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Perry J. Blackshear - One of the best experts on this subject based on the ideXlab platform.
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tip60 and sirtuin 2 regulated MARCKS acetylation and phosphorylation are required for diabetic embryopathy
Nature Communications, 2019Co-Authors: Penghua Yang, Albert E Reece, Jianxiang Zhong, Perry J. Blackshear, Min Zhan, Deborah J. Stumpo, Cheng Xu, Xi Chen, Peixin YangAbstract:Failure of neural tube closure results in severe birth defects and can be induced by high glucose levels resulting from maternal diabetes. MARCKS is required for neural tube closure, but the regulation and of its biological activity and function have remained elusive. Here, we show that high maternal glucose induced MARCKS acetylation at lysine 165 by the acetyltransferase Tip60, which is a prerequisite for its phosphorylation, whereas Sirtuin 2 (SIRT2) deacetylated MARCKS. Phosphorylated MARCKS dissociates from organelles, leading to mitochondrial abnormalities and endoplasmic reticulum stress. Phosphorylation dead MARCKS (PD-MARCKS) reversed maternal diabetes-induced cellular organelle stress, apoptosis and delayed neurogenesis in the neuroepithelium and ameliorated neural tube defects. Restoring SIRT2 expression in the developing neuroepithelium exerted identical effects as those of PD-MARCKS. Our studies reveal a new regulatory mechanism for MARCKS acetylation and phosphorylation that disrupts neurulation under diabetic conditions by diminishing the cellular organelle protective effect of MARCKS. Neural tube defects can arise from high glucose levels caused by maternal diabetes, and MARCKS is required for neural tube closure. Here, Yang et al. show that acetylation and phosphorylation of MARCKS in hyperglycemic conditions causes mitochondrial and ER stress, leading to neural tube defects.
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myristoylated alanine rich protein kinase substrate MARCKS regulates small gtpase rac1 and cdc42 activity and is a critical mediator of vascular smooth muscle cell migration in intimal hyperplasia formation
Journal of the American Heart Association, 2015Co-Authors: Dan Yu, Perry J. Blackshear, Deborah J. Stumpo, George Makkar, Dudley K Strickland, Thomas A Blanpied, Rajabrata Sarkar, Thomas S MonahanAbstract:Background Transcription of the myristoylated alanine‐rich C kinase substrate (MARCKS) is upregulated in animal models of intimal hyperplasia. MARCKS knockdown inhibits vascular smooth muscle cell (VSMC) migration in vitro; however, the mechanism is as yet unknown. We sought to elucidate the mechanism of MARCKS‐mediated motility and determine whether MARCKS knockdown reduces intimal hyperplasia formation in vivo. Methods and Results MARCKS knockdown blocked platelet‐derived growth factor (PDGF)‐induced translocation of cortactin to the cell cortex, impaired both lamellipodia and filopodia formation, and attenuated motility of human coronary artery smooth muscle cells (CASMCs). Activation of the small GTPases, Rac1 and Cdc42, was prevented by MARCKS knockdown. Phosphorylation of MARCKS resulted in a transient shift of MARCKS from the plasma membrane to the cytosol. MARCKS knockdown significantly decreased membrane‐associated phosphatidylinositol 4,5‐bisphosphate (PIP2) levels. Cotransfection with an intact, unphosphorylated MARCKS, which has a high binding affinity for PIP2, restored membrane‐associated PIP2 levels and was indispensable for activation of Rac1 and Cdc42 and, ultimately, VSMC migration. Overexpression of MARCKS in differentiated VSMCs increased membrane PIP2 abundance, Rac1 and Cdc42 activity, and cell motility. MARCKS protein was upregulated early in the development of intimal hyperplasia in the murine carotid ligation model. Decreased MARKCS expression, but not total knockdown, attenuated intimal hyperplasia formation. Conclusions MARCKS upregulation increases VSMC motility by activation of Rac1 and Cdc42. These effects are mediated by MARCKS sequestering PIP2 at the plasma membrane. This study delineates a novel mechanism for MARCKS‐mediated VSMC migration and supports the rational for MARCKS knockdown to prevent intimal hyperplasia.
