Myristoylated Alanine-rich C Kinase Substrate Research Articles

Differential interaction of β2e with phosphoinositides: A comparative study between β2e and MARCKS

ABSTRACTVoltage-gated calcium (CaV) channels are responsible for Ca2+ influx in excitable cells. As one of the auxiliary subunits, the CaV β subunit plays a pivotal role in the membrane expression and receptor modulation of CaV channels. In particular, the subcellular localization of the β subunit is critical for determining the biophysical properties of CaV channels. Recently, we showed that the β2e isotype is tethered to the plasma membrane. Such a feature of β2e is due to the reversible electrostatic interaction with anionic membrane phospholipids. Here, we further explored the membrane interaction property of β2e by comparing it with that of myristoylated alanine-rich C kinase substrate (MARCKS). First, the charge neutralization of the inner leaf of the plasma membrane induced the translocation of both β2e and MARCKS to the cytosol, while the transient depletion of poly-phosphoinositides (poly-PIs) by translocatable pseudojanin (PJ) systems induced the cytosolic translocation of β2e but not MARCKS. Second, the activation of protein kinase C (PKC) induced the translocation of MARCKS but not β2e. We also found that after the cytosolic translocation of MARCKS by receptor activation, depletion of poly-PIs slowed the recovery of MARCKS to the plasma membrane. Together, our data demonstrate that both β2e and MARCKS bind to the membrane through electrostatic interaction but with different binding affinity, and thus, they are differentially regulated by enzymatic degradation of membrane PIs.

Involvement of myristoylated alanine-rich C kinase substrate phosphorylation and translocation in cholecystokinin-induced amylase release in rat pancreatic acini.

Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKC-δ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.

MARCKS is involved in methylmercury-induced decrease in cell viability and nitric oxide production in EA.hy926 cells.

Methylmercury (MeHg) is a persistent environmental contaminant that has been reported worldwide. MeHg exposure has been reported to lead to increased risk of cardiovascular diseases; however, the mechanisms underlying the toxic effects of MeHg on the cardiovascular system have not been well elucidated. We have previously reported that mice exposed to MeHg had increased blood pressure along with impaired endothelium-dependent vasodilation. In this study, we investigated the toxic effects of MeHg on a human endothelial cell line, EA.hy926. In addition, we have tried to elucidate the role of myristoylated alanine-rich C kinase substrate (MARCKS) in the MeHg toxicity mechanism in EA.hy926 cells. Cells exposed to MeHg (0.1–10 µM) for 24 hr showed decreased cell viability in a dose-dependent manner. Treatment with submaximal concentrations of MeHg decreased cell migration in the wound healing assay, tube formation on Matrigel and spontaneous nitric oxide (NO) production of EA.hy926 cells. MeHg exposure also elicited a decrease in MARCKS expression and an increase in MARCKS phosphorylation. MARCKS knockdown or MARCKS overexpression in EA.hy926 cells altered not only cell functions, such as migration, tube formation and NO production, but also MeHg-induced decrease in cell viability and NO production. These results suggest the broad role played by MARCKS in endothelial cell functions and the involvement of MARCKS in MeHg-induced toxicity.

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Marcks Marks Resistance to Proteasome Inhibitors: Exocytosis of Polyubiquitinated Proteins in Bortezomib-Resistant Leukemia Cells

