Substrate For Protein Kinase Research Articles

Inhibition of MARCKS phosphorylation attenuates of dendritic cell migration in a murine model of acute asthma

BackgroundMARCKS (myristoylated alanine-rich C kinase substrates) serves as a substrate for protein kinase C, residing in the plasma membrane while acts as an actin filament crosslinking protein. This investigation aims to elucidate phosphorylated MARCKS (p-MARCKS) levels and activity in allergic asthma patients and explore the therapeutic potential of peptide inhibitors targeting p-MARCKS in an acute mouse model of allergic asthma. MethodsImmunohistochemistry and histology staining were employed on lung tissue slides to evaluate p-MARCKS expression and allergic asthma symptoms. Airway resistance was measured using invasive whole-body plethysmography. Flow cytometry detected lung dendritic cell migration, and migration/maturation assays were conducted on isolated murine bone marrow-derived dendritic cells (BM-DCs). ResultsElevated p-MARCKS expression was observed in both human asthmatic tissues and animal models immunized with ovalbumin or Alternaria alternata. Remarkably, asthmatic individuals showed elevated high p-MARCKS expression in lung tissues. Intraperitoneal injection of the peptide MPS, targeting the MARCKS phosphorylation site domain, before allergen challenged, effectively suppressed MARCKS phosphorylation in murine lung tissues. MPS inhibited both in vivo and in vitro migration and maturation of dendritic cells (BM-DCs) and reduced Th2-related lymphocyte activation in bronchoalveolar lavage fluid (BALF). MPS pretreatment additionally suppressed all symptoms associated with allergic airway asthma, including a reduction in inflammatory cell influx, airway mucous cell metaplasia, and airway hyperreactivity. ConclusionThese findings suggest that phosphorylated MARCKS occurs in asthmatic lung tissue, and the inhibition of MARCKS phosphorylation by the MPS peptide reduces dendritic cell migration and Th2-related lymphocytes in the lungs in a murine model of acute asthma.

DNA-encoded probe-based assay for profiling plant kinase activities.

Elucidating kinase-substrate relationships is pivotal for deciphering cellular signaling mechanisms, yet it remains challenging due to the complexity of kinase networks. Herein, we report the development of a versatile DNA-based kinase assay platform for high-throughput profiling of plant protein kinase activities and substrate preferences. Our approach employs DNA-linked peptide substrates, facilitating quantitative and specific kinase activity detection through next-generation DNA sequencing. Leveraging DNA barcodes as quantitative readouts, our approach establishes a high-throughput, sensitive, and specific platform for dissecting kinase-substrate networks in plants, representing a powerful tool for elucidating signaling mechanisms in plants.

TBC1D1 is an energy-responsive polarization regulator of macrophages via governing ROS production in obesity.

Energy status is linked to the production of reactive oxygen species (ROS) in macrophages, which is elevated in obesity. However, it is unclear how ROS production is upregulated in macrophages in response to energy overload for mediating the development of obesity. Here, we show that the Rab-GTPase activating protein (RabGAP) TBC1D1, a substrate of the energy sensor AMP-activated protein kinase (AMPK), is a critical regulator of macrophage ROS production and consequent adipose inflammation for obesity development. TBC1D1 deletion decreases, whereas an energy overload-mimetic non-phosphorylatable TBC1D1S231A mutation increases, ROS production and M1-like polarization in macrophages. Mechanistically, TBC1D1 and its downstream target Rab8a form an energy-responsive complex with NOX2 for ROS generation. Transplantation of TBC1D1S231A bone marrow aggravates diet-induced obesity whereas treatment with an ultra-stable TtSOD for removal of ROS selectively in macrophages alleviates both TBC1D1S231A mutation- and diet-induced obesity. Our findings therefore have implications for drug discovery to combat obesity.

Phosphodiesterases 4B and 4D Differentially Regulate cAMP Signaling in Calcium Handling Microdomains of Mouse Hearts.

The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.

Brain-specific serine/threonine-protein kinase 1 is a substrate of protein kinase C epsilon involved in sex-specific ethanol and anxiety phenotypes.

