The macrophage--a cell for all seasons.
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Animals↗
Biomedical subjects
A Aderem
Publications and source records attributed to A Aderem.
Mechanisms of phagocytosis in macrophages.
Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive response. In order to discriminate between infectious agents and self, macrophages have evolved a restricted number of phagocytic receptors, like the mannose receptor, that recognize conserved motifs on pathogens. Pathogens are also phagocytosed by complement receptors after relatively nonspecific opsonization with complement and by Fc receptors after specific opsonization with antibodies. All these receptors induce rearrangements in the actin cytoskeleton that lead to the internalization of the particle. However, important differences in the molecular mechanisms underlying phagocytosis by different receptors are now being appreciated. These include differences in the cytoskeletal elements that mediate ingestion, differences in vacuole maturation, and differences in inflammatory responses. Infectious agents, such as M. tuberculosis, Legionella pneumophila, and Salmonella typhimurium, enter macrophages via heterogeneous pathways and modify vacuolar maturation in a manner that favors their survival. Macrophages also play an important role in the recognition and clearance of apoptotic cells; a notable feature of this process is the absence of an inflammatory response.
Animals↗
MacMARCKS is not essential for phagocytosis in macrophages.
MacMARCKS (also known as myristoylated alanine-rich protein kinase C substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates. MacMARCKS contains within it a basic effector domain that contains the serine residues that are phosphorylated by protein kinase C, as well as a calcium/calmodulin and actin-binding site. Two previous reports demonstrated that a macrophage cell line expressing a mutant form of MacMARCKS that lacks the effector domain is defective in phagocytosis and cell adhesion (Zhu, Z., Bao, Z., and Li, J. (1995) J. Biol. Chem. 270, 17652-17655; Li, J., Zhu, Z., and Bao, Z. (1996) J. Biol. Chem. 271, 12985-12990). We report here that macrophages from MacMARCKS null mice phagocytose and spread normally. Thus, although MacMARCKS is recruited to phagosomes, it is not absolutely required for phagocytosis.
Animals↗
Identification of the basolateral targeting determinant of a peripheral membrane protein, MacMARCKS, in polarized cells.
BACKGROUND: Although the molecular determinants that specify the targeting of transmembrane proteins to the apical or basolateral membrane domains within polarized epithelial cells have been well characterized, very little is known about the targeting of peripheral membrane proteins within these cells. MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. This myristoylated protein regulates actin structure at cell membranes and is essential for the morphogenic movement of neuroepithelial cells during the formation of the neural tube. RESULTS: MacMARCKS was specifically targeted to sites of cell-cell contact in the basolateral domain of polarized Madin-Darby canine kidney (MDCK) epithelial cells and was displaced from this location upon activation of PKC. We defined the basolateral targeting determinant of MacMARCKS to be the effector domain, a basic region spanning 24 amino acids and containing the PKC phosphorylation sites as well as binding sites for calmodulin and actin. This domain, in conjunction with a myristoyl moiety, was sufficient to target a non-membrane-associated protein--green fluorescent protein--specifically to the basolateral surface of polarized MDCK cells. CONCLUSIONS: This is the first description of a specific amino acid sequence that specifies targeting of a peripheral membrane protein to the basolateral membrane in polarized epithelial cells.
Animals↗
MARCKS regulates membrane ruffling and cell spreading.
The dynamic rearrangement of the actin cytoskeleton is fundamental to most biological processes including embryogenesis, morphogenesis, cell movement, wound healing and metastasis [1]. Membrane ruffling and reversible cell-substratum interactions underlie actin-driven cell movement. Protein kinase C (PKC) stimulates membrane ruffling and adhesion [2], but the mechanism by which this occurs is unknown. Myristoylated alaninerich C kinase substrate (MARCKS) is a PKC substrate that cycles on and off membranes by a mechanism termed the myristoyl-electrostatic switch [3-6]. While at the membrane, MARCKS binds to and sequesters acidic phospholipids including phosphatidyl-inositol-4,5-bisphosphate (PIP2) [7]. MARCKS also binds and cross-links filamentous actin, an activity which is regulated by PKC-dependent phosphorylation and calcium-calmodulin [3]. In this report, we demonstrate that expression, in fibroblasts, of MARCKS containing a mutation which abrogates the myristoyl-electrostatic switch prevents cell spreading. The MARCKS mutant arrests the cell during an early stage of spreading, characterized by profuse membrane blebbing, and prevents the formation of membrane ruffles and lamellae usually found at the leading edge of spreading cells. This defect in the regulation of the actin cytoskeleton is accompanied by a decrease in cell-substratum adhesion. Our results provide direct evidence that MARCKS and PKC regulate actin-dependent membrane ruffling and cell adhesion, perhaps via a PIP2-dependent mechanism.
