Kim J, Blackshear P J, Johnson J D, McLaughlin S
Department of Physiology and Biophysics, HSC, SUNY, Stony Brook 11794.
Biophys J. 1994 Jul;67(1):227-37. doi: 10.1016/S0006-3495(94)80473-4.
Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.
多个研究小组观察到，磷酸化会导致MARCKS（肉豆蔻酰化富含丙氨酸的蛋白激酶底物）蛋白从细胞膜和磷脂囊泡上脱离。我们的工作假设是，MARCKS与膜的有效结合既需要N端肉豆蔻酸酯疏水插入双层膜，也需要蛋白中单个碱性残基簇与酸性脂质的静电结合，而磷酸化会逆转这种静电结合。用肉豆蔻酰化肽和磷脂囊泡进行的膜结合测量表明，这种疏水部分充其量只能勉强将蛋白质附着于质膜。我们在此报告了与MARCKS和神经调节蛋白磷酸化结构域相对应的碱性肽的膜结合测量结果。这些肽的结合随磷脂囊泡中酸性脂质的百分比呈S形增加，并且可以用 Gouy-Chapman/质量作用理论来描述，该理论解释了静电作用和维度降低如何产生明显的协同性。MARCKS肽对含有10%酸性磷脂的膜的静电亲和力（10⁴ M⁻¹ = χ/[P]，其中χ是结合到囊泡外层单分子层的肽的摩尔比，[P]是水相中肽的浓度）与肉豆蔻酸部分对双层膜的疏水亲和力相同。磷酸化使MARCKS肽对含有15%酸性脂质的膜的亲和力降低约1000倍，并使肽从磷脂囊泡中快速解离（t₁/₂ < 30秒）。
