Cloning and Characterization of Phospholipase D from Rat Brain

Abstract

The regulation of phospholipase D cloned from rat brain (rPLD) was examined in vivo and in vitro. The enzyme was a shorter splice variant of human phospholipase D 1 (Hammond, S. M., Altshuller, Y. M. , Sung, T.-C., Rudge, S. M., Rose, K., Engebrecht, J. A., Morris, A. J., and Frohman, M. A. (1995) J. Biol. Chem. 270, 29640-29643). Its expression in COS-7 cells led to increased phospholipase D (PLD) activity that was further stimulated by constitutively active V14RhoA. V14RhoA had no effect on the endogenous PLD of the COS-7 cells, but constitutively active L71ARF3 increased its activity. In contrast, L71ARF3 did not activate rPLD expressed in the cells. Addition of phorbol ester markedly increased the endogenous PLD activity of COS-7 cells, and there was a further increase in the cells expressing rPLD. In membranes from COS-7 cells expressing rPLD, addition of myristoylated ADP-ribosylation factor (ARF) and RhoA in vitro stimulated PLD activity. The effect of ARF was greater than that of RhoA, although the concentrations for half-maximal stimulation (0.08-0.2 microM) were similar. Membranes isolated from cells expressing rPLD plus L71ARF3 and/or V14RhoA also showed higher PLD activity but no synergism between the two G proteins. Addition of phorbol ester and protein kinase C alpha (PKCalpha) also stimulated PLD activity in membranes from COS-7 cells expressing rPLD, but it had no effect on the activity in control (vector) membranes and did not enhance the effects of constitutively active ARF or Rho. The stimulation by PKCalpha did not require ATP and was not increased by addition of this nucleotide. No synergism between ARF and Rho and between these and PKCalpha on PLD activity was observed when these were added to membranes from cells expressing rPLD. Oleate inhibited the PLD activity of membranes from both control and rPLD-expressing cells. In summary, these results indicate that in vitro, rPLD is stimulated by ARF, RhoA, and PKCalpha and inhibited by oleate. However, in intact COS-7 cells, ARF activates endogenous PLD but not rPLD, whereas the reverse is true for RhoA. In addition, the effects of phorbol ester are much greater in the intact cells. It is concluded that the regulation of rPLD in intact COS-7 cells differs significantly from that seen in vitro; possible reasons for this are discussed.