Imaging the Raf-MEK-ERK Signaling Cascade in Living Cells.

Conventional biochemical methods for studying cellular signaling cascades have relied on destructive cell disruption. In contrast, the live cell imaging of fluorescent-tagged transfected proteins offers a non-invasive approach to understanding signal transduction events. One strategy involves monitoring the phosphorylation-dependent shuttling of a fluorescent-labeled kinase between the nucleus and cytoplasm using nuclear localization, export signals, or both. In this paper, we introduce a simple method to visualize intracellular signal transduction in live cells by exploring the translocation properties of PKC from the cytoplasm to the membrane. We fused bait protein to PKC, allowing the bait (RFP-labeled) and target (GFP-labeled) proteins to co-translocate from the cytoplasm to the membrane. However, in non-interacting protein pairs, only the bait protein was translocated to the plasma membrane. To verify our approach, we examined the Raf-MEK-ERK signaling cascade (ERK pathway). We successfully visualized direct Raf1/MEK2 interaction and the KSR1-containing ternary complex (Raf1/MEK2/KSR1). However, the interaction between MEK and ERK was dependent on the presence of the KSR1 scaffold protein under our experimental conditions.

Potential Prognostic Role of Protein Kinase D Isoforms in Head and Neck Cancers.

Head and neck squamous cell carcinomas (HNSCC) are among the most common malignancies in men worldwide. Nevertheless, their clinical management is hampered by the limited availability of reliable predictive and prognostic biomarkers. Protein kinase D (PKD) isoforms contribute to major cellular processes. However, their potential role in HNSCC has not been studied systematically, which is the focus of this study. A total of 63 therapy-naive patients with squamous cell carcinoma were consecutively enrolled. Tissue microarray duplicate cores from each case were tested in situ for PKD1, PKD2, and PKD3 expression using immunohistochemistry, and the results were correlated with clinicopathological parameters. We found a high frequency of PKD1/PKD2 positive cases in oropharyngeal and PKD2 positive cases in laryngeal localizations. Only high PKD2 levels were statistically linked to elevated tumor grades, more advanced TNM (3-4) tumor stages, and p16INK4a expression, while elevated PKD3 levels were associated with favorable disease-specific survival. Both PKD2 and PKD3 have been proposed to promote tumor cell proliferation, migration/invasion, and angiogenesis. However, the role of PKD3 was elusive in some cancers. Our findings suggest that testing for PKD isotypes with immunohistochemistry may support the diagnostic estimation of tumor progression and prognosis in HNSCC with a potential therapeutic relevance.

Protein Kinase C and Matrix Metalloproteinases Expression Using Phorbol Myristate Acetate in Degenerative Intervertebral Disc Cells.

Background: Degeneration of nucleus pulposus (NP) cells involves multiple factors. The relationship between the canonical Wnt/ß-catenin signaling pathway and matrix metalloproteinases (MMPs) is important in cellular senescence. Protein kinase C (PKC), an intermediate of the non-canonical Wnt pathway stimulated by phorbol myristate acetate (PMA), possibly prevents NP cell senescence, although not yet demonstrated in human-based studies. This study aimed to investigate the effect of PMA stimulation on the non-canonical and canonical Wnt pathways and MMP expression in human NP cells to ascertain its inhibitory effects on the senescence of NP cells. Methods: Human disc tissues of Pfirrmann grades 1 and 2 were collected from patients during spinal surgery and subsequently cultured. Protein and ribonucleic acid (RNA) were isolated from NP cells treated with PMA (400 nM) for 24 hours. Expression of MMP1, MMP13, tissue inhibitor of matrix metalloproteinase 1 (TIMP1), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), transient receptor potential vanilloid 4 (TRPV4), interleukin-6 (IL-6), and ß-catenin were detected using western blot analysis. Messenger RNA (mRNA) expression of type II collagen and glycosaminoglycan (GAG) were analyzed using reverse transcription polymerase chain reaction. IL-6 and prostaglandin E2 (PGE2) levels were measured using enzyme-linked immunosorbent assay. Results: Expression of PKC-δ (intermediate of the non-canonical Wnt pathway) and ß-catenin (intermediate of the canonical Wnt pathway) was increased by PMA treatment. The mRNA levels of type II collagen and GAG increased; however, their protein levels were not altered. PMA treatment increased the expression of MMP1, TIMP1, ADAMTS5, IL-6, PGE2, and TRPV4; however, the expression of MMP13 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was unaltered. Conclusions: PMA activated PKC-δ, affecting the non-canonical Wnt pathway; however, its effect on ß-catenin in the canonical Wnt pathway was limited. ß-catenin activation through the TRPV4 channel led to increased expression of MMP1 and ADAMTS5 and that of IL-6 and PGE2 owing to NF-κB expression. Consequently, the degeneration of NP cells was not prevented.

