Location of the myristoylated alanine-rich C-kinase substrate (MARCKS) effector domain in negatively charged phospholipid bicelles

The effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS-ED) is a highly basic, unstructured protein segment that is responsible for attaching MARCKS reversibly to the membrane interface. When attached to the interface, it also has the capacity to sequester phosphoinosities, such as PI(4,5)P(2), within the plane of the bilayer. Here, the position of the MARCKS-ED was determined when bound to phospholipid bicelles using high-resolution NMR methods. Two sets of data indicate that the phenylalanine residues of the MARCKS-ED are positioned within the membrane hydrocarbon a few angstroms from the aqueous-hydrocarbon interface. First, short-range nuclear Overhauser effects are detected between the aromatic side chains and the lipid acyl chain methylenes. Second, paramagnetic enhancements of nuclear relaxation, produced by molecular oxygen, are similar for the phenylalanine aromatic protons and those observed for protons in the upper portion of the acyl chain. The rates of amide-water proton exchange are fast and only slightly hindered when the peptide is bound to bicelles, indicating that the backbone does not lie within the membrane hydrocarbon. These results indicate that highly charged peptides such as the MARCKS-ED penetrate the membrane interface with aromatic amino acid side chains inserted into the hydrocarbon and the peptide backbone lying within the bilayer interface. This position may serve to enhance the electrostatic fields produced by this basic domain at the membrane interface and may play a role in the ability of the MARCKS-ED to sequester polyphosphoinositides.     

Sequestration of phosphoinositides by mutated MARCKS effector domain inhibits stimulated Ca(2+) mobilization and degranulation in mast cells

Protein kinase C β (PKCβ) participates in antigen-stimulated mast cell degranulation mediated by the high-affinity receptor for immunoglobulin E, FcεRI, but the molecular basis is unclear. We investigated the hypothesis that the polybasic effector domain (ED) of the abundant intracellular substrate for protein kinase C known as myristoylated alanine-rich protein kinase C substrate (MARCKS) sequesters phosphoinositides at the inner leaflet of the plasma membrane until MARCKS dissociates after phosphorylation by activated PKC. Real-time fluorescence imaging confirms synchronization between stimulated oscillations of intracellular Ca(2+) concentrations and oscillatory association of PKCβ-enhanced green fluorescent protein with the plasma membrane. Similarly, MARCKS-ED tagged with monomeric red fluorescent protein undergoes antigen-stimulated oscillatory dissociation and rebinding to the plasma membrane with a time course that is synchronized with reversible plasma membrane association of PKCβ. We find that MARCKS-ED dissociation is prevented by mutation of four serine residues that are potential sites of phosphorylation by PKC. Cells expressing this mutated MARCKS-ED SA4 show delayed onset of antigen-stimulated Ca(2+) mobilization and substantial inhibition of granule exocytosis. Stimulation of degranulation by thapsigargin, which bypasses inositol 1,4,5-trisphosphate production, is also substantially reduced in the presence of MARCKS-ED SA4, but store-operated Ca(2+) entry is not inhibited. These results show the capacity of MARCKS-ED to regulate granule exocytosis in a PKC-dependent manner, consistent with regulated sequestration of phosphoinositides that mediate granule fusion at the plasma membrane.     

Influence of the effector peptide of MARCKS-related protein on actin polymerization: a kinetic analysis

The members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with membranes, protein kinase C, and calmodulin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. This domain is also involved in the interaction between MARCKS/MRP and actin. In this article we show that a peptide corresponding to the effector domain of MRP, the effector peptide, strongly influences the dynamics of actin polymerization. Depending on the stoichiometric ratio of effector peptide to actin the peptide either accelerates or retards the actin polymerization process, which takes place in the presence of near-physiological salt concentrations. A model is developed in which this phenomenon is explained by two independent nucleation processes involving free actin monomers and peptide-bound actin monomers, respectively. As a control, a possible regulatory mechanism has been investigated: we show that calmodulin inhibits the actin polymerizing activity of the MRP effector peptide, thereby validating our model approach.     

