Membrane Association of the Myristoylated Alanine-rich C Kinase Substrate (MARCKS) Protein.

Swierczynski, Sharon L.; Blackshear, Perry J.

doi:10.1074/jbc.270.22.13436

Cited by 100 publications

(115 citation statements)

References 59 publications

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“…Thus, correlation between MARCKS phosphorylation and translocation induced by PKC activation seems to be dependent on cell types, which was why the correlation was not perfectly observed in 293T cells. Similar conclusion has been reported in several groups (Byers et al, 1993;Swierczynski and Blackshear, 1995). In any case, Sprouty4 inhibited PLC-mediated MARCKS translocation, which is dependent on PKC activation.…”

Section: Sprouty4 Inhibits Pip 2 Hydrolysissupporting

confidence: 91%

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

et al. 2009

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Section: Sprouty4 Inhibits Pip 2 Hydrolysissupporting

confidence: 91%

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

et al. 2009

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“…PIP 2 is involved in the membrane binding of proteins such as phospholipase C-␦ 1 (51,52), gelsolin, and profilin (53). Ca 2ϩ ⅐CaM produces translocation of MARCKS-(151-175) from vesicles (19), just as it produces translocation of MARCKS from phospholipid vesicles (10) and some biological membranes (11). In the phospholipid vesicle system (22), translocation of MARCKS-(151-175) to the aqueous phase breaks up the domains, thereby releasing PIP 2 that then can be hydrolyzed by phosphoinositide-specific phospholipase C. The results obtained here and in studies on domains in phospholipid vesicles (22) suggest that an increase in the level of calcium ions, and hence Ca 2ϩ ⅐CaM, in biological cells could lead to rapid desorption of the effector region of MARCKS from the membrane and a subsequent rapid release of PIP 2 .…”

Section: Discussionmentioning

confidence: 99%

“…First, membrane binding of both the protein and the peptide depends strongly on the mole fraction of acidic lipid in the membrane and the ionic strength of the solution (10, 19). Second, CaM binds with high affinity to both the peptide and the effector region of the protein, producing desorption of both molecules from membranes (2,10,11,20,21). Third, protein kinase C phosphorylation inhibits the membrane binding of both MARCKS and the peptide (9,10,18,19).…”

mentioning

confidence: 99%

“…17) introduces three negatively charged phosphates into the effector region, which weakens its electrostatic interaction with acidic lipids and produces desorption of MARCKS from membranes (9 -11, 18). Ca 2ϩ -calmodulin (Ca 2ϩ ⅐CaM) binds with high (nanomolar) affinity to the effector region, producing desorption of MARCKS from both phospholipid vesicles and plasma membranes (10,11).…”

mentioning

confidence: 99%

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Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

Arbuzova

Wang

Murray

et al. 1997

Journal of Biological Chemistry

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Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, k on ‫؍‬ 10 11 M ؊1 s ؊1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, k on is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (k off ) is ϳ10 s ؊1 (lifetime ‫؍‬ 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca 2؉ -calmodulin (Ca 2؉ ⅐CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 M Ca 2؉ ⅐CaM. Our results suggest that Ca 2؉ ⅐CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca 2؉ -calmodulin could rapidly release phosphatidylinositol 4,5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).The myristoylated alanine-rich C kinase substrate (MARCKS), 1 a major substrate of protein kinase C in many cell types, is essential for brain development (1). Although the exact function of MARCKS is not known, this peripheral protein binds to calmodulin (2) and actin (3) and may play a role in secretion, membrane trafficking, and cell motility (reviewed in Refs. 4 and 5). In the plasma membranes of macrophages, MARCKS is concentrated in lateral domains (6): it colocalizes with actin filaments and protein kinase C-␣ in nascent phagosomes (7). Binding of MARCKS to either phospholipid vesicles or biological membranes requires both the insertion of its myristate chain into the hydrocarbon interior of the membrane and interaction of its basic effector region with acidic phospholipids (Refs. 8 -13; reviewed in Refs. 14 -16). Phosphorylation by protein kinase C (reviewed in Ref. 17) introduces three negatively charged phosphates into the effector region, which weakens its electrostatic interaction with acidic lipids and produces desorption of MARCKS from membranes (9 -11, 18). Ca 2ϩ -calmodulin (Ca 2ϩ ⅐CaM) binds with high (nanomolar) affinity to the effector region, producing desorption of MARCKS from both phospholipid vesicles and plasma membranes (10, 11).Several experiments suggest that a peptide corre...

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“…The mutant cDNA was subcloned into Bluescript, and the correct sequence was confirmed by automated sequencing using an Applied Biosystems DNA sequencer, model 373A. Following linearization of the Bluescript/p40 chimera with EcoRI, mRNA was synthesized in vitro using T3 RNA polymerase (Promega) (16). The resulting mRNA was translated in vitro as described previously (15).…”

Section: Methodsmentioning

confidence: 99%

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Spizz¹,

Blackshear

1997

Journal of Biological Chemistry

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The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH 4 Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 ؋ g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time-and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.The myristoylated alanine-rich C kinase substrate (MARCKS) 1 is a prominent cellular substrate for protein kinase C (PKC) (1, 2). Expression of this heat-stable, acidic protein is essential for life, as demonstrated by the perinatal death of mice that are completely deficient in MARCKS (3). Complete lack of expression leads to gross abnormalities of central nervous system development; however, heterozygous mice, which express MARCKS at 50% wild-type levels, appear to be normal.Cellular levels of MARCKS are regulated by both transcriptional and translational mechanisms (4 -14). In addition, we recently demonstrated that the cellular concentrations of MARCKS can also be regulated by a proteolytic event. This proteolytic cleavage results in amino-and carboxyl-terminal fragments of MARCKS that co-exist in cells with the full-length protein. This cleavage of MARCKS was inhibited in intact fi...

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Membrane Association of the Myristoylated Alanine-rich C Kinase Substrate (MARCKS) Protein.

Cited by 100 publications

References 59 publications

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

Sprouty4 negatively regulates protein kinase C activation by inhibiting phosphatidylinositol 4,5-biphosphate hydrolysis

Kinetics of Interaction of the Myristoylated Alanine-rich C Kinase Substrate, Membranes, and Calmodulin

Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

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