid peroxidation and metabolic dysfunction in male mice. Pgam5 depletion protects mitochondrial function in alcohol-exposed male mouse cardiomyocytes. Pgam5 depletion normalises MQC in ACM. EtOH impairs MQC through inducing Phb2 dephosphorylation at Ser91. Pgam5 interacts with Phb2 and induces Phb2 dephosphorylation. Transgenic mice expressing a Ser91 phospho-mimetic Phb2 mutant are resistant to ACM., (© 2024 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Background: Lymph node metastasis (LNM) is the primary mode of metastasis in gastric cancer (GC). However, the precise mechanisms underlying this process remain elusive. Tumor cells necessitate lipid metabolic reprogramming to facilitate metastasis, yet the role of lipoprotein lipase (LPL), a pivotal enzyme involved in exogenous lipid uptake, remains uncertain in tumor metastasis. Therefore, the aim of this study was to investigate the presence of lipid metabolic reprogramming during LNM of GC as well as the role of LPL in this process., Methods: Intracellular lipid levels were quantified using oil red O staining, BODIPY 493/503 staining, and flow cytometry. Lipidomics analysis was employed to identify alterations in intracellular lipid composition following LPL knockdown. Protein expression levels were assessed through immunohistochemistry, Western blotting, and enzyme-linked immunosorbent assays. The mouse popliteal LNM model was utilized to investigate differences in LNM. Immunoprecipitation and mass spectrometry were employed to examine protein associations. In vitro phosphorylation assays and Phos-tag sodium dodecyl-sulfate polyacrylamide gel electrophoresis assays were conducted to detect angiopoietin-like protein 4 (ANGPTL4) phosphorylation., Results: We identified that an elevated intracellular lipid level represents a crucial characteristic of node-positive (N+) GC and further demonstrated that a high-fat diet can expedite LNM. LPL was found to be significantly overexpressed in N+ GC tissues and shown to facilitate LNM by mediating dietary lipid uptake within GC cells. Leptin, an obesity-related hormone, intercepted the effect exerted by ANGPTL4/Furin on LPL cleavage. Circulating leptin binding to the leptin receptor could induce the activation of inositol-requiring enzyme-1 (IRE1) kinase, leading to the phosphorylation of ANGPTL4 at the serine 30 residue and subsequently reducing its binding affinity with LPL. Moreover, our research revealed that LPL disrupted lipid homeostasis by elevating intracellular levels of arachidonic acid, which then triggered the cyclooxygenase-2/prostaglandin E2 (PGE2) pathway, thereby promoting tumor lymphangiogenesis., Conclusions: Leptin-induced phosphorylation of ANGPTL4 facilitates LPL-mediated lipid uptake and consequently stimulates the production of PGE2, ultimately facilitating LNM in GC., (© 2024 The Author(s). Cancer Communications published by John Wiley & Sons Australia, Ltd on behalf of Sun Yat‐sen University Cancer Center.)

Idiopathic pulmonary fibrosis treatments are limited, often with severe side effects, highlighting the need for novel options. Taraxerone has diverse biomedical properties, but its mechanism remains unclear. This study investigates taraxerone's impact and the mechanisms involved in bleomycin-induced pulmonary fibrosis in mice. After establishing a pulmonary fibrosis mouse model, taraxerone was intraperitoneally injected continuously for 14-28 days. The in vivo antifibrotic and antioxidative stress effects of taraxerone were assessed. In vitro, the influence of taraxerone on transforming growth factor-β1-induced myofibroblast transformation and oxidative stress was investigated. Subsequently, quantitative polymerase chain reaction screened the histone deacetylase and Sirtuin family, and taraxerone's effects on SIRT1 were assessed. After SIRT1 siRNA treatment, changes in myofibroblast transformation and antioxidant capacity in response to taraxerone were observed. Acetylation and phosphorylation levels of Smad3 were evaluated. We also examined the binding levels of SIRT1 with Pho-Smad3 and Smad3, as well as the nuclear localization of Smad2/3. EX527 confirmed SIRT1's in vivo action in response to taraxerone. In vitro experiments suggested that taraxerone inhibited myofibroblast differentiation by activating SIRT1 and reducing oxidative stress. We also observed a new interaction between SIRT1 and the Smad complex. Taraxerone activates SIRT1, enabling it to bind directly to Smad3. This leads to reduced Smad complex phosphorylation and limited nuclear translocation. As a result, the transcription of fibrotic factors is reduced. In vivo validation confirms taraxerone's SIRT1-mediated antifibrotic effectiveness. This suggests that targeting SIRT1-mediated inhibition of myofibroblast differentiation could be a key strategy in taraxerone-based therapy for pulmonary fibrosis., (© 2024 John Wiley & Sons Ltd.)

