Regulation of phospholipase C-beta(1) activity by phosphatidic acid.
The role of phosphatidic acid (PA) in regulating phospholipase C-beta(1) (PLC-beta(1)) activity was determined. PA promoted the binding of PLC-beta(1) to sucrose-loaded unilamellar vesicles (SLUV) containing phosphatidylcholine. PA increased enzymatic activity over a range of Ca(2+) concentrations and reduced the Ca(2+) concentration required for half-maximal stimulation of activity. PA did not affect the apparent K(m) for phosphatidylinositol 4, 5-bisphosphate. Lysophosphatidic acid also enhanced the binding of PLC-beta(1) to SLUV but was less effective in stimulating enzymatic activity. Diacylglycerol, phosphatidylserine, and oleic acid had little effect on activity. Anionic and neutral detergents did not stimulate activity. PA stimulation was relatively independent of acyl chain length. Dipalmitoyl-PA (16:0) was comparable to PA from egg lecithin and dimyristoyl-PA (C14:0) in stimulating activity, while dilauroyl-PA (C12:0) was slightly less effective. A 100 kDa catalytic fragment of PLC-beta(1) lacking amino acid residues C-terminal to His(880) did not bind to PA and was insensitive to stimulation by 7-15 mol % PA. Stimulation of 100 kDa enzymatic activity required 30 mol % PA. PA increased receptor-G protein stimulation of PLC-beta(1) activity in membranes. These results demonstrate that PA stimulates basal and receptor-G protein-regulated PLC-beta(1) activity. PA stimulation occurs through both a C-terminal-dependent and an independent mechanism. The C-terminal-mediated mechanism for stimulation may constitute an important pathway for conferring specific regulation of PLC-beta(1) in response to increases in cellular PA levels.
Litosch
I
I
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101, USA. ilitosch@med.miami.edu
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002417:Cattle; D018699:Coated Vesicles / Q000378:metabolism; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000378:metabolism*; D054799:Phospholipase C beta; D051381:Rats; D010738:Type C Phospholipases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D010712:Phosphatidic Acids; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins