Myristoylated Alanine-rich C Kinase Substrate Research Articles

Myristoylated alanine-rich C-kinase substrate effector domain phosphorylation regulates the growth and radiation sensitization of glioblastoma.

Glioblastoma harbors frequent alterations in receptor tyrosine kinases, phosphatidylinositol-3 kinase (PI3K) and phosphatase and tensin homolog (PTEN) that dysregulate phospholipid signaling driven tumor proliferation and therapeutic resistance. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a 32 kDa intrinsically unstructured protein containing a polybasic (+13) effector domain (ED), which regulates its electrostatic sequestration of phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP2), and its binding to phosphatidylserine, calcium/calmodulin, filamentous actin, while also serving as a nuclear localization sequence. MARCKS ED is phosphorylated by protein kinase C (PKC) and Rho-associated protein kinase (ROCK) kinases; however, the impact of MARCKS on glioblastoma growth and radiation sensitivity remains undetermined. In the present study, using a tetracycline-inducible system in PTEN-null U87 cells, we demonstrate that MARCKS overexpression suppresses growth and enhances radiation sensitivity in vivo. A new image cytometer, Xcyto10, was utilized to quantify differences in MARCKS ED phosphorylation on localization and its association with filamentous actin. The overexpression of the non-phosphorylatable ED mutant exerted growth-suppressive and radiation-sensitizing effects, while the pseudo-phosphorylated ED mutant exhibited an enhanced colony formation and clonogenic survival ability. The identification of MARCKS protein-protein interactions using co-immunoprecipitation coupled with tandem mass spectrometry revealed novel MARCKS-associated proteins, including importin-β and ku70. On the whole, the findings of this study suggest that the determination of the MARCKS ED phosphorylation status is essential to understanding the impact of MARCKS on cancer progression.

The myristoylated alanine-rich C kinase substrate differentially regulates kinase interacting with stathmin in vascular smooth muscle and endothelial cells and potentiates intimal hyperplasia formation

ObjectiveRestenosis limits the durability of all cardiovascular reconstructions. Vascular smooth muscle cell (VSMC) proliferation drives this process, but an intact, functional endothelium is necessary for vessel patency. Current strategies to prevent restenosis employ antiproliferative agents that affect both VSMCs and endothelial cells (ECs). Knockdown of the myristoylated alanine-rich C kinase substrate (MARCKS) arrests VSMC proliferation and paradoxically potentiates EC proliferation. MARCKS knockdown decreases expression of the kinase interacting with stathmin (KIS), increasing p27kip1 expression, arresting VSMC proliferation. Here, we seek to determine how MARCKS influences KIS protein expression in these two cell types. MethodsPrimary human coronary artery VSMCs and ECs were used for in vitro experiments. MARCKS was depleted by transfection with small interfering RNA. Messenger RNA was quantitated with the real-time reverse transcription polymerase chain reaction. Protein expression was determined by Western blot analysis. Ubiquitination was determined with immunoprecipitation. MARCKS and KIS binding was assessed with co-immunoprecipitation. Intimal hyperplasia was induced in CL57/B6 mice with a femoral artery wire injury. MARCKS was knocked down in vivo by application of 10 μM of small interfering RNA targeting MARCKS suspended in 30% Pluronic F-127 gel. Intimal hyperplasia formation was assessed by measurement of the intimal thickness on cross sections of the injured artery. Re-endothelialization was determined by quantitating the binding of Evans blue dye to the injured artery. ResultsMARCKS knockdown did not affect KIS messenger RNA expression in either cell type. In the presence of cycloheximide, MARCKS knockdown in VSMCs decreased KIS protein stability but had no effect in ECs. The effect of MARCKS knockdown on KIS stability was abrogated by the 26s proteasome inhibitor MG-132. MARCKS binds to KIS in VSMCs but not in ECs. MARCKS knockdown significantly increased the level of ubiquitinated KIS in VSMCs but not in ECs. MARCKS knockdown in vivo resulted in decreased KIS expression. Furthermore, MARCKS knockdown in vivo resulted in decreased 5-ethynyl-2′-deoxyuridine integration and significantly reduced intimal thickening. MARCKS knockdown enhanced endothelial barrier function recovery 4 days after injury. ConclusionsMARCKS differentially regulates the KIS protein stability in VSMCs and ECs. The difference in stability is due to differential ubiquitination of KIS in these two cell types. The differential interaction of MARCKS and KIS provides a possible explanation for the observed difference in ubiquitination. The effect of MARCKS knockdown on KIS expression persists in vivo, potentiates recovery of the endothelium, and abrogates intimal hyperplasia formation.