Interaction between actin and the effector peptide of MARCKS-related protein. Identification of functional amino acid segments

It is widely assumed that the members of the MARCKS protein family, MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) and MARCKS-related protein (MRP), interact with actin via their effector domain, a highly basic segment composed of 24-25 amino acid residues. To clarify the mechanisms by which this interaction takes place, we have examined the effect of a peptide corresponding to the effector domain of MRP, the so-called effector peptide, on both the dynamic and the structural properties of actin. We show that in the absence of cations the effector peptide polymerizes monomeric actin and causes the alignment of the formed filaments into bundle-like structures. Moreover, we document that binding of calmodulin or phosphorylation by protein kinase C both inhibit the actin polymerizing activity of the MRP effector peptide. Finally, several effector peptides were synthesized in which positively charged or hydrophobic segments were deleted or replaced by alanines. Our data suggest that a group of six positively charged amino acid residues at the N-terminus of the peptide is crucial for its interaction with actin. While its actin polymerizing activity critically depends on the presence of all three positively charged segments of the peptide, hydrophobic amino acid residues rather modulate the polymerization velocity.     

Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins

The basic effector domain of myristoylated alanine-rich C kinase substrate (MARCKS), a major protein kinase C substrate, binds electrostatically to acidic lipids on the inner leaflet of the plasma membrane; interaction with Ca2+/calmodulin or protein kinase C phosphorylation reverses this binding. Our working hypothesis is that the effector domain of MARCKS reversibly sequesters a significant fraction of the L-alpha-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PIP2) on the plasma membrane. To test this, we utilize three techniques that measure the ability of a peptide corresponding to its effector domain, MARCKS(151-175), to sequester PIP2 in model membranes containing physiologically relevant fractions (15-30%) of the monovalent acidic lipid phosphatidylserine. First, we measure fluorescence resonance energy transfer from Bodipy-TMR-PIP2 to Texas Red MARCKS(151-175) adsorbed to large unilamellar vesicles. Second, we detect quenching of Bodipy-TMR-PIP2 in large unilamellar vesicles when unlabeled MARCKS(151-175) binds to vesicles. Third, we identify line broadening in the electron paramagnetic resonance spectra of spin-labeled PIP2 as unlabeled MARCKS(151-175) adsorbs to vesicles. Theoretical calculations (applying the Poisson-Boltzmann relation to atomic models of the peptide and bilayer) and experimental results (fluorescence resonance energy transfer and quenching at different salt concentrations) suggest that nonspecific electrostatic interactions produce this sequestration. Finally, we show that the PLC-delta1-catalyzed hydrolysis of PIP2, but not binding of its PH domain to PIP2, decreases markedly as MARCKS(151-175) sequesters most of the PIP2.     

Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - effects beyond electrostatic

The presence of charged lipids in the cell membrane constitutes the background for the interaction with numerous membrane proteins. As a result, the valence of the lipids plays an important role concerning their lateral organization in the membrane and therefore the very manner of this interaction. This present study examines this aspect, particularly regarding to the interaction of the anionic lipid DPPS with the highly basic charged effector domain of the MARCKS protein, examined in monolayer model systems. Film balance, fluorescence microscopy and X-ray reflection/diffraction measurements were used to study the behavior of DPPS in a mixture with DPPC for its dependance on the presence of MARCKS (151-175). In the mixed monolayer, both lipids are completely miscible therefore DPPS is incorporated in the ordered crystalline DPPC domains as well. The interaction of MARCKS peptide with the mixed monolayer leads to the formation of lipid/peptide clusters causing an elongation of the serine group of the DPPS up to 7? in direction to surface normal into the subphase. The large cationic charge of the peptide pulls out the serine group of the interface which simultaneously causes an elongation of the phosphodiester group of the lipid fraction too. The obtained results were used to compare the interaction of MARCKS peptide with the polyvalent PIP(2) in mixed monolayers. On this way we surprisingly find out, that the relative small charge difference of the anionic lipids causes a significant different interaction with MARCKS (151-175). The lateral arrangement of the anionic lipids depends on their charge values and determines the diffusion of the electrostatic binding clusters within the membrane.     

Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters spin-labeled phosphatidylinositol 4,5-bisphosphate in lipid bilayers

The myristoylated alanine-rich protein kinase C substrate (MARCKS) may function to sequester phosphoinositides within the plane of the bilayer. To characterize this interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), a novel spin-labeled derivative, proxyl-PIP(2), was synthesized and characterized. In the presence of molecules known to bind PI(4,5)P(2) the EPR spectrum of this label exhibits an increase in line width because of a decrease in label dynamics, and titration of this probe with neomycin yields the expected 1:1 stoichiometry. Thus, this probe can be used to quantitate the interactions made by the PI(4,5)P(2) head group within the bilayer. In the presence of a peptide comprising the effector domain of MARCKS the EPR spectrum broadens, but the changes in line shape are modulated by both changes in label correlation time and spin-spin interactions. This result indicates that at least some proxyl-PIP(2) are in close proximity when bound to MARCKS and that MARCKS associates with multiple PI(4,5)P(2) molecules. Titration of the proxyl-PIP(2) EPR signal by the MARCKS-derived peptide also suggests that multiple PI(4,5)P(2) molecules interact with MARCKS. Site-directed spin labeling of this peptide shows that the position and conformation of this protein segment at the membrane interface are not altered significantly by binding to PI(4,5)P(2). These data are consistent with the hypothesis that MARCKS functions to sequester multiple PI(4,5)P(2) molecules within the plane of the membrane as a result of interactions that are driven by electrostatic forces.     

MARCKS regulates membrane targeting of Rab10 vesicles to promote axon development

Axon development requires membrane addition from the intracellular supply, which has been shown to be mediated by Rab10-positive plasmalemmal precursor vesicles (PPVs). However, the molecular mechanisms underlying the membrane trafficking processes of PPVs remain unclear. Here, we show that myristoylated alanine-rich C-kinase substrate (MARCKS) mediates membrane targeting of Rab10-positive PPVs, and this regulation is critical for axon development. We found that the GTP-locked active form of Rab10 binds to membrane-associated MARCKS, whose affinity depends on the phosphorylation status of the MARCKS effector domain. Either genetic silencing of MARCKS or disruption of its interaction with Rab10 inhibited axon growth of cortical neurons, impaired docking and fusion of Rab10 vesicles with the plasma membrane, and consequently caused a loss of membrane insertion of axonal receptors responsive to extracellular axon growth factors. Thus, this study has identified a novel function of MARCKS in mediating membrane targeting of PPVs during axon development.     

Activity-dependent mobilization of the adhesion molecule polysialic NCAM to the cell surface of neurons and endocrine cells.

The alpha-2,8-linked sialic acid polymer (PSA) on the neural cell adhesion molecule (NCAM) is an important regulator of cell surface interactions. We have examined the translocation of PSA-NCAM to the surface of cultured cortical neurons and insulin secreting beta cells under different conditions of cell activity. Endoneuraminidase N, an enzyme that specifically cleaves PSA chains, was used to remove pre-existing PSA from the plasma membrane and the re-expression of the molecule was monitored by immunocytochemistry. Punctate PSA immunostaining was restored on the surface of 68% of neurons within 1 h. This recovery was almost completely prevented by tetrodotoxin, suggesting that spontaneous electrical activity is required. K+ depolarization (50 mM) allowed recovery of PSA surface staining in the presence of tetrodotoxin and this effect required the presence of extracellular Ca2+. Rapid redistribution of PSA-NCAM to the surface of beta cells was observed under conditions that stimulate insulin secretion. Ca2+ channel inhibition decreased both PSA-NCAM expression and insulin secretion to control, non-stimulated levels. Finally, subcellular fractionation of an insulin-secreting cell line showed that the secretory vesicle fraction is highly enriched in PSA-NCAM. These results suggest that PSA-NCAM can be translocated to the cell surface via regulated exocytosis. Taken together, our results provide unprecedented evidence linking cell activity and PSA-NCAM expression, and suggest a mechanism for rapid modulation of cell surface interactions.     