Background: The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway regulates a variety of cellular processes. A major activation event in this pathway involves the phosphorylation of a tyrosine of STAT, converting unphosphorylated STAT (uSTAT) to phosphorylated STAT (pSTAT), an active transcription factor. In a noncanonical role, uSTAT contributes to the maintenance of heterochromatin stability. As such, an increase in pSTAT concurrently reduces uSTAT, resulting in heterochromatin loss, as observed in Drosophila somatic tissues. Paradoxically, an opposing phenomenon occurs in Drosophila male germline stem cells (GSCs), where the JAK/STAT pathway remains persistently active due to a continuous supply of ligands. Here, computational simulations were employed to dissect JAK/STAT pathway activation under different cellular contexts, mimicking somatic and germline cells. In these simulations, ordinary differential equations were leveraged to replicate the chemical reactions governing JAK/STAT signaling under different conditions., Results: The outcomes indicate that transient ligand stimulation, typical in somatic tissues, led to a momentary reduction in uSTAT levels. Conversely, sustained ligand stimulation, a characteristic feature of the GSC niche, resulted in elevated uSTAT levels at equilibrium., Conclusion: The simulation suggests that the duration of ligand exposure could explain the observed opposite effects of JAK/STAT activation on heterochromatin in somatic versus GSCs., (© 2023 American Association for Anatomy.)

Aberrant cellular signaling pathways are a hallmark of cancer and other diseases. One of the most important signaling mechanisms involves protein phosphorylation/dephosphorylation. Protein phosphorylation is catalyzed by protein kinases, and over 530 protein kinases have been identified in the human genome. Aberrant kinase activity is one of the drivers of tumorigenesis and cancer progression and results in altered phosphorylation abundance of downstream substrates. Upstream kinase activity can be inferred from the global collection of phosphorylated substrates. Mass spectrometry-based phosphoproteomic experiments nowadays routinely allow identification and quantitation of >10k phosphosites per biological sample. This substrate phosphorylation footprint can be used to infer upstream kinase activities using tools like Kinase Substrate Enrichment Analysis (KSEA), Posttranslational Modification Substrate Enrichment Analysis (PTM-SEA), and Integrative Inferred Kinase Activity Analysis (INKA). Since the topic of kinase activity inference is very active with many new approaches reported in the past 3 years, we would like to give an overview of the field. In this review, an inventory of kinase activity inference tools, their underlying algorithms, statistical frameworks, kinase-substrate databases, and user-friendliness is presented. The most widely-used tools are compared in-depth. Subsequently, recent applications of the tools are described focusing on clinical tissues and hematological samples. Two main application areas for kinase activity inference tools can be discerned. (1) Maximal biological insights can be obtained from large data sets with group comparisons using multiple complementary tools (e.g., PTM-SEA and KSEA or INKA). (2) In the oncology context where personalized treatment requires analysis of single samples, INKA for example, has emerged as tool that can prioritize actionable kinases for targeted inhibition., (© 2022 The Authors. Mass Spectrometry Reviews published by John Wiley & Sons Ltd.)