Protein kinase D1 in myeloid lineage cells contributes to the accumulation of CXCR3+CCR6+ nonconventional Th1 cells in the lungs and potentiates hypersensitivity pneumonitis caused by S. rectivirgula.

Introduction: Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that leads to granuloma formation. We previously found that activation of protein kinase D1 (PKD1) in the lungs following exposures to Saccharopolyspora rectivirgula contributes to the acute pulmonary inflammation, IL-17A expression in the lungs, and development of HP. In the present study, we investigated whether PKD1 in myeloid-lineage cells affects the pathogenic course of the S. rectivirgula-induced HP. Methods: Mice were exposed intranasally to S. rectivirgula once or 3 times/week for 3 weeks. The protein and mRNA expression levels of cytokines/chemokines were detected by enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. Flow cytometry was used to detect the different types of immune cells and the levels of surface proteins. Lung tissue sections were stained with hematoxylin and eosin, digital images were captured, and immune cells influx into the interstitial lung tissue were detected. Results: Compared to control PKD1-sufficient mice, mice with PKD1 deficiency in myeloid-lineage cells (PKD1mKO) showed significantly suppressed expression of TNFα, IFNγ, IL-6, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula. Substantially reduced levels of alveolitis and granuloma formation were observed in the PKD1mKO mice repeatedly exposed to S. rectivirgula for 3 weeks. In addition, expression levels of the Th1/Th17 polarizing cytokines and chemokines such as IFNγ, IL-17A, CXCL9, CXCL10, CXCL11, and CCL20 in lungs were significantly reduced in the PKD1mKO mice repeatedly exposed to S. rectivirgula. Moreover, accumulation of CXCR3+CCR6+ nonconventional Th1 in the lungs were significantly reduced in PKD1mKO mice repeatedly exposed to S. rectivirgula. Discussion: Our results demonstrate that PKD1 in myeloid-lineage cells plays an essential role in the development and progress of HP caused by repeated exposure to S. rectivirgula by contributing Th1/Th17 polarizing proinflammatory responses, alveolitis, and accumulation of pathogenic nonconventional Th1 cells in the lungs.

Mycobacterium avium inhibits protein kinase C and MARCKS phosphorylation in human cystic fibrosis and non-cystic fibrosis cells.

In cystic fibrosis (CF), there is abnormal translocation and function of the cystic fibrosis transmembrane conductance regulator (CFTR) and an upregulation of the epithelial sodium channel (ENaC). This leads to hyperabsorption of sodium and fluid from the airway, dehydrated mucus, and an increased risk of respiratory infections. In this study, we performed a proteomic assessment of differentially regulated proteins from CF and non-CF small airway epithelial cells (SAEC) that are sensitive to Mycobacterium avium. CF SAEC and normal non-CF SAEC were infected with M. avium before the cells were harvested for protein. Protein kinase C (PKC) activity was greater in the CF cells compared to the non-CF cells, but the activity was significantly attenuated in both cell types after infection with M. avium compared to vehicle. Western blot and densitometric analysis showed a significant increase in cathepsin B protein expression in M. avium infected CF cells. Myristoylated alanine rich C-kinase substrate (MARCKS) protein was one of several differentially expressed proteins between the groups that was identified by mass spectrometry-based proteomics. Total MARCKS protein expression was greater in CF cells compared to non-CF cells. Phosphorylation of MARCKS at serine 163 was also greater in CF cells compared to non-CF cells after treating both groups of cells with M. avium. Taken together, MARCKS protein is upregulated in CF cells and there is decreased phosphorylation of the protein due to a decrease in PKC activity and presumably increased cathepsin B mediated proteolysis of the protein after M. avium infection.

