Actin directly interacts with phospholipase D, inhibiting its activity
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- Actin directly interacts with phospholipase D, inhibiting its activity
- Lee, Sukmook; Park, Jong Bae; Kim, Jong Hyun; Kim, Yong; Kim, Jung Hwan; Shin, Kum-Joo; Lee, Jun Sung; Ha, Sang Hoon; Suh, Pann-Ghill; Ryu, Sung Ho
- Issue Date
- AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
- JOURNAL OF BIOLOGICAL CHEMISTRY, v.276, no.30, pp.28252 - 28260
- 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 phosphaticlylinositol-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., Sub, 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 a-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions
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