Myristoylated alanine rich protein kinaseC substrate is a potential cancer prognostic factor that regulates cell migration and invasion in glioblastoma.

Myristoylated alanine‑rich C‑kinase substrate (MARCKS) serves an important role in various pathological processes in several malignancies. However, little is known about the specific role and molecular mechanism of MARCKS in glioblastoma (GBM). In the present study, it was found that the expression of MARCKS was significantly upregulated in GBM, and was associated with a poor clinical outcome in patients with GBM. Knockdown of MARCKS suppressed the migration and invasion of GBM cells invitro. Western blotting showed that the knockdown of MARCKS reduced the expression of phosphorylated phosphoinositide3‑kinase and protein kinaseB, as well as zinc finger protein SNAI1 expression, thereby modulating the expression of its downstream epithelial‑mesenchymal transition (EMT)‑associated factors, including E‑cadherin, vimentin, N‑cadherin and β‑catenin in GBM cells. These results indicate that MARCKS functioned in the migration and invasion of GBM, and therefore may provide a potential therapeutic target in GBM therapy.

Prognostic Value of Upregulation of Myristoylated Alanine-Rich C-Kinase Substrate in Gastric Cancer.

BackgroundAccumulating evidence suggests a connection of Myristoylated alanine-rich C-kinase substrate (MARCKS) with several physiological and pathological processes. However, the relevance of MARCKS in gastric cancer (GC) needs to be elucidated.Material/MethodsThe abundance of MARCKS in GC tissues was assessed using techniques of immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR). Moreover, the MARCKS expression profile in the TCGA database was analyzed through an online website analysis. We also investigated MARCKS function using cell wounding and Matrigel invasion assays.ResultsTCGA analysis and our data suggest that transcript abundance and protein level of MARCKS was higher in GC tumor samples compared with peri-tumor tissues. There was a remarkable association of upregulated MARCKS with the cell differentiation (P<0.001), T stage (P=0.034), and N stage (P=0.002) followed by advanced TNM phase (P=0.008). Furthermore, it was predicted that higher expression of MARCKS is linked to poor overall survival (P=0.015) and disease-free survival (P=0.020), and that high levels of MARCKS function as an independent prognostic marker, as shown by multivariate Cox regression analysis in prediction of poor overall (HR=0.408; 95% confidence interval=0.247–0.674; P<0.001) and disease-free survival rates (HR=0.525; 95% confidence interval=0.216–0.584; P<0.001). GC cells showed significant reduction in cell migration and invasion upon depletion of MARCKS as noted through Matrigel invasion and cell wounding assays. Further analyses showed that silencing MARCKS impeded the epithelial-mesenchymal transition (EMT).ConclusionsOur study indicates that elevated expression of MARCKS is significantly associated with metastatic capability of GC cells, and MARCKS overexpression can serve as a biomarker of GC poor prognosis.

MARCKS and Lung Disease.

MARCKS (myristoylated alanine-rich C kinase substrate) is a prominent PKC substrate expressed in all eukaryotic cells. It is known to bind to and cross-link actin filaments, to serve as a bridge between Ca2+/calmodulin and PKC signaling, and to sequester the signaling molecule phosphatidylinositol 4,5-bisphosphate in the plasma membrane. Since the mid-1980s, this evolutionarily conserved and ubiquitously expressed protein has been associated with regulating cellular events that require dynamic actin reorganization, including cellular adhesion, migration, and exocytosis. More recently, translational studies have implicated MARCKS in the pathophysiology of a number of airway diseases, including chronic obstructive pulmonary disease, asthma, lung cancer, and acute lung injury/acute respiratory distress syndrome. This article summarizes the structure and cellular function of MARCKS (also including MARCKS family proteins and MARCKSL1 [MARCKS-like protein 1]). Evidence for MARCKS's role in several lung diseases is discussed, as are the technological innovations that took MARCKS-targeting strategies from theoretical to therapeutic. Descriptions and updates derived from ongoing clinical trials that are investigating inhalation of a MARCKS-targeting peptide as therapy for patients with chronic bronchitis, lung cancer, and ARDS are provided.