Topological Heterogeneity of Protein Kinase C Modulators in Human T-Cells Resolved with In-Cell Dynamic Nuclear Polarization NMR Spectroscopy.

Phorbol ester analogs are a promising class of anticancer therapeutics and HIV latency reversing agents that interact with cellular membranes to recruit and activate protein kinase C (PKC) isoforms. However, it is unclear how these esters interact with membranes and how this might correlate with the biological activity of different phorbol ester analogs. Here, we have employed dynamic nuclear polarization (DNP) NMR to characterize phorbol esters in a native cellular context. The enhanced NMR sensitivity afforded by DNP and cryogenic operation reveals topological heterogeneity of 13C-21,22-phorbol-myristate-acetate (PMA) within T cells utilizing 13C-13C correlation and double quantum filtered NMR spectroscopy. We demonstrate the detection of therapeutically relevant amounts of PMA in T cells down to an upper limit of ∼60.0 pmol per million cells and identify PMA to be primarily localized in cellular membranes. Furthermore, we observe distinct 13C-21,22-PMA chemical shifts under DNP conditions in cells compared to model membrane samples and homogenized cell membranes, that cannot be accounted for by differences in conformation. We provide evidence for distinct membrane topologies of 13C-21,22-PMA in cell membranes that are consistent with shallow binding modes. This is the first of its kind in-cell DNP characterization of small molecules dissolved in the membranes of living cells, establishing in-cell DNP-NMR as an important method for the characterization of drug-membrane interactions within the context of the complex heterogeneous environment of intact cellular membranes. This work sets the stage for the identification of the in-cell structural interactions that govern the biological activity of phorbol esters.

An Ingenane-Type Diterpene from Euphorbia kansui Promoted Cell Apoptosis and Macrophage Polarization via the Regulation of PKC Signaling Pathways.

Euphorbia kansui, a toxic Chinese medicine used for more than 2000 years, has the effect of "purging water to promote drinking" and "reducing swelling and dispersing modules". Diterpenes and triterpenes are the main bioactive components of E. kansui. Among them, ingenane-type diterpenes have multiple biological activities as a protein kinase C δ (PKC-δ) activator, which have previously been shown to promote anti-proliferative and pro-apoptotic effects in several human cancer cell lines. However, the activation of PKC subsequently promoted the survival of macrophages. Recently, we found that 13-hydroxyingenol-3-(2,3-dimethylbutanoate)-13-dodecanoate (compound A) from E. kansui showed dual bioactivity, including the inhibition of tumor-cell-line proliferation and regulation of macrophage polarization. This study identifies the possible mechanism of compound A in regulating the polarization state of macrophages, by regulating PKC-δ-extracellular signal regulated kinases (ERK) signaling pathways to exert anti-tumor immunity effects in vitro, which might provide a new treatment method from the perspective of immune cell regulation.

Scar matrix drives Piezo1 mediated stromal inflammation leading to placenta accreta spectrum.

Scar tissue formation is a hallmark of wound repair in adults and can chronically affect tissue architecture and function. To understand the general phenomena, we sought to explore scar-driven imbalance in tissue homeostasis caused by a common, and standardized surgical procedure, the uterine scar due to cesarean surgery. Deep uterine scar is associated with a rapidly increasing condition in pregnant women, placenta accreta spectrum (PAS), characterized by aggressive trophoblast invasion into the uterus, frequently necessitating hysterectomy at parturition. We created a model of uterine scar, recapitulating PAS-like invasive phenotype, showing that scar matrix activates mechanosensitive ion channel, Piezo1, through glycolysis-fueled cellular contraction. Piezo1 activation increases intracellular calcium activity and Protein kinase C activation, leading to NF-κB nuclear translocation, and MafG stabilization. This inflammatory transformation of decidua leads to production of IL-8 and G-CSF, chemotactically recruiting invading trophoblasts towards scar, initiating PAS. Our study demonstrates aberrant mechanics of scar disturbs stroma-epithelia homeostasis in placentation, with implications in cancer dissemination.

