title: Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters spin-labeled phosphatidylinositol 4,5-bisphosphate in lipid bilayers.
abstract: 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.
authors.affiliation: Department of Chemistry and Biophysics Program, University of Virginia, Charlottesville, Virginia 22904-4319, USA.
authors.forename: Michelle E
authors.initials: ME
authors.lastname: Rauch
authors.forename: Colin G
authors.initials: CG
authors.lastname: Ferguson
authors.forename: Glenn D
authors.initials: GD
authors.lastname: Prestwich
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D002135:Calcium-Binding Proteins; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D005959:Glucosidases; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers / Q000378:metabolism*; D008055:Lipids / Q000737:chemistry; D008565:Membrane Proteins; D008698:Mesylates / Q000494:pharmacology; D008954:Models, Biological; D008956:Models, Chemical; D019814:Myristic Acid / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013113:Spin Labels
chemical_list: D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008051:Lipid Bilayers; D008055:Lipids; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D008698:Mesylates; D019269:Phosphatidylinositol 4,5-Diphosphate; D010750:Phosphoproteins; D011994:Recombinant Proteins; D013113:Spin Labels; D019814:Myristic Acid; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; C074502:methanethiosulfonate; D005959:Glucosidases; C070368:PRKCSH protein, human; D003545:Cysteine