Actins↗
Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.
The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.
Animals↗
Molecular determinants of the myristoyl-electrostatic switch of MARCKS.
MARCKS is a protein kinase C (PKC) substrate which binds calcium/calmodulin and actin, and which has been implicated in cell motility, phagocytosis, membrane traffic, and mitogenesis. MARCKS cycles on and off the membrane via a myristoyl electrostatic switch (McLaughlin, S., and Aderem, A.(1995) Trends Biochem. Sci. 20, 272-276). Here we define the molecular determinants of the myristoyl-electrostatic switch. Mutation of the N-terminal glycine results in a nonmyristoylated form of MARCKS which does not bind membranes and is poorly phosphorylated. This indicates that myristic acid targets MARCKS to the membrane, where it is efficiently phosphorylated by PKC. A chimeric protein in which the N terminus of MARCKS is replaced by a sequence, which is doubly palmitoylated, is phosphorylated by PKC but not released from the membrane. Thus two palmitic acid moieties confer sufficient membrane binding energy to render the second, electrostatic membrane binding site superfluous. Mutation of the PKC phosphorylation sites results in a mutant which does not translocate from the membrane to the cytosol. A mutant in which the intervening sequence between the myristoyl moiety and the basic effector domain is deleted, is not displaced from the membrane by PKC dependent phosphorylation, fulfilling a theoretical prediction of the model. In addition to the nonspecific membrane binding interactions conferred by the myristoyl-electrostatic switch, indirect immunofluorescence microscopy demonstrates that specific protein-protein interactions also specify the intracellular localization of MARCKS.
Animals↗
Molecular definition of distinct cytoskeletal structures involved in complement- and Fc receptor-mediated phagocytosis in macrophages.
It has long been known from the results of ultrastructural studies that complement- and immunoglobulin G (IgG)-opsonized particles are phagocytosed differently by macrophages (Kaplan. G. 1977. Scand. J. Immunol. 6:797-807). Complement-opsonized particles sink into the cell, whereas IgG-coated particles are engulfed by lamellipodia, which project from the cell surface. The molecular basis for these differences is unknown. We used indirect immunofluorescence and confocal microscopy to examine how cytoskeletal proteins associate with phagosomes containing complement-opsonized zymosan (COZ) particles or IgG beads in phorbol-myristateacetate-treated peritoneal macrophages. During ingestion of COZ, punctate structures rich in F-actin, vinculin, alpha-actinin, paxillin, and phosphotyrosine-containing proteins are distributed over the phagosome surface. These foci are detected beneath bound COZ within 30 s of warming the cells to 37 degrees C, and their formation requires active protein kinase C. By contrast, during Fc receptor-mediated phagocytosis, all proteins examined were uniformly distributed on or near the phagosome surface. Moreover, ingestion of IgG beads was blocked by tyrosine kinase inhibitors, whereas phagocytosis of COZ was not. Thus, the signals required for particle ingestion, and the arrangement of cytoskeletal proteins on the phagosome surface, vary depending upon which phagocytic receptor is engaged. Moreover, complement receptor (CR)-mediated internalization required intact microtubules and was accompanied by the accumulation of vesicles beneath the forming phagosome, suggesting that membrane trafficking plays a key role in CR-mediated phagocytosis.
Actinin↗
Disruption of the MacMARCKS gene prevents cranial neural tube closure and results in anencephaly.
MacMARCKS is a member of the MARCKS family of protein kinase C (PKC) substrates. Biochemical evidence demonstrates that these proteins integrate calcium and PKC-dependent signals to regulate actin structure at the membrane. We report here that deletion of the MacMARCKS gene prevents cranial neural tube closure in the developing brain, resulting in anencephaly. This suggests a central role for MacMARCKS and the PKC signal transduction pathway in the folding of the anterior neural plate during the early phases of brain formation, and supports the hypothesis that actin-based motility directs cranial neural tube closure.
Anencephaly↗
Stimulus-dependent phosphorylation of MacMARCKS, a protein kinase C substrate, in nerve termini and PC12 cells.