Protein kinase C epsilon (PKCε) regulates behavioural responses to ethanol and plays a role in anxiety-like behaviour, but knowledge is limited on downstream substrates of PKCε that contribute to these behaviours. We recently identified brain-specific serine/threonine-protein kinase 1 (BRSK1) as a substrate of PKCε. Here, we test the hypothesis that BRSK1 mediates responses to ethanol and anxiety-like behaviours that are also PKCε dependent. We used in vitro kinase assays to further validate BRSK1 as a substrate of PKCε and used Brsk1-/- mice to assess the role of BRSK1 in ethanol- and anxiety-related behaviours and in physiological responses to ethanol. We found that BRSK1 is phosphorylated by PKCε at a residue identified in a chemical genetic screen of PKCε substrates in mouse brain. Like Prkce-/- mice, male and female Brsk1-/- mice were more sensitive than wild-type to the acute sedative-hypnotic effect of alcohol. Unlike Prkce-/- mice, Brsk1-/- mice responded like wild-type to ataxic doses of ethanol. Although in Prkce-/- mice ethanol consumption and reward are reduced in both sexes, they were reduced only in female Brsk1-/- mice. Ex vivo slice electrophysiology revealed that ethanol-induced facilitation of GABA release in the central amygdala was absent in male Brsk1-/- mice similar to findings in male Prkce-/- mice. Collectively, these results indicate that BRSK1 is a target of PKCε that mediates some PKCε-dependent responses to ethanol in a sex-specific manner and plays a role distinct from PKCε in anxiety-like behaviour.

A resource database for protein kinase substrate sequence-preference motifs based on large-scale mass spectrometry data

BackgroundProtein phosphorylation is one of the most prevalent posttranslational modifications involved in molecular control of cellular processes, and is mediated by over 520 protein kinases in humans and other mammals. Identification of the protein kinases responsible for phosphorylation events is key to understanding signaling pathways. Unbiased phosphoproteomics experiments have generated a wealth of data that can be used to identify protein kinase targets and their preferred substrate sequences.MethodsThis study utilized prior data from mass spectrometry-based studies identifying sites of protein phosphorylation after in vitro incubation of protein mixtures with recombinant protein kinases. PTM-Logo software was used with these data to generate position-dependent Shannon information matrices and sequence motif ‘logos’. Webpages were constructed for facile access to logos for each kinase and a new stand-alone application was written in Python that uses the position-dependent Shannon information matrices to identify kinases most likely to phosphorylate a particular phosphorylation site.ResultsA database of kinase substrate target preference logos allows browsing, searching, or downloading target motif data for each protein kinase (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/). These logos were combined with phylogenetic analysis of protein kinase catalytic sequences to reveal substrate preference patterns specific to particular groups of kinases (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinaseTree.html). A stand-alone program, KinasePredictor, is provided (https://esbl.nhlbi.nih.gov/Databases/Kinase_Logos/KinasePredictor.html). It takes as input, amino-acid sequences surrounding a given phosphorylation site and generates a ranked list of protein kinases most likely to phosphorylate that site.ConclusionsThis study provides three new resources for protein kinase characterization. It provides a tool for prediction of kinase-substrate interactions, which in combination with other types of data (co-localization, etc.), can predict which kinases are likely responsible for a given phosphorylation event in a given tissue.-q3N7pjxaL8wVBVRsJ9wBTVideo

Stimulation of soluble guanylyl cyclase in erythrocytes induces export of cGMP and cardioprotection in type 2 diabetes