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functional role of the interaction between polysialic acid and myristoylated alanine rich c kinase substrate at the plasma membrane
Journal of Biological Chemistry, 2013Co-Authors: Thomas Theis, Perry J. Blackshear, Bibhudatta Mishra, Maren Von Der Ohe, Gabriele Loers, Maksymilian Prondzynski, Ole Pless, Melitta SchachnerAbstract:Polysialic acid (PSA) is a homopolymeric glycan that plays crucial roles in the developing and adult nervous system. So far only a few PSA-binding proteins have been identified. Here, we identify myristoylated alanine-rich C kinase substrate (MARCKS) as novel PSA binding partner. Binding assays showed a direct interaction between PSA and a peptide comprising the effector domain of MARCKS (MARCKS-ED). Co-immunoprecipitation of PSA-carrying neural cell adhesion molecule (PSA-NCAM) with MARCKS and co-immunostaining of MARCKS and PSA at the cell membrane of hippocampal neurons confirm the interaction between PSA and MARCKS. Co-localization and an intimate interaction of PSA and MARCKS at the cell surface was seen by confocal microscopy and fluorescence resonance energy transfer (FRET) analysis after the addition of fluorescently labeled PSA or PSA-NCAM to live CHO cells or hippocampal neurons expressing MARCKS as a fusion protein with green fluorescent protein (GFP). Cross-linking experiments showed that extracellularly applied PSA or PSA-NCAM and intracellularly expressed MARCKS-GFP are in close contact, suggesting that PSA and MARCKS interact with each other at the plasma membrane from opposite sides. Insertion of PSA and MARCKS-ED peptide into lipid bilayers from opposite sides alters the electric properties of the bilayer confirming the notion that PSA and the effector domain of MARCKS interact at and/or within the plane of the membrane. The MARCKS-ED peptide abolished PSA-induced enhancement of neurite outgrowth from cultured hippocampal neurons indicating an important functional role for the interaction between MARCKS and PSA in the developing and adult nervous system.
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myristoylated alanine rich c kinase substrate MARCKS heterozygous mutant mice exhibit deficits in hippocampal mossy fiber ca3 long term potentiation
Hippocampus, 2006Co-Authors: Rifat J Hussain, Perry J. Blackshear, Deborah J. Stumpo, Robert H Lenox, Ted Abel, Robert K McnamaraAbstract:The myristoylated alanine-rich C kinase substrate (MARCKS) is a primary protein kinase C (PKC) substrate in brain thought to transduce PKC signaling into alterations in the filamentous (F) actin cytoskeleton. Within the adult hippocampus, MARCKS is highly expressed in the dentate gyrus (DG)-CA3 mossy fiber pathway, but is expressed at low levels in the CA3-CA1 Schaffer collateral-CA1 pathway. We have previously demonstrated that 50% reductions in MARCKS expression in heterozygous MARCKS mutant mice produce robust deficits in spatial reversal learning, but not contextual fear conditioning, suggesting that only specific aspects of hippocampal function are impaired by reduction in MARCKS expression. To further elucidate the role of MARCKS in hippocampal synaptic plasticity, in the present study we examined basal synaptic transmission, paired-pulse facilitation, post-tetanic potentiation, and long-term potentiation (LTP) in the hippocampal mossy fiber-CA3 and Schaffer collateral-CA1 pathways of heterozygous MARCKS mutant and wild-type mice. We found that LTP is significantly impaired in the mossy fiber-CA3 pathway, but not in the Schaffer collateral-CA1 pathway, in heterozygous MARCKS mutant mice, whereas basal synaptic transmission, paired-pulse facilitation, and post-tetanic potentiation are unaffected in both pathways. These findings indicate that a 50% reduction in MARCKS expression impairs processes required for long-term, but not short-term, synaptic plasticity in the mossy fiber-CA3 pathway. The implications of these findings for the role of the mossy fiber-CA3 pathway in hippocampus-dependent learning processes are discussed.