Despite the proven efficacy of proteasome inhibitors like bortezomib (BTZ) in multiple myeloma, mantle cell lymphoma, and in an experimental setting in pediatric acute leukemia, development of drug resistance remains a primary hindrance. To further understand the molecular basis underlying this chemoresistance phenomenon, various leukemia cell line models with acquired resistance to BTZ were developed and characterized. One common characteristic was that acquisition of point mutations in PSMB5 and upregulation of the β5-subunit of the proteasome were key determinants of BTZ-resistance in vitro. However, it remains unclear how these drug resistance modalities translate to the overcoming of proteolytic stress imposed by proteasome inhibition. From this perspective, we here undertook a multi-modality (DNA, mRNA, miRNA) array-based analyses of human CCRF-CEM acute leukemia cells and two BTZ-resistant subclones [one with a low resistance level [(10-fold, CEM/BTZ7) and another subline with a high resistance level (140-fold, CEM/BTZ200)] to determine whether or not complementary mechanisms contribute to BTZ resistance. Gene expression profiling studies revealed markedly reduced proteolytic stress induction in drug resistant cells over a broad BTZ concentration range. Moreover, several genes involved in cytoskeleton regulation and vesicle migration were increased in resistant cells. Of all genes, myristoylated alanine-rich C-kinase substrate (MARCKS) was the most differentially overexpressed gene with 25- to 42-fold upregulation in CEM/BTZ7 and CEM/BTZ200 cells, respectively. These observations were corroborated at the protein level and solely included unphosphorylated MARCKS rather than phosphorylated MARCKS, which was marginally expressed in CEM/BTZ cells. Interestingly, MARCKS upregulation was also observed in other BTZ-resistant and leukemia cells (CEM and THP1) with acquired resistance to the proteasome inhibitor salinosporamide A and the immunoproteasome inhibitor PR924. Given the overexpression of MARCKS in proteasome inhibitor-resistant leukemia cells, we further explored whether or not MARCKS overexpression may serve as a predictive marker of BTZ resistance in clinical samples of acute leukemia patients. To this end, we examined primary patient specimens from a phase II childhood refractory/relapsed ALL trial in which BTZ is administered in two intensive re-induction regimens containing vincristine, prednisone, PEG-asparaginase, doxorubicin or cyclophosphamide and etoposide followed by methotrexate treatment. MARCKS expression was demonstrated in 64% of therapy-refractory pediatric leukemia specimens (n=44) wherein higher MARCKS expression trended (p=0.09) towards a dismal response to BTZ-containing chemotherapy. Finally, from a mechanistic perspective, we showed a concentration-dependent association of MARCKS protein with the emergence of ubiquitin-containing vesicles in the resistant cells. This association with MARCKS protein was lost upon exocytosis of these vesicles, which were found to be extruded and taken up in co-cultures with recipient HeLa cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance through vesicular exocytosis of ubiquitinated proteins in BTZ-resistant cells to overcome proteolytic stress over a broad range of cytotoxic BTZ concentrations. DisclosuresKaspers:Janssen-Cilag: Research Funding. Smeets:Novartis: Employment. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cloos:Takeda: Honoraria.

MARCKS ED Inhibits Fibrin Formation By Blocking Coagulation Protein Complex Assembly on Phosphatidylserine

Introduction: Prothrombinase is an essential enzyme of the coagulation cascade that is made up of a heterodimer of coagulation factor Xa and Va. The complex most readily forms in the presence of lipid membranes containing the anionic lipid phosphatidylserine. The phosphatidylserine lipid head groups provide a surface for the prothrombinase complex to assemble and significantly increase the enzyme's turnover number. Here, we investigate how truncation and modification of the naturally occurring myristoylated alanine-rich C-kinase substrate (MARCKS) protein into small peptide and peptidomimetic fragments (MARCKS ED) forms lipid probes that bind to phosphatidylserine, and block the assembly and enzymatic activity of the prothrombinase complex. Compared to known phosphatidylserine binding proteins such as annexin V or lactadherin, the MARCKS ED probes are less than a tenth of the molecular weight and do not require calcium to bind to phosphatidylserine.Methods: Using biophysical assays, we examine how the MARCKS ED probes assemble on lipid membranes containing phosphatidylserine and how this assembly alters the binding of factor Xa to these surfaces. Effects on the enzymatic activity of the prothrombinase complex in the presence of activated platelets and other phosphatidylserine exposing membranes are explored using a modified prothrombinase assay. The binding and anticoagulant activity of the MARCKS ED probes during whole blood coagulation are examined by quantifying fluorescently labeled MARCKS ED probe localization, platelet accumulation, and fibrin formation in microfluidic flow assays.Results: We find that the MARCKS ED probes can antagonize factor Xa from binding phosphatidylserine by measuring reduced association of factor Xa to lipid membranes treated with the MARCKS ED probes. Pre-treatment of activated platelets with the MARCKS ED probes significantly reduced the assembly and enzymatic activity of prothrombinase (100% activity with vehicle control vs. 51% with 1 μM MARCKS ED peptide treatment, P < 0.05). Similar inhibitory activity was also observed when activated platelets were replaced with other phosphatidylserine exposing lipid membranes, such as synthetic liposomes or biologic microparticles. In the whole blood microfluidic flow assay, the MARCKS ED probes colocalized with adherent platelets and inhibited fibrin formation (100% peak fibrin intensity with vehicle control vs. 17% peak fibrin intensity with 1 μM MARCKS ED peptide treatment, P < 0.05). The MARCKS ED probes did not alter platelet surface area coverage. Electron microscopy images of the final clots formed in the microfluidic flow assay show an absence of fibrin formation with MARCKS ED probe treatment.Conclusions: We demonstrate that the MARCKS ED probes can inhibit the assembly and enzymatic activity of prothrombinase, and that this activity translates to significantly reduced fibrin formation in whole blood. The MARCKS ED peptide and peptidomimetic probes may provide a new therapeutic tool for inhibiting the assembly of phosphatidylserine dependent coagulation factors, as well as a research tool for identifying platelet phosphatidylserine exposure. DisclosuresKastelowitz:University of Colorado Boulder: Patents & Royalties. Yin:University of Colorado Boulder: Patents & Royalties.