GDNF facilitates cognitive function recovery following neonatal surgical-induced learning and memory impairment via activation of the RET pathway and modulation of downstream effectors PKMζ and Kalirin in rats.

OBJECTIVE: The aim of this study is to elucidate the underlying mechanism through which glial cell line-derived neurotrophic factor (GDNF) improves cognitive deficits in adults resulting from neonatal surgical interventions. METHODS: Newborn Sprague-Dawley rats, regardless of gender, were randomly allocated into seven groups on postnatal day 7 as follows (n=15): (1) Control group (not subjected to anesthesia, surgery, or any pharmaceutical interventions); (2) GDNF group (received intracerebroventricular injection of GDNF); (3) Surgery group (underwent right carotid artery exposure under anesthesia with 3 % sevoflurane); (4) Surgery plus GDNF group; (5) Surgery plus GDNF and type II JAK inhibitor NVP-BBT594 (BBT594) group (administered intraperitoneal injection of BBT594); (6) BBT group; and (7) Surgery plus BBT group. Starting from postnatal day 33, all rats underwent Barnes maze and fear conditioning tests, followed by decapitation under sevoflurane anesthesia for subsequent analyses. The left hemibrains underwent Golgi staining, while the right hemibrains were used for hippocampal protein extraction to assess Protein kinase Mζ (PKMζ) and Kalirin expression through western blotting. RESULTS: GDNF demonstrated a mitigating effect on spatial learning and memory impairment, as well as context-related fear memory impairment, reductions in dendritic total lengths, and spinal density within the hippocampus induced by surgical intervention. Notably, all of these ameliorative effects of GDNF were reversed upon administration of the RET inhibitor BBT594. Additionally, GDNF alleviated the downregulation of protein expression of PKMζ and Kalirin in the hippocampus of rats subjected to surgery, subsequently reversed by BBT594. CONCLUSION: The effective impact of GDNF on learning and memory impairment caused by surgical intervention appears to be mediated through the RET pathway. Moreover, GDNF may exert its influence by upregulating the expression of PKMζ and Kalirin, consequently enhancing the development of dendrites and dendritic spines.

Role of NOX1 and NOX5 in protein kinase C/reactive oxygen species­mediated MMP­9 activation and invasion in MCF­7 breast cancer cells.

NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF­7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)­9 were assessed by reverse transcription­quantitative PCR and western blotting, and the activity of MMP­9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12­O­tetradecanoylphorbol­13­acetate (TPA)­induced MMP­9 expression and invasion of MCF­7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA­induced MMP­9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA­induced MMP­9 expression and invasion in MCF­7 cells. NOX1­ and NOX5­specific small interfering RNAs attenuated TPA­induced MMP­9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA­induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA­induced intracellular ROS levels, MMP­9 expression and NOX activity in MCF­7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA­induced MMP­9 expression and invasion of MCF­7 breast cancer cells. Furthermore, the present study indicated that TPA­induced MMP­9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti­invasive effects in breast cancer.

Genetic polymorphism in untranslated regions of PRKCZ influences mRNA structure, stability and binding sites.