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Membrane Association of the Myristoylated Alanine-rich C Kinase Substrate (MARCKS) Protein. MUTATIONAL ANALYSIS PROVIDES EVIDENCE FOR COMPLEX INTERACTIONS
The Journal of biological chemistry, 1995Co-Authors: Sharon L. Swierczynski, Perry J. BlackshearAbstract:Abstract The myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent cellular substrate for protein kinase C, is associated with membranes in various cell types. MARCKS is myristoylated at its amino terminus; this modification is thought to play the major role in anchoring MARCKS to cellular membranes. Recent studies have suggested that the protein's basic phosphorylation site/calmodulin binding domain may also be involved in the membrane association of MARCKS through electrostatic interactions. The present studies used mutations in the primary structure of the protein to investigate the nature of the association between MARCKS and cell membranes. In chick embryo fibroblasts, activation of protein kinase C led to a decrease in MARCKS membrane association as determined by cell fractionation techniques. Cell-free assays revealed that nonmyristoylated MARCKS exhibited almost no affinity for fibroblast membranes, despite readily demonstrable binding of the wild-type protein. Similar experiments in which the four serines in the phosphorylation site domain were mutated to aspartic acids, mimicking phosphorylation, decreased, but did not eliminate, membrane binding when compared to either the wild-type protein or a comparable tetra-asparagine mutant. Addition of calmodulin in the presence of Ca also inhibited binding of the wild-type protein to membranes, presumably by neutralizing the phosphorylation site domain, or by physically interfering with its membrane association. Surprisingly, expression of a nonmyristoylatable mutant form of MARCKS in intact cells led to only a 46% decrease in its plasma membrane association, as determined by cell fractionation and immunoelectron microscopy. These results are consistent with a complex model of the interaction of MARCKS with cellular membranes, in which the myristoyl moiety, the positively charged phosphorylation site domain, and possibly other domains make independent contributions to membrane binding in intact cells.
David M Byers - One of the best experts on this subject based on the ideXlab platform.
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The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2004Co-Authors: Meenakshi Sundaram, Harold W Cook, David M ByersAbstract:Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.
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induction of protein kinase c substrates myristoylated alanine rich c kinase substrate MARCKS and MARCKS related protein mrp by amyloid β protein in mouse bv 2 microglial cells
Neuroscience Letters, 2003Co-Authors: Anne Murphy, Neale D Ridgway, Harold W Cook, Janet R Sunohara, Meenakshi Sundaram, Christopher R Mcmaster, David M ByersAbstract:Abstract Microglial activation by amyloid β-protein in senile plaques contributes to neurodegeneration in Alzheimer disease. In BV-2 microglial cells, amyloid β-protein 1–40 (Aβ 1–40) elicited a dose-dependent increase (3–4 fold) of Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP), two protein kinase C substrates implicated in membrane-cytoskeletal alterations underlying microglial adhesion, migration, secretion, and phagocytosis. Neither MARCKS nor MRP was induced by the amyloid fragment Aβ 25–35, although both Aβ 1–40 and Aβ 25–35 caused extensive aggregation of BV-2 cells. Interferon-γ synergistically enhanced the induction by Aβ 1–40 of inducible nitric oxide synthase, but not MARCKS or MRP. Our results suggest that MARCKS and MRP may play important roles in microglia activated by amyloid peptides.
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regulation of MARCKS and MARCKS related protein expression in bv 2 microglial cells in response to lipopolysaccharide
Journal of Neurochemistry, 2001Co-Authors: Janet R Sunohara, Neale D Ridgway, Harold W Cook, David M ByersAbstract:Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-kappaB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines.
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inhibitors of actin polymerization and calmodulin binding enhance protein kinase c induced translocation of MARCKS in c6 glioma cells
Biochimica et Biophysica Acta, 1997Co-Authors: Donna N Douglas, Horstsiegfried Fink, S D Rose, Neale D Ridgway, Harold W Cook, David M ByersAbstract:Abstract MARCKS ( m yristoylated a lanine- r ich C - k inase s ubstrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4 β -12- O -tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC α to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC α in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.
Ching-hsien Chen - One of the best experts on this subject based on the ideXlab platform.