Abstract Background Red blood cells (RBCs) are known to export cardioprotective nitric oxide (NO) bioactivity. Although, RBCs contain a functional soluble guanylyl cyclase (sGC), the signalling behind the export of NO bioactivity remains unclear. Impairment of NO signalling in RBCs has been suggested to contribute to augmented cardiac ischemia-reperfusion injury in type 2 diabetes (T2D). We hypothesized that increased NO signaling by stimulation of sGC in RBCs protects from myocardial ischemia-reperfusion injury by release of a cardioprotective signal. Purpose To investigate whether stimulation of sGC, the intracellular NO receptor, in RBCs from T2D patients induces export of a cardioprotective factor and to determine its nature. Methods RBCs were collected from patients with T2D and from gender- and age-matched healthy subjects and incubated with different pharmacological compounds affecting the NO-signalling pathway. The RBCs or their supernatant were then administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure and infarct size were measured. ELISA and immunofluorescence were used to determine levels of cyclic guanosine monophosphate (cGMP) and substrate of protein kinase G activation. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85-23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results RBCs from T2D patients impaired post-ischemic cardiac function and increased infarct size compared to RBCs from healthy subjects, and these effects were prevented by pre-incubation of the RBCs with the sGC stimulator CYR715. Administration of the supernatant from RBCs incubated with the sGC stimulator resulted in similar cardioprotection, indicating release of a cardioprotective factor from the RBCs (Figure 1A and B). The cardioprotection was abolished by inhibition of transport of cGMP with MK571, an inhibitor of cyclic nucleotide transport. Extracellular cGMP levels in the supernatant from RBCs were increased following sGC stimulation, and administration of exogenous cGMP induced cardioprotection (Figure 1C and D). Expression of protein kinase G-dependent phosphorylated vasodilator-specific phosphoprotein in cardiomyocytes was increased following administration of supernatant from RBCs incubated with the sGC stimulator (Figure 1E). Conclusions This study shows that stimulation of sGC in RBCs leads to release of a cardioprotective mediator, identified as cGMP, that activates cardiac protein kinase G and protects from ischemia-reperfusion injury. It further identifies RBC sGC as a novel therapeutic target to prevent cardiovascular injury in T2D.Figure 1

Commentary on: The actin bundling activity of ITPKA mainly accounts for its migration-promoting effect in lung cancer cells.

1,4,5-triphosphate 3-kinase A (ITPKA) was first described and characterized by Irvine et al. in 1986 and cloned by Takazawa et al. in 1990. It is one of the components of the Ca2+ and calmodulin signaling pathway and a substrate for cAMP-dependent kinase (PKA) and protein kinase C (PKC), and is mainly involved in the regulation of intracellular inositol polyphosphate signaling molecules. Through a series of studies, Sabine's team has found that ITPKA expression was up-regulated in a variety of cancer cells, and silencing ITPKA inhibited while overexpressing ITPKA promoted cancer cell migration in vitro and metastasis in vivo. The latest research from Sabine's team has demonstrated that in H1299 lung cancer cells, the mechanism by which ITPKA promoted migration and invasion was predominantly depending on the ability of binding to F-actin, which will induce cancer cells to form a tight flexible actin networks. Small molecule compounds targeting the IP3 kinase activity of ITPKA protein may only inhibit the migration and invasion of cancer cells caused by the enhanced ITPKA kinase activity under ATP stimulation, but not the cytoskeletal remodeling caused by the binding of ITPKA protein to F-actin and the driven migration and invasion of cancer cells. Therefore, targeted therapeutic strategy focusing on blocking the binding of ITPKA to F-actin is indispensable when designing the inhibitors targeting ITPKA protein.

Deciphering the regulatory network of the NAC transcription factor FvRIF, a key regulator of strawberry (Fragaria vesca) fruit ripening.

The NAC transcription factor ripening inducing factor (RIF) was previously reported to be necessary for the ripening of octoploid strawberry (Fragaria × ananassa) fruit, but the mechanistic basis of RIF-mediated transcriptional regulation and how RIF activity is modulated remains elusive. Here, we show that FvRIF in diploid strawberry, Fragaria vesca, is a key regulator in the control of fruit ripening and that knockout mutations of FvRIF result in a complete block of fruit ripening. DNA affinity purification sequencing coupled with transcriptome deep sequencing suggests that 2,080 genes are direct targets of FvRIF-mediated regulation, including those related to various aspects of fruit ripening. We provide evidence that FvRIF modulates anthocyanin biosynthesis and fruit softening by directly regulating the related core genes. Moreover, we demonstrate that FvRIF interacts with and serves as a substrate of MAP kinase 6 (FvMAPK6), which regulates the transcriptional activation function of FvRIF by phosphorylating FvRIF at Thr-310. Our findings uncover the FvRIF-mediated transcriptional regulatory network in controlling strawberry fruit ripening and highlight the physiological significance of phosphorylation modification on FvRIF activity in ripening.

