RhoA and a Cytosolic 50-kDa Factor Reconstitute GTPγS-dependent Phospholipase D Activity in Human Neutrophil Subcellular Fractions

Abstract

Receptor activation of phospholipase D has been implicated in signal transduction in a variety of cells. Reconstitution of cell-free guanosine 5 -O-(3-thiotriphosphate)(GTP␥S)-dependent phospholipase D activity from human neutrophils requires protein factors in both the plasma membrane and the cytosol. We previously proposed that one of the factors is a Ras-family small molecular weight GTPase of the Rho subtype (Bowman, E. P., Uhlinger, D. J., and Lambeth, J. D. (1993) J. Biol. Chem. 268, 21509 -21512). Herein, we have used RhoGDI (GDP dissociation inhibitor), an inhibitory Rho-binding protein, to selectively extract Rhotype GTPases from the plasma membrane, and have used immunoprecipitation as well as chromatographic methods to remove cytosolic Rho. Depletion of RhoA from either the plasma membrane or the cytosol resulted in a partial loss in GTP␥S dependent activity, while removal of RhoA from both fractions resulted in a nearly complete loss in activity. Activity was nearly completely restored by adding purified recombinant RhoA, which showed an EC 50 of 52 nM, while Rac1 showed little activity. Cytosol fractionated using DEAEcellulose chromatography separated ADP-ribosylation factor and Rho from the major activating fraction. Gel exclusion chromatography of this fraction revealed an activating factor of 50 kDa apparent molecular mass. Using RhoA-depleted membranes, reconstitution of phospholipase D activity required both RhoA and the 50-kDa factor. Thus, RhoA along with a non-Rho, non-ADP-ribosylation factor 50-kDa cytosolic factor are both required to reconstitute GTP␥S-dependent phospholipase D activity by neutrophil plasma membranes.Phospholipase D (PLD) 1 is activated via receptor-coupled mechanisms and by phorbol esters in a variety of cells (1-3). The enzyme catalyzes the hydrolysis of phosphatidylcholine to generate free choline and phosphatidic acid. Phosphatidic acid can be further metabolized via phosphatidic acid phosphohydrolase to form diacylglycerol, and is the major source of signaling diacylglycerol in some cell types such as neutrophil (2, 4). Phosphatidic acid and diacylglycerol have been implicated as second messengers involved in regulation of cell growth, differentiation, inflammation, and in a variety of cell-specific responses such as the respiratory burst of granulocytes (4, 5).While the occurrence of receptor-activated PLD has been widely documented, its molecular mechanism of activation remains poorly understood. GTP␥S activates PLD in cell-free systems including liver plasma membranes (6) and in lysates from human neutrophils and HL-60 cells (7,8). Unlike the liver system, reconstitution of GTP␥S-and phorbol myristate acetate-stimulated PLD from neutrophils requires protein factors in both the cytosol and the plasma membrane (7, 8). The calcium-dependent (7) activation by either GTP␥S or phorbol myristate acetate has been reported to require the participation of a 50-kDa cytosolic factor (9) as well as unknown membrane components which include the PLD catalytic moiety (10). Howeve...

Reconstitution of GTP‐γ‐S‐dependent phospholipase D activity with ARF, RhoA, and a soluble 36‐kDa protein

“…The 36-kDa protein may differ from the 50-kDa protein in h ~matopoietic cell lines [13][14][15] 7 he 36-kDa protein containing fraction (1 mg protein) prepared as i~ the legend to Table 2 was applied to a Superose 12 column x~hich had been equilibrated with buffer A [18], and eluted with the s,tme buffer at a flow rate 0.5 ml/min using an FPLC system (Pharmacia). PtdEtOH produced (dpm x 10 -3) Supernatant and particulate fractions were prepared from various rat tissues [18].…”

“…More recently, an additional protein factor has been reported 1 aat is needed for guanosine 5'-O-(3-thiotriphosphate) 43TP-~t-S)-dependent PLD activity. PLD obtained from lematopoietic cell lines is shown to be activated by a 50-~Da soluble protein and either ARF [13,14] or RhoA [15]. ];rain PLD is also activated by a cytosolic factor in a synerr istic manner with ARF [16], and this factor has recently been ~Corresponding author.…”

ADP-ribosylation Factor and Rho Proteins Mediate fMLP-dependent Activation of Phospholipase D in Human Neutrophils

“…A 50-kDa factor has been shown to act synergistically with both ARF (11,12) and Rho in stimulating PLD (13). Similarly, both G proteins also act synergistically with protein kinase C␣ to activate PLD.…”

The C Terminus of Mammalian Phospholipase D Is Required for Catalytic Activity

“…Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) was the first activator and a key element in establishing assays for the enzymes (6,(11)(12)(13). This was followed by the elucidation of activation by members of both the Arf (6,14) and Rho (15)(16)(17)(18)(19) families of small monomeric GTPases and by classical isoforms of protein kinase C (PKC) via a phosphorylation independent pathway (20,21). Subsequent studies with recombinant proteins in vitro have shown that PLD1 responds to all of these activators (22), while PLD2 is only stimulated by PIP 2 and Arf (9,23,24).…”