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abstract 2006 tobacco smoke induced MARCKS phosphorylation promotes inflammatory and stemness properties in lung cancer
Cancer Research, 2018Co-Authors: David C. Yang, Mujen Huang, Kent E Pinkerton, Ching-hsien ChenAbstract:Exposure to tobacco smoke is a major risk factor associated with lung cancer development and progression; however, the mechanisms of how tobacco smoke activates pathways promoting cancer malignancy remain to be established. Previously, phosphorylation of MARCKS at Ser159/163 (phospho-MARCKS) was shown to promote malignancy of neoplastic disease, but it is still unclear how phospho-MARCKS is upregulated in lung cancer. Herein, we investigated if phospho-MARCKS is elevated in response to smoke exposure and plays a key role in smoke-related lung cancer. In a screen of 200 patients with lung cancer, we confirmed that strong phospho-MARCKS staining was observed in lung cancer specimens from smokers and was positively correlated, as compared to non-smokers. In addition, we demonstrated that increased smoking pack-year was noted in lung cancer patients with high phospho-MARCKS. Surprisingly, there is an apparent trend that patients with both tobacco use and high phospho-MARCKS levels had the worst overall survival. MARCKS was found to become activated in airway epithelium and lung cancer in response to smoke both in vitro and in vivo. MARCKS inhibition significantly attenuated smoke-induced oncosphere formation and migratory capability. Mechanistically, upregulation of phospho-MARCKS by smoke occurred in parallel with increased inflammatory cytokine expression (e.g. TNF-α, IL-8) and stemness marker expression (e.g. Oct4, Nanog, CD133, Sox2). Conversely, targeting of MARCKS phosphorylation with the MPS peptide resulted in down-regulation of smoke-induced stemness and cytokine genes. Our data suggests that phospho-MARCKS plays a role in modulating smoke-mediated lung cancer progression and is a potential prognostic biomarker in smoke-related lung cancer. Citation Format: David C. Yang, Yichin Wu, Mu-Jen Huang, Kent E. Pinkerton, Reen Wu, Ching-Hsien Chen. Tobacco smoke-induced MARCKS phosphorylation promotes inflammatory and stemness properties in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2006.
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Myristoylated alanine-rich C kinase substrate (MARCKS): a multirole signaling protein in cancers
Cancer and Metastasis Reviews, 2017Co-Authors: Lon Wolf R. Fong, David C. Yang, Ching-hsien ChenAbstract: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, Ca^2+ 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.
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abstract 1810 dysregulation of myristoylated alanine rich c kinase substrate MARCKS a novel therapeutic target in renal cell carcinoma
Cancer Research, 2017Co-Authors: Ching-hsien Chen, Muhammad Arif, Wenhsin Chang, R H WeissAbstract:Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/VEGF signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably VEGF-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multi-kinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multi-kinase inhibitors. Citation Format: Ching-Hsien Chen, Wen-Hsin Chang, Eric Yu, Muhammad S. Arif, Reen Wu, Robert H. Weiss. Dysregulation of myristoylated alanine-rich C kinase substrate (MARCKS): A novel therapeutic target in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1810. doi:10.1158/1538-7445.AM2017-1810
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elevated MARCKS phosphorylation contributes to unresponsiveness of breast cancer to paclitaxel treatment
Oncotarget, 2015Co-Authors: Ching-hsien Chen, Lon Wolf R. Fong, Yuan Yuan, Chun Ting Cheng, Jing Zhai, Muhammad Arif, David K AnnAbstract:// Ching-Hsien Chen 1 , Chun-Ting Cheng 2,3 , Yuan Yuan 4 , Jing Zhai 5 , Muhammad Arif 1 , Lon Wolf R. Fong 1 , Reen Wu 1 and David K. Ann 2,3 1 Department of Internal Medicine, Division of Pulmonary and Critical Care Medicine and Center for Comparative Respiratory Biology and Medicine, University of California Davis, California, USA 2 Department of Molecular Pharmacology, Beckman Research Institute, City of Hope, Duarte, California, USA 3 Irell and Manella Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, Duarte, California, USA 4 Department of Medical Oncology and Experimental Therapeutics, City of Hope Comprehensive Cancer Center, Duarte, California, USA 5 Department of Pathology, City of Hope Comprehensive Cancer Center, Duarte, California, USA Correspondence to: David K. Ann, email: // Reen Wu, email: // Keywords : phospho-MARCKS, MANS peptide, paclitaxel, mitotic inhibitor, breast cancer Received : December 23, 2014 Accepted : March 26, 2015 Published : April 14, 2015 Abstract 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.
Kenneth B Adler - One of the best experts on this subject based on the ideXlab platform.
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MARCKS and lung disease
American Journal of Respiratory Cell and Molecular Biology, 2019Co-Authors: Mary Katherine Sheats, Anne L. Crews, Shijing Fang, Joungjoa Park, Qi Yin, Indu Parikh, Brian Dickson, Kenneth B AdlerAbstract: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.
