Actin Directly Interacts with Phospholipase D, Inhibiting Its Activity

Sukmook Lee, Jong Bae Park, Jong Hyun Kim, Yong Kim, Jung Hwan Kim, Kum Joo Shin, Jun Sung Lee, Sang Hoon Ha, Pann Ghill Suh, Sung Ho Ryu

Research output: Contribution to journalArticlepeer-review

103 Scopus citations

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, β-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 β-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified β-actin potently inhibited both phosphatidylinositol-4,5-bisphosphate- and oleate-dependent PLD2 activities in a concentration-dependent manner (IC 50 = 5 nM). In a previous paper, we reported that α-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 β-actin could displace α-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 β-actin on PLD2. We also observed that β-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, β-actin and α-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions.

Original languageEnglish (US)
Pages (from-to)28252-28260
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number30
DOIs
StatePublished - Jul 27 2001
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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