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p6O0, the transforming protein kinase of Rous sarcoma virus, contains the 14-carbon saturated fatty acid, myristic acid, linked through an amide bond to the a-amino group of its NH2-terminal glycine residue. Myristic acid is known to be attached to four other eukaryotic proteins. In each case the fatty acid is also linked through an amide bond to an NH2-terminal glycine. We have used oligonucleotidedirected mutagenesis to examine the amino acid specificity of the enzyme that myristoylates the NH2 terminus of these proteins. Replacement of the NH2-terminal glycine in p6O1 with either alanine or glutamic acid prevented myristoylation completely. This indicates that the myristoylating enzyme may have an absolute specificity for glycine. Strikingly, neither nonmyristoylated mutant src protein induced morphological transformation of infected cells, even though wild-type levels of phosphorylation of cellular proteins on tyrosine were observed in these cells. Since conversion of the NH2-terminal residue from glycine to alanine should have little effect on the conformation of p6O1, the inability of this mutant p6Owr protein to induce morphological transformation suggests that the myristoyl moiety is essential for the transforming activity of the protein.Cellular transformation by Rous sarcoma virus results from the expression of a single viral protein designated p6011 (1). p60src functions as a tyrosine-specific protein kinase (2, 3) in vivo and is also reported to phosphorylate phosphatidylinositol in vitro (4). Tyrosine phosphorylation may be crucial in the control of cellular proliferation. The mitogens epidermal growth factor (5), platelet-derived growth factor (6), and insulin-like growth factor (7) all stimulate tyrosine protein kinase activity when they bind to their cell-surface receptors. Immunofluorescence (8), immunoelectron microscopy (9), and cell fractionation (10-12) all suggest that a significant fraction of the p60'" in transformed cells is associated with the cytoplasmic face of the plasma membrane. p60'1 may therefore deliver an unregulated mitogenic signal through the continuous phosphorylation ofone or more proteins involved in the normal regulation of proliferation.p60src is bound firmly to cellular membranes yet contains no large cluster of hydrophobic amino acids similar to those which are responsible for anchoring membrane-bound proteins such as the HLA and H-2 glycoproteins (human and murine major histocompatibility proteins) to a lipid bilayer. p60s' does, however, contain covalently bound myristic acid, a rare 14-carbon saturated fatty acid (13). This myristic acid moiety is attached by an amide linkage to the a-amino group of the NH2-terminal glycine residue of p6src (14). Consequently, an attractive hypothesis is that the hydrophobic myristoyl group plays a role in binding p6OSrc to membranes.Myristoylation is an uncommon form of protein modification. Nevertheless, the amino acid to which myristic acid is attached has been identified unambiguously in four additional proteins. The c...

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