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MARCKS regulates neuritogenesis and interacts with a cdc42 signaling network
Scientific Reports, 2018Co-Authors: Jon Brudvig, Kenneth B Adler, Jacob T Cain, R M Sears, G G Schmidtgrimminger, Erika S Wittchen, H T Ghashghaei, Jill M WeimerAbstract: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.
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Fibroblast Migration Is Regulated by Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) Protein.
PloS one, 2013Co-Authors: Laura E. Ott, Kenneth B Adler, Mary K. Sheats, Eui Jae Sung, Adam T. Melvin, Jason M. Haugh, Samuel L. JonesAbstract:Myristoylated alanine-rich C-kinase substrate (MARCKS) is a ubiquitously expressed substrate of protein kinase C (PKC) that is involved in reorganization of the actin cytoskeleton. We hypothesized that MARCKS is involved in regulation of fibroblast migration and addressed this hypothesis by utilizing a unique reagent developed in this laboratory, the MANS peptide. The MANS peptide is a myristoylated cell permeable peptide corresponding to the first 24-amino acids of MARCKS that inhibits MARCKS function. Treatment of NIH-3T3 fibroblasts with the MANS peptide attenuated cell migration in scratch wounding assays, while a myristoylated, missense control peptide (RNS) had no effect. Neither MANS nor RNS peptide treatment altered NIH-3T3 cell proliferation within the parameters of the scratch assay. MANS peptide treatment also resulted in inhibited NIH-3T3 chemotaxis towards the chemoattractant platelet-derived growth factor-BB (PDGF-BB), with no effect observed with RNS treatment. Live cell imaging of PDGF-BB induced chemotaxis demonstrated that MANS peptide treatment resulted in weak chemotactic fidelity compared to RNS treated cells. MANS and RNS peptides did not affect PDGF-BB induced phosphorylation of MARCKS or phosphoinositide 3-kinase (PI3K) signaling, as measured by Akt phosphorylation. Further, no difference in cell migration was observed in NIH-3T3 fibroblasts that were transfected with MARCKS siRNAs with or without MANS peptide treatment. Genetic structure-function analysis revealed that MANS peptide-mediated attenuation of NIH-3T3 cell migration does not require the presence of the myristic acid moiety on the amino-terminus. Expression of either MANS or unmyristoylated MANS (UMANS) C-terminal EGFP fusion proteins resulted in similar levels of attenuated cell migration as observed with MANS peptide treatment. These data demonstrate that MARCKS regulates cell migration and suggests that MARCKS-mediated regulation of fibroblast migration involves the MARCKS amino-terminus. Further, this data demonstrates that MANS peptide treatment inhibits MARCKS function during fibroblast migration and that MANS mediated inhibition occurs independent of myristoylation.
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MARCKS and hsp70 interactions regulate mucin secretion by human airway epithelial cells in vitro
American Journal of Physiology-lung Cellular and Molecular Physiology, 2013Co-Authors: Shijing Fang, Anne L. Crews, Joungjoa Park, Wei Chen, Kenneth B AdlerAbstract:Myristoylated alanine-rich C kinase substrate (MARCKS) protein has been recognized as a key regulatory molecule controlling mucin secretion by airway epithelial cells in vitro and in vivo. We recently showed that two intracellular chaperones, heat shock protein 70 (HSP70) and cysteine string protein (CSP), associate with MARCKS in the secretory mechanism. To elucidate more fully MARCKS-HSP70 interactions in this process, studies were performed in well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture utilizing specific pharmacological inhibition of HSP70 with pyrimidinone MAL3-101 and siRNA approaches. The results indicate that HSP70 interaction with MARCKS is enhanced after exposure of the cells to the protein kinase C activator/mucin secretagogue, phorbol 12-myristate 13-acetate (PMA). Pretreatment of NHBEs with MAL3-101 attenuated in a concentration-dependent manner PMA-stimulated mucin secretion and interactions among HSP70, MARCKS, and CSP. In additional studies, trafficking of MARCKS in living NHBE cells was investigated after transfecting cells with fluorescently tagged DNA constructs: MARCKS-yellow fluorescent protein, and/or HSP70-cyan fluorescent protein. Cells were treated with PMA 48 h posttransfection, and trafficking of the constructs was examined by confocal microscopy. MARCKS translocated rapidly from plasma membrane to cytoplasm, whereas HSP70 was observed in the cytoplasm and appeared to associate with MARCKS after PMA exposure. Pretreatment of cells with either MAL3-101 or HSP70 siRNA inhibited translocation of MARCKS. These results provide evidence of a role for HSP70 in mediating mucin secretion via interactions with MARCKS and that these interactions are critical for the cytoplasmic translocation of MARCKS upon its phosphorylation.