Novel Functional Features of cGMP Substrate Proteins IRAG1 and IRAG2.

The inositol triphosphate-associated proteins IRAG1 and IRAG2 are cGMP kinase substrate proteins that regulate intracellular Ca2+. Previously, IRAG1 was discovered as a 125 kDa membrane protein at the endoplasmic reticulum, which is associated with the intracellular Ca2+ channel IP3R-I and the PKGIβ and inhibits IP3R-I upon PKGIβ-mediated phosphorylation. IRAG2 is a 75 kDa membrane protein homolog of IRAG1 and was recently also determined as a PKGI substrate. Several (patho-)physiological functions of IRAG1 and IRAG2 were meanwhile elucidated in a variety of human and murine tissues, e.g., of IRAG1 in various smooth muscles, heart, platelets, and other blood cells, of IRAG2 in the pancreas, heart, platelets, and taste cells. Hence, lack of IRAG1 or IRAG2 leads to diverse phenotypes in these organs, e.g., smooth muscle and platelet disorders or secretory deficiency, respectively. This review aims to highlight the recent research regarding these two regulatory proteins to envision their molecular and (patho-)physiological tasks and to unravel their functional interplay as possible (patho-)physiological counterparts.

On-Slide Lambda Protein Phosphatase-Mediated Dephosphorylation of Fixed Samples.

Protein phosphorylation is a ubiquitous post-translational modification that regulates a plethora of intracellular processes, making its analysis crucial for understanding intracellular dynamics. The commonly used methods, such as radioactive labeling and gel electrophoresis, do not provide information about subcellular localization. Immunofluorescence using phospho-specific antibodies and subsequent analysis via microscopy allows researchers to assess subcellular localization, but it typically lacks validation whether the observed fluorescent signal is phosphorylation specific. In this study, an on-slide dephosphorylation assay coupled with immunofluorescence staining using phospho-specific antibodies on fixed samples is proposed as a fast and simple approach to validate phosphorylated proteins in their native subcellular context. The assay was validated using antibodies against two different phosphorylated target proteins, connexin 43 phosphorylated at serine 373, and phosphorylated substrates of protein kinase A, with a dramatic reduction in the signal upon dephosphorylation. The proposed approach provides a convenient way to validate phosphorylated proteins without the need for additional sample preparation steps, reducing the time and effort required for analysis, while minimizing the risk of protein loss or alteration.

MARCKS is a New Prognostic Biomarker in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common type of cancers, but there is still a lack of known biomarkers for the effective diagnosis or prognosis of HCC. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a substrate of protein kinase C, which was located in the cell plasma membrane. The purpose of our study was to evaluate the role of MARCKS in HCC. The role of MARCKS in HCC was explored by bioinformatics and experiment. We demonstrated that MARCKS expression was significantly elevated in HCC datasets of TCGA. MARCKS was up-regulated in tumor sample in HCC. Functional enrichment indicated that MARCKS-related differentially expressed genes (DEGs) were mainly enriched in cell junction tissue, response to growth factors and cell population proliferation. Tumor and ECM-receptor interactions related pathways were enriched by the KEGG. MARCKS expression in HCC patients was higher in females, younger individuals, and those at worse clinical stages. Cox regression analysis showed that MARCKS expression was a risk factor for overall survival and disease-specific survival of patients. MARCKS was up-regulated in HCC, may play a crucial role in HCCs, and has prognostic value for clinical outcomes.

Phosphoproteomic Approaches for Identifying Phosphatase and Kinase Substrates.

Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here, we survey common LC-MS/MS-based phosphoproteomic workflows for identifying protein kinase and phosphatase substrates, noting key advantages and limitations of each. We conclude by discussing the value of inducible degradation technologies coupled with phosphoproteomics as a new approach that overcomes some limitations of current methods for substrate identification of kinases, phosphatases, and other regulatory enzymes.

MAPKAP Kinase-2 phosphorylation of PABPC1 controls its interaction with 14-3-3 proteins after DNA damage: A combined kinase and protein array approach.

