MARCKS Peptide (151-175), Phosphorylated

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.

Both the myristoylated alanine-rich protein kinase C substrate protein (MARCKS) and a peptide corresponding to its basic effector domain, MARCKS-(151-175), inhibit phosphoinositide-specific phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP(2)) in vesicles (Glaser, M., Wanaski, S., Buser, C. A., Boguslavsky, V., Rashidzada, W., Morris, A., Rebecchi, M., Scarlata, S. F., Runnels, L. W., Prestwich, G. D., Chen, J., Aderem, A., Ahn, J., and McLaughlin, S. (1996) J. Biol. Chem. 271, 26187-26193). We report here that adding 10-100 nm MARCKS-(151-175) to a subphase containing either PLC-delta or -beta inhibits hydrolysis of PIP(2) in a monolayer and that this inhibition is due to the strong binding of the peptide to PIP(2). Two direct binding measurements, based on centrifugation and fluorescence, show that approximately 10 nm PIP(2), in the form of vesicles containing 0.01%, 0.1%, or 1% PIP(2), binds 50% of MARCKS-(151-175). Both electrophoretic mobility measurements and competition experiments suggest that MARCKS-(151-175) forms an electroneutral complex with approximately 4 PIP(2). MARCKS-(151-175) binds equally well to PI(4,5)P(2) and PI(3,4)P(2). Local electrostatic interactions of PIP(2) with MARCKS-(151-175) contribute to the binding energy because increasing the salt concentration from 100 to 500 mm decreases the binding 100-fold. We hypothesize that the effector domain of MARCKS can bind a significant fraction of the PIP(2) in the plasma membrane, and release the bound PIP(2) upon interaction with Ca(2+)/calmodulin or phosphorylation by protein kinase C.

In vitro hippocampal studies by Gay et al. (2008) demonstrated that a myristoylated alanine-rich C kinase substrate (MARCKS) peptide comprising the phosphorylation site or effector domain of the protein acts as a powerful inhibitor of alpha7 nicotinic acetylcholine receptors (nAChRs), which are known to be critically involved in memory function. However, behavioral consequences of hippocampal MARCKS peptide infusions have not been investigated. The purpose of the current study was to determine if local infusions in the rat ventral hippocampus of long (comprising amino acids 151-175) and short (amino acids 159-165) forms of MARCKS peptides could affect memory performance in the 16-arm radial maze. Our results demonstrated a dramatic impairment of both working (changing) and reference (constant) memory with MARCKS(151-175) only. The shorter MARCKS peptide did not affect memory performance. This is in line with in vitro results reported by Gay et al. (2008) that long, but not short, MARCKS peptides inhibit alpha7 nAChRs. We also found that the effect of the MARCKS(151-175) peptide was dose-dependent, with a robust memory impairment at 10 microg/side, and smaller inconsistent effects at lower doses. Our present behavioral study, together with the earlier in vitro study by Gay et al. (2008), suggests that effector domain MARCKS peptides could play a significant role in memory regulation and impairment.