Translocation Of Myristoylated Alanine-rich C Kinase Substrate Research Articles

Restoration of MARCK enhances chemosensitivity in cancer

PurposeIncreased ATP-binding-cassette (ABC) transporter activity is a major cause of chemotherapy resistance in cancer. The ABC transporter family member ABCB1 is often overexpressed in colorectal cancer (CRC). Phosphatidylinositol-4,5-bisphosphat (PI(4,5)P2)-dependent pathways are involved in the regulation of ABCB1 function. The protein Myristoylated Alanine-Rich C-Kinase Substrate (MARCKS) is a pivotal regulator of PI(4,5)P2 and inactivated in many CRC cancers via genetic deletion or hyperphosphorylation. Therefore, MARCKS may critically impact ABCB1.MethodsCRC samples as well as CRC cell lines were tested for a connection between MARCKS and ABCB1 via immunofluorescence and Western-blot analysis. ABCB1 function was studied via calcein influx assay under treatment with known ABCB1 inhibitors (verapamil, tariquidar) as well as the kinase inhibitor bosutinib. ABCB1 internalization and MARCKS translocation was analyzed via confocal microscopy exploiting the endocytosis inhibitors chlorpromazine and dynasore. Abundance of PI(4,5)P2 was monitored by intramolecular fluorescence resonance energy transfer (FRET). Reproductive cell survival was studied via colorimetric WST-1 and clonogenic assays in combination with exposure to the chemotherapeutics doxorubicin and 5-fuorouracil (5-FU).ResultsWe found increased ABCB1 expression in MARCKS negative CRC patient tumor samples and established CRC cell lines. Mechanistically, the reconstitution of MARCKS function via recombinant expression or the pharmacological inhibition of MARCKS phosphorylation led to a substantial decrease in ABCB1 activity. In CRC cells, bosutinib treatment resulted in a MARCKS translocation from the cytosol to the plasma membrane, while simultaneously, ABCB1 was relocated to intracellular compartments. Inhibition of MARCKS phosphorylation via bosutinib rendered cells more sensitive to the chemotherapeutics doxorubicin and 5-FU.ConclusionsCells devoid of MARCKS function showed incomplete ABCB1 internalization, leading to higher ABCB1 activity enhancing chemoresistance. Vice versa our data suggest the prevention of MARCKS inhibition by reversing hyperphosphorylation or genomic restoration after deletion as two promising approaches to overcome tumor cell resistance towards chemotherapeutic ABCB1 substrates.

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 protein mediates hydrogen peroxide regulation of endothelial permeability

Impairment of endothelial barrier function is implicated in many vascular and inflammatory disorders. One prevalent mechanism of endothelial dysfunction is an increase in reactive oxygen species under oxidative stress. Previous reports have demonstrated that hydrogen peroxide (H(2)O(2)), a highly stable reactive oxygen species that modulates physiological signaling pathways, also enhances endothelial permeability, but the mechanism of this effect is unknown. Here, we identify the actin-binding protein myristoylated alanine-rich C-kinase substrate (MARCKS) as a key mediator of the H(2)O(2)-induced permeability change in bovine aortic endothelial cells. MARCKS knockdown and H(2)O(2) treatment alter the architecture of the actin cytoskeleton in endothelial cells, and H(2)O(2) induces the phosphorylation and translocation of MARCKS from the cell membrane to the cytosol. Using pharmacological inhibitors and small interference RNA constructs directed against specific proteins, we uncover a signaling cascade from Rac1 to Abl1, phospholipase Cγ1, and PKCδ that is triggered by H(2)O(2) and leads to MARCKS phosphorylation. Our findings establish a distinct role for MARCKS in the regulation of H(2)O(2)-induced permeability change in endothelial cells, and suggest potential new therapeutic targets for the treatment of disorders involving oxidative stress and altered endothelial permeability.

Directed migration of mouse macrophages in vitro involves myristoylated alanine-rich C-kinase substrate (MARCKS) protein

A role for MARCKS protein in directed migration of macrophages toward a chemoattractant was investigated. A peptide identical to the N-terminus of MARCKS (the MANS peptide), shown previously to inhibit the function of MARCKS in various cell types, was used. We investigated whether this MARCKS-related peptide could affect migration of macrophages, using the mouse macrophage-like J774A.1 cell line and primary murine macrophages. Both of these cell types migrated in response to the chemoattractants macrophage/MCPs, MCP-1 (25-100 ng/ml) or C5a (5-20 ng/ml). Cells were preincubated (15 min) with MANS or a mis-sense control peptide (RNS), both at 50 μM, and effects on migration determined 3 h after addition of chemoattractants. The movement and interactions of MARCKS and actin also were followed visually via confocal microscopy using a fluorescently labeled antibody to MARCKS and fluorescently tagged phalloidin to identify actin. MANS, but not RNS, attenuated migration of J774A.1 cells and primary macrophages in response to MCP-1 or C5a, implicating MARCKS in the cellular mechanism of directed migration. Exposure of cells to MCP-1 resulted in rapid phosphorylation and translocation of MARCKS from plasma membrane to cytosol, whereas actin appeared to spread through the cell and into cell protrusions; there was visual and biochemical evidence of a transient interaction between MARCKS and actin during the process of migration. These results suggest that MARCKS is involved in directed migration of macrophages via a process involving its phosphorylation, cytoplasmic translocation, and interaction with actin.