BACKGROUND: Variations in untranslated regions (UTR) alter regulatory pathways impacting phenotype, disease onset, and course of disease. Protein kinase C Zeta (PRKCZ), a serine-threonine kinase, is implicated in cardiovascular, neurological and oncological disorders. Due to limited research on PRKCZ, this study aimed to investigate the impact of UTR genetic variants' on binding sites for transcription factors and miRNA. RNA secondary structure, eQTLs, and variation tolerance analysis were also part of the study. METHODS: The data related to PRKCZ gene variants was downloaded from the Ensembl genome browser, COSMIC and gnomAD. The RegulomeDB database was used to assess the functional impact of 5' UTR and 3'UTR variants. The analysis of the transcription binding sites (TFBS) was done through the Alibaba tool, and the Kyoto Encyclopaedia of Genes and Genomes (KEGG) was employed to identify pathways associated with PRKCZ. To predict the effect of variants on microRNA binding sites, PolymiRTS was utilized for 3' UTR variants, and the SNPinfo tool was used for 5' UTR variants. RESULTS: The results obtained indicated that a total of 24 variants present in the 3' UTR and 25 variants present in the 5' UTR were most detrimental. TFBS analysis revealed that 5' UTR variants added YY1, repressor, and Oct1, whereas 3' UTR variants added AP-2alpha, AhR, Da, GR, and USF binding sites. The study predicted TFs that influenced PRKCZ expression. RNA secondary structure analysis showed that eight 5' UTR and six 3' UTR altered the RNA structure by either removal or addition of the stem-loop. The microRNA binding site analysis highlighted that seven 3' UTR and one 5' UTR variant altered the conserved site and also created new binding sites. eQTLs analysis showed that one variant was associated with PRKCZ expression in the lung and thyroid. The variation tolerance analysis revealed that PRKCZ was an intolerant gene. CONCLUSION: This study laid the groundwork for future studies aimed at targeting PRKCZ as a therapeutic target.

Phenylephrine Enhances the Mitogenic Effect of S-Allyl-L-cysteine on Primary Cultured Hepatocytes through Protein Kinase C-Induced B-Raf Phosphorylation.

The co-mitogenic effects of the α1-adrenoceptor agonist phenylephrine on S-allyl-L-cysteine (SAC)-induced hepatocyte proliferation were examined in primary cultures of adult rat hepatocytes. The combination of phenylephrine (10-10-10-6 M) and SAC (10-6 M) exhibited a significant dose-dependent increase in the number of hepatocyte nuclei and viable cells compared to SAC alone. This combination also increased the progression of hepatocyte nuclei into the S-phase. The potentiating effect of phenylephrine on SAC-induced cell proliferation was counteracted by prazosin (an α1-adrenergic receptor antagonist) and GF109203X (selective protein kinase C (PKC) inhibitor). In addition, PMA (direct PKC activator) potentiated the proliferative effects of SAC similarly to phenylephrine. In essence, these findings suggest that PKC activity plays a crucial role in enhancing SAC-induced cell proliferation. Moreover, the effects of phenylephrine on SAC-induced Ras activity, Raf phosphorylation, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation were investigated. Phenylephrine (or PMA) in combination with SAC did not augment Ras activity, but further increased ERK2 phosphorylation and its upstream B-Raf phosphorylation. These results indicate that PKC activation, triggered by stimulating adrenergic α1 receptors, further amplifies SAC-induced cell proliferation through enhanced ERK2 phosphorylation via increased B-Raf-specific phosphorylation in primary cultured hepatocytes.

Protein kinase C iota promotes glycolysis via PI3K/AKT/mTOR signalling in high grade serous ovarian cancer.