Vinorelbine and epirubicin share common features with polysialic acid and modulate neuronal and glial functions.

Polysialic acid (PSA), a large, linear glycan composed of 8 to over 100 α2,8-linked sialic acid residues, modulates development of the nervous system by enhancing cell migration, axon pathfinding, and synaptic targeting and by regulating differentiation of progenitor cells. PSA also functions in developing and adult immune systems and is a signature of many cancers. In this study we identified vinorelbine, a semi-synthetic third generation vinca alkaloid, and epirubicin, an anthracycline and 4'-epimer of doxorubicin, as PSA mimetics. Similar to PSA, vinorelbine and epirubicin bind to the PSA-specific monoclonal antibody 735 and compete with the bacterial analog of PSA, colominic acid in binding to monoclonal antibody 735. Vinorelbine and epirubicin stimulate neurite outgrowth of cerebellar neurons via the neural cell adhesion molecule, via myristoylated alanine-rich C kinase substrate, and via fibroblast growth factor receptor, signaling through Erk pathways. Furthermore, the two compounds enhance process formation of Schwann cells and migration of cerebellar neurons in culture, and reduce migration of astrocytes after injury. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds vinorelbine and epirubicin, thus raising the possibility to re-target drugs used in treatment of cancers to nervous system repair. Vinorelbine and epirubicin, identified as PSA mimetics, enhance, like PSA, neuronal migration, neuritogenesis, and formation of Schwann cell processes, and reduce astrocytic migration. Ablating NCAM, inhibiting fibroblast growth factor (FGFR) receptor, or adding the effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) minimize the vinorelbine and epirubicin effects, indicating that they are true PSA mimetics triggering PSA-mediated functions.

The Effector Domain of MARCKS Is a Nuclear Localization Signal that Regulates Cellular PIP2 Levels and Nuclear PIP2 Localization

Translocation to the nucleus of diacylglycerol kinase (DGK)– ζ is dependent on a sequence homologous to the effector domain of Myristoylated Alanine Rich C-Kinase Substrate (MARCKS). These data would suggest that MARCKS could also localize to the nucleus. A single report demonstrated immunofluorescence staining of MARCKS in the nucleus; however, further experimental evidence confirming the specific domain responsible for this localization has not been reported. Here, we report that MARCKS is present in the nucleus in GBM cell lines. We then over-expressed wild-type MARCKS (WT) and MARCKS with the effector domain deleted (ΔED), both tagged with V5-epitope in a GBM cell line with low endogenous MARCKS expression (U87). We found that MARCKS-WT localized to the nucleus, while the MARCKS construct without the effector domain remained in the cytoplasm. We also found that over-expression of MARCKS-WT resulted in a significant increase in total cellular phosphatidyl-inositol (4,5) bisphosphate (PIP2) levels, consistent with prior evidence that MARCKS can regulate PIP2 levels. We also found increased staining for PIP2 in the nucleus with MARCKS-WT over-expression compared to MARCKS ΔED by immunofluorescence. Interestingly, we observed MARCKS and PIP2 co-localization in the nucleus. Lastly, we found changes in gene expression when MARCKS was not present in the nucleus (MARCKS ΔED). These data indicate that the MARCKS effector domain can function as a nuclear localization signal and that this sequence is critical for the ability of MARCKS to regulate PIP2 levels, nuclear localization, and gene expression. These data suggests a novel role for MARCKS in regulating nuclear functions such as gene expression.

