Phosphatidylcholine metabolism and intracellular signal transduction

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Phosphatidylcholine plays an important role in the signal transduction of cells. Its metabolism is induced upon the occupancy of membrane receptors by agonists, resulting in the generation of various lipid second messengers. In this project, we studied two major enzymes in phosphatidylcholine metabolism, phospholipase D and lysophospholipase.Phospholipase D contains two groups, fatty acid-dependent and G-protein-dependent types. We had previously succeeded in highly purifying the fatty acid-dependent enzyme from pig lung microsomes. Here, we obtained three different types of rat phospholipase D clones using PCR probes synthesized based on the consensus among yeast, castor bean, and human enzymes, and expressed them in the fission yeast Schizosaccharomyces pombe. One enzyme was identical to human PLD1 and thus designated rPLD1. The secondr enzyme was an isoform of rPLD1 generated by alternative splicing. Both enzymes were stimulated by the small G proteins, Art and RhoA,and phosphatidylinositol 4,5-bisphosphate. The third enzyme was a new isoform encoded by a different gene and desinated rPLD2.rPLD2 was stimulated by phosphatidylinositol 4,5-bisphosphate, but was not affected by the small G proteins.Lysophospholipase hydrolyzes lysophosphatidylcholine which is known to modulate the activity of protein kinase C and thus is considered to be a down-regulator of the signal transduction pathway of cells. Here, we obtained four enzyme preparations from rat liver and pig gastric mucosa and then raised antibodies. Using the antibodies we showed that there are at least three isoforms of lysophospholipase with different antigenicity and tissue distribution : type I low molecular weight form (24kd), type II low molecular weight form (23kd), and type III high molecular weight form (60 kd) with transacylase activity. Type I enzyme was cloned and shown to be posttranscriptionally induced during differentiation of HL-60 promyelocytic cells into granulocytes.