BACKGROUND: Epithelial ovarian cancer, especially high grade serous ovarian cancer (HGSOC) is by far, the most lethal gynecological malignancy with poor prognosis and high relapse rate. Despite of availability of several therapeutic interventions including poly-ADP ribose polymerase (PARP) inhibitors, HGSOC remains unmanageable and identification of early detection biomarkers and therapeutic targets for this lethal malady is highly warranted. Aberrant expression of protein kinase C iota (PKCί) is implicated in many cellular and physiological functions involved in tumorigenesis including cell proliferation and cell cycle deregulation. METHODS AND RESULTS: Two high grade serous ovarian cancer cells SKOV3 and COV362 were employed in this study. PKCί was genetically knocked down or pharmacologically inhibited and several functional and biochemical assays were performed. We report that PKCί is overexpressed in HGSOC cells and patient tissue samples with a significant prognostic value. Pharmacological inhibition of PKCί by Na-aurothiomalate or its shRNA-mediated genetic knockdown suppressed HGSOC cell proliferation, EMT and induced apoptosis. Moreover, PKCί positively regulated GLUT1 and several other glycolytic genes including HK1, HK2, PGK1, ENO1 and LDHA to promote elevated glucose uptake and glycolysis in HGSOC cells. Mechanistically, PKCί drove glycolysis via PI3K/AKT/mTOR signalling. Na-aurothiomalate and highly selective, dual PI3K/mTOR inhibitor dactolisib could serve as novel anti-glycolytic drugs in HGSOC. CONCLUSION: Taken together, our results indicate PKCί/PI3K/AKT/mTOR signalling cascade could be a novel therapeutic target in a lethal pathology like HGSOC.

Phosphorylation mapping of laminin γ1-chain: Kinases, functional interaction sequences, and phosphorylationinterfering cancer mutations.

We computationally predicted all phosphorylation sites in the sequence of the human laminin γ1-chain (LAMC1), and computationally identified, for the first time, all kinases for experimentally observed phosphorylated residues of the LAMC1 and all missense deleterious LAMC1 mutations found in different cancer types that interfere with LAMC1 phosphorylation. Also, we mapped the above data to all the biologically functional interaction sequences of the LAMC1. Five kinases (CKII, GPCRK1, PKA, PKC, and CKI) are most enriched for LAMC1 phosphorylation, and the significance of ecto-kinases in this process was emphasized. PKA and PKC targeted more residues inside and close to functional interaction sequences compared with other kinases and in the functional interaction sequence RPESFAIYKRTR. Most phosphorylation-interfering mutations were found in cutaneous melanoma and uterine endometrioid carcinoma. The mutation R255H interfered with the experimentally observed phosphorylation of LAMC1 inside the functional interaction sequence TDIRVTLNRLNTF, while the mutations S181Y and S213Y interfered with the experimentally observed phosphorylation of LAMC1 outside the functional interaction sequences. Mutations R359C,H, R589H, R657C,H, R663I,G, and T1207 interfered with the predicted phosphorylation inside or close to the functional interaction sequences, whereas other mutations interfered outside. PKA- and PKC-predicted phosphorylation was mostly interfered with by mutations inside functional interaction sequences. Phosphorylation- interfering mutations and functional interaction sequences were suggested to promote specific cancer types or cancer progression in general.

CREB-regulated transcription during glycogen synthesis in astrocytes.

Glycogen storage, conversion and utilization in astrocytes play an important role in brain energy metabolism. The conversion of glycogen to lactate through glycolysis occurs through the coordinated activities of various enzymes and inhibition of this process can impair different brain processes including formation of long-lasting memories. To replenish depleted glycogen stores, astrocytes undergo glycogen synthesis, a cellular process that has been shown to require transcription and translation during specific stimulation paradigms. However, the detail nuclear signaling mechanisms and transcriptional regulation during glycogen synthesis in astrocytes remains to be explored. In this report, we study the molecular mechanisms of vasoactive intestinal peptide (VIP)-induced glycogen synthesis in astrocytes. VIP is a potent neuropeptide that triggers glycogenolysis followed by glycogen synthesis in astrocytes. We show evidence that VIP-induced glycogen synthesis requires CREB-mediated transcription that is calcium dependent and requires conventional Protein Kinase C but not Protein Kinase A. In parallel to CREB activation, we demonstrate that VIP also triggers nuclear accumulation of the CREB coactivator CRTC2 in astrocytic nuclei. Transcriptome profiles of VIP-induced astrocytes identified robust CREB transcription, including a subset of genes linked to glucose and glycogen metabolism. Finally, we demonstrate that VIP-induced glycogen synthesis shares similar as well as distinct molecular signatures with glucose-induced glycogen synthesis, including the requirement of CREB-mediated transcription. Overall, our data demonstrates the importance of CREB-mediated transcription in astrocytes during stimulus-driven glycogenesis.