X MARCKS the spot: myristoylated alanine-rich C kinase substrate in neuronal function and disease.

Intracellular protein-protein interactions are dynamic events requiring tightly regulated spatial and temporal checkpoints. But how are these spatial and temporal cues integrated to produce highly specific molecular response patterns? A helpful analogy to this process is that of a cellular map, one based on the fleeting localization and activity of various coordinating proteins that direct a wide array of interactions between key molecules. One such protein, myristoylated alanine-rich C-kinase substrate (MARCKS) has recently emerged as an important component of this cellular map, governing a wide variety of protein interactions in every cell type within the brain. In addition to its well-documented interactions with the actin cytoskeleton, MARCKS has been found to interact with a number of other proteins involved in processes ranging from intracellular signaling to process outgrowth. Here, we will explore these diverse interactions and their role in an array of brain-specific functions that have important implications for many neurological conditions.

Abstract A87: Myristoylated alanine rich C-kinase substrate (MARCKS) expression in lung cancer cells influences immune cell populations in tumor microenvironment in murine models

Abstract Purpose: To determine if MARCKS expression in lung cancer cells alters the recruitment of immune cells to the tumor microenvironment while influencing outcome in in vivo models. Background: Lung cancer is the leading cause of cancer related deaths in the United States. There is increasing support that aberrant immune cell recruitment and activation in the tumor microenvironment leads to pro-tumor behavior. Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) is an intracellular protein that has been described to alter cellular proliferation as well as be involved in lung epithelial cytokine secretion and immune cell migration. Depending on the type of cancer, MARCKS has either pro-tumor or anti-tumor properties. Currently, it is unknown whether MARCKS expression is beneficial or detrimental in lung cancer. We wanted to investigate if MARCKS expression was able to delay tumor burden as well as influence the immune cell populations being recruited to the tumor microenvironment. Methods: The C57BL/6 murine lung cancer cell line Lewis Lung Carcinoma (LLC) was engineered with lentiviral particles to over-express MARCKS (LLC-MARCKS). Western blot confirmed over-expression of MARCKS. Tumors were implanted into the flank of C57BL/6 mice and survival data was collected comparing LLC-MARCKS over-expression tumors versus empty lentiviral plasmid containing LLC tumors (LLC-Ctrl). Survival data was graphed by Kaplan-Meier and statistics calculated with log-rank survival test. To analyze immune cells in the tumor microenvironment, orthotopic lung nodules were established by tail-vein injections. Three weeks after tail-vein injections, lungs were resected from LLC-MARCKS and LLC-Ctrl tumors bearing mice for flow cytometry analysis. Differences in immune cells between groups were calculated by student's t-test. Results: Mice with LLC-MARCKS tumors had an increase in median survival time compared to LLC-Ctrl tumors (31 days vs. 26 days) and a trend toward increase survival (p=0.058). While collecting lungs, it was grossly apparent that mice with LLC-Ctrl tumors had greater tumor burden and signs of disease, such as bloody exudates, compared to mice with LLC-MARCKS tumors. H &amp; E staining confirmed that LLC-Ctrl tumors had higher tumor burden then LLC-MARCKS tumors. Flow cytometry identified a decrease in F4/80+ macrophages (p&amp;lt;0.05) and neutrophils (p&amp;lt;0.05) in LLC-MARCKS cells compared to LLC-Ctrl. There was no difference in MDSC, DC subpopulations, CD4 T-cells, CD8 T-cells, B-cells, or T-regs. Conclusion: MARCKS expression in the murine lung cancer LLC cell line prolonged survival while also decreasing F4/80+ macrophages and neutrophils in the tumor microenvironment. Citation Format: Timothy D. Rohrbach, Travis D. Hull, John S. Jarboe, Nicholas Eustace, Yong Wang, Jessy S. Deshane, Christopher D. Willey. Myristoylated alanine rich C-kinase substrate (MARCKS) expression in lung cancer cells influences immune cell populations in tumor microenvironment in murine models. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A87.

Calmodulin and CaMKII modulate ENaC activity by regulating the association of MARCKS and the cytoskeleton with the apical membrane.