Biologically active milli-calpain associated with caveolae is involved in a spatially compartmentalised signalling involving protein kinase C alpha and myristoylated alanine-rich C-kinase substrate (MARCKS)

We have previously shown that calpain promotes myoblast fusion by acting on protein kinase C-alpha and the cytosolic phosphorylated form of MARCKS. In other cell types, various isoforms of calpain, PKC alpha and MARCKS were found associated with caveolae. These vesicular invaginations of the plasma membrane are essential for myoblast fusion and differentiation. We have isolated caveolae from myoblasts and studied the presence of calpain isoforms and their possible effects on signalling mediated by caveolae-associated PKC. Our results show that milli-calpain co-localizes with myoblast caveolae. Futhermore we provide evidence, using a calcium ionophore and a specific inhibitor of calpains (calpastatin peptide), that milli-calpain reduces the PKC alpha and MARCKS content in these structures. Purified milli-calpain causes the appearance of the active catalytic fragment of PKC alpha (PKM), without having an effect on MARCKS. Addition of phorbol myristate acetate, an activator of PKC, induces tranlocation of PKC alpha towards caveolae and results in a significant reduction of MARCKS associated with caveolae. This phenomenon is not observed when a PKC alpha inhibitor is added at the same time. We conclude that the presence of biologically active milli-calpain within myoblast caveolae induces, in a PKC alpha-dependent manner, MARCKS translocation towards the cytosol. Such a localised signalling event may be essential for myoblast fusion and differentiation.

Moving MARCKS Maintains Spreading

Disatnik et al. report studies dissecting the role of the protein kinase C (PKC) substrate MARCKS (myristoylated alanine-rich C-kinase substrate) in integrin-induced cell spreading. Their results indicate that MARCKS has two sequential roles: one to promote attachment of the cells to the substrate, and the other to promote cell spreading associated with polymerization and cross-linking of actin. The two phases appear to result from distinct localizations of the MARCKS protein. Within 30 min of plating of mouse myoblasts on fibronectin-coated dishes, α5ß1 integrin-mediated signals cause activation of PKC; dissociation of MARCKS, which is initially associated with the plasma membrane; and movement to the cytosol. When this process was blocked in cells ectopically expressing a MARCKS mutant lacking PKC phosphorylation sites, there was a nearly complete loss of attachment and spreading of cells. At later time points, MARCKS becomes dephosphorylated and returns to the plasma membrane, a process that requires an N-terminal myristoylation domain. Expression of MARCKS in which the myristoylation site was mutated yielded cells that underwent initial attachment but failed to spread. MARCKS is known to directly and indirectly influence actin cross-linking. Thus, the authors propose that the bidirectional translocation of MARCKS provides a key link between integrin signaling and control of cytoskeletal dynamics. M. H. Disatnik, S. C. Boutet, W. Pacio, A. Y. Chan, L. B. Ross, C. H. Lee, T. A. Rando, The bi-directional translocation of MARCKS between membrane and cytosol regulates integrin-mediated muscle cell spreading. J. Cell Sci . 117 , 4469-4479 (2004). [Abstract] [Full Text]

Spatiotemporal interactions of myristoylated alanine-rich C kinase substrate (MARCKS) protein with the actin cytoskeleton and exocytosis of oxytocin upon prostaglandin F2alpha stimulation of bovine luteal cells.

