Low Molecular Weight GTP-binding Proteins in HL-60 Granulocytes

Abstract

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors in both the plasma membrane and the cytosol. HL-60 cytosol was fractionated by ammonium sulfate and gel-permeation chromatography. Two cytosolic protein fractions were found to reconstitute the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-stimulated PLD in a reconstitution assay consisting of 3H-labeled HL-60 membranes and eluted column fractions. The major peak of reconstituting activity was in the region of 50 kDa, and a second discrete peak of PLD reconstitution activity was observed in the region of 18 kDa. Rho GDP/GTP exchange inhibitor, Rho GDI, comigrated with Rac2 and RhoA, but not Rac1. RhoA and Rac2 were entirely complexed with Rho GDI and eluted with an apparent molecular mass of 43 kDa by gel filtration chromatography. The partial overlap between cytosolic Rac2 and RhoA with the 50-kDa peak of reconstituting activity was not consistent with the participation of cytosolic Rho-related GTPases in the activation of PLD by guanine nucleotides. However, recombinant Rho GDI, which inhibits nucleotide exchange on the Rho family of small GTP-binding proteins, reduced GTP gamma S-stimulated PLD activity in HL-60 homogenates. The stimulatory exchange factor, Smg GDS, which is active on Rho and Rac, could be partially separated from the PLD-stimulating factor(s) by gel-permeation chromatography. Moreover, recombinant Smg GDS failed to stimulate GTP-dependent PLD activity. Cytosolic ADP-ribosylation factor (ARF) was exclusively located in the 18-kDa peak of reconstitution activity. Faint amounts of membrane-bound ARF were also detected using the monoclonal antibody 1D9. The effects of the 50-kDa and 18-kDa PLD-inducing factors on the salt-extracted PLD activity were synergistic. The weak stimulatory effect of ARF alone suggested that the GTP gamma S-stimulated PLD activity is dependent on the presence of another protein(s), presumably ARF-regulatory proteins. We propose that a membrane-bound GTP-binding protein, possibly ARF, may be involved in the activation of PLD when combined with the component(s) of the 50-kDa fraction.

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.…”

Ras protein is involved in the physiological regulation of phospholipase D by platelet derived growth factor

“…In fact, several molecules has been implicated in PLD activation, including members of the family of small GTP-binding proteins such as Ras (Carnero et al, 1994a,b;Jiang et al, 1995a,b;del Peso et al, 1996;1997), ARF (Bourgoin et al, 1995;Brown et al, 1993;Hammond et al, 1995), Rho (Bowman et al, 1993;Malcolm et al, 1994) and Ral (Jiang et al, 1995b). The implication of various distinct small GTP-binding proteins in PLD activation most probably re¯ects the existence of several dierent PLD isoenzymes.…”

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.…”