Identification and cDNA cloning of 35-kDa phosphatidic acid phosphatase (type 2) bound to plasma membranes. Polymerase chain reaction amplification of mouse H2O2-inducible hic53 clone yielded the cDNA encoding phosphatidic acid phosphatase.
We previously described the purification of an 83-kDa phosphatidic acid phosphatase (PAP) from the porcine thymus membranes (Kanoh, H., Imai, S.-i., Yamada, K. and Sakane, F.(1992) J. Biol. Chem. 267, 25309-25314). However, we found that a minor 35-kDa protein could account for the PAP activity when the purified enzyme preparation was further analyzed. We thus determined the N-terminal sequence of the 35-kDa candidate protein and prepared antipeptide antibody against the determined sequence, MFDKTRLPYVALDVL. The antibody almost completely precipitated the purified enzyme activity. Furthermore, the antibody precipitated from the radioiodinated enzyme preparation a single 35-kDa protein, which was converted to a 29-kDa form when treated with N-glycanase. We also found that the immunoprecipitable PAP activity was exclusively associated with the plasma membranes of porcine thymocytes. These results indicated that the 35-kDa glycosylated protein represents the plasma membrane-bound (type 2) PAP. We surprisingly noted that the N-terminal sequence of the porcine PAP was almost completely conserved in the internal sequence encoded by a mouse partial cDNA clone, hic53, reported as a H2O2-inducible gene (Egawa, K., Yoshiwara, M., Shibanuma, M., and Nose, K.(1995) FEBS Lett. 372, 74-77). We thus amplified from the mouse kidney RNA the hic53 clone by polymerase chain reaction, and obtained a cDNA encoding a novel protein of 283 amino acid residues with a calculated Mr of 31,894. Methionine reported as an internal residue was found to serve as an initiator, and the C-terminal 64 residues were lacking in hic53. The protein contains several putative membrane-spanning domains and two N-glycosylation sites. When transfected into 293 cells, the cDNA gave more than 10-fold increase of the membrane-bound PAP activity, which could be precipitated by the antipeptide antibody. In [35S]methionine-labeled cells, the translational product was confirmed to be a 35-kDa protein, which became 30 kDa in cells treated with tunicamycin, an inhibitor of N-glycosylation. We thus succeeded first in identifying the porcine type 2 PAP and subsequently in determining the primary structure of a mouse homolog of the PAP.
Kai
M
M
Department of Biochemistry, Sapporo Medical University School of Medicine, West-17, South-1, Chuo-Ku, Sapporo 060 Japan.
Wada
I
I
Imai
S
S
Sakane
F
F
Kanoh
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D007120:Immunochemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010711:Phosphatidate Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013950:Thymus Gland / Q000201:enzymology
D018076:DNA, Complementary; C095904:Hic-53 protein, mouse; D008565:Membrane Proteins; D010711:Phosphatidate Phosphatase