14-3-3 proteins play critical roles in controlling multiple aspects of the cellular response to stress and DNA damage including regulation of metabolism, cell cycle progression, cell migration, and apoptotic cell death by binding to protein substrates of basophilic protein kinases following their phosphorylation on specific serine/threonine residues. Although over 200 mammalian proteins that bind to 14-3-3 have been identified, largely through proteomic studies, in many cases the relevant protein kinase responsible for conferring 14-3-3-binding to these proteins is not known. To facilitate the identification of kinase-specific 14-3-3 clients, we developed a biochemical approach using high-density protein filter arrays and identified the translational regulatory molecule PABPC1 as a substrate for Chk1 and MAPKAP Kinase-2 (MK2) in vitro, and for MK2 in vivo, whose phosphorylation results in 14-3-3-binding. We identify Ser-470 on PABPC1 within the linker region connecting the RRM domains to the PABC domain as the critical 14-3-3-binding site, and demonstrate that loss of PABPC1 binding to 14-3-3 results in increased cell proliferation and decreased cell death in response to UV-induced DNA damage.

Abstract 5980: Identification of CDK15 activating cyclins by a recombinant co-expression approach

Abstract Cyclin dependent kinases (CDK) form a family of 20 different CDKs with an additional 6 CDK-like proteins. Members of this family have been shown to be involved in the regulation of many critical cellular processes, including the regulation of cell cycle progression, gene transcription and neuronal function. Deregulated CDK activity has been observed frequently in human cancer and other diseases. Enzymatic kinase activity of CDKs has been found to depend on the formation of complexes with regulatory proteins of the Cyclin family and requires in some cases the presence of additional complex partners, like MAT1 for CDK7/Cyclin H. To date, at least 15 different Cyclins have been described. For some CDKs it has been shown that they can also be activated by binding to proteins distinct from classical Cyclins. However, even while the 20 canonical CDKs share significant structural homology, they regulate very different functions of cell growth and development, partially due to their inherent substrate specificity but also regulated by their respective Cyclin partners. During the past 30 years several CDKs have been subject to extensive study resulting in a multitude of publications. This includes specifically CDK1, CDK2 and CDK4/6 and to a lesser extend CDK7 and CDK9. Other CDKs have been studied much less extensively and consequently the accumulated knowledge about their biological function and regulation is very limited. CDK15 (also known as PFTK2 or ALS2CR7) is one of these understudied members of the CDK family, with less than 20 publications in PubMed and the search term "CDK15" in contrast to more than 8600 hits for "CDK2". Its biological function is largely obscure even while evidence was presented that CDK15 is involved in the regulation of breast and colorectal cancer. Comparable to all other members of the CDK family, recombinantly overexpressed purified CDK15 does not exhibit detectable in-vitro activity and to date no Cyclin or other activating complex partner for CDK15 has been published. Since CDK inhibitors have been in the focus of interest at least since the approval of the first candidate (Palbociclib) for the treatment of ER+/HER+ breast cancer, testing the selectivity of other drug candidates within the complete group of CDKs is critically important to avoid unwanted off-target effects and toxicity. In order to enable in-vitro activity studies with CDK15 we evaluated different CDK15 constructs in combination with a comprehensive panel of different Cyclins using the baculo virus expression system. CDK15 and the respective Cyclins were co-expressed in insect cells, purified by affinity chromatography and tested for in-vitro activity using a panel of generic, broad-spectrum protein kinase substrates. We will present data on the identification of CDK15 activating Cyclins and the biochemical characterization of the in-vitro kinase activity of such CDK15/Cyclin complexes including a panel of kinase inhibitors. Citation Format: Daniel Mueller, Constance Ketterer, Diane Kraemer, Carolin Heidemann-Dinger, Lena Pilgermayer, Thomas Weber, Andreas Gericke, Frank Totzke, Michael H. Kubbutat. Identification of CDK15 activating cyclins by a recombinant co-expression approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5980.

Protein S-acylation controls the subcellular localization and biological activity of PHYTOCHROME KINASE SUBSTRATE.

