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

Abstract

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...

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

“…That ions with masses identical to those predicted for c 18 (and a 18 ) fragments are seen in the experimental spectrum indicates that some portion of the sample is phosphorylated at either Ser 19 or Ser 23. Similar observation of ions with masses identical to those predicted for c 12 and c 13 indicates that some fraction of the sample is phosphorylated at either Ser 16, Ser 19, or Ser 23.…”

“…This protein also binds calmodulin [7], acidic membrane phospholipids [8 -10], and actin filaments [11]. These interactions are believed to be regulated by protein phosphorylation [3,4,12,13]. The MARCKS protein is also a major substrate for protein kinase C (PKC) [14].…”

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

“…MARCKS-(151-175) or Lys-13) to PC/PIP 2 (99:1) vesicles where Ͼ10 nM peptide changes the charge of the vesicle. The fluorescence assay using the acrylodan probe typically requires higher peptide concentrations (Ն50 nM) for a good signal/noise ratio, and the probe introduces some binding energy because of its hydrophobic insertion (44). The equilibrium dialysis assay requires a longer time (ϳ24 h), and the loss of peptide to the dialysis membrane and the cell is significant.…”

“…Kinetic Measurements-Kinetic measurements of MARCKS-(151-175) binding were performed on an SLM-Aminco spectrofluorometer with a stopped-flow attachment as described previously (44). Briefly, 100 nM of acrylodan-MARCKS-(151-175) was mixed rapidly with varying concentrations of PC/PIP 2 LUVs (diameter 100 nm) in a stoppedflow chamber.…”

“…Peptide Labeling-We used a protocol modified from Molecular Probes (Conjugation with Thiol-reactive Probes) to label peptides with a thiol-reactive fluorescent acrylodan probe, as described in detail elsewhere (44). Briefly, we mixed 1 ml of ϳ1 mM peptide in 10 mM K 2 HPO 4 / KH 2 PO 4 , pH 7.0, with acrylodan probe dissolved in DMF (mole ratio of 1.5:1 acrylodan/peptide) for 1 h, purified the labeled peptide using high pressure liquid chromatography, and checked its purity with MALDI mass spectrometry (CASM, State University of New York, Stony Brook).…”