Background and Purpose: Affinity-based, selective orthosteric ligands for the muscarinic acetylcholine receptors (mAChRs) are difficult to develop due to high sequence homology across the five subtypes. Selectivity can also be achieved via the selective activation of a particular subtype or signalling pathway. Promisingly, a prior study identified compounds 6A and 7A as functionally selective and G i biased compounds at the M 2 mAChR. Here, we have investigated the activation of individual G protein subfamilies and the downstream signalling profiles of 6A and 7A at the M 2 mAChR., Experimental Approach: G protein activation was measured with the TRUPATH assay in M 2 mAChR FlpIn CHO cells. Activity in downstream signalling pathways was determined using the cAMP CAMYEL BRET sensor and assay of ERK 1/2 phosphorylation., Key Results: M 2 mAChRs coupled to Gɑ i1 , Gɑ oA and Gɑ s , but not Gɑ q , in response to canonical orthosteric agonists. Compounds 6A and 7A did not elicit any G protein activation, cAMP inhibition or stimulation, or ERK 1/2 phosphorylation. Instead, a Schild analysis indicates a competitive, antagonistic interaction of compounds 6A and 7A with ACh in the Gɑ i1 activation assay. Overexpression of the M 2 mAChR may suggest an expression-dependent activation profile of compounds 6A and 7A., Conclusions and Implications: These data confirm that the M 2 mAChR preferentially couples to Gɑ i/o and to a lesser extent to Gɑ s in response to canonical orthosteric ligands. However, this study was not able to detect Gɑ i bias of compounds 6A and 7A, highlighting the importance of cellular background when classifying new ligands., Linked Articles: This article is part of a themed issue Therapeutic Targeting of G Protein-Coupled Receptors: hot topics from the Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists 2021 Virtual Annual Scientific Meeting. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.14/issuetoc., (© 2022 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Tyrosine phosphorylation is a crucial posttranslational modification that is involved in various aspects of cell biology and often has functions in cancers. It is necessary not only to identify the specific phosphorylation sites but also to quantify their phosphorylation levels under specific pathophysiological conditions. Because of its high sensitivity and accuracy, mass spectrometry (MS) has been widely used to identify endogenous and synthetic phosphotyrosine proteins/peptides across a range of biological systems. However, phosphotyrosine-containing proteins occur in extremely low abundance and they degrade easily, severely challenging the application of MS. This review highlights the advances in both quantitative analysis procedures and enrichment approaches to tyrosine phosphorylation before MS analysis and reviews the differences among phosphorylation, sulfation, and nitration of tyrosine residues in proteins. In-depth insights into tyrosine phosphorylation in a wide variety of biological systems will offer a deep understanding of how signal transduction regulates cellular physiology and the development of tyrosine phosphorylation-related drugs as cancer therapeutics., (© 2023 John Wiley & Sons Ltd.)

Abstract Chemoresistance remains a major obstacle to successful cancer therapy, especially for advanced cancers. It used to be recognised as a stable outcome resulting from genetic changes. However, recent studies showed that chemoresistance can also be unstable and reversible with the involvement of non‐genetic alterations. In the present study, we found that activating transcription factor 4 (ATF4) is downregulated in chemoresistant gastric cancer cells. The over‐expression of ATF4 reversed chemoresistance by activating CHOP transcription to enhance drug‐induced apoptosis, and vice versa. Moreover, casein kinase 1 delta (CK1δ) was identified as the kinase responsible for ATF4‐S219 phosphorylation, which triggered βTrCP‐mediated ATF4 polyubiquitination to promote its proteasomal degradation subsequently. Interestingly, drug withdrawal gradually restored chemosensitivity as well as ATF4 expression in chemoresistant cells, highlighting the dependence of dynamic drug resistance on ATF4 protein expression. In line with these findings, the inhibition of ATF4 protein degradation by CK1δ or proteasome inhibitors overcame chemoresistance both in vitro and in vivo. Taken together, these results indicate that CK1δ stimulates βTrCP‐dependent ATF4 polyubiquitination and subsequent proteasomal degradation to promote chemoresistance in gastric cancer. Stabilisation of the ATF4 protein with bortezomib (BTZ), an anticancer drug that inhibits proteasomal degradation, might be a rational strategy to improve chemotherapeutic efficacy in gastric cancer.

