Abstract
In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid, human promyelocytic leukemic HL60 cells differentiate into granulocyte-like cells. In cell lysate and in vitro reconstitution system, phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was up-regulated by dbcAMP or all-trans retinoic acid treatment. In the present study, the mechanism(s) for increased PLD activity during differentiation was examined. Western blot analysis revealed that the contents of ADP-ribosylation factor, Rac2, and Cdc42Hs but not RhoA and Rac1 in the cytosolic fraction were elevated during differentiation. However, the cytosolic fraction from undifferentiated cells was almost equally potent as the cytosolic fraction from differentiated cells in the ability to stimulate membrane PLD activity. It was shown that the GTPgammaS-dependent PLD activity in membranes from differentiated cells was much higher than that in membranes from undifferentiated cells, suggesting that the increased PLD activity during differentiation was due to alterations in some membrane component(s). Clostridium botulinum ADP-ribosyltransferase C3 and C. difficile toxin B, which are known as inhibitors of RhoA and Rho family proteins, respectively, effectively suppressed PLD activity in membranes from differentiated cells. In fact, the amount of membrane-associated RhoA was increased during differentiation. Furthermore, the extent of GTPgammaS-dependent PLD activity partially purified from membranes from differentiated cells was greater than that from membranes from undifferentiated cells in the presence of recombinant ADP-ribosylation factor 1. The PLD (hPLD1) mRNA level was observed to be up-regulated during differentiation, as inferred by reverse transcription-polymerase chain reaction. Our results suggest the possibility that the increased Rho proteins in membranes and the changed level of PLD itself may be, at least in part, responsible for the increase in GTPgammaS-dependent PLD activity during granulocytic differentiation of HL60 cells.
Highlights
Increasing evidence has indicated that phospholipase D (PLD)1 plays an important role in signal transduction in many
To gain further insight into the mechanism underlying the enhancement of PLD activity during differentiation, we have studied guanosine 5-O-(3thiotriphosphate) (GTP␥S)-dependent PLD activation in the lysate and the reconstitution system prepared from undifferentiated and differentiated HL60 cells
GTP␥S-dependent membrane PLD activity is higher in differentiated cells than in undifferentiated cells, comparable amounts of ADP-ribosylation factor (Arf) and Rho family proteins were present in the cytosolic fractions from both undifferentiated and differentiated HL60 cells
Summary
Materials—RPMI 1640 medium, penicillin, streptomycin, and recombinant reverse transcriptase were obtained from Life Technologies, Inc. [3H]Oleic acid and [palmitoyl-3H]dipalmitoyl phosphatidylcholine (DPPC) were from DuPont NEN. dbcAMP, all-trans retinoic acid (ATRA), nitroblue tetrazolium (NBT), NAD, UDP-glucose, brefeldin A, phosphatidylinositol 4,5-bisphosphate, egg PC, and phosphatidylethanolamine were from Sigma. [3H]Oleic acid-labeled HL60 cell lysates (200 g of protein/assay) or membranes (20 g of protein/assay) were incubated in buffer A containing 1 mM MgATP and CaCl2 to give a final free Ca2ϩ concentration of 1 M (total, 0.l ml) and stimulated with 10 M GTP␥S at 37 °C for 30 min in the presence of butanol (0.3%, v/v). Mixed-lipid vesicles (phosphatidylethanolamine/phosphatidylinositol 4,5-bisphosphate/egg PC, 10:1.5:1 molar ratio) containing [palmitoyl-3H]DPPC (3 Ci/ml) were added to membranes and cytosolic and extracted PLD fractions in a reaction mixture containing 50 mM Na-HEPES (pH 7.5), 80 mM KCl, 1 mM dithiothreitol, 3 mM MgCl2, 3 mM EGTA, and 2 mM CaCl2 to give a final free Ca2ϩ concentration of 300 nM (total, 0.1 ml) and stimulated with 10 M GTP␥S at 37 °C for 30 min in the presence of butanol (0.3%, v/v). The intensity of the bands was quantified by a densitometer (Densitograph; ATTO, Japan)
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