Protein kinase C-mediated phosphorylation and calmodulin binding of recombinant myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein.

Verghese, George M.; Johnson, J D; Vasulka, C; Haupt, D M; Stumpo, Deborah J.; Blackshear, Perry J.

doi:10.1016/s0021-9258(17)37116-8

Cited by 91 publications

(20 citation statements)

References 29 publications

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“…MARCKS is a major substrate of PKCα, and other conventional PKCs, that becomes phosphorylated during leukocyte activation. ,, Unphosphorylated MARCKS possesses a high affinity for anionic and PIP 2 lipids while this interaction is disrupted upon specific phosphorylation by Ca 2+ -activated PKC at multiple sites within its PIP 2 binding region, leading to PIP 2 release. − Previous work has shown that MARCKS inhibits the activity of PI3Kα by sequestering its target and substrate lipid PIP 2 and that such inhibition is reversed by Ca 2+ –PKC phosphorylation of MARCKS, whereupon the newly released PIP 2 recruits PI3K to the membrane and thereby increases the surface density of active lipid kinase, yielding faster net PIP 3 production . Release of PIP 2 from MARCKS also enhances actin cytoskeleton–membrane interactions, because many actin binding proteins employ PIP 2 as a membrane anchor. − …”

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confidence: 99%

Regulation of a Coupled MARCKS–PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module

et al. 2016

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Amoeboid cells that employ chemotaxis to travel up an attractant gradient possess a signaling network assembled on the leading edge of the plasma membrane that senses the gradient and remodels the actin mesh and cell membrane to drive movement in the appropriate direction. In leukocytes such as macrophages and neutrophils, and perhaps in other amoeboid cells as well, the leading edge network includes a positive feedback loop in which the signaling of multiple pathway components is cooperatively coupled. Cytoplasmic Ca2+ is a recently recognized component of the feedback loop at the leading edge where it stimulates phosphoinositide-3-kinase (PI3K) and the production of its product signaling lipid phosphatidylinositol 3,4,5-trisphosphate (PIP3). A previous study implicated Ca2+-activated protein kinase C (PKC) and the phosphatidylinositol 4,5-bisphosphate (PIP2) binding protein MARCKS as two important players in this signaling, because PKC phosphorylation of MARCKS releases free PIP2 that serves as the membrane binding target and substrate for PI3K. This study asks whether calmodulin (CaM), which is known to directly bind MARCKS, also stimulates PIP3 production by releasing free PIP2. Single-molecule fluorescence microscopy is used to quantify the surface density and enzyme activity of key protein components of the hypothesized Ca2+–CaM–MARCKS–PIP2–PI3K–PIP3 circuit. The findings show that CaM does stimulate PI3K lipid kinase activity by binding MARCKS and displacing it from PIP2 headgroups, thereby releasing free PIP2 that recruits active PI3K to the membrane and serves as the substrate for the generation of PIP3. The resulting CaM-triggered activation of PI3K is complete in seconds and is much faster than PKC-triggered activation, which takes minutes. Overall, the available evidence implicates both PKC and CaM in the coupling of Ca2+ and PIP3 signals and suggests these two different pathways have slow and fast activation kinetics, respectively.

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Regulation of a Coupled MARCKS–PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module

et al. 2016

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“…In addition to binding PIP2 and F-actin, the ED also binds calcium/calmodulin. Three to four serine residues (depending on species) within the ED are targets for phosphorylation by PKC (Stumpo et al, 1989;Verghese et al, 1994). Either phosphorylation by PKC or binding of calcium/calmodulin abolishes the electrostatic interaction between MARCKS and the membrane, thereby inducing a translocation of MARCKS to the cytosolic compartment; the F-actin crosslinking activity of MARCKS in vitro also is diminished (reviewed in Arbuzova et al, 2002).…”

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Essential Role for the PKC Target MARCKS in Maintaining Dendritic Spine Morphology

Calabrese

Halpain

2005

Neuron

168

180

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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.

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“…k ad and k de denote the rate constants of membrane association and dissociation of the unphosphorylated protein and k ki and k ph denote the enzymatic activities of the kinase and phosphatase. For the kinase activity we have used a Michaelis-Menten type kinetics [22], whereas we have neglected this property for the phosphatase, assuming that the concentration of phosphorylated proteins is well below the respective Michaelis-Menten constant [23]. Based on the properties of protein kinase C we assume, that the kinase needs lipids for full activation [24,25] and that membrane bound proteins decrease the available membrane space and thus the kinase activity [6].…”

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“…2: (a) Phase diagrams with tie-lines for the phase separation in model I and (b) phase diagram for the active phase separation in model II. Parameters in (a) are N = 25 and u = 1.0 and in (b) are N = 25, Dm = 0.04 µm 2 s −1[26,27], Dc = 20 µm 2 s −1 , k ad = 1 s −1[28], k de = 0.005 s −1[28], km = 0.01[22], k ph = 0.2 s −1[21]..…”

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Alternative Mechanisms of Structuring Biomembranes: Self-Assembly versus Self-Organization

John

Bär

2005

Phys. Rev. Lett.

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We study two mechanisms for the formation of protein patterns near membranes of living cells by mathematical modelling. Self-assembly of protein domains by electrostatic lipid-protein interactions is contrasted with self-organization due to a nonequilibrium biochemical reaction cycle of proteins near the membrane. While both processes lead eventually to quite similar patterns, their evolution occurs on very different length and time scales. Self-assembly produces periodic protein patterns on a spatial scale below 0.1 microm in a few seconds followed by extremely slow coarsening, whereas self-organization results in a pattern wavelength comparable to the typical cell size of 100 microm within a few minutes suggesting different biological functions for the two processes.

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Protein kinase C-mediated phosphorylation and calmodulin binding of recombinant myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein.

Cited by 91 publications

References 29 publications

Regulation of a Coupled MARCKS–PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module

Regulation of a Coupled MARCKS–PI3K Lipid Kinase Circuit by Calmodulin: Single-Molecule Analysis of a Membrane-Bound Signaling Module

Essential Role for the PKC Target MARCKS in Maintaining Dendritic Spine Morphology

Alternative Mechanisms of Structuring Biomembranes: Self-Assembly versus Self-Organization

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