PHYTOCHROME KINASE SUBSTRATE (PKS) proteins are involved in light-modulated changes in growth orientation. They act downstream of phytochromes to control hypocotyl gravitropism in the light and act early in phototropin signaling. Despite their importance for plant development, little is known about their molecular mode of action, except that they belong to a protein complex comprising phototropins at the plasma membrane. Identifying evolutionary conservation is one approach to reveal biologically important protein motifs. Here, we show that PKS sequences are restricted to seed plants and that these proteins share six motifs (A to F from the N to the C terminus). Motifs A and D are also present in BIG GRAIN, while the remaining four are specific to PKSs. We provide evidence that motif C is S-acylated on highly conserved cysteines, which mediates the association of PKS proteins with the plasma membrane. Motif C is also required for PKS4-mediated phototropism and light-regulated hypocotyl gravitropism. Finally, our data suggest that the mode of PKS4 association with the plasma membrane is important for its biological activity. Our work therefore identifies conserved cysteines contributing to plasma membrane association of PKS proteins and strongly suggests that this is their site of action to modulate environmentally regulated organ positioning.

Abstract P6-10-15: Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity

Abstract Background. Because of its high metastatic potential, inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer. We previously demonstrated that Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein overexpression was associated with shorter metastasis-free survival (MFS) in IBC patients, but not in non-IBC (nIBC) patients. However, the mechanism of action of MARCKS and its particular association to poorer outcome in IBC compared to nIBC are poorly understood. Methods. We evaluated in vitro the inhibitory effect of MPS (MARCKS phosphorylation site domain), a peptide targeting MARCKS phosphorylation site domain (PSD) in single and in combination with paclitaxel treatment, on cell proliferation and cell motility in two cell lines of different phenotype (SUM149 for IBC and MDA-MB-231 for nIBC), as well as its distinct molecular mechanisms of action. We also assessed the clinical relevance of the protein expression of MARCKS and phosphatase and tensin homolog (PTEN) in a large series of IBC vs. nIBC patients. Results. In vitro, the treatment with MPS peptide impaired cell proliferation, migration, and invasion in SUM149 compared to MDA-MB-231 cells. More important, MARCKS inhibition increased paclitaxel sensitivity when using combination therapy in SUM149 cells compared to MDA-MB-231 cells. Interestingly, we could partially explain this specific inhibitory effect in IBC cells using western blot: MARCKS inhibition in single and in combination induced up and downregulation of the PTEN/AKT signaling pathway respectively in IBC compared to nIBC cells. Importantly, a negative correlation of MARCKS and PTEN was only found in the clinical IBC samples (180 patients) compared to nIBC samples (355 patients). More importantly, the group of patients with negative MARCKS and positive PTEN protein expression was associated to better 5-year MFS only in IBC patients. Conclusion. These results indicate two major findings: first, the important prognostic value of the negative correlation of MARCKS and PTEN expression in IBC patients, and second the specific role of MARCKS in regulating different downstream pathways and increasing the paclitaxel response in combination treatment in IBC compared to non-IBC. They suggest a potential IBC-specific targetable biomarker, the inhibition of which might impair disease aggressiveness and perhaps enhance patients’ survival. Citation Format: Maroua Manai, Ines Bini, Pascal Finetti, Haifa Bichiou, Carolina Reduzzi, Naziha Ben Hamida, Marc Lopez, Khaled Rahal, Karima Mrad, Mohamed Manai, Massimo Cristofanilli, Hamouda Boussen, Raoudha Doghri, Maher Kharrat, François Bertucci. Targeting MARCKS in inflammatory breast cancer increased paclitaxel sensitivity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-15.

The GSTP1/MAPKs/BIM/SMAC modulatory actions of nitazoxanide: Bioinformatics and experimental evidence in subcutaneous solid Ehrlich carcinoma-inoculated mice