Abstract Because there is no effective treatment for late‐stage prostate cancer (PCa) at this moment, identifying novel targets for therapy of advanced PCa is urgently needed. A new network‐based systems biology approach, XDeath, is developed to detect crosstalk of signaling pathways associated with PCa progression. This unique integrated network merges gene causal regulation networks and protein‐protein interactions to identify novel co‐targets for PCa treatment. The results show that polo‐like kinase 1 (Plk1) and DNA methyltransferase 3A (DNMT3a)‐related signaling pathways are robustly enhanced during PCa progression and together they regulate autophagy as a common death mode. Mechanistically, it is shown that Plk1 phosphorylation of DNMT3a leads to its degradation in mitosis and that DNMT3a represses Plk1 transcription to inhibit autophagy in interphase, suggesting a negative feedback loop between these two proteins. Finally, a combination of the DNMT inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza) with inhibition of Plk1 suppresses PCa synergistically.

Abstract The MM500 meta‐study aims to establish a knowledge basis of the tumor proteome to serve as a complement to genome and transcriptome studies. Somatic mutations and their effect on the transcriptome have been extensively characterized in melanoma. However, the effects of these genetic changes on the proteomic landscape and the impact on cellular processes in melanoma remain poorly understood. In this study, the quantitative mass‐spectrometry‐based proteomic analysis is interfaced with pathological tumor characterization, and associated with clinical data. The melanoma proteome landscape, obtained by the analysis of 505 well‐annotated melanoma tumor samples, is defined based on almost 16 000 proteins, including mutated proteoforms of driver genes. More than 50 million MS/MS spectra were analyzed, resulting in approximately 13,6 million peptide spectrum matches (PSMs). Altogether 13 176 protein‐coding genes, represented by 366 172 peptides, in addition to 52 000 phosphorylation sites, and 4 400 acetylation sites were successfully annotated. This data covers 65% and 74% of the predicted and identified human proteome, respectively. A high degree of correlation (Pearson, up to 0.54) with the melanoma transcriptome of the TCGA repository, with an overlap of 12 751 gene products, was found. Mapping of the expressed proteins with quantitation, spatiotemporal localization, mutations, splice isoforms, and PTM variants was proven not to be predicted by genome sequencing alone. The melanoma tumor molecular map was complemented by analysis of blood protein expression, including data on proteins regulated after immunotherapy. By adding these key proteomic pillars, the MM500 study expands the knowledge on melanoma disease.

Malaria, a disease caused by infection with parasites of the genus Plasmodium, causes millions of deaths worldwide annually. Of the five Plasmodium species that can infect humans, Plasmodium falciparum causes the most serious parasitic infection. The emergence of drug resistance and the ineffectiveness of old therapeutic regimes against malaria mean there is an urgent need to better understand the basic biology of the malaria parasite. Previously, we have reported the presence of parasite‐specific helicases identified through genome‐wide analysis of the P. falciparum (3D7) strain. Helicases are involved in various biological pathways in addition to nucleic acid metabolism, making them an important target of study. Here, we report the detailed biochemical characterization of P. falciparum parasite‐specific helicase 1 (PfPSH1) and the effect of phosphorylation on its biochemical activities. The C‐terminal of PfPSH1 (PfPSH1C) containing all conserved domains was used for biochemical characterization. PfPSH1C exhibits DNA‐ or ribonucleic acid (RNA)‐stimulated ATPase activity, and it can unwind DNA and RNA duplex substrates. It shows bipolar directionality because it can translocate in both (3′–5′ and 5′–3′) directions. PfPSH1 is mainly localized to the cytoplasm during early stages (including ring and trophozoite stages of intraerythrocytic development), but at late stages, it is partially located in the cytoplasm. The biochemical activities of PfPSH1 are upregulated after phosphorylation with PKC. The detailed biochemical characterization of PfPSH1 will help us understand its functional role in the parasite and pave the way for future studies.