Phosphatidylinositol bisphosphate (PIP2) regulates epithelial sodium channel (ENaC) open probability. In turn, myristoylated alanine-rich C kinase substrate (MARCKS) protein or MARCKS-like protein 1 (MLP-1) at the plasma membrane regulates the delivery of PIP2 to ENaC. MARCKS and MLP-1 are regulated by changes in cytosolic calcium; increasing calcium promotes dissociation of MARCKS from the membrane, but the calcium-regulatory mechanisms are unclear. However, it is known that increased intracellular calcium can activate calmodulin and we show that inhibition of calmodulin with calmidazolium increases ENaC activity presumably by regulating MARCKS and MLP-1. Activated calmodulin can regulate MARCKS and MLP-1 in two ways. Calmodulin can bind to the effector domain of MARCKS or MLP-1, inactivating both proteins by causing their dissociation from the membrane. Mutations in MARCKS that prevent calmodulin association prevent dissociation of MARCKS from the membrane. Calmodulin also activates CaM kinase II (CaMKII). An inhibitor of CaMKII (KN93) increases ENaC activity, MARCKS association with ENaC, and promotes MARCKS movement to a membrane fraction. CaMKII phosphorylates filamin. Filamin is an essential component of the cytoskeleton and promotes association of ENaC, MARCKS, and MLP-1. Disruption of the cytoskeleton with cytochalasin E reduces ENaC activity. CaMKII phosphorylation of filamin disrupts the cytoskeleton and the association of MARCKS, MLP-1, and ENaC, thereby reducing ENaC open probability. Taken together, these findings suggest calmodulin and CaMKII modulate ENaC activity by destabilizing the association between the actin cytoskeleton, ENaC, and MARCKS, or MLP-1 at the apical membrane.

Association of MARCKS phosphorylation with breast cancer malignancy and paclitaxel unresponsiveness.

e22007 Background: Accumulating evidence has suggested that myristoylated alanine-rich C-kinase substrate (MARCKS) is critical for regulating multiple pathophysiological processes. However, the mol...

Elevated intraocular pressure induces Rho GTPase mediated contractile signaling in the trabecular meshwork

Rho GTPase regulated contractile signaling in the trabecular meshwork (TM) has been shown to modulate aqueous humor (AH) outflow and intraocular pressure (IOP). To explore whether elevated IOP, a major risk factor for primary open angle glaucoma (POAG) influences Rho GTPase signaling in the TM, we recorded AH outflow in enucleated contralateral porcine eyes perfused for 4–5 h at either 15 mm or 50 mm Hg pressure. After perfusion, TM tissue extracted from perfused eyes was evaluated for the activation status of Rho GTPase, myosin light chain (MLC), myosin phosphatase target substrate 1 (MYPT1), myristoylated alanine-rich C-kinase substrate (MARCKS) and paxillin. Eyes perfused at 50 mm Hg exhibited a significant decrease in AH outflow facility compared with those perfused at 15 mm Hg. Additionally, TM tissue from eyes perfused at 50 mm Hg revealed significantly increased levels of activated RhoA and phosphorylated MLC, MYPT1, MARCKS and paxillin compared to TM tissue derived from eyes perfused at 15 mm Hg. Taken together, these observations indicate that elevated IOP-induced activation of Rho GTPase-dependent contractile signaling in the TM is associated with increased resistance to AH outflow through the trabecular pathway, and demonstrate the sensitivity of Rho GTPase signaling to mechanical force in the AH outflow pathway.

Mechanism for phosphoinositide selectivity and activation of TRPV1 ion channels.

Although PI(4,5)P2 is believed to play an essential role in regulating the activity of numerous ion channels and transporters, the mechanisms by which it does so are unknown. Here, we used the ability of the TRPV1 ion channel to discriminate between PI(4,5)P2 and PI(4)P to localize the region of TRPV1 sequence that interacts directly with the phosphoinositide. We identified a point mutation in the proximal C-terminal region after the TRP box, R721A, that inverted the selectivity of TRPV1. Although the R721A mutation produced only a 30% increase in the EC50 for activation by PI(4,5)P2, it decreased the EC50 for activation by PI(4)P by more than two orders of magnitude. We used chemically induced and voltage-activated phosphatases to determine that PI(4)P continued to support TRPV1 activity even after depletion of PI(4,5)P2 from the plasma membrane. Our data cannot be explained by a purely electrostatic mechanism for interaction between the phosphoinositide and the protein, similar to that of the MARCKS (myristoylated alanine-rich C kinase substrate) effector domain or the EGF receptor. Rather, conversion of a PI(4,5)P2-selective channel to a PI(4)P-selective channel indicates that a structured phosphoinositide-binding site mediates the regulation of TRPV1 activity and that the amino acid at position 721 likely interacts directly with the moiety at the 5' position of the phosphoinositide.

