Cardiac phospholipase D2 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 Ca(2+) mobilization and contractile performance in the heart. However, the molecular identity of this myocardial PLD and the mechanisms that regulate it are not well understood. Using subcellular fractionation and Western blot analysis, we found that PLD2 is the major myocardial PLD and that it localizes primarily to sarcolemmal membranes. A 100-kDa PLD2-interacting cardiac protein was detected using a protein overlay assay employing purified PLD2 and then identified as alpha-actinin using peptide-mass fingerprinting with matrix-assisted laser desorption/ionization mass spectroscopy. The direct association between PLD2 and alpha-actinin was confirmed using an in vitro binding assay and localized to PLD2's N-terminal 185 amino acids. Purified alpha-actinin potently inhibits PLD2 activity (IC(50) = 80 nm) in an interaction-dependent and ADP-ribosylation factor-reversible manner. Finally, alpha-actinin co-localizes with actin and with PLD2 in the detergent-insoluble fraction from sarcolemmal membranes. These results suggest that PLD2 is reciprocally regulated in sarcolemmal membranes by alpha-actinin and ARF1 and accordingly that a major role for PLD2 in cardiac function may involve reorganization of the actin cytoskeleton.
Park
J B
JB
Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
Kim
J H
JH
Kim
Y
Y
Ha
S H
SH
Yoo
J S
JS
Du
G
G
Frohman
M A
MA
Suh
P G
PG
Ryu
S H
SH
D000185:Actinin / Q000737:chemistry / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D006352:Heart Ventricles; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology* / Q000648:ultrastructure; D010739:Phospholipase D / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D012508:Sarcolemma / Q000201:enzymology* / Q000648:ultrastructure; D018411:Spodoptera; D014162:Transfection
D011994:Recombinant Proteins; D000185:Actinin; D000246:Adenosine Diphosphate Ribose; C105606:phospholipase D2; D010739:Phospholipase D