In the bovine corpus luteum (CL) phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) protein in response to prostaglandin F2alpha (PGF2alpha) is correlated with the secretion of oxytocin. The present study was conducted to 1) examine the intracellular translocation characteristics of wild-type and mutant forms of a green fluorescent protein (GFP)-conjugated MARCKS (MARCKS-GFP) after PGF2alpha treatment and 2) evaluate PGF2alpha-induced temporal changes in MARCKS-GFP and actin cortex associated with exocytosis of oxytocin. In experiment 1, cells of the bovine CL were cultured on coverslips overnight. Then, wild-type and mutant MARCKS-GFP constructs were transfected separately into cells and expression was detected through fluorescence microscopy. Forty-eight hours after transfection, cells were treated with vehicle, PGF2alpha (56 nM), or a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA], 1 microM). Treatment of cells expressing wild-type MARCKS-GFP with PGF2alpha and TPA resulted in translocation of MARCKS from the plasma membrane to the cytoplasm within 2.5 min. Phosphorylation mutant MARCKS-GFP (m3) protein was localized on the plasma membrane, and treatments did not cause its translocation to the cytoplasm. Myristoylation mutant MARCKS-GFP (G2A) was observed solely in the cytoplasm, and no changes were detected in the intracellular location of this mutant MARCKS after treatment. In experiment 2, luteal cells were transfected with one of the three MARCKS-GFP constructs. Cells were then fixed and probed sequentially for oxytocin and filamentous actin. Results revealed that only wild-type MARCKS-GFP transfected large luteal cells contained advanced signs of exocytosis (peripheral movement of oxytocin vesicles; shorter actin filaments) with translocation of MARCKS-GFP from membrane to cytoplasm in response to PGF2alpha treatment. These data demonstrate that phosphorylation of membrane-bound MARCKS protein is requisite for exocytosis of oxytocin to occur in bovine large luteal cells.

MARCKS protein is a key molecule regulating mucin secretion by human airway epithelial cells in vitro.

Hypersecretion of airway mucin characterizes numerous respiratory diseases. Although diverse pathological stimuli can provoke exocytotic release of mucin from secretory cells of the airway epithelium, mechanisms involved remain obscure. This report describes a new paradigm for the intracellular signaling mechanism regulating airway mucin secretion. Direct evidence is provided that the myristoylated alanine-rich C kinase substrate (MARCKS) is a central regulatory molecule linking secretagogue stimulation at the cell surface to mucin granule release by differentiated normal human bronchial epithelial cells in vitro. Down-regulation of MARCKS expression or disruption of MARCKS function in these cells inhibits the secretory response to subsequent stimulation. The intracellular mechanism controlling this secretory process involves cooperative action of two separate protein kinases, protein kinase C and cGMP-dependent protein kinase. Upon stimulation, activated protein kinase C phosphorylates MARCKS, causing translocation of MARCKS from the plasma membrane to the cytoplasm, where it is then dephosphorylated by a protein phosphatase 2A that is activated by cGMP-dependent protein kinase, and associates with both actin and myosin. Dephosphorylated cytoplasmic MARCKS would also be free to interact with mucin granule membranes and thus could link granules to the contractile cytoskeleton, mediating their movement to the cell periphery and subsequent exocytosis. These findings suggest several novel intracellular targets for pharmacological intervention in disorders involving aberrant secretion of respiratory mucin and may relate to other lesions involving exocytosis of membrane-bound granules in various cells and tissues.

Involvement of the θ-type protein kinase C in translocation of myristoylated alanine-rich C kinase substrate (MARCKS) during myogenesis of chick embryonic myoblasts

The phosphorylation pattern of numerous proteins in the soluble extracts of chick embryonic muscle cells changes dramatically during myogenesis. One of these proteins, the 63 kDa protein, whose phosphorylation state declines during the differentiation process, was identified as the myristoylated alanine-rich C kinase substrate (MARCKS), a major, specific substrate of protein kinase C (PKC). This decrease in the phosphorylation state of MARCKS was due to a decrease in the level of protein in the cytosol with a simultaneous increase in its level in the membrane fraction. Immunostaining of the cultured myoblasts also revealed that MARCKS translocated from the cytosol to the plasma membrane and to the peripheral region of nuclei as the mononucleated myoblasts fused to form multinucleated myotubes. Immunoprecipitation with an anti-PKC-θ antibody, but not with the antibodies against the other PKC isoforms, such as conventional PKC-α, novel PKC-∆, and novel PKC-ϵ, inhibited phosphorylation of MARCKS. Moreover, expression of PKC-θ was found to be down-regulated during the course of myogenic differentiation. In addition, treatment of the cells with PMA, which activates PKC-θ and hence increases the phosphorylation state of MARCKS, reversibly inhibited both MARCKS translocation and myoblast fusion. These results suggest that MARCKS is preferentially phosphorylated by PKC-θ in cultured myoblasts and that the down-regulation of PKC-θ is partly responsible for MARCKS translocation during myogenesis. These results also suggest that PKC-θ-controlled MARCKS translocation is associated with, or a requisite event for, myoblast fusion.