AimsEhrlich ascites carcinoma and its subcutaneous inoculated solid tumour form (SEC) are reliable models for chemotherapeutic molecular targets exploration. Novel chemotherapeutic approaches are identified as molecular targets for intrinsic apoptosis, like the modulation of the second mitochondria-derived activator of caspases (SMAC). SMAC is a physiological substrate of mitogen-activated protein kinases (MAPKs). Glutathione-S-transferase P1 (GSTP1) and its close association with MAPKs play an important role in malignant cell proliferation, metastasis, and resistance to chemotherapeutics. Nitazoxanide (NTZ) is an emerging cancer therapy and its targeted GSTP1 evidence remains a knowledge need. Main methodsIn the present mice-established SEC, the chemotherapeutic roles of oral NTZ (200 mg/kg/day) and 5-fluorouracil (5-FU; 20 mg/kg/day, intraperitoneally) regimens were evaluated by measuring changes in tumour mass, the tumour MAPKs, cytochrome c, Bcl-2 interacting mediator of cell death (BIM), and SMAC signalling pathway in addition to its molecular downstream; caspases 3 and 9. Key findingsComputational analysis for these target protein interactions showed direct-ordered interactions. After individual therapy with NTZ and 5-FU regimens, the histological architecture of the extracted tumour discs revealed decreases in viable tumour regions with significant necrosis surrounds. These findings were consistent with gross tumour sizes. Each separate regimen lowered the remarkable GSTP1 and elevated the low MAPKs expressions, cytochrome c, BIM, SMAC, and caspases 3, and 9 in EST tissues. SignificanceThe chemotherapeutic activity of NTZ in SEC was proven. Additionally, NTZ possesses a SMAC modulatory activity that, following thorough research, should be taken into consideration as a chemotherapeutic approach in solid tumours.

Phosphoinositides regulation of hyperpolarization-activated cyclic-nucleotide gated (HCN) channels.

Several ion channels are regulated by protein-lipid interactions. Hyperpolarization-activated cyclic-nucleotide gated (HCN) channels specifically, have been shown to be regulated by phosphoinositides (PIPs). PIPs enhance HCN activation via a rightward shift in voltage-dependency and has importance applications in neuronal and cardiac function. Using computational and electrophysiological approaches, we aim to examine putative sites for HCN-PIP interactions. Computational docking and coarse-grained simulations indicate that PIP binding to HCN1 and HCN4 channels are not well coordinated, but rather occurs over a broad surface of charged residues primarily in the HCN-domain, S2 and S3 helices that can be loosely organized in 2 or 3 overlapping clusters. Thus, HCN-PIP interactions are more resembling of electrostatic interactions that occur in myristoylated alanine-rich C kinase substrate (MARCKS) proteins, than the specifically coordinated interactions that occur in pleckstrin homology domains (PH domains) or ion channels such as inward rectifier potassium (Kir) channels. Our results also indicate that phosphatidylinositol (PI) interactions with HCN1 are even lower affinity, explaining why unphosphorylated PI have no effect on HCN1 activation unlike phosphorylated PIPs. Experimental validation of putative binding sites are addressed by electrophysiological approaches.

Defining the interactions between HCN4 and the regulatory proteins IRAG and LRMP.

Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are the molecular determinants of the funny (If) current, a mixed Na+/K+ current that controls pacemaking in cardiac cells. In the sinoatrial node (SAN), an increase in If in response to cAMP contributes to an increase in action potential (AP) firing rate and consequently heart rate. We recently discovered two related modulators of HCN4 function: lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG). Despite homology between these two proteins, LRMP inhibits the depolarizing shift in the voltage dependence of activation of HCN4 in response to cAMP while IRAG shifts the basal voltage dependence in the depolarizing direction. Even with a clear picture of the functional effects that these two proteins exert on HCN4, the domains responsible for the interactions are completely unknown. Here, we pair a FRET two-hybrid approach with patch clamp electrophysiology to determine which domains of LRMP, IRAG, and HCN4 are involved in complex formation. Both LRMP and IRAG have small ER luminal domains, ER transmembrane segments, and a large cytoplasmic domain that likely interacts with an intracellular portion of HCN4 channels. By co-expressing cerulean-tagged cytosolic fragments of HCN4 with citrine-tagged fragments of LRMP and IRAG, we determine which regions on each protein are required for complex formation using FRET. We then test the functional effect of removing these critical domains on the regulation of the channel by each protein using patch clamp electrophysiology. Our preliminary results suggest that the N-terminus of HCN4 is critical for both LRMP and IRAG regulation of the channel. Moreover, the first 230 amino acids of LRMP is sufficient to bind HCN4. These results shed light on a novel amino terminus-dependent mechanism for regulating the gating of HCN4 channels.