title: Actin directly interacts with phospholipase D, inhibiting its activity.
abstract: Mammalian phospholipase D (PLD) plays a key role in several signal transduction pathways and is involved in many diverse functions. To elucidate the complex molecular regulation of PLD, we investigated PLD-binding proteins obtained from rat brain extract. Here we report that a 43-kDa protein in the rat brain, beta-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry. We also determined that the region between amino acids 613 and 723 of PLD2 is required for the direct binding of beta-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified beta-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC50 = 5 nm). In a previous paper, we reported that alpha-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 (ARF1)-reversible manner (Park, J. B., Kim, J. H., Kim, Y., Ha, S. H., Kim, J. H., Yoo, J.-S., Du, G., Frohman, M. A., Suh, P.-G., and Ryu, S. H. (2000) J. Biol. Chem. 275, 21295-21301). In vitro binding analyses showed that beta-actin could displace alpha-actinin binding to PLD2, demonstrating independent interaction between cytoskeletal proteins and PLD2. Furthermore, ARF1 could steer the PLD2 activity in a positive direction regardless of the inhibitory effect of beta-actin on PLD2. We also observed that beta-actin regulates PLD1 and PLD2 with similar binding and inhibitory potencies. Immunocytochemical and co-immunoprecipitation studies demonstrated the in vivo interaction between the two PLD isozymes and actin in cells. Taken together, these results suggest that the regulation of PLD by cytoskeletal proteins, beta-actin and alpha-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.
authors.affiliation: Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
authors.forename: S
authors.initials: S
authors.lastname: Lee
authors.forename: J B
authors.initials: JB
authors.lastname: Park
authors.forename: J H
authors.initials: JH
authors.lastname: Kim
authors.forename: Y
authors.initials: Y
authors.lastname: Kim
authors.forename: J H
authors.initials: JH
authors.lastname: Kim
authors.forename: K J
authors.initials: KJ
authors.lastname: Shin
authors.forename: J S
authors.initials: JS
authors.lastname: Lee
authors.forename: S H
authors.initials: SH
authors.lastname: Ha
authors.forename: P G
authors.initials: PG
authors.lastname: Suh
authors.forename: S H
authors.initials: SH
authors.lastname: Ryu
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D007313:Insecta; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
chemical_list: D000199:Actins; D018076:DNA, Complementary; D019269:Phosphatidylinositol 4,5-Diphosphate; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010739:Phospholipase D