Membrane association is required for cell transformation by pp60v-src (v-Src), the product of the v-src oncogene of Rous sarcoma virus. Previous experiments have identified two NH2-terminal membrane-binding motifs: a myristate (14-carbon acyl chain) attached to the NH2-terminal glycine and three basic residues at positions 5, 7, and 9 of Src. We examined the membrane binding of each motif using myristylated (myr-src) and nonmyristylated (nonmyr-src) peptides corresponding to the NH2 terminus of Src. All myristylated peptides partitioned equally well onto electrically neutral phosphatidylcholine vesicles (K1 = 10(4) M-1). Identical binding has been observed for simple myristylated peptides (e.g., myr-Gly) and arises from the hydrophobic insertion of the myristate into the bilayer. A nonmyristylated peptide corresponding to residues 2-16 of Src [nonmyr-src(2-16), net charge = +5] bound to vesicles containing 33% monovalent acidic phospholipids with K1 = 10(3) M-1. Penta(lysine) (+5 net charge) exhibits the same binding behavior, which is due to the electrostatic interaction between basic residues and acidic lipids. The corresponding myristylated peptide, myr-src(2-16), binds 3 orders of magnitude more strongly to vesicles containing 33% acidic lipids than to neutral vesicles. The resulting apparent association constant, K1 = 10(7) M-1, is approximately equal to the product of the partition coefficients for the two individual interactions. This 10(7) M-1 binding is sufficiently strong to anchor the Src protein to biological membranes. We propose a simple model that explains the observed synergism between the two peptide-membrane interactions.

Membrane targeting of pp6w0 (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids.

The binding of Src to phospholipid membranes requires both hydrophobic insertion of its myristate into the hydrocarbon interior of the membrane and nonspecific electrostatic interaction of its N-terminal cluster of basic residues with acidic phospholipids. We provide a theoretical description of the electrostatic partitioning of Src onto phospholipid membranes. Specifically, we use molecular models to represent a nonmyristoylated peptide corresponding to residues 2-19 of Src [nonmyr-Src(2-19); GSSKSKPKDPSQRRRSLE-NH2] and a phospholipid bilayer, calculate the electrostatic interaction by solving the nonlinear Poisson-Boltzmann equation, and predict the molar partition coefficient using statistical thermodynamics. The theoretical predictions agree with experimental data obtained by measuring the partitioning of nonmyr-Src(2-19) onto phospholipid vesicles: membrane binding increases as the mole percent of acidic lipid in the vesicles is increased, the ionic strength of the solution is decreased, or the net positive charge of the peptide is increased. The theoretical model also correctly predicts the measured partitioning of the myristoylated peptide, myr-Src(2-19); for example, adding 33% acidic lipid to electrically neutral vesicles increases the partitioning of myr-Src(2-19) 100-fold. Phosphorylating either serine 12 (by protein kinase C) or serine 17 (by cAMP-dependent protein kinase) decreases the partitioning of myr-Src(2-19) onto vesicles containing acidic lipid 10-fold. We investigated the effect of phosphorylation on the localization of Src to biological membranes by expressing fusion constructs of Src's N terminus with a soluble carrier protein in COS-1 cells; phosphorylation produces a small shift in the distribution of the Src chimeras from the plasma membrane to the cytosol.