Downregulation of tRF-Cys-GCA-029 by hyperglycemia promotes tumorigenesis and glycolysis of diabetic breast cancer through upregulating PRKCG translation.

BACKGROUND: Diabetes mellitus (DM) affects up to one-third of breast cancer (BC) patients. Patients with co-existing BC and DM (BC-DM) have worsened BC prognosis. Nevertheless, the molecular mechanisms orchestrating BC-DM prognosis remain poorly understood. tRNA-derived fragments (tRFs) have been shown to regulate cancer progression. However, the biological role of tRFs in BC-DM has not been explored. METHODS: tRF levels in tumor tissues and cells were detected by tRF sequencing and qRT-PCR. The effects of tRF on BC cell malignancy were assessed under euglycemic and hyperglycemic conditions in vitro. Metabolic changes were assessed by lactate, pyruvate, and extracellular acidification rate (ECAR) assays. Diabetic animal model was used to evaluate the impacts of tRF on BC tumor growth. RNA-sequencing (RNA-seq), qRT-PCR, Western blot, polysome profiling, luciferase reporter assay, and rescue experiments were performed to explore the regulatory mechanisms of tRF in BC-DM. RESULTS: We identified that tRF-Cys-GCA-029 was downregulated in BC-DM tissues and under hyperglycemia conditions in BC cells. Functionally, downregulation of tRF-Cys-GCA-029 promoted BC cell proliferation and migration in a glucose level-dependent manner. tRF-Cys-GCA-029 knockdown also enhanced glycolysis metabolism in BC cells, indicated by increasing lactate/pyruvate production and ECAR levels. Notably, injection of tRF-Cys-GCA-029 mimic significantly suppressed BC tumor growth in diabetic-mice. Mechanistically, tRF-Cys-GCA-029 regulated BC cell malignancy and glycolysis via interacting with PRKCG in two ways: binding to the coding sequence (CDS) of PRKCG mRNA to regulate its transcription and altering polysomal PRKCG mRNA expression to modify its translation. CONCLUSIONS: Hyperglycemia-downregulated tRF-Cys-GCA-029 enhances the malignancy and glycolysis of BC cells. tRF-Cys-GCA-029-PRKCG-glycolysis axis may be a potential therapeutic target against BC-DM.

Molecular insights into the regulatory landscape of PKC-related kinase-2 (PRK2/PKN2) using targeted small compounds.

The PKC-related kinases (PRKs, also termed PKNs) are important in cell migration, cancer, hepatitis C infection, and nutrient sensing. They belong to a group of protein kinases called AGC kinases that share common features like a C-terminal extension to the catalytic domain comprising a hydrophobic motif. PRKs are regulated by N-terminal domains, a pseudosubstrate sequence, Rho-binding domains, and a C2 domain involved in inhibition and dimerization, while Rho and lipids are activators. We investigated the allosteric regulation of PRK2 and its interaction with its upstream kinase PDK1 using a chemical biology approach. We confirmed the phosphoinositide-dependent protein kinase 1 (PDK1)-interacting fragment (PIF)-mediated docking interaction of PRK2 with PDK1 and showed that this interaction can be modulated allosterically. We showed that the polypeptide PIFtide and a small compound binding to the PIF-pocket of PRK2 were allosteric activators, by displacing the pseudosubstrate PKL region from the active site. In addition, a small compound binding to the PIF-pocket allosterically inhibited the catalytic activity of PRK2. Together, we confirmed the docking interaction and allostery between PRK2 and PDK1 and described an allosteric communication between the PIF-pocket and the active site of PRK2, both modulating the conformation of the ATP-binding site and the pseudosubstrate PKL-binding site. Our study highlights the allosteric modulation of the activity and the conformation of PRK2 in addition to the existence of at least two different complexes between PRK2 and its upstream kinase PDK1. Finally, the study highlights the potential for developing allosteric drugs to modulate PRK2 kinase conformations and catalytic activity.