Myristoylated alanine-rich C kinase substrate (MARCKS) regulates the expression of proinflammatory cytokines in macrophages through activation of p38/JNK MAPK and NF-κB

MARCKS, a substrate of protein kinase C, is involved in various processes associated with cytoskeletal movement. Although the expression of MARCKS is highly induced in macrophages, its role in macrophage function has not been studied in detail. Notably, the suppression of MARCKS expression in macrophage cell lines blocked LPS-induced expression of TNF-α at the transcriptional level. Treatment of macrophages with MARCKS N-terminus sequence (MANS) and effector domain (ED) peptides, which mimic functional domains and block the phosphorylation of MARCKS, suppressed the LPS-induced expression of TNF-α through suppression of p38 and JNK MAPKs and NF-κB. Treatment of mice with MANS peptide reduced serum TNF-α and IL-6 levels and resulted in 40% survival of mice after the administration of a lethal dose of LPS. These data demonstrate that MARCKS is involved in the regulation of proinflammatory cytokine expression in macrophages and that MARCKS-derived peptides can be used to suppress inflammatory responses.

Elevated MARCKS phosphorylation contributes to unresponsiveness of breast cancer to paclitaxel treatment.

Accumulating evidence has suggested that myristoylated alanine-rich C-kinase substrate (MARCKS) is critical for regulating multiple pathophysiological processes. However, the molecular mechanism underlying increased phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) and its functional consequence in neoplastic disease remain to be established. Herein, we investigated how phospho-MARCKS is regulated in breast carcinoma, and its role in the context of chemotherapy. In a screen of patients with breast tumors, we find that the abundance of phospho-MARCKS, not MARCKS protein per se, increased in breast cancers and positively correlated with tumor grade and metastatic status. Among chemotherapeutic agents, mitotic inhibitors, including paclitaxel, vincristine or eribulin, notably promoted phospho-MARCKS accumulation in multiple breast cancer cells. We further show that phospho-MARCKS acted upstream of Src activation upon paclitaxel exposure. Reduction of phospho-MARCKS by knockdown of MARCKS or pharmacological agents increased paclitaxel sensitivity. Particularly, a known phospho-MARCKS inhibitor, MANS peptide, was demonstrated to increase paclitaxel efficacy and attenuate angiogenesis/metastasis of xenografted breast cancer cells by decreasing abundance of phospho-MARCKS and messages of inflammatory mediators. Our data suggest that unresponsiveness of breast cancer to paclitaxel treatment is, at least in part, mediated by phospho-MARCKS and also provide an alternative therapeutic strategy against breast cancer by improving taxanes sensitivity.

Calpain‐2 Proteolysis of MARCKS is a Negative Feedback Regulator of ENaC

Proteolytic and hormonal regulation of ENaC activity is important for total body sodium homeostasis and blood pressure control. We recently identified a role for myristoylated alanine‐rich C kinase substrate (MARCKS) in the phosphotidylinositol 4,5‐bisphosphate (PIP2) dependent regulation of ENaC. Like ENaC, MARCKS is regulated by proteases. Calpains are intracellular calcium‐dependent cysteine proteases involved in many physiological functions. The purpose of this study was to examine the ability of calpain‐2 to serve as a negative feedback regulator of ENaC by cleaving MARCKS. Immunoprecipitation and Western blot studies showed the interaction between ENaC and MARCKS in Xenopus 2F3 cells decreased in response to the calcium ionophore A23187, and this effect was attenuated when cells were pretreated with the pharmacological inhibitor of calpain‐2 A6060. A difference in MARCKS expression in sucrose density gradient fractions was observed for cells treated with the calcium ionophore and the calpain‐2 inhibitor compared to untreated cells. Similarly, amiloride‐sensitive transepithelial current across cell monolayers was altered. Taken together, calcium‐dependent calpain‐2 proteolysis of MARCKS prevents constitutive stimulation of ENaC activity at the level of its open probability. This research was supported by a National Institutes of Health grant (1K01DK099617‐01A1).