Investigation into the mechanism regulating MRP localization

The major PKC substrates MARCKS and MacMARCKS (MRP) are membrane-binding proteins implicated in cell spreading, integrin activation and exocytosis. According to the myristoyl-electrostatic switch model the co-operation between the myristoyl moiety and the positively charged effector domain (ED) is an essential mechanism by which proteins bind to membranes. Loss of the electrostatic interaction between the ED and phospholipids, such as Ptdins(4,5)P2, results in the translocation of such proteins to the cytoplasm. While this model has been extensively tested for the binding of MARCKS far less is known about the mechanisms regulating MRP localization. We demonstrate that after phosphorylation, MRP is relocated to the intracellular membranes of late endosomes and lysosomes. MRP binds to all membranes via its myristoyl moiety, but for its localization at the plasma membrane the ED is also required. Although the ED of MRP can bind to Ptdins(4,5)P2 in vitro, this binding is not essential for its retention at or targeting to the plasma membrane. We conclude that the co-operation between the myristoyl moiety and the ED is not required for the binding to membranes in general but that it is essential for the targeting of MRP to the plasma membrane in a Ptdins(4,5)P2-independent manner.     

Polysialic acid and activity-dependent synapse remodeling

Polysialic acid (PSA) is a large carbohydrate added post-translationally to the extracellular domain of the Neural Cell Adhesion Molecule (NCAM) that influences its adhesive and other functional properties. PSA-NCAM is widely distributed in the developing nervous system where it promotes dynamic cell interactions, like those responsible for axonal growth, terminal sprouting and target innervation. Its expression becomes restricted in the adult nervous system where it is thought to contribute to various forms of neuronal and glial plasticity. We here review evidence, obtained mainly from hypothalamic neuroendocrine centers and the olfactory system, that it intervenes in structural synaptic plasticity and accompanying neuronal-glial transformations, making possible the formation and elimination of synapses that occur under particular physiological conditions.     

Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions

A peptide corresponding to the basic (+13), unstructured effector domain of myristoylated alanine-rich C kinase substrate (MARCKS) binds strongly to membranes containing phosphatidylinositol 4,5-bisphosphate (PIP(2)). Although aromatic residues contribute to the binding, three experiments suggest the binding is driven mainly by nonspecific local electrostatic interactions. First, peptides with 13 basic residues, Lys-13 and Arg-13, bind to PIP(2)-containing vesicles with the same high affinity as the effector domain peptide. Second, removing basic residues from the effector domain peptide reduces the binding energy by an amount that correlates with the number of charges removed. Third, peptides corresponding to a basic region in GAP43 and MARCKS effector domain-like regions in other proteins (e.g. MacMARCKS, adducin, Drosophila A kinase anchor protein 200, and N-methyl-d-aspartate receptor) also bind with an energy that correlates with the number of basic residues. Kinetic measurements suggest the effector domain binds to several PIP(2). Theoretical calculations show the effector domain produces a local positive potential, even when bound to a bilayer with 33% monovalent acidic lipids, and should thus sequester PIP(2) laterally. This electrostatic sequestration was observed experimentally using a phospholipase C assay. Our results are consistent with the hypothesis that MARCKS could reversibly sequester much of the PIP(2) in the plasma membrane.     