Marcks Inhibition By a Peptide Inhibitor, MPS, Cooperates with Bortezomib to Effectively Eliminate Drug Resistance in Multiple Myeloma

Overexpression of myristoylated alanine-rich C kinase substrate (MARCKS) has been implicated in the progression of multiple cancer types including multiple myeloma (MM). Our previous study showed that overexpression of MARCKS in MM cells plays an important role in drug resistance. However; the mechanism(s) underlying MARCKS overexpression and its association with drug resistance in MM remain undefined. Using miRNA target scan algorithms, we identified miR-34a as a regulator of MARCKS, which is underexpressed in drug-resistant MM cells (8226-R5 and MM1R) in comparison to parental cells (8226 and MM1S, respectively). Using luciferase assays, we demonstrated direct targeting of MARCKS by miR-34a. Notably, targeting of MARCKS by miR-34a overexpression re-sensitized the resistant cells to anti-myeloma drugs. Moreover, since all known biological functions of MARCKS including its oncogenic effects are conducted by its phosphorylated form, we investigated whether inhibitor of MARCKS phosphorylation could antagonize drug resistance in MM cells. We showed that a MARCKS PSD peptide inhibitor (MPS) had a dose dependent cytotoxic effect on drug resistant MM cells in comparison to its mutated (inactive) form, but minimal cytotoxicity on normal peripheral blood mononuclear cells (PBMCs). Furthermore, MPS treatment sensitized drug-resistant MM cells to bortezomib (BTZ), a first line proteasome inhibitor that is widely used in the treatment of MM. We further investigated the effect of MPS on tumorigenesis in mouse xenograft models of MM resistant cells. Combination of BTZ plus MPS significantly suppressed tumor growth and prolonged overall survival as compared with MPS alone, BTZ alone or vehicle treated controls. IHC analysis of xenograft tumor sections revealed that combination treatment of MPS and BTZ resulted in decreased proliferation (Ki67) and increased apoptosis (TUNEL), compared to either BTZ or MPS alone. These results support that targeting of phospho-MARCKS by MPS contributes to drug sensitivity in MM resistant cells. Furthermore, we investigated the mechanisms by which MPS induces cell death and overcome bortezomib resistance. Following MPS treatment, PUMA was accumulated both in MM1R and 8226R5 MM cell lines, indicating that MPS treatment may lead to apoptosis. Interestingly, the level of LC3BII, a marker of autophagy, was also significantly upregulated, suggesting enhanced autophagic flux after MARCKS inhibition. The significant upregulation of LC3BII observed in both MM1R and 8226R5 MM cell lines after MARCKS silencing further confirmed the correlation between MARCKS inhibition and autophagy induction. Remarkably, MARCKS-silenced drug resistant MM cells were more vulnerable than negative control cells to the autophagy inhibitor chloroquine (CQ). More significant cytotoxicity of the BTZ plus CQ combo was also detected in MARCKS-inhibited MM cells than in negative control cells. Mechanistically, co-immuno-precipitation assays revealed increased interactions of PUMA with both Mcl-1 and Bcl-xL in MARCKS-silenced 8226R5 cells, while the interaction between Beclin-1 and Bcl-xL in MARCKS silenced 8226R5 cells was dampened. These results suggest that PUMA upregulation after MRCKS inhibition enhances the initiation of autophagic response by sequestering Bcl-2 family proteins like Bcl-xL from Beclin-1/Vps34 complexes. Taken together, our results demonstrate that the MPS peptide can sensitize drug resistant MM cell to anti-myeloma drugs by inhibiting MARCKS phosphorylation in vitro and in vivo. These findings underscore the importance of MARCKS suppression in antagonizing adaptive BTZ resistance and provide novel venues to treat MM. DisclosuresNo relevant conflicts of interest to declare.

