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, ␤-actin, acts as a major PLD2 direct-binding protein as revealed by peptide mass fingerprinting in combination with matrixassisted 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 ␤-actin, using bacterially expressed glutathione S-transferase fusion proteins of PLD2 fragments. Intriguingly, purified ␤-actin potently inhibited both phosphatidylinositol-4,5-bisphosphateand oleate-dependent PLD2 activities in a concentrationdependent manner (IC 50 ‫؍‬ 5 nM). In a previous paper, we reported that ␣-actinin inhibited PLD2 activity in an interaction-dependent and an ADP-ribosylation factor 1 In vitro binding analyses showed that ␤-actin could displace ␣-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 ␤-actin on PLD2. We also observed that ␤-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, ␤-actin and ␣-actinin, and ARF1 may play an important role in cytoskeleton-related PLD functions. Mammalian phospholipase D (PLD)1 hydrolyzes phosphatidylcholine (PC) to generate phosphatidic acid and choline in response to a variety of signals, which can include hormones, neurotransmitters, and growth factors (1). phosphatidic acid itself has been shown to be an intracellular lipid second messenger and to be involved in multiple physiological events such as the promotion of mitogenesis, stimulation of respiratory bursts, secretory processes, actin cytoskeletal reorganization, and the activation of Raf-1 kinase and phosphatidylinositol 4-phosphate (PtdIns4P) 5-kinase isoforms in a large number of cells. These relationships suggest that agonist-induced PLD activation may play roles in multiple signaling events (2-7). The mammalian PLD isozymes identified thus far, PLD1 and PLD2, share a sequence homology of ϳ50%, but they have very different regulatory properties. PLD1 has low basal activity in the presence of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and can be activated by several cytosolic factors including protein kinase C ␣ and small GTP-binding proteins such as Rho A, Rac-1, ARF1, RalA, and CDC42 (8 -15). PLD2 also depends on PIP 2 but has a higher basal activity than PLD1 (16), and it has been proposed that PLD2 may be closely associated with different cellular inhibitors. Alth...

Lysophosphatidylcholine suppresses apoptosis and induces neurite outgrowth in PC12 cells through activation of phospholipase D2

“…Actin cytoskeletal reorganization plays a key role in neurite outgrowth and guidance of primary cultured neurons (Daniels et al, 1998;Dickson, 2002), and PA directly interacts with several actin cytoskeleton-regulatory proteins, including PI4P-5-kinase, protein phosphatase 1 , and -COP. Moreover, PLD2 localizes in actin cytoskeleton through direct interaction with actin in PC12 cells (Lee et al, 2001). In the present study, we demonstrated that overexpression of PLD2 increased LPCinduced neurite outgrowth, and that inhibition of LPC-induced generation of PA by 1-butanol abrogated the LPC-induced neurite outgrowth.…”

Phospholipases D1 and D2 Coordinately Regulate Macrophage Phagocytosis

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Phosphatidic Acid Is a Leukocyte Chemoattractant That Acts through S6 Kinase Signaling

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