PubMed · 10881187
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T Smith. 2000. Fixing mistakes.. https://doi.org/10.1038/75838
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Neurocalcin is a member of a novel family of neuronal calcium sensors that belongs to the superfamily of EF-hand Ca(2+)-binding proteins. Neurocalcin is myristoylated on its N-terminus in vivo and can associate with biological membranes in a calcium and myristoyl-dependent manner. This process known as "Ca(2+)-myristoyl switch" has been best described for the photoreceptor specific protein, recoverin, as well as for several other neuronal calcium sensors. Here, we used reversed micelles to chemically acylate nonmyristoylated neurocalcin at its N-terminus with fatty acids of different lengths (from C12 to C16). This approach allowed us to prepare neurocalcin derivatives in which a single fatty acid is selectively linked to the N-terminal glycine of the polypeptide chain through an amide bond. The membrane binding properties of the monoacylated neurocalcins were then examined by cosedimentation with phospholipid vesicles and direct binding to lipid monolayers by surface plasmon resonance spectroscopy (Biacore). Our results show that neurocalcins monoacylated with lauric, myristic, or palmitic acid were able to associate with membrane in a calcium-dependent manner. This indicates that the Ca(2+)-myristoyl switch can function with different lipid moieties and is not strictly restricted to myristate. The ability to modify at will the fatty acid linked to the N-terminal glycine should be useful to analyze the contribution of the fatty acid moiety to the biological function of this family of neuronal calcium sensors.
Human transmembrane tumor necrosis factor (pro-TNF) was examined for protein acylation. The cDNA encoding pro-TNF was expressed in both COS-1 cells and Sf9 cells and metabolic labeling with [(3)H]myristic or [(3)H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro-TNF was specifically labeled with [(3)H]palmitic acid. The [(3)H]palmitic acid labeling of pro-TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site-directed mutagenesis of the two cysteine residues residing in the leader sequence of pro-TNF demonstrated that palmitoylation of pro-TNF occurs solely at Cys-47, located at the boundary between the transmembrane and cytoplasmic domains of pro-TNF. Thus, pro-TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.
Fatty acylation of Src family kinases is essential for localization of the modified proteins to the plasma membrane and to plasma membrane rafts. It has been suggested that the presence of saturated fatty acyl chains on proteins is conducive for their insertion into liquid ordered lipid domains present in rafts. The ability of unsaturated dietary fatty acids to be attached to Src family kinases has not been investigated. Here we demonstrate that heterogeneous fatty acylation of Src family kinases occurs and that the nature of the attached fatty acid influences raft-mediated signal transduction. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, we show that in addition to 14:0 (myristate), 14:1 and 14:2 fatty acids can be attached to the N-terminal glycine of the Src family kinase Fyn when the growth media are supplemented with these dietary fatty acids. Moreover, we synthesized novel iodinated analogs of oleate and stearate, and we showed that heterogeneous S-acylation can occur on cysteine residues within Fyn as well as Galpha, GAP43, and Ras. Modification of Fyn with unsaturated or polyunsaturated fatty acids reduced its raft localization and resulted in decreased T cell signal transduction. These studies establish that heterogeneous fatty acylation is a widespread occurrence that serves to regulate signal transduction by membrane-bound proteins.