Identification of DKK-1 as a novel mediator of statin effects in human endothelial cells

This study shows that DKK-1, a member of the Dickkopf family and a regulator of the Wnt pathways, represents a novel target of statins which, through the inhibition of HMG-CoA reductase and of non-steroidal isoprenoid intermediates, exert extra-beneficial effect in preventing atherosclerosis beyond their effect on the lipid profile. We found that atorvastatin downregulates DKK-1 protein (−88.3 ± 4.1%) and mRNA expression (−90 ± 4.2%) through the inhibition of Cdc42, Rho and Rac geranylgeranylated proteins. Further, a combined approach based on the integration of label-free quantitative mass spectrometry based-proteomics and gene silencing allowed us to demonstrate that DKK-1 itself mediates, at least in part, statin effects on human endothelial cells. Indeed, DKK-1 is responsible for the regulation of the 21% of the statin-modulated proteins, which include, among others, clusterin/apoJ, plasminogen activator inhibitor type 1 (PAI-1), myristoylated alanine-rich C-kinase substrate (MARCKS), and pentraxin 3 (PTX3). The Gene Ontology enrichment annotation revealed that DKK-1 is also a potential mediator of the extracellular matrix organization, platelet activation and response to wounding processes induced by statin. Finally, we found that plasma level of DKK-1 from cholesterol-fed rabbits treated with atorvastatin (2.5 mg/kg/day for 8 weeks) was lower (−42 ± 23%) than that of control animals. Thus, DKK-1 is not only a target of statin but it directly regulates the expression of molecules involved in a plethora of biological functions, thus expanding its role, which has been so far restricted mainly to cancer.

MARCKS regulates neuritogenesis and interacts with a CDC42 signaling network

Through the process of neuronal differentiation, newly born neurons change from simple, spherical cells to complex, sprawling cells with many highly branched processes. One of the first stages in this process is neurite initiation, wherein cytoskeletal modifications facilitate membrane protrusion and extension from the cell body. Hundreds of actin modulators and microtubule-binding proteins are known to be involved in this process, but relatively little is known about how upstream regulators bring these complex networks together at discrete locations to produce neurites. Here, we show that Myristoylated alanine-rich C kinase substrate (MARCKS) participates in this process. Marcks−/− cortical neurons extend fewer neurites and have less complex neurite arborization patterns. We use an in vitro proteomics screen to identify MARCKS interactors in developing neurites and characterize an interaction between MARCKS and a CDC42-centered network. While the presence of MARCKS does not affect whole brain levels of activated or total CDC42, we propose that MARCKS is uniquely positioned to regulate CDC42 localization and interactions within specialized cellular compartments, such as nascent neurites.

Overexpression of MARCKS indicates a poor prognosis of oral squamous cell carcinoma.

Myristoylated alanine-rich C kinase substrate (MARCKS) is a protein kinase C substrate functioning in different physiological and pathological mechanisms. Previous studies have suggested that MARCKS is capable of influencing tumorigenesis and progression. However, a limited number of studies are available regarding the role of MARCKS in oral squamous cell carcinoma (OSCC). The present study primarily examined MARCKS expression in the OSCC tissues. Furthermore, increased expression of MARCKS was confirmed in the majority of OSCC tissues. Increased MARCKS expression was correlated with more advanced tumor stages, lymphatic metastasis and a poorer overall patient survival. Further molecular mechanistic examinations revealed that downregulated MARCKS expression inhibited the proliferation and migration of OSCC cells in vitro through interruption of MARCKS expression. In addition, the present study demonstrated that MARCKS aggravated OSCC progression via the phosphoinositide 3-kinase/protein kinase B pathway. Accordingly, the present study considered MARCKS to be a promoter of OSCC tumorigenesis and progression, with the potential utility as a biomarker of a poor prognosis.

Ser46-Phosphorylated MARCKS Is a Marker of Neurite Degeneration at the Pre-aggregation Stage in PD/DLB Pathology.

Phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) reflects neurite degeneration at the early stage of Alzheimer's disease (AD), before extracellular Aβ aggregates are histologically detectable. Here, we demonstrate that similar changes in MARCKS occur in Parkinson's disease (PD) and dementia with Lewy bodies (DLB) pathologies in both mouse models and human patients. The increase in the level of pSer46-MARCKS began before α-synuclein aggregate formation, at a time when human α-Syn-BAC-Tg/GBA-hetero-KO mice exhibited no symptoms, and was sustained during aging, consistent with the pattern in human postmortem brains. The results strongly imply a common mechanism of pre-aggregation neurite degeneration in AD and PD/DLB pathologies.

MARCKS phosphorylation and amylase release in GLP-1-stimulated acini isolated from rat pancreas.

