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

doi:10.1074/jbc.270.7.3172

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

ASSESSMENT OF THE ROLE OF ARF AND OF A 50-kDa CYTOSOLIC PROTEIN IN PHOSPHOLIPASE D ACTIVATION (*)

From the (1)Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Sainte-Foy, Québec G1V 4G2, Canada and the
(2)Department of Biochemistry, Kobe University School of Medicine, Kobe 650, Japan

^§ To whom correspondence should be addressed:
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, 2705 Blvd. Laurier, Sainte-Foy, Québec G1V 4G2, Canada.
Tel.: 418-654-2772; Fax: 418-656-2765.

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 GTPS (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 GTPS-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 GTPS-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.

Footnotes

↵* This work was supported in part by grants and fellowships from the National Cancer Institute of Canada and the Fonds de la Recherche en Santé du Québec (to S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PLD

phospholipase D

PEt

phosphatidylethanol

G protein

GTP-binding protein

GTPS

guanosine 5′-3-O-(thio)triphosphate

GDI

GDP/GTP dissociation inhibitor

GDS

GDP/GTP dissociation stimulator

ARF

ADP-ribosylation factor

Pipes

piperazine-N,N′-bis(2-ethanesulfonic acid)

HSA

human serum albumin

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride.
↵² P. A. Randazzo and R. A. Kahn, personal communication.
- Received July 21, 1994.
- Revision received December 19, 1994.