Cardiac phospholipase D-2 localizes to sarcolemmal membranes and is inhibited by alpha-actinin in an ADP-ribosylation factor-reversible manner

Myocardial phospholipase D (PLD) has been implicated in the regulation of C a2+ mobilization and contractile performance in the heart. However, the mol ecular identity of this myocardial PLD and the mechanisms that regulate it are not well understood. Using subcellular fractionation and Western blot a nalysis, we found that PLD2 is the major myocardial PLD and that it localiz es primarily to sarcolemmal membranes. A 100-kDa PLD2-interacting cardiac p rotein was detected using a protein overlay assay employing purified PLD2 a nd then identified as alpha-actinin using peptide-mass fingerprinting with matrix-assisted laser desorption/ionization mass spectroscopy. The direct a ssociation between PLD2 and alpha-actinin was confirmed using an in vitro b inding assay and localized to PLD2's N-terminal 185 amino acids. Purified a lpha-actinin potently inhibits PLD2 activity (Ic(50) = 80 nM) in an interac tion-dependent and ADP-ribosylation factor-reversible manner. Finally, alph a-actinin co-localizes with actin and with PLD2 in the detergent-insoluble fraction from sarcolemmal membranes. These results suggest that PLD2 is rec iprocally regulated in sarcolemmal membranes by alpha-actinin and ARF1 and accordingly that a major role for PLD2 in cardiac function may involve reor ganization of the actin cytoskeleton.