Electrostatics and the Membrane Association of Src: Theory and Experiment ² Diana Murray, ‡ Luz Hermida-Matsumoto, § Carolyn A. Buser, ‡,| James Tsang, ‡ Catherine T. Sigal, §,⊥ Nir Ben-Tal, ∇,# Barry Honig, ∇ Marilyn D. Resh, § and Stuart McLaughlin* ,‡ Department of Physiology and Biophysics, State UniVersity of New York at Stony Brook, Stony Brook, New York 11794-8661, Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and Department of Biochemistry and Molecular Biophysics, Columbia UniVersity, New York, New York 10032 ReceiVed August 13, 1997; ReVised Manuscript ReceiVed NoVember 20, 1997 ABSTRACT: 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-NH 2 ] 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. The v-Src oncoprotein (pp60 v-src ) 1 and its normal cellular homologue c-Src (pp60 c-src ) are membrane-bound, nonre- ceptor tyrosine kinases (1-4). The N terminus of Src contains two motifs essential for membrane localization: a myristate attached cotranslationally to the N-terminal glycine by N-myristoyltransferase (5-9) and a stretch of hydrophilic amino acids rich in positively charged residues (10, 11). Mutations that either prevent myristoylation (12-14) or reduce the net positive charge in the N-terminal cluster of basic residues (10, 11) inhibit the association of Src with the plasma membrane, producing nontransforming pheno- types. Studies with either the intact Src protein (11) or myris- toylated peptides corresponding to the N terminus of Src (15) support the hypothesis that the myristate inserts hydropho- bically into the hydrocarbon interior of the bilayer and the basic residues interact electrostatically with acidic phospho- lipids. Myristate alone provides barely enough energy to attach the peptide or protein to membranes (16-18); the myristate and basic residues act synergistically (or with apparent cooperativity) to effect the membrane binding of Src. For example, adding 33% acidic lipid to electrically neutral vesicles increases the membrane binding of both Src and the N-terminal peptide myr-Src(2-16) (myristate- GSSKSKPKDPSQRRR-NH 2 ) by 3 orders of magnitude (11, 15). Buser et al. (15) described the synergism between Src’s two membrane binding motifs with a “ball and string” model that represents the myristoylated peptide or protein as two dimensionless binding sites, A (acyl chain) and B (basic residues), joined by a flexible, electrically neutral string of length r. Following Crothers and Metzger (19) and Perutz ² This research was supported by Grant MCB-9419175 from the NSF and Grant GM-24971 from the NIH to S.M., Grant BE235 from the American Cancer Society and Grant CA-72309 from the NIH to M.D.R., Grant MCB-9304127 from the NSF and Grant MCA95C015 from the NCSA to B.H., Grant 5T32 NSO 7372-03 from the NIH and a Helen Hay Whitney Foundation Postdoctoral Fellowship to D.M., and the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation Fellowship to C.A.B. * Address correspondence to this author. Telephone: 516-444-3039. Fax: 516-444-3432. E-mail: smcl@informatics.informatics.sunysb.edu. ‡ State University of New York at Stony Brook. § Memorial Sloan-Kettering Cancer Center. | Present address: Cancer Research Department, Merck and Co., West Point, PA 19486. ⊥ Present address: Laboratory of Cellular and Molecular Recognition, National Institute of Mental Health, Bethesda, MD 20892. ∇ Columbia University. # Present address: Department of Biochemistry, Tel Aviv University, Ramat Aviv 69978, Israel. 1 Abbreviations: c-Src, pp60 c-src ; CD, circular dichroism; PKA, cAMP-dependent protein kinase; DAG, diacylglycerol; EPR, electron paramagnetic resonance; -Gal, -galactosidase; LUV, large unilamellar vesicles; myr, myristoylated; nonmyr, nonmyristoylated; PC, phos- phatidylcholine; PG, phosphatidylglycerol; PH, pleckstrin homology; PIP 2, phosphatidylinositol 4,5-bisphosphate; PS, phosphatidylserine; PKC, protein kinase C; SUV, sonicated unilamellar vesicles; v-Src, pp60 v-src . 2145 Biochemistry 1998, 37, 2145-2159 S0006-2960(97)02012-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/06/1998