Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles.

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major cellular substrate of protein kinase C (PKC), and PKC phosphorylation produces translocation of MARCKS from membrane to cytoplasm in many cells. Our working hypothesis is that binding of MARCKS to biological membranes requires both hydrophobic insertion of its myristoyl chain into the lipid bilayer and electrostatic interaction of its basic domain with acidic lipids. We tested this hypothesis by measuring the binding of murine MARCKS to large unilamellar phospholipid vesicles (LUVs). We estimated the partition coefficient of the myristoyl moiety of MARCKS (KH) by measuring the binding of MARCKS to electrically neutral LUVs (KH = 3 x 10(3) M-1). We examined the effect of electrostatic interactions by measuring the binding of MARCKS to LUVs containing 20% acidic lipid and obtained four results. First, incorporating 20% acidic lipid into the LUVs increased binding of MARCKS about 100-fold. Second, PKC phosphorylation, which added 3 negatively charged phosphate groups to the basic domain, reduced 20-fold the binding of MARCKS to these negatively charged vesicles. Third, increasing the KCl concentration from 0.1 to 0.5 M reduced the binding 15-fold. Fourth, Ca(2+)-calmodulin reduced the binding 20-fold. We present a simple theoretical model that explains these results, which are all consistent with the working hypothesis.