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calpain and MARCKS protein regulation of airway mucin secretion
Pulmonary Pharmacology & Therapeutics, 2012Co-Authors: Randall W Lampe, Anne L. Crews, Shijing Fang, Joungjoa Park, Kenneth B AdlerAbstract:Hypersecretion of mucin plays an important role in the pathophysiology of many inflammatory airway diseases, including asthma, chronic bronchitis, and cystic fibrosis. Myristoylated alanine-rich C-kinase substrate (MARCKS) protein has been shown to play an important role in regulation of airway mucin secretion, as peptides analogous to the amino (N)-terminus of MARCKS attenuate mucin secretion by airway epithelium in vitro and in vivo. Here, we investigated a potential role for the protease Calpain, a calcium-dependent cysteine protease that can cleave MARCKS, in the MARCKS-related secretory mechanism. We theorized that Calpain might cleave MARCKS near the N-terminus, thereby attenuating the ability of MARCKS to bind to membranes and/or creating a small N-terminal peptide that could act as a competitive intracellular inhibitor to remaining endogenous full-length MARCKS molecules. Primary normal human bronchial epithelial (NHBE) cells and the virally-transformed human bronchial epithelial HBE1 cell line were exposed to phorbol-12-myristate-13-acetate (PMA) to stimulate the Protein Kinase C (PKC) pathway, leading to enhanced mucin secretion, and Calpain activity within the cells was measured with a fluorescent cleavage assay. Calpain activity was increased by PMA, and pretreatment of the cells with Calpain inhibitors reduced both Calpain activity and mucin secretion in a concentration-dependent manner. Thus, as opposed to the original hypothesis, inactivating Calpain caused a decrease rather than an increase in secretion. HBE1 cells transfected with DNA constructs encoding a MARCKS-YFP fusion protein showed cleavage at a putative site near the N-terminus in response to PMA. Cleavage of MARCKS by Calpain may have an important role in regulation of the PKC/MARCKS pathway regulating airway mucin secretion.
Harold W Cook - One of the best experts on this subject based on the ideXlab platform.
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The MARCKS family of phospholipid binding proteins: regulation of phospholipase D and other cellular components.
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2004Co-Authors: Meenakshi Sundaram, Harold W Cook, David M ByersAbstract:Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) are essential proteins that are implicated in coordination of membrane-cytoskeletal signalling events, such as cell adhesion, migration, secretion, and phagocytosis in a variety of cell types. The most prominent structural feature of MARCKS and MRP is a central basic effector domain (ED) that binds F-actin, Ca2+-calmodulin, and acidic phospholipids; phosphorylation of key serine residues within the ED by protein kinase C (PKC) prevents the above interactions. While the precise roles of MARCKS and MRP have not been established, recent attention has focussed on the high affinity of the MARCKS ED for phosphatidylinositol 4,5-bisphosphate (PIP2), and a model has emerged in which calmodulin- or PKC-mediated regulation of these proteins at specific membrane sites could in turn control spatial availability of PIP2. The present review summarizes recent progress in this area and discusses how the above model might explain a role for MARCKS and MRP in activation of phospholipase D and other PIP2-dependent cellular processes.