Little is known about the effects of glucagon-like peptide 1 (GLP-1) on the pancreatic exocrine gland. In the gland, secretagogues induce amylase release. That signal transduction is evoked mainly by an increase in intracellular Ca2+ levels and activation of protein kinase C (PKC). We previously demonstrated that myristoylated alanine-rich C kinase substrate (MARCKS), a PKC substrate, is involved in pancreatic amylase release. Here, we studied the effects of GLP-1 on MARCKS phosphorylation and amylase release in rat pancreatic acini. GLP-1 induced amylase release and MARCKS phosphorylation in isolated pancreatic acini. Inhibitors of cAMP-dependent protein kinase (PKA) suppressed those effects. Furthermore, a MARCKS-related peptide inhibited the GLP-1-induced amylase release. These findings suggest that GLP-1 induces amylase release through MARCKS phosphorylation via activation of PKA in isolated pancreatic acini.

The Role of Myristoylated, Alanine-rich C-kinase Substrate (MARCKS) in a Murine Model of Chronic Airway Inflammation

Abstract Severe asthma, a complicate chronic lung inflammation. Several chronic inflammatory cells that involved in the inflammatory responses, such as macrophages, neutrophils, and eosinophils, produce large amounts of ROS, the excess level of ROS would increase lung inflammation and lung injury. In fact, this inflammation is well controlled by steroid, but some subset of patients these glucocorticoids are less effective. Neutrophil recruitment is a prominent feature of acute exacerbations of chronic asthma. In patients with difficult-to-control asthma, neutrophilic inflammation rather than eosinophils often predominate. Myristoylated alanine-rich C kinase substrate (MARCKS), is a protein kinase C (PKC) substrate. MARCKS also plays a crucial role that phosphorylation of MARCKS by PKC leads its release from plasma membrane into the cytosol, participating in regulating cell migration such as macrophage and neutrophil. We found that increase of airway MARCKS phosphorylation in asthmatic patients and murine model of chronic steroid-resistance allergic asthma. In this report, our observations indicate that 1) targeting of MARCKS phosphorylation inhibited chronic asthmatic symptoms, such as eosinophil and neutrophil infiltrate into lung. MPS peptide inhibits mucus production, collagen deposition and airway hyperresponsiveness rather than steroid-resistance group; 2) targeting of MARCKS phosphorylation decreased IFN-r, IL-4, IL-17A, IL-33, eotaxin and KC production. MPS peptide also inhibit NADPH oxidase DUOX-1, 2 protein expression and end product 3-nitrotyrosine production; We found that MPS peptide shows a therapeutic potential for steroid-resistance allergic asthma.

Carnosic acid protects mice from high-fat diet-induced NAFLD by regulating MARCKS.

Non-alcoholic fatty liver disease (NAFLD) comprises a spectrum of liver damage characterized by abnormal hepatic fat accumulation and inflammatory response. Although the molecular mechanisms responsible for the disease are not yet fully understood, the pathogenesis of NAFLD likely involves multiple signals. The identification of effective therapeutic strategies to target these signals is of utmost importance. Carnosic acid (CA), as a phenolic diterpene with anticancer, anti-bacterial, anti-diabetic and neuroprotective properties, is produced by many species of the Lamiaceae family. Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. In the present study, wild-type C57BL/6 and MARCKS-deficient mice were randomly divided into the normal chow- or high-fat (HF) diet-fed groups. The HF diet increased the fasting glucose and insulin levels, and promoted glucose intolerance in the wild-type mice. MARCKS deficiency further upregulated intolerance, fasting glucose and insulin. The HF diet also promoted hepatic steatosis, serum alanine transaminase (ALT) and aspartate transaminase (AST) activity, inflammation and lipid accumulation in the wild-type mice. These responses were accelerated in the MARCKS-deficient mice. Importantly, increased inflammation and lipid accumulation were associated with phosphoinositide 3-kinase (PI3K)/AKT, NLR family pyrin domain containing 3 (NLRP3)/nuclear factor-κB (NF-κB) and sterol regulatory element binding protein-1c (SREBP-1c) signaling pathway activation. The mice treated with CA exhibited a significantly improved glucose and insulin tolerance. The production of pro-inflammatory cytokines and lipid accumulation were suppressed by CA. Significantly, MARCKS was reduced in mice fed the HF diet. CA treatment upregulated MARCKS expression compared to the HF group. Furthermore, the activation of the PI3K/AKT, NLRP3/NF-κB and SREBP-1c signaling pathways was inhibited by CA. Taken together, our data suggest that CA suppresses inflammation and lipogenesis in mice fed a HF diet through MARCKS regulation. Thus, CA may be prove to be a useful anti-NAFLD agent.