Myristoylated Alanine-Rich C Kinase Substrate Accelerates TNF-&amp;lt;i&amp;gt;α&amp;lt;/i&amp;gt;-Induced Apoptosis in SH-SY5Y Cells in a Caspases-6 and/or -7-Dependent Manner

Cell proliferation, differentiation, and the elimination of unnecessary cells by apoptosis occur in the development of the nervous system. It is reported that brain dysplasia appears as the results of myristoylated alanine-rich C kinase substrate (MARCKS) knockout or the mutant mouse. We therefore expect that MARCKS participates in the development of the nervous system. However, the mechanism underlying such participation has not been identified. In this study, we observed the effects of the overexpression of MARCKS or unphosphorylatable MARCKS on cell proliferation and TNF-α-induced apoptosis in neuroblastoma SH-SY5Y cells. Furthermore, we restrained MARCKS expression by the RNAi method. In the results, MARCKS-overexpressing cells and not unphosphorylatable MARCKS-overexpressing cells showed increased cell proliferation rates. On the other hand, the RNAi decreased the proliferation of MARCKS-knocked down SH-SY5Y cells. These results indicated that MARCKS-overexpressing cells were more sensitive to TNF-α than normal SH-SY5Y cells. Moreover, in MARCKS-overexpressing cells TNF-α-induced apoptosis was inhibited by caspase-6 and -7 inhibitors but not by caspase-3 inhibitor. These results suggested that MARCKS participated in TNF-α-induced apoptosis in a caspase-6 and/or -7-dependent manner.

Targeting the effector domain of the myristoylated alanine rich C-kinase substrate enhances lung cancer radiation sensitivity.

Lung cancer is the leading cause of cancer related deaths. Common molecular drivers of lung cancer are mutations in receptor tyrosine kinases (RTKs) leading to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pro-growth, pro-survival signaling pathways. Myristoylated alanine rich C-kinase substrate (MARCKS) is a protein that has the ability to mitigate this signaling cascade by sequestering the target of PI3K, phosphatidylinositol (4,5)-bisphosphate (PIP2). As such, MARCKS has been implicated as a tumor suppressor, though there is some evidence that MARCKS may be tumor promoting in certain cancer types. Since the MARCKS function depends on its phosphorylation status, which impacts its subcellular location, MARCKS role in cancer may depend highly on the signaling context. Currently, the importance of MARCKS in lung cancer biology is limited. Thus, we investigated MARCKS in both clinical specimens and cell culture models. Immunohistochemistry scoring of MARCKS protein expression in a diverse lung tumor tissue array revealed that the majority of squamous cell carcinomas stained positive for MARCKS while other histologies, such as adenocarcinomas, had lower levels. To study the importance of MARCKS in lung cancer biology, we used inducible overexpression of wild-type (WT) and non-phosphorylatable (NP)-MARCKS in A549 lung cancer cells that had a low level of endogenous MARCKS. We found that NP-MARCKS expression, but not WT-MARCKS, enhanced the radiosensitivity of A549 cells in part by inhibiting DNA repair as evidenced by prolonged radiation-induced DNA double strand breaks. We confirmed the importance of MARCKS phosphorylation status by treating several lung cancer cell lines with a peptide mimetic of the phosphorylation domain, the effector domain (ED), which effectively attenuated cell growth as measured by cell index. Thus, the MARCKS ED appears to be an important target for lung cancer therapeutic development.

In Vitro Neutrophil Migration Requires Protein Kinase C-Delta (δ-PKC)-Mediated Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Phosphorylation.

Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that myristoylated alanine-rich C-kinase substrate (MARCKS), a well-known actin binding protein and protein kinase C (PKC) substrate, is a key regulator of neutrophil functions. In the current study, we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates f-Met-Leu-Phe (fMLF)-induced MARCKS phosphorylation (IC50=5.709 μM), adhesion (IC50=8.4 μM), and migration (IC50=6.7 μM), while α-, β-, and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC-mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC-mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils.