Characterization of the human LPIN1-encoded phosphatidate phosphatase isoforms

Abstract

The human LPIN1 gene encodes the protein lipin 1, which possesses phosphatidate (PA) phosphatase (3-sn-phosphatidate phosphohydrolase; EC 3.1.3.4) activity (Han, G.-S., Wu, W.-I., and Carman, G. M. (2006) J. Biol. Chem. 281, 9210-9218). In this work, we characterized human lipin 1 alpha, beta, and gamma isoforms that were expressed in Escherichia coli and purified to near homogeneity. PA phosphatase activities of the alpha, beta, and gamma isoforms were dependent on Mg(2+) or Mn(2+) ions at pH 7.5 at 37 degrees C. The activities were inhibited by concentrations of Mg(2+) and Mn(2+) above their optimums and by Ca(2+), Zn(2+), N-ethylmaleimide, propranolol, and the sphingoid bases sphingosine and sphinganine. The activities were thermally labile at temperatures above 40 degrees C. The alpha, beta, and gamma activities followed saturation kinetics with respect to the molar concentration of PA (K(m) values of 0.35, 0.24, and 0.11 mm, respectively) but followed positive cooperative (Hill number approximately 2) kinetics with respect to the surface concentration of PA (K(m) values of 4.2, 4.5, and 4.3 mol %, respectively) in Triton X-100/PA-mixed micelles. The turnover numbers (k(cat)) for the alpha, beta, and gamma isoforms were 68.8 + or - 3.5, 42.8 + or - 2.5, and 5.7 + or - 0.2 s(-1), respectively, whereas their energy of activation values were 14.2, 15.5, and 18.5 kcal/mol, respectively. The isoform activities were dependent on PA as a substrate and required at least one unsaturated fatty acyl moiety for maximum activity.

FIGURE 1.

PA phosphatase reaction and its roles in lipid synthesis and signaling. The reaction catalyzed by PA phosphatase (highlighted) is shown. The reaction product DAG is used for the synthesis of triacylglycerol (TAG) and the synthesis of phosphatidylethanolamine (PE) or phosphatidylcholine (PC). The reaction substrate PA is used for the synthesis of phosphatidylinositol (PI) or phosphatidylglycerol (PG) via CDP-DAG. The substrate and product of the PA phosphatase reaction play roles in lipid signaling.

FIGURE 2.

Schematic representation of the human LPIN1 gene and its protein products. A, the exon-intron organization of the human LPIN1 gene is shown with respect to coding exons. The exons numbered 1–19 comprise LPIN1α, whereas those combined with an additional exon (indicated as β or γ) constitute LPIN1β and LPIN1γ, respectively. B, the human lipin 1 isoforms (α, β, and γ) are represented with the number of amino acids and the conserved lipin domains NLIP and haloacid dehalogenase-like (HAD-like). The specific sequences for lipin 1β and lipin 1γ are indicated as β and γ, respectively. NLS, nuclear localization signal. C, the human lipin 1β- and lipin 1γ-specific sequences are aligned with the mouse counterparts. *, conserved amino acid.

FIGURE 3.

SDS-PAGE analysis of the E. coli-expressed and purified human LPIN1-encoded isoforms. A, Rosetta 2(DE3)pLysS cells bearing a LPIN1 plasmid (pGH321, pGH322, or pGH327) were grown to A_{600 nm} = 0.5. After induction of LPIN1 expression with 1 mm isopropyl β-d-1-thiogalactopyranoside for the indicated time intervals, an equal number of the induced cells were lysed with Laemmli sample buffer and resolved on an 8% SDS-polyacrylamide gel. B, the E. coli-expressed lipin 1 isoforms were subjected to affinity purification with Ni²⁺-nitrilotriacetic acid resin. The purified His₆-tagged lipin 1 isoforms (5 μg of protein) were subjected to SDS-PAGE (8% slab gel). Proteins on the SDS-polyacrylamide gels were visualized with Coomassie Blue. The positions of the full-length lipin 1 isoforms and molecular mass markers are indicated.

FIGURE 4.

Effects of pH and Triton X-100 on the human LPIN1-encoded PA phosphatase isoform activities. A, PA phosphatase activity was measured at the indicated pH values with 50 mm Tris-maleate-glycine buffer. B, the enzyme activity was measured in 50 mm Tris-HCl (pH 7.5) buffer with the indicated concentrations of Triton X-100. The highest activity of the α isoform was set at 100%, and the activities of the β and γ isoforms were calculated relative to the activity of the α isoform. The data shown are means ± S.D. from triplicate enzyme determinations.

FIGURE 5.

Effects of Mg²⁺ and Mn²⁺ on the human LPIN1-encoded PA phosphatase isoform activities. PA phosphatase activity was measured in the absence and presence of the indicated concentrations of MgCl₂ (A) or MnCl₂ (B). Relative activities were presented as described in the legend to Fig. 3. For B, the highest activity of the α isoform was calculated relative to its activity measured with MgCl₂ in A. The data shown are means ± S.D. from triplicate enzyme determinations.

FIGURE 6.

Effects of N-ethylmaleimide and 2-mercaptoethanol on the human LPIN1-encoded PA phosphatase isoform activities. PA phosphatase activity was measured in the presence of the indicated concentrations of N-ethylmaleimide (A) or 2-mercaptoethanol (B). In A, 2-mercaptoethanol was not present. Relative activities were presented as described in the legend to Fig. 3. The data shown are means ± S.D. from triplicate enzyme determinations.

FIGURE 7.

Effect of temperature on the activities and stabilities of the human LPIN1-encoded PA phosphatase isoforms. A, PA phosphatase activity was measured at the indicated temperatures for 20 min in a temperature-controlled water bath. Relative activities were presented as described in the legend to Fig. 3. B, the enzyme samples were first incubated for 20 min at the indicated temperatures. After incubation, the samples were cooled in an ice bath for 10 min to allow for enzyme renaturation, and PA phosphatase activity was then measured for 20 min at 37 °C. The highest activity of each isoform was set at 100%. The data shown are means ± S.D. from triplicate enzyme determinations.

FIGURE 8.

Dependence of the human LPIN1-encoded PA phosphatase isoform activities on the molar and surface concentrations of PA. PA phosphatase activity was measured as a function of the indicated molar concentrations of PA (A) and as a function of the indicated surface concentrations of PA (B). For the experiment shown in A, the molar ratio of Triton X-100 to PA was maintained at 10:1 (9.1 mol % PA). For the experiment shown in B, the molar concentration of PA was held constant at 1 mm, and the Triton X-100 concentration was varied to obtain the indicated surface concentrations. The data shown are means ± S.D. from triplicate enzyme determinations. The best fit curves were derived from the kinetic analysis of the data.

FIGURE 9.

Fatty acyl specificity of the human LPIN1-encoded PA phosphatase isoform activities. PA phosphatase activity was measured with 9.1 mol % PA with the indicated fatty acyl moieties. The amount of phosphate released from PA in the enzyme reactions was determined by the colorimetric assay using the malachite green-molybdate reagent. Relative activities were presented as described in Fig. 3. The data shown are means ± S.D. from triplicate enzyme determinations.