The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type

Abstract

H-Rev107 is a protein that was previously cloned as a negative regulator of proto-oncogene Ras and classified as a class II tumor suppressor. Its structural similarity to lecithin retinol acyltransferase and Ca2+-independent phosphatidylethanolamine (PE) N-acyltransferase led us to analyze H-Rev107 as an enzyme involved in phospholipid metabolism. Here, we show that recombinant H-Rev107s from rat, human, and mouse possess phospholipase (PL) A1 or A2 activity toward phosphatidylcholine (PC). Further examination with purified recombinant protein revealed that H-Rev107 functions as a cytosolic Ca2+-independent PLA(1/2) for PC and PE with higher PLA1 activity than PLA2 activity. Dithiothreitol and iodoacetic acid exhibited stimulatory and inhibitory effects, respectively. Histidine-21 and cysteine-111 of rat H-Rev107 were presumed to form a catalytic dyad based on database analysis, and their single mutants were totally inactive. These results suggested that H-Rev107 is a hydrolase of the thiol type. The N-terminal proline-rich and C-terminal hydrophobic domains of H-Rev107 were earlier reported to be responsible for the regulation of cell proliferation. Analysis of deletion mutants indicated that these domains are also catalytically essential, suggesting relevance of the catalytic activity to the anti-proliferative activity.

Fig. 1.

Alignment of the deduced amino acid sequences of human, mouse, and rat H-Rev107s. The sequences were aligned using the program GENETYX-MAC (version 10). Closed and shaded boxes indicate identity in all three or any two polypeptides, respectively. Gaps introduced for maximal alignment are indicated by dashes. The highly conserved histidine and cysteine residues in the LRAT family are indicated by asterisks. The N-terminal proline-rich domain (A) and C-terminal hydrophobic domain (B) are indicated by lines. The N-terminal (ΔN1 and ΔN2) or C-terminal (ΔC1) residues of the deletion mutants of rat H-Rev107, which were used in the experiment shown in Fig. 8, are also shown.

Fig. 2.

Transient expression and enzymatic activities of human, mouse, and rat H-Rev107s. COS-7 cells were transfected with the insert-free vector (Mock) or the expression vector harboring FLAG-tagged H-Rev107 of either human, mouse, or rat, or FLAG-tagged Ca²⁺-independent N-acyltransferase (iNAT) of rat. The cell homogenates (10 μg protein) were analyzed by Western blotting with an anti-FLAG antibody (A). The cell homogenates (10 μg protein) were also assayed for phosphatidylethanolamine (PE) N-acylation activity (B) and for the phospholipase (PL) A_1/2 activity (C) as described under Experimental Procedures. The products were separated by TLC. The positions of authentic compounds on the TLC plate are indicated by arrows. NPPE, N-[¹⁴C]palmitoyl-PE; C16:0, [¹⁴C]palmitic acid; PC, phosphatidylcholine.

Fig. 3.

Subcellular distribution of H-Rev107. The cell homogenates (Hom), cytosolic fractions (Cyto), and membrane fractions (Mem) (10 μg protein) from COS-7 cells expressing rat H-Rev107 were analyzed by Western blotting (A) or assayed for PLA_1/2 activity (B). The homogenates of COS-7 cells transfected with the insert-free vector (Mock) were also analyzed. Enzyme activity is shown as mean values ± SD (n = 3).

Fig. 4.

SDS-PAGE and enzyme assay with purified H-Rev107. A: Purified FLAG-tagged rat H-Rev107 (2.0 μg protein) was analyzed by SDS-PAGE on 14% gel, followed by Coomassie staining. The band of H-Rev107 is indicated by an arrowhead. The positions of molecular markers are also shown. B: Purified H-Rev107 (0.2 μg protein) was assayed for PE N-acylation activity and PLA_1/2 activity. Enzyme activity is shown as mean values ± SD (n = 3).

Fig. 5.

Properties of purified H-Rev107 as PLA_1/2. A: Purified rat H-Rev107 (0.2 μg protein) was assayed for PLA_1/2 activity at the indicated pH. The buffers used were HEPES-NaOH (closed circles), Tris-HCl (open circles), and glycine-NaOH (closed triangles). B: The assay was performed with different concentrations of 1,2-[¹⁴C]dipalmitoyl-PC as a substrate. C, D: The assay was performed in the presence of the indicated substances. In D, H-Rev107 was preincubated with the substances at 4°C for 3 min before the assay. Enzyme activity is shown as mean values ± SD (n = 3). BEL, bromoenol lactone; MAFP, methyl arachidonyl fluorophosphonate.

Fig. 6.

PLA₁ and PLA₂ activities of H-Rev107 toward various glycerophospholipids. The purified rat H-Rev107 (0.2 μg protein) was allowed to react with 200 μM of various PCs or PEs. A: A thin-layer chromatogram is shown. B: PLA₁-like activity (black bars) and PLA₂-like activity (gray bars) for each substrate are shown. The substrates used were 1,2-[¹⁴C]dipalmitoyl-PC (*P*P-PC), 1-[¹⁴C]palmitoyl-2-palmitoyl-PC (*PP-PC), 1-palmitoyl-2-[¹⁴C]palmitoyl-PC (P*P-PC), 1-palmitoyl-2-[¹⁴C]oleoyl-PC (P*O-PC), 1-palmitoyl-2-[¹⁴C]arachidonoyl-PC (P*A-PC), 1-palmitoyl-2-[¹⁴C]linoleoyl-PE (P*L-PE), and 1-palmitoyl-2-[¹⁴C]arachidonoyl-PE (P*A-PE). Enzyme activity is shown as mean values ± SD (n = 3).

Fig. 7.

Analysis of the mutants H21L and C111S of H-Rev107. COS-7 cells were transfected with the insert-free vector (Mock) or expression vectors harboring either the wild type of rat H-Rev107 (WT) or its single mutant H21L or C111S. The cell homogenates (10 μg protein) were analyzed by Western blotting (A) and assayed for PLA_1/2 activity (B) and PE N-acylation activity (C). Enzyme activity is shown as mean values ± SD (n = 3).

Fig. 8.

Analysis of deletion mutants of H-Rev107. COS-7 cells were transfected with the insert-free vector (Mock) or the expression vectors harboring either the wild type of rat H-Rev107 (WT) or its deletion mutants ΔN1, ΔN2, or ΔC1. The cell homogenates (10 μg protein) were analyzed by Western blotting (A) or assayed for PLA_1/2 activity (B). Enzyme activity is shown as mean values ± SD (n = 3).

Fig. 9.

Tissue distribution of rat H-Rev107 mRNA. Total RNA was isolated from the indicated rat organs and analyzed by RT-PCR using specific primers for H-Rev107 (upper panel) and GAPDH (lower panel).