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Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids involved in signal transduction and in lipid biosynthesis in cells. LPA acyltransferase (LPAAT), also known as 1-acyl sn-glycerol-3-phosphate acetyltransferase (EC 2.3.1.51), catalyzes the conversion of LPA to PA. In this study, we describe the isolation and characterization of two human cDNAs that encode proteins possessing LPAAT activities. These two proteins, designated here as LPAAT-alpha and LPAAT-beta, contain extensive sequence sequence similarities to microbial or plant LPAAT sequences. LPAAT-alpha mRNA was detected in all tissues with highest expression in skeletal muscle whereas LPAAT-beta was expressed predominantly in heart and liver tissues. Expression of these two cDNAs in an Escherichia coli strain with a mutated LPAAT gene (plsC) complements its growth defect and shifts the equilibrium of cellular lipid content from LPA to PA and other lipids. Overexpression of these two cDNAs in mammalian cells leads to increased LPAAT activity in cell-free extracts using an in vitro assay that measures the conversion of fluorescently labeled LPA to PA. This increase in LPAAT activity correlates with enhancement of transcription and synthesis of tumor necrosis factor-alpha and interleukin-6 from cells upon stimulation with interleukin-1beta, suggesting LPAAT overexpression may amplify cellular signaling responses from cytokines.

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The cytokine interleukin la (IL-la) is a critical mediator of the immune and inflammatory responses.A unique determinant of its activity as compared with IL-1f may be its association with the plasma membrane. While the biologic activity of "membrane IL-1" has been extensively reported, the mechanism of membrane binding remains unclear. We report that the N terminus of the 31-kDa IL-la precursor is myristoylated on specific internal lysine residues. Immunoprecipitation of [3HJmyristic acid-radiolabeled human monocyte lysates with IgG antibodies to the 31-kDa IL-la precursor recovered a protein with the physicochemical properties of the IL-la N-terminal propiece (16 kDa, pl 4.45). Glycyl N-myristoylation of this protein is precluded by the absence of a glycine residue at position 2, suggesting that the propiece is myristoylated on e-amino groups of lysine. To determine which lysine(s) are acylated, a series of synthetic peptides containing all lysines found in the IL-la N-terminal propiece were used in an in vitro myristoylation assay containing peptide, myristoyl-CoA, and monocyte lysate as enzyme source. Analysis of the reaction products by reverse-phase HPLC and gas-phase sequencing demonstrated the specific myristoylation of Lys-82 and Lys-83, yielding predominantly monoacylated product. A conserved sequence in the IL-113 propiece was myristoylated with at least 8-fold less efficiency. Acylation of the IL-la precursor by a previously unrecognized lysyl e-amino N-myristoyltransferase activity may facilitate its specific membrane targeting. N-myristoylation of newly translated proteins has received significant attention as a major determinant of protein targeting and function (for review, see ref. 1). A variety of viral and mammalian membrane-associated proteins are myristoylated; when mutated to nonmyristoylated forms these become soluble, cytosolic proteins with significantly altered function (2-8). For most of these proteins, cotranslational acylation is performed by the enzyme myristoyl CoA:protein N-myristoyltransferase, which forms an amide bond between myristic acid and an N-terminal glycine residue. However, a few myristoylated proteins, including the insulin receptor, the ,u immunoglobulin heavy chain, tumor necrosis factor a, and the interleukin la and 1(3 (IL-la and IL-113) precursors, lack glycine residues correctly positioned for N-myristoylation (9-12). An alternative mechanism for myristoylation of these proteins would be the acylation of internal lysine residues, in which the free E-amino groups form the characteristic amide bonds. While an enzymatically catalyzed fatty acid (octanoyl) acylation of internal lysine E-amino groups has been documented for Agistrodon phospholipase A2 (13), discrete cotranslational myristoylation of internal lysine residues has not been demonstrated.IL-la and IL-1p8 are cytokines with important roles in inflammation and the immune response. Both IL-la and IL-1X3 are translated as 31-to 33-kDa precursors which are subsequently proteolytically processed to the e...

SummaryNH2-terminal glycine myristyl acylation is a cotranslational modification that affects both protein localization and function. However, several proteins that lack NH2-terminal glycine residues, including the interleukin 1 (Ibl) precursors, also contain covalently linked myristate. To date, the site(s) of acylation of these proteins has not been determined. During an evaluation of Ibl acylation, it was observed that [3H]myristate-labded human monocyte lysates contained a prominent 26-kD myristylated protein, which was identified as the tumor necrosis factor ce (TNF) precursor protein on the basis of specific immune precipitation. Radioimmunoprecipitates from the supernates of labeled monocytes indicated that the processed or mature 17-kD form of TNF does not contain myristate, suggesting that the site of acylation occurs within the 76-amino acid propiece of the precursor molecule. As the TNF precursor does not contain an NH2-terminal glycine, we hypothesized that myristyl acylation occurs on the N-e-NH2 groups of lysine, of which two are present in the propiece (K19K20). Synthetic peptides were designed to include all seven lysine residues present within the entire 26-kD TNF precursor, and used in an in vitro myristyl acylation assay containing peptide, myristyl-CoA, and monocyte lysate as a source of enzyme. Analysis of reaction products by reverse phase high performance liquid chromatography and gas phase sequencing demonstrated the exclusive myristyl acylation of K19 and K20, consistent with the presence in monocytes of a specific lysyl N-e-NH2-myristyl transferase activity. The acylated lysine residues are located immediately downstream from a hydrophobic, probable membrane-spanning segment of the propiece. Specific myristyl acylation of the TNF propiece may facilitate membrane insertion or anchoring of this critical inflammatory mediator.mong the many modifications of newly synthesized proteins, cotranslational acylation with myristic acid has received considerable attention as an important determinant of protein function and intracellular localization (for review, see reference 1). For most myristylated proteins studied thus far, acyhtion occurs via the formation of an amide bond linking the fatty acid to an NHz-terminal glycine residue after the removal of the initiator methionine. This process has been weU characterized and the enzyme responsible, myristyl CoA: protein N-myristyl transferase (NMT), 1 has been cloned (2). However, in a few cases myristylated proteins have been identified that lack the correctly positioned NHz-terminal glycine strictly required for acylation by NMT. These proteins include the insulin receptor, the/~ Ig heavy chain, and 1 Abbreviation used in this paper: NMT, N-myristyl transferase.the Ibl oc and B precursors (3-5). All of these proteins are myristylated by an undescribed enzymatic mechanism that does not involve acylation on NH2-terminal glycines. One potential alternative mechanism for myristyl acylation would be the myristylation of internal lysine residues, using ...