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.

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.Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) 1 plays many important roles in cells (reviewed in Refs. 1-8). Not only is PIP 2 the source of 3 second messengers, its hydrolysis via phospholipase Cs (PLCs) (9, 10) produces inositol 1,4,5-trisphosphate and diacylglycerol (11,12), and its phosphorylation via phosphoinositide 3-kinases produces phosphatidylinositol 3,4,5-trisphosphate (6, 13-15), but it also can be a second messenger itself (4,8,16,17). Moreover, PIP 2 helps regulate cytoskeletal attachment (18 -20), exo-and endocytosis (1-3), enzyme activity (21), and ion channel function (17,22). Several groups (2,8,16,23) have suggested that these myriad functions can be explained if there are different pools of PIP 2 in the plasma membrane. One hypothesis is that proteins act as reversible buffers to bind much of the PIP 2 and then release it locally in response to specific signals (8,24,25). These proteins would have to be present at a concentration comparable with PIP 2 , be localized to the plasma membrane, bind PIP 2 with high affinity, and release it in response to physiological stimuli. Myristoylated alanine-rich C kinase substrate (MARCKS) satisfies these criteria.MARCKS (reviewed in Refs. 26 -30), a ubiquitous protein kinase C (PKC) (31) substrate, is present at high concentration in many cell types (e.g. ϳ10 M in brain tissue (27, 32)) and is localized to the plasma membrane in quiescent cells. The binding of MARCKS to plasma membranes requires both hydrophobic insertion of its N-terminal myristate into the bilayer and electrostatic interactions between its effecto...

No abstract