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 phosphor ylation produces translocation of MARCKS from membrane to cytoplasm in many cells. Our working hypothesis is that binding of MARCKS to biolo gical membranes requires both hydrophobic insertion of its myristoyl c hain 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). me estimated the partition coefficient of the myristoyl moiety of MARCKS (K-H) by measuring the binding of MARCKS to electrically neu tral LUVs (K-H = 3 x 10(3) M(-1)). We examined the effect of electrost atic interactions by measuring the binding of MARCKS to LUVs containin g 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 phosph ate groups to the basic domain, reduced 20-fold the binding of MARCKS to these negatively charged vesicles. Third, increasing the KCl concen tration from 0.1 to 0.5 M reduced the binding 15-fold. Fourth, Ca2+-ca lmodulin reduced the binding 20-fold We present a simple theoretical m odel that explains these results, which are all consistent with the wo rking hypothesis.