Crystal structure of a MARCKS peptide containing the calmodulin-binding domain in complex with Ca2+-calmodulin

The calmodulin-binding domain of myristoylated alanine-rich C kinase substrate (MARCKS), which interacts with various targets including calmodulin, actin and membrane lipids, has been suggested to function as a crosstalk point among several signal transduction pathways. We present here the crystal structure at 2 A resolution of a peptide consisting of the MARCKS calmodulin (CaM)-binding domain in complex with Ca2+-CaM. The domain assumes a flexible conformation, and the hydrophobic pocket of the calmodulin N-lobe, which is a common CaM-binding site observed in previously resolved Ca2+-CaM-target peptide complexes, is not involved in the interaction. The present structure presents a novel target-recognition mode of calmodulin and provides insight into the structural basis of the flexible interaction module of MARCKS.     

Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth

Lecticans, a family of chondroitin sulfate proteoglycans, represent the largest group of proteoglycans expressed in the nervous system. We previously showed that the C-type lectin domains of lecticans bind two classes of sulfated cell surface glycolipids, sulfatides and HNK-1-reactive sulfoglucuronylglycolipids (SGGLs). In this paper, we demonstrate that the interaction between the lectin domain of brevican, a nervous system-specific lectican, and cell surface SGGLs acts as a novel cell recognition system that promotes neuronal adhesion and neurite outgrowth. The Ig chimera of the brevican lectin domain bind to the surface of SGGL-expressing rat hippocampal neurons. The substrate of the brevican chimera promotes adhesion and neurite outgrowth of hippocampal neurons. The authentic, full-length brevican also promotes neuronal cell adhesion and neurite outgrowth. These activities of brevican substrates are neutralized by preincubation of cells with HNK-1 monoclonal antibodies and by pretreatment of the brevican substrates with purified SGGLs. Brevican and HNK-1 carbohydrates are coexpressed in specific layers of the developing hippocampus where axons from entorhinal neurons elongate. Our observations suggest that cell surface SGGLs and extracellular lecticans comprise a novel cell-substrate recognition system operating in the developing nervous system.     

Essential role for the PKC target MARCKS in maintaining dendritic spine morphology

Spine morphology is regulated by intracellular signals, like PKC, that affect cytoskeletal and membrane dynamics. We investigated the role of MARCKS (myristoylated, alanine-rich C-kinase substrate) in dendrites of 3-week-old hippocampal cultures. MARCKS associates with membranes via the combined action of myristoylation and a polybasic effector domain, which binds phospholipids and/or F-actin, unless phosphorylated by PKC. Knockdown of endogenous MARCKS using RNAi reduced spine density and size. PKC activation induced similar effects, which were prevented by expression of a nonphosphorylatable mutant. Moreover, expression of pseudophosphorylated MARCKS was, by itself, sufficient to induce spine loss and shrinkage, accompanied by reduced F-actin content. Nonphosphorylatable MARCKS caused spine elongation and increased the mobility of spine actin clusters. Surprisingly, it also decreased spine density via a novel mechanism of spine fusion, an effect that required the myristoylation sequence. Thus, MARCKS is a key factor in the maintenance of dendritic spines and contributes to PKC-dependent morphological plasticity.     

Distinctive PSA-NCAM and NCAM Hallmarks in Glutamate-Induced Dendritic Atrophy and Synaptic Disassembly

