Electrostatic Sequestration of PIP2 on Phospholipid Membranes by Basic/Aromatic Regions of Proteins

Abstract

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.

Determining selectivity of phosphoinositide-binding domains

“…These experiments gave a mean K D for PtdIns(4,5)P 2 binding by the PLC 1 PH domain of 1.66 ± 0.8 µM. Measurements using different techniques by several other laboratories, including those of McLaughlin [38], Rebecchi [40] and Hirose [41] all fall within a range of 1 µM -2.8 µM. This is perhaps the most well characterized PH domain/phosphoinositide interaction.…”

“…Corbin et al [37] have followed vesicle association of the Grp1 PH domain by monitoring FRET between tryptophans in the PH domain and dansylated lipid incorporated into the vesicles. A similar FRET approach has also been employed for assessing the binding of Texas Red labeled MARCKS peptide to vesicles containing fluorescent PtdIns(4,5)P 2 [38]. These approaches are very powerful, but require the appropriate labeled lipids and/or proteins.…”

Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

“…MARCKS shows high affinity binding to phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and plays an important role in regulating the availability of PIP 2 by sequestration of PIP 2 (39), which has been implicated in signal transduction (40). The MARCKS-ED peptide binds PIP 2 with high affinity like the fulllength MARCKS and inhibits PIP 2 hydrolysis in vitro (41), indicating that the effector domain mediates the sequestration of PIP 2 .…”

“…In addition, MARCKS is involved in cell adhesion, lamellipodia formation, initiation of neuritogenesis, neurite outgrowth, growth cone adhesion, pathfinding, dendrite branching, dendritic spine morphogenesis, and synaptic plasticity (13,28,39,41,(61)(62)(63)(64)(65)(66)(67)(68)(69)(70)(71). Knockdown of MARCKS by RNA interference and overexpression of MARCKS mutants revealed that non-phosphorylated MARCKS stabilizes growth cone adhesion, enhances branching and growth of dendrites, elongates dendritic spines, and enhances initiation of neuritogenesis and neurite outgrowth (65,66,68,69).…”

The Ras–Membrane Interface: Isoform-Specific Differences in the Catalytic Domain

“…The crowding of acidic lipids, such as PtdSer and PtdIns, around proteins with polybasic membrane-binding domains induces a nonuniform surface potential and potentially promotes a unique K-Ras signaling nanocluster (31). K-Ras could serve as a hub for acidic secondary messengers, like phosphatidylinositol 4,5-bisphosphate (PIP2), to promote rapid signal activation and transduction, prevent constitutive signal propagation, and cause dissociation of electrostatically stabilized proteins from the plasma membrane (35)(36)(37). The implication of these events in isoform-specific signaling has been observed in the Ras/Raf/ MEK/ERK pathway.…”