Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle.
Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.
Tate
R J
RJ
Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, UK.
Tolan
D
D
Pyne
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D015870:Gene Expression; D006168:Guinea Pigs; D006801:Humans; D008274:Magnesium; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010711:Phosphatidate Phosphatase / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010711:Phosphatidate Phosphatase; D008274:Magnesium