MARCKS Is Necessary for Netrin-DCC Signaling and Corpus Callosum Formation.

Axons of the corpus callosum (CC), the white matter tract that connects the left and right hemispheres of the brain, receive instruction from a number of chemoattractant and chemorepulsant cues during their initial navigation towards and across the midline. While it has long been known that the CC is malformed in the absence of Myristoylated alanine-rich C-kinase substrate (MARCKS), evidence for a direct role of MARCKS in axon navigation has been lacking. Here, we show that MARCKS is necessary for Netrin-1 (NTN1) signaling through the DCC receptor, which is critical for axon guidance decisions. Marcks null (Marcks-/-) neurons fail to respond to exogenous NTN1 and are deficient in markers of DCC activation. Without MARCKS, the subcellular distributions of two critical mediators of NTN1-DCC signaling, the tyrosine kinases PTK2 and SRC, are disrupted. Together, this work establishes a novel role for MARCKS in axon dynamics and highlights the necessity of MARCKS as an organizer of DCC signaling at the membrane.

Mechanism of inhibition of taurolithocholate-induced retrieval of plasma membrane MRP2 by cyclic AMP and tauroursodeoxycholate.

Taurolithocholate (TLC) produces cholestasis by inhibiting biliary solute secretion in part by retrieving MRP2 from the plasma membrane (PM). Tauroursodeoxycholate (TUDC) and cAMP reverse TLC‐induced cholestasis by inhibiting TLC‐induced retrieval of MRP2. However, cellular mechanisms for this reversal are incompletely understood. Recently, we reported that TLC decreases PM‐MRP2 by activating PKCε followed by phosphorylation of myristoylated alanine‐rich C kinase substrate (MARCKS). Thus, cAMP and TUDC may reverse TLC‐induced cholestasis by inhibiting the TLC/PKC ε/MARCKS phosphorylation pathway. We tested this hypothesis by determining whether TUDC and/or cAMP inhibit TLC‐induced activation of PKCε and phosphorylation of MARCKS. Studies were conducted in HuH‐NTCP cell line and rat hepatocytes. Activation of PKC ε was determined from the translocation of PKC ε to PM using a biotinylation method. Phosphorylation of MARCKS was determined by immunoblotting with a phospho‐MARCKS antibody. TLC, but not cAMP and TUDC, activated PKC ε and increased MARCKS phosphorylation in HuH‐NTCP as well in rat hepatocytes. Treatment with TUDC or cAMP inhibited TLC‐induced activation of PKCε and increases in MARCKS phosphorylation in both cell types. Based on these results, we conclude that the reversal of TLC‐induced cholestasis by cAMP and TUDC involves, at least in part, inhibition of TLC‐mediated activation of the PKCε/MARCKS phosphorylation pathway.

Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers.

Emerging evidence implicates myristoylated alanine-rich C-kinase substrate (MARCKS), a major substrate of protein kinase C (PKC), in a critical role for cancer development and progression. MARCKS is tethered to the plasma membrane but can shuttle between the cytosol and plasma membrane via the myristoyl-electrostatic switch. Phosphorylation of MARCKS by PKC leads to its translocation from the plasma membrane to the cytosol where it functions in actin cytoskeletal remodeling, Ca2+ signaling through binding to calmodulin, and regulation of exocytic vesicle release in secretory cells such as neurons and airway goblet cells. Although the contribution of MARCKS to various cellular processes has been extensively studied, its roles in neoplastic disease have been conflicting. This review highlights the molecular and functional differences of MARCKS that exist between normal and tumor cells. We also discuss the recent advances in the potential roles of MARCKS in tumorigenesis, metastasis, and resistance to anti-cancer therapies, with a focus on addressing the inconsistent results regarding the function of MARCKS as a promoter or inhibitor of oncogenesis.

A spectroscopic sensing platform for MARCKS protein monolayers

We developed a highly sensitive silicon platform, suitable to assess the molecular organization of protein samples. Prototype platforms were obtained using different electrochemical protocols for the electrodeposition of Ag-nanoparticles onto the hydrogenated silicon surface. A platform with high Surface Enhanced Raman Scattering efficiency was selected based on the surface coverage and the number density of particles size distribution. The performance of the platform was determined by studying the interaction of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS) protein with the substrate according to its molecular organization. The chemical and structural characteristics of MARCKS molecules were examined under two configurations: i) a disordered distribution given by a MARCKS solution drop deposited onto the platform and, ii) a compact monolayer transferred to the platform by the Langmuir-Blodgett method. Raman spectra show vibrational bands of Phenylalanine and Lysine residues specific for the protein effector domain, and evidence the presence of alpha helix structure in both configurations. Moreover, we distinguished the supramolecular order between the compact monolayer and random molecular distribution. The platforms containing Ag-nanoparticles are suitable for studies of protein structure and interactions, advancing a methodological strategy for our long term goal, which is to explore the interaction of proteins with model membranes.