Involvement of the θ-type protein kinase C in translocation of myristoylated alanine-rich C kinase substrate (MARCKS) during myogenesis of chick embryonic myoblasts

The phosphorylation pattern of numerous proteins in the soluble extracts of chick embryonic muscle cells changes dramatically during myogenesis. One of these proteins, the 63 kDa protein, whose phosphorylation state declines during the differentiation process, was identified as the myristoylated alanine-rich C kinase substrate (MARCKS), a major, specific substrate of protein kinase C (PKC). This decrease in the phosphorylation state of MARCKS was due to a decrease in the level of protein in the cytosol with a simultaneous increase in its level in the membrane fraction. Immunostaining of the cultured myoblasts also revealed that MARCKS translocated from the cytosol to the plasma membrane and to the peripheral region of nuclei as the mononucleated myoblasts fused to form multinucleated myotubes. Immunoprecipitation with an anti-PKC-theta antibody, but not with the antibodies against the other PKC isoforms, such as conventional PKC-alpha, novel PKC-delta, and novel PKC-epsilon, inhibited phosphorylation of MARCKS. Moreover, expression of PKC-theta was found to be down-regulated during the course of myogenic differentiation. In addition, treatment of the cells with PMA, which activates PKC-theta and hence increases the phosphorylation state of MARCKS, reversibly inhibited both MARCKS translocation and myoblast fusion. These results suggest that MARCKS is preferentially phosphorylated by PKC-theta in cultured myoblasts and that the down-regulation of PKC-theta; is partly responsible for MARCKS translocation during myogenesis. These results also suggest that PKC-theta-controlled MARCKS translocation is associated with, or a requisite event for, myoblast fusion.

PKC-epsilon regulates basolateral endocytosis in human T84 intestinal epithelia: role of F-actin and MARCKS.

Protein kinase C (PKC) and the actin cytoskeleton are critical effectors of membrane trafficking in mammalian cells. In polarized epithelia, the role of these factors in endocytic events at either the apical or basolateral membrane is poorly defined. In the present study, phorbol 12-myristate 13-acetate (PMA) and other activators of PKC selectively enhanced basolateral but not apical fluid-phase endocytosis in human T84 intestinal epithelia. Stimulation of basolateral endocytosis was blocked by the conventional and novel PKC inhibitor Gö-6850, but not the conventional PKC inhibitor Gö-6976, and correlated with translocation of the novel PKC isoform PKC-epsilon. PMA treatment induced remodeling of basolateral F-actin. The actin disassembler cytochalasin D stimulated basolateral endocytosis and enhanced stimulation of endocytosis by PMA, whereas PMA-stimulated endocytosis was blocked by the F-actin stabilizers phalloidin and jasplakinolide. PMA induced membrane-to-cytosol redistribution of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS). Cytochalasin D also induced MARCKS translocation and enhanced PMA-stimulated translocation of MARCKS. A myristoylated peptide corresponding to the phosphorylation site domain of MARCKS inhibited both MARCKS translocation and PMA stimulation of endocytosis. MARCKS translocation was inhibited by Gö-6850 but not Gö-6976. The results suggest that a novel PKC isoform, likely PKC-epsilon, stimulates basolateral endocytosis in model epithelia by a mechanism that involves F-actin and MARCKS.

Activation of protein kinase C-alpha and translocation of the myristoylated alanine-rich C-kinase substrate correlate with phorbol ester-enhanced noradrenaline release from SH-SY5Y human neuroblastoma cells.

The aim of this study was to investigate the mechanism by which short-term pretreatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 nM) enhances noradrenaline (NA) release from the human neuroblastoma cell line SH-SY5Y. Subcellular fractionation and immunocytochemical studies demonstrated that an 8-min TPA treatment caused translocation of the alpha-subtype of protein kinase C (PKC) from the cytosol to the plasma membrane. In contrast, TPA altered the distribution of PKC-epsilon from cytosolic and membrane-associated to cytoskeleton- and membrane-associated. TPA had no effect on the cytosolic location of PKC-zeta. Subcellular fractionation studies also showed that the myristoylated alanine-rich C-kinase substrate (MARCKS), a major neuronal PKC substrate that has been implicated in the mechanism of neurotransmitter release, translocated from membranes to cytosol in response to an 8-min TPA treatment. Under these conditions the level of phosphorylation of MARCKS increased threefold. The ability of TPA to enhance NA release and to cause the translocation and phosphorylation of MARCKS was inhibited by the PKC inhibitor Ro 31-8220 (10 microM). Selective down-regulation of PKC subtypes by prolonged exposure to phorbol 12,13-dibutyrate (100 nM) attenuated the TPA-induced enhancement of NA release and the translocation of MARCKS over an interval similar to that of down-regulation of PKC-alpha (but not -epsilon or -zeta). Thus, we have demonstrated a strong correlation between the translocation of MARCKS and the enhancement of NA release from SH-SY5Y cells due to the TPA-induced activation of PKC-alpha.