MacMARCKS (also known as myristoylated alanine-rich C kinase substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates, which binds Ca2+/calmodulin in a phosphorylation-dependent manner. Immunoprecipitation demonstrated that MacMARCKS is present in both PC12 cells and in neurons. Upon depolarization of PC12 cells with 60 mM KCl, MacMARCKS phosphorylation increased 4-fold over basal levels in a Ca(2+)-dependent manner. By immunofluorescence microscopy, MacMARCKS was colocalized in PC12 cells to neurite tips with the synaptic vesicle membrane protein synaptophysin and to vesicles in the perinuclear region. Subcellular fractionation demonstrated that MacMARCKS associates tightly with membranes in PC12 cells. In Percoll-purified rat cerebrocortical synaptosomes, depolarization with 60 mM KCl in the presence of exogenous Ca2+ transiently increased MacMARCKS phosphorylation, whereas phorbol ester promoted a sustained increase in MacMARCKS phosphorylation. Subcellular fractionation of rat brain indicated that MacMARCKS was present in both soluble and particulate fractions; particulate MacMARCKS was associated with both small vesicles and highly purified synaptic vesicles. These results are consistent with a role for MacMARCKS in integrating Ca(2+)-calmodulin and protein kinase C-dependent signals in the regulation of neurosecretion.
Animals↗
Mechanisms of phagocytosis.
Recent advances in research on phagocytosis include a better appreciation of the cross-talk between phagocytic receptors, the definition of multiple signaling domains within these receptors, and a deeper understanding of the downstream effector pathways leading to actin polymerization and particle internalization. Phagosome maturation in macrophages proceeds via a series of membrane fusion and fission events, which modify the phagosome in small increments, and appears to be regulated, in part, by GTP-binding proteins and perhaps by protein kinase C. The isolation of dysphagic mutants of Dictyostelium discoideum presages the identification of new genes required for phagocytosis.
Actins↗
Microtubule-active agents mimic lipopolysaccharides in priming macrophages for enhanced arachidonic acid metabolism.
Macrophages play a crucial role in inflammation and host defense, in part, by secreting metabolites of arachidonic acid (20:4). Bacterial lipopolysaccharides (LPS) are poor agonists of the 20:4 cascade, but do have the capacity to prime macrophages for greatly increased 20:4 metabolism upon subsequent stimulation with activators of protein kinase C (PKC). The microtubule-stabilizing agent, taxol, mimics many of the effects of LPS in macrophages. We demonstrate in this study that taxol, like LPS, primes murine peritoneal macrophages for an enhanced release of 20:4 in response to both phorbol 12-myristate 13-acetate (PMA) and zymosan. Taxol and LPS, when used at maximum concentrations, acted additively to prime macrophages for PMA-stimulated release of 20:4, suggesting that the two agents signal through different pathways. Interestingly, agents that stimulate the depolymerization of microtubules, colchicine and nocodazole, also primed macrophages for an enhanced release of 20:4 in response to PMA, however, they did not prime when zymosan was the stimulus. We conclude that agents that disrupt the microtubule network prime resident peritoneal macrophages for enhanced release of 20:4.
Animals↗
A role for MARCKS, the alpha isozyme of protein kinase C and myosin I in zymosan phagocytosis by macrophages.
Myristoylated, alanine-rich C-kinase substrate (MARCKS) is a lipopolysaccharide-induced protein kinase C (PKC) substrate that has been proposed to regulate actin-membrane interactions, as well as actin structure at the membrane. We studied the distribution of MARCKS, the alpha isozyme of PKC (PKC alpha), and myosin I in lipopolysaccharide-treated peritoneal macrophages ingesting zymosan particles. MARCKS, PKC alpha, and myosin I colocalized with F-actin and talin in the cortical cytoplasm adjacent to forming phagocytic cups. After particle ingestion was completed, myosin I, F-actin, and talin were no longer enriched in the vicinity of the phagosome. By contrast, MARCKS and PKC alpha remained associated with the phagosome membrane until after acquisition of the lysosomal marker Lamp-1. Vinculin was not detected on phagosomes at any time point examined. Phagocytosis of zymosan was accompanied by rapid and sustained phosphorylation of MARCKS. Inhibitors of PKC reduced zymosan binding to the macrophage surface and blocked the focal accumulation of F-actin, talin, phosphotyrosine-containing proteins, MARCKS, and PKC alpha beneath attached particles. We propose that PKC-dependent phosphorylation is an early signal required for zymosan phagocytosis and that MARCKS and PKC alpha have a role in phagosome maturation. The colocalization of F-actin and MARCKS at the cytoplasmic face of the nascent phagosome reinforces the hypothesis that MARCKS regulates actin structure at the membrane. Our data also suggest that myosin I functions as a mechanical motor during particle uptake.
Actins↗
Protein kinase C regulates MARCKS cycling between the plasma membrane and lysosomes in fibroblasts.