Novel direct factor Xa inhibitory compounds from Tenebrio molitor with anti-platelet aggregation activity

Tenebrio molitor is an edible insect that has antimicrobial, anticancer, and antihypertensive effects. The aim of this study was to identify the unreported bioactive compounds from T. molitor larvae with inhibitory activities against factor Xa (FXa) and platelet aggregation. Isolated compounds were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. A diketopiperazine (1, cyclo(L-Pro-L-Tyr)) and a phenylethanoid (2, N-acetyltyramine) were isolated and inhibited the catalytic activity of FXa in a mixed inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619. They inhibited ADP- and U46619-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) and the expression of P-selectin and PAC-1 in platelets. They also improved the production of nitric oxide and inhibited the oversecretion of endothelin-1 compared to that of the ADP- or U46619-treated group. In an animal model of arterial and pulmonary thrombosis, the isolated compounds showed enhanced antithrombotic effects. They also elicited anticoagulant effects in mice. Compounds 1–2 inhibited ADP-, collagen-, or U46619-induced platelet aggregation and showed similar anti-thrombotic efficacy to rivaroxaban, a positive control. Therefore, 1–2 could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs.

Generation and intracellular trafficking of a polysialic acid-carrying fragment of the neural cell adhesion molecule NCAM to the cell nucleus

Polysialic acid (PSA) and its major protein carrier, the neural cell adhesion molecule NCAM, play important roles in many nervous system functions during development and in adulthood. Here, we show that a PSA-carrying NCAM fragment is generated at the plasma membrane by matrix metalloproteases and transferred to the cell nucleus via endosomes and the cytoplasm. Generation and nuclear import of this fragment in cultured cerebellar neurons is induced by a function-triggering NCAM antibody and a peptide comprising the effector domain (ED) of myristoylated alanine-rich C kinase substrate (MARCKS) which interacts with PSA within the plane of the plasma membrane. These treatments lead to activation of the fibroblast growth factor (FGF) receptor, phospholipase C (PLC), protein kinase C (PKC) and phosphoinositide-3-kinase (PI3K), and subsequently to phosphorylation of MARCKS. Moreover, the NCAM antibody triggers calmodulin-dependent activation of nitric oxide synthase, nitric oxide (NO) production, NO-dependent S-nitrosylation of matrix metalloprotease 9 (MMP9) as well as activation of matrix metalloprotease 2 (MMP2) and MMP9, whereas the ED peptide activates phospholipase D (PLD) and MMP2, but not MMP9. These results indicate that the nuclear PSA-carrying NCAM fragment is generated by distinct and functionally defined signal transducing mechanisms.

Inhibition of AKT1 signaling promotes invasion and metastasis of non-small cell lung cancer cells with K-RAS or EGFR mutations

Accumulating evidence supports a role of the PI3K-AKT pathway in the regulation of cell motility, invasion and metastasis. AKT activation is known to promote metastasis, however under certain circumstances, it also shows an inhibitory activity on metastatic processes, and the cause of such conflicting results is largely unclear. Here we found that AKT1 is an important regulator of metastasis and down-regulation of its activity is associated with increased metastatic potential of A549 cells. Inhibition of AKT1 enhanced migration and invasion in KRAS- or EGFR-mutant non-small cell lung cancer (NSCLC) cells. The allosteric AKT inhibitor MK-2206 promoted metastasis of KRAS-mutated A549 cells in vivo. We next identified that the phosphorylation of Myristoylated alanine-rich C-kinase substrate (MARCKS) and LAMC2 protein level were increased with AKT1 inhibition, and MARCKS or LAMC2 knockdown abrogated migration and invasion induced by AKT1 inhibition. This study unravels an anti-metastatic role of AKT1 in the NSCLC cells with KRAS or EGFR mutations, and establishes an AKT1-MARCKS-LAMC2 feedback loop in this regulation.