INCREASED ACTIVITY OF SMALL GTP-BINDING PROTEIN-DEPENDENT PHOSPHOLIPASE-D DURING DIFFERENTIATION IN HUMAN PROMYELOCYTIC LEUKEMIC HL60 CELLS

In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic ac id, human promyelocytic leukemic HL60 cells differentiate into granulo cyte-like cells. In cell lysate and in vitro reconstitution system, ph ospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotrip hosphate) (GTP gamma S) was up regulated by db-cAMP or all-trans retin oic acid treatment. In the present study, the mechanism(s) for increas ed PLD activity during differentiation was examined. Western blot anal ysis revealed that the contents of ADP-ribosylation factor, Rac2, and Cdc42Hs but not RhoA and Rad in the cytosolic fraction were elevated d uring differentiation. However, the cytosolic fraction from undifferen tiated 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 GTP gamma S-dependent PLD activity in membrane s from differentiated cells was much higher than that in membranes fro m undifferentiated cells, suggesting that the increased PLD activity d uring differentiation was due to alterations in some membrane componen t(s). Clostridium botulinum ADP-ribosyltransferase C3 and C. difficile toxin B, which are known as inhibitors of RhoA and Rho family protein s, 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 GTP gamma S-dependent PLD activity partially purified from membranes from differentiated cells was greater than that from membranes from undiffe rentiated 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 l east in part, responsible for the increase in GTP gamma S-dependent PL D activity during granulocytic differentiation of HL60 cells.