Extraction optimization and reactivity of 7α-acetoxy-6ß-hydroxyroyleanone and ability of its derivatives to modulate PKC isoforms.

Protein kinase C is a family of kinases that play important roles in carcinogenesis. Medicinal plants from Plectranthus spp. (Lamiaceae) are a well-known source of interesting abietanes, such as 7α-acetoxy-6ß-hydroxyroyleanone (Roy). This study aimed to extract and isolate Roy from P. grandidentatus Gürke, comparing two extraction methods (CO2 supercritical and ultrasound-assisted acetonic extraction), and design new royleanone derivatives for PKC modulation focusing on breast cancer therapy. The concentration of Roy in the extracts was determined by HPLC-DAD. The supercritical extraction method yielded 3.6% w/w, with the presence of 42.7 µg mg-1 of Roy (yield of 0.13%), while ultrasound-assisted acetonic extraction yielded 2.3% w/w, with the presence of 55.2 µg mg-1 of Roy (yield of 0.15%). The reactivity of Roy was investigated aiming at synthetizing new ester derivatives through standard benzoylation and esterification reactions. The benzoylated (Roy-12-Bz) and acetylated (Roy-12-Ac) derivatives in the C12 position were consistently prepared with overall good yields (33-86%). These results indicate the 12-OH position as the most reactive for esterification, affording derivatives under mild conditions. The reported di-benzoylated (RoyBz) and di-acetylated (RoyAc) derivatives were also synthesized after increasing the temperature (50 °C), reaction time, and using an excess of reagents. The cytotoxic potential of Roy and its derivatives was assessed against breast cancer cell lines, with RoyBz emerging as the most promising compound. Derivatization at position C-12 did not offer advantages over di-esterification at positions C-12 and C-6 or over the parent compound Roy and the presence of aromatic groups favored cytotoxicity. Evaluation of royleanones as PKC-α, ßI, δ, ε, and ζ activators revealed DeRoy's efficacy across all isoforms, while RoyPr showed promising activation of PKC-δ but not PKC-ζ, highlighting the influence of slight structural changes on isoform selectivity. Molecular docking analysis emphasized the importance of microenvironmental factors in isoform specificity, underscoring the complexity of PKC modulation and the need for further exploration.

EPAC1 and 2 inhibit K+ currents via PLC/PKC and NOS/PKG pathways in rat ventricular cardiomyocytes.

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2'-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.

Evaluating anticancer activity of emodin by enhancing antioxidant activities and affecting PKC/ADAMTS4 pathway in thioacetamide-induced hepatocellular carcinoma in rats.

Emodin is a naturally occurring anthraquinone derivative with a wide range of pharmacological activities, including neuroprotective and anti-inflammatory activities. We aim to assess the anticancer activity of emodin against hepatocellular carcinoma (HCC) in rat models using the proliferation, invasion, and angiogenesis biomarkers. After induction of HCC, assessment of the liver impairment and the histopathology of liver sections were investigated. Hepatic expression of both mRNA and protein of the oxidative stress biomarkers, HO-1, Nrf2; the mitogenic activation biomarkers, ERK5, PKCδ; the tissue destruction biomarker, ADAMTS4; the tissue homeostasis biomarker, aggregan; the cellular fibrinolytic biomarker, MMP3; and of the cellular angiogenesis biomarker, VEGF were measured. Emodin increased the survival percentage and reduced the number of hepatic nodules compared to the HCC group. Besides, emodin reduced the elevated expression of both mRNA and proteins of all PKC, ERK5, ADAMTS4, MMP3, and VEGF compared with the HCC group. On the other hand, emodin increased the expression of mRNA and proteins of Nrf2, HO-1, and aggrecan compared with the HCC group. Therefore, emodin is a promising anticancer agent against HCC preventing the cancer prognosis and infiltration. It works through many mechanisms of action, such as blocking oxidative stress, proliferation, invasion, and angiogenesis.