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 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.