MARCKS is a protein kinase C (PKC) substrate that is phosphorylated during neurosecretion, phagocyte activation and growth factor-dependent mitogenesis. MARCKS binds calcium/calmodulin and crosslinks F-actin, and both these activities are regulated by PKC-dependent phosphorylation. We present evidence here that PKC-dependent phosphorylation also regulates the cycling of MARCKS between the plasma membrane and Lamp-1-positive lysosomes. Immuno-fluorescence and immunoelectron microscopy, and subcellular fractionation, demonstrated that MARCKS was predominantly associated with the plasma membrane of resting fibroblasts. Activation of PKC resulted in MARCKS phosphorylation and its displacement from the plasma membrane to Lamp-1-positive lysosomes. MARCKS phosphorylation is required for its translocation to lysosomes since mutating either the serine residues phosphorylated by PKC (phos-) or the PKC inhibitor staurosporine, prevented MARCKS phosphorylation, its release from the plasma membrane, and its subsequent association with lysosomes. In the presence of lysosomotropic agents or nocodazole, MARCKS accumulated on lysosomes and returned to the plasma membrane upon drug removal, further suggesting that the protein cycles between the plasma membrane and lysosomes. In contrast to wild-type MARCKS, the phos- mutant did not accumulate on lysosomes in cells treated with NH4Cl, suggesting that basal phosphorylation of MARCKS promotes its constitutive cycling between these two compartments.
Alkaloids↗
The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions.
Hydrophobic insertion of the acyl chain into the bilayer is necessary but not sufficient for the membrane binding of a myristoylated protein. The myristoylated alanine-rich C kinase substrate (MARCKS), Src, ADP-ribosylation factor and human immunodeficiency virus-1 matrix proteins also contain a cluster of basic residues that bind to acidic phospholipids; the hydrophobic and electrostatic interactions act together to anchor the protein to a membrane. For MARCKS, and perhaps other proteins, phosphorylation of serines within its basic cluster reduces the electrostatic attraction, producing translocation of the protein from the membrane to the cytosol by a simple 'electrostatic switch' mechanism.
Cell Membrane↗
The MARCKS family of protein kinase-C substrates.
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Amino Acid Sequence↗
Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles.
The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major cellular substrate of protein kinase C (PKC), and PKC phosphorylation produces translocation of MARCKS from membrane to cytoplasm in many cells. Our working hypothesis is that binding of MARCKS to biological membranes requires both hydrophobic insertion of its myristoyl chain into the lipid bilayer and electrostatic interaction of its basic domain with acidic lipids. We tested this hypothesis by measuring the binding of murine MARCKS to large unilamellar phospholipid vesicles (LUVs). We estimated the partition coefficient of the myristoyl moiety of MARCKS (KH) by measuring the binding of MARCKS to electrically neutral LUVs (KH = 3 x 10(3) M-1). We examined the effect of electrostatic interactions by measuring the binding of MARCKS to LUVs containing 20% acidic lipid and obtained four results. First, incorporating 20% acidic lipid into the LUVs increased binding of MARCKS about 100-fold. Second, PKC phosphorylation, which added 3 negatively charged phosphate groups to the basic domain, reduced 20-fold the binding of MARCKS to these negatively charged vesicles. Third, increasing the KCl concentration from 0.1 to 0.5 M reduced the binding 15-fold. Fourth, Ca(2+)-calmodulin reduced the binding 20-fold. We present a simple theoretical model that explains these results, which are all consistent with the working hypothesis.
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Correlation between protein kinase C binding proteins and substrates in REF52 cells.
We have used a blot overlay assay to identify phosphatidylserine-dependent interactions between protein kinase C (PKC) and PKC binding proteins. The purpose of the present studies was to compare the properties of PKC binding proteins and PKC substrates detected by in vivo and in vitro phosphorylation assays. The major binding proteins and substrates in REF52 cells shared similar properties including enrichment by calmodulin-Sepharose chromatography, binding to phosphatidylserine, and resistance to heat denaturation. In addition, several of the major binding proteins and substrates were coordinately down modulated in SV40-transformed REF52 cells. The major PKC substrate, MARCKS, was also detected as a PKC binding protein. These results emphasize that the phosphatidylserine-dependent interactions between PKC and several substrates are of sufficient affinity to be detected in a blot overlay. Down modulation of binding proteins/substrates in transformed cells may reflect either decreased expression or increased basal phosphorylation of the target proteins and is likely to be involved in maintenance of the transformed phenotype.
Blotting, Western↗