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Is PRG-1 a new lipid phosphatase?

Nature Neuroscience volume 7, page 789 (2004)Cite this article

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Brauer et al.1 recently described an integral membrane protein, PRG-1, with partial sequence similarity to the lipid phosphate phosphatases (LPPs)—cell surface–localized 'ecto phosphatases' that dephosphorylate lysophosphatidic acid (LPA) and related substrates2. The title identifies PRG-1 as a “new lipid phosphatase,” and the article says it contains “highly conserved phosphatase sequences,” but these were not identified explicitly1. Alignment with the invariant tripartite consensus sequence (denoted C1, C2 and C3) that defines LPPs and related phosphatases reveals non-conservative substitution in PRG-1 of three residues that are critical in catalysis (Supplementary Fig. 1 online) and directly involved in substrate hydrolysis2,3,4,5,6. The lysine and arginine residues from the C1 motif are critical in substrate/transition state orientation in the active site, and the imidazole group of the missing C3 histamine residue functions as a nucleophile in the first step of the hydrolysis reaction2,3,4,5,6. Thus PRG-1 could not catalyze a phosphatase reaction using the reaction chemistry shared by these enzymes.

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, 27599-7090, North Carolina, USA

    Mark I McDermott, Yury J Sigal & Andrew J Morris

  2. Howard Hughes Medical Institute, University of California, San Diego, La Jolla, 92093-0668, California, USA

    Vicki A Sciorra

Authors
  1. Mark I McDermott
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  2. Yury J Sigal
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  3. Vicki A Sciorra
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  4. Andrew J Morris
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Corresponding author

Correspondence to Andrew J Morris.

Supplementary information

Supplementary Fig. 1

Alignment of catalytic homology domain of PRG1 with the phosphatase consensus sequence and catalytic domain sequences from representative members of the phosphatase family. CPO: C. inequalis vanadium chloroperoxidase; PgpB: E. coli phosphatidylglycerolphosphatase; DPP1: S. cerevisiae diacylglycerol pyrophosphate phosphatase; hSPP1: H. sapiens sphingosine 1-phosphate phosphatase; hLPP1: H. sapiens lipid phosphate phosphatase 1. hLPR1: H. sapiens lipid phosphatase related protein 1 (Genbank accession # AAH22465). Residues involved in substrate binding/ transition state stabilization are in blue, charge relay residues in red. Non conserved catalytic domain residues in LPR1 and PRG1 are underlined.

Supplementary Fig. 2

Western blot analysis of GFP-tagged LPP3, LPR1 and PRG1 expressed in HEK293 cells. HEK293 cells were transfected with vectors for expression of the indicated proteins using lipofectamine and cultured for 48 hours. Transfection efficiencies were estimated to be 50-70% based on examination of GFP fluorescence in live cells. Cells were harvested and total membrane fractions prepared as described in the methods section. Equal amounts of protein (∼50 μg) were separated on a 10% SDS PAGE gel and analyzed by western blotting using an anti GFP monoclonal antibody. Immunoreactive species corresponding to the expected molecular weights of GFPLPP3, LPR1 and PRG-1 are arrowed. Similar expression patterns were observed when these proteins were expressed in N1E 115 cells.

Supplementary Table 1

LPA phosphatase activity of LPP3 and PRF1 and LPR1 expressed in HEK293 and N1E 115 cells. HEK 293 and N1E 115 cells were transfected with plasmid vectors for expression of GFP, GFP-LPP3, GFP-PRG1 and GFP-LPR1. LPA phosphatase activities were determined using the procedures described in the methods section. Substrates were presented to either intact cells or crude membrane preparations as sonicated dispersions prepared in the presence or absence of 0.1 mg/ml fatty acid free BSA or in mixed micelles with Triton X-100. For assays using uncomplexed or BSA complexed LPA the substrate concentration was 10 μM. For assays using Triton X-100 solublilized substrates the LPA concentration was 100 μM 8 and the Triton X-100 concentration was 3.2 mM. Assays contained equivalent numbers of intact cells (∼2.5 x 106 cells) or membrane protein (∼25 μg). The data shown are means ± SD of triplicate determinations. Phosphatase activities were determined by quantitation of the reaction products, either PO42− or in some cases MAG as indicated. In assays conducted using intact cells reaction products were quantitated in the entire incubation (i.e. incubation medium plus cells). The data shown are means ± SD of triplicate determinations.

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McDermott, M., Sigal, Y., Sciorra, V. et al. Is PRG-1 a new lipid phosphatase?. Nat Neurosci 7, 789 (2004). https://doi.org/10.1038/nn0804-789a

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