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induction of protein kinase c substrates myristoylated alanine rich c kinase substrate MARCKS and MARCKS related protein mrp by amyloid β protein in mouse bv 2 microglial cells
Neuroscience Letters, 2003Co-Authors: Anne Murphy, Neale D Ridgway, Harold W Cook, Janet R Sunohara, Meenakshi Sundaram, Christopher R Mcmaster, David M ByersAbstract:Abstract Microglial activation by amyloid β-protein in senile plaques contributes to neurodegeneration in Alzheimer disease. In BV-2 microglial cells, amyloid β-protein 1–40 (Aβ 1–40) elicited a dose-dependent increase (3–4 fold) of Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP), two protein kinase C substrates implicated in membrane-cytoskeletal alterations underlying microglial adhesion, migration, secretion, and phagocytosis. Neither MARCKS nor MRP was induced by the amyloid fragment Aβ 25–35, although both Aβ 1–40 and Aβ 25–35 caused extensive aggregation of BV-2 cells. Interferon-γ synergistically enhanced the induction by Aβ 1–40 of inducible nitric oxide synthase, but not MARCKS or MRP. Our results suggest that MARCKS and MRP may play important roles in microglia activated by amyloid peptides.
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regulation of MARCKS and MARCKS related protein expression in bv 2 microglial cells in response to lipopolysaccharide
Journal of Neurochemistry, 2001Co-Authors: Janet R Sunohara, Neale D Ridgway, Harold W Cook, David M ByersAbstract:Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP) have been implicated in membrane-cytoskeletal events underlying cell adhesion, migration, secretion, and phagocytosis. In BV-2 microglial cells, lipopolysaccharide (LPS) elicited a dose-dependent increase in mRNA of both MRP (sixfold) and MARCKS (threefold) with corresponding increases in [3H]myristoylated and immunoreactive protein levels. LPS also produced significant increases in protein kinase C (PKC)-beta twofold and PKC-epsilon (1.5-fold). Pro-inflammatory cytokines produced by activated microglia (IL-1beta, IL-6, TNF-alpha) did not mimic LPS effects on MARCKS or MRP expression when added individually or in combination. LPS and IFN-gamma produced a synergistic induction of iNOS but not MARCKS or MRP. Induction of MARCKS and MRP by LPS was completely blocked by inhibitors of NF-kappaB (PDTC) and protein tyrosine kinases (herbimycin A), partially blocked by the p38 kinase inhibitor SB203580, and unaffected by the MEK inhibitor PD98059. LPS induction of iNOS was considerably more sensitive to all these inhibitors. The Src kinase inhibitor PP2 had no effect, while the closely related inhibitor PP1 actually increased LPS induction of MARCKS and MRP. Our results suggest that MARCKS and MRP may play an important role in LPS-activated microglia, but are not part of the neuroinflammatory response produced by cytokines.
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inhibitors of actin polymerization and calmodulin binding enhance protein kinase c induced translocation of MARCKS in c6 glioma cells
Biochimica et Biophysica Acta, 1997Co-Authors: Donna N Douglas, Horstsiegfried Fink, S D Rose, Neale D Ridgway, Harold W Cook, David M ByersAbstract:Abstract MARCKS ( m yristoylated a lanine- r ich C - k inase s ubstrate) is known to interact with calmodulin, actin filaments, and anionic phospholipids at a central basic domain which is also the site of phosphorylation by protein kinase C (PKC). In the present study, cytochalasin D (CD) and calmodulin antagonists were used to examine the influence of F-actin and calmodulin on membrane interaction of MARCKS in C6 glioma cells. CD treatment for 1 h disrupted F-actin filaments, increased membrane bound immunoreactive MARCKS (from 51% to 62% of total), yet markedly enhanced the amount of MARCKS translocated to the cytosolic fraction in response to the phorbol ester 4 β -12- O -tetradecanoylphorbol 13-acetate. In contrast, CD treatment had no effect on phorbol ester-stimulated phosphorylation of MARCKS or on translocation of PKC α to the membrane fraction. Staurosporine also increased membrane association of MARCKS in a PKC-independent manner, as no change in MARCKS phosphorylation was noted and bis-indolylmaleimide (a more specific PKC inhibitor) did not alter MARCKS distribution. Staurosporine inhibited the phorbol ester-induced translocation of MARCKS but not of PKC α in both CD pretreated and untreated cells. Calmodulin antagonists (trifluoperazine, calmidazolium) had little effect on the cellular distribution or phosphorylation of MARCKS, but were synergistic with phorbol ester in translocating MARCKS from the membrane without a further increase in its phosphorylation. We conclude that cytoskeletal integrity is not required for phosphorylation and translocation of MARCKS in response to activated PKC, but that interaction with both F-actin and calmodulin might serve to independently modulate PKC-regulated localization and function of MARCKS at cellular membranes.