Dendritic and synapse remodeling are forms of structural plasticity that play a critical role in normal hippocampal function. Neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) participate in neurite outgrowth and synapse formation and plasticity. However, it remains unclear whether they contribute to dendritic retraction and synaptic disassembly. Cultured hippocampal neurons exposed to glutamate (5 µM) showed a reduced MAP-2 (+) area in the absence of neuronal death 24 h after the insult. Concomitantly, synapse loss, revealed by decreased synaptophysin and post-synaptic density-95 cluster number and area, together with changes in NCAM and PSA-NCAM levels were found. Dendritic atrophy and PSA-NCAM reduction proved NMDA-receptor dependent. Live-imaging experiments evidenced dendritic atrophy 4 h after the insult; this effect was preceded by smaller NCAM clusters (1 h) and decreased surface and total PSA-NCAM levels (3 h). Simultaneously, total NCAM cluster number and area remained unchanged. The subsequent synapse disassembly (6 h) was accompanied by reductions in total NCAM cluster number and area. A PSA mimetic peptide prevented both the dendritic atrophy and the subsequent synaptic changes (6 h) but had no effect on the earliest synaptic remodeling (3 h). Thus, NCAM-synaptic reorganization and PSA-NCAM level decrease precede glutamate-induced dendritic atrophy, whereas the NCAM level reduction is a delayed event related to synapse loss. Consequently, distinctive stages in PSA-NCAM/NCAM balance seem to accompany glutamate-induced dendritic atrophy and synapse loss.     

多聚唾液酸(PSA)及其修饰的神经粘附分子(PSA-NCAM)对肿瘤及细胞信号通路的影响

神经粘附分子(Neural cell adhesion molecule, NCAM)是免疫球蛋白家族中的一员,在细胞粘附和细胞通信,尤其是神经系统的生长和塑型中起重要作用。而多聚唾液酸(Polysialic acid, PSA)则是控制NCAM粘附能力形成与神经系统分化的重要因素。研究发现,多种肿瘤细胞中存在PSA以及多聚唾液酸化的神经粘附分子(PSA-NCAM)再表达的现象,预示PSA及PSA-NCAM与多种肿瘤细胞的粘附性、迁移性和侵袭性等特性密切相关,影响肿瘤细胞的生长与转移,并通过介导多种细胞信号通路影响癌症的发生与发展。文章综述了NCAM以及PSA对癌症的发生与发展、预后的作用及其功能对细胞下游信号传导的影响。     

Interactions controlling the membrane binding of basic protein domains: phenylalanine and the attachment of the myristoylated alanine-rich C-kinase substrate protein to interfaces.

Basic residues are known to play a critical role in the attachment of protein domains to membrane interfaces. Many of these domains also contain hydrophobic residues that may alter the binding and the position of the domain on the interface. In the present study, the role of phenylanine in determining the membrane position, dynamics and free energy of a peptide derived from the effector domain of the myristoylated alanine-rich C-kinase substrate (MARCKS) protein was examined. Deuterium NMR in membranes containing phosphatidylcholine (PC) and phosphatidylserine (PS) indicates that this peptide, MARCKS(151-175), partially penetrates the membrane interface when bound and alters the effective charge density on the membrane interface by approximately 2 charges per bound peptide. However, a derivative of this peptide in which the five phenylalanines are replaced by alanine, MARCKS-Ala, does not penetrate the interface when membrane-bound. This result was confirmed by depth measurements by electron paramagnetic resonance spectroscopy on several spin-labeled derivatives of the Phe-less derivative. In contrast to nitroxides on MARCKS(151-175), nitroxides on the derivative lacking Phe do not reside within the bilayer but are in the aqueous phase when the peptide is bound to the membrane. The Phe to Ala substitutions shift the position of the labeled side chains by approximately 10-15 A. The side-chain dynamics of MARCKS-Ala are strongly influenced by membrane charge density and indicate that this peptide is drawn closer to the membrane interface at higher charge densities. As expected, MARCKS-Ala binds more weakly to membranes composed of PS/PC (1:9) than does the native MARCKS peptide; however, each phenylalanine contributes only 0.2 kcal/mol to the binding energy difference, far less than the 1.3 kcal/mol expected for the binding of phenylalanine to the membrane interface. This energetic discrepancy and the differences in membrane position of these peptides can be accounted for by a dehydration energy that is encountered as the peptide approaches the membrane interface. This energy likely includes a Born repulsion acting between the charged peptide and the low dielectric membrane interior. The interplay between the long-range attractive Coulombic force, the short-range repulsive force and the hydrophobic effect controls the position and energetics of protein domains on acidic membrane interfaces.