Ha, V.L.; (2004) The coordinated regulation of phospholipase D by ADP-ribosylation factors and their exchange factors. Doctoral thesis , University of London.

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Abstract

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme, the activation of which has effects on cellular events including cell growth and membrane trafficking in mammalian cells. In a reconstitution assay consisting of permeabilised HL-60 cells (human myeloid leukemic cells), experiments confirmed that members of the ADP- ribosylation factor (ARF) family proteins including ARFl and ARF6 were efficient PLD activators. However, ARFl was a stronger activator of PLD than ARF6, a result that was also found in in vitro PLD assays. Moreover, the myristoylated amino terminal ?-helix of ARF was essential for the activation of PLD. The activation of ARF is in turn regulated by a specific family of small guanine nucleotide exchange factors (GEFs) comprising ARNO, GRP-1 and cytohesin-1. The GEFs catalyze the release of bound GDP and its replacement by GTP, leading to ARF activation. Using the reconstitution assay, the PLD activation mediated by ARF was enhanced by the GEFs. However, the GEFs did not improve the stimulation of PLD by ARF in vitro. Interestingly, these GEFs activated PLD with high potency but none of the three GEFs was more potent than the others in their regulation of PLD in cell- based assays. It seems that this pattern of PLD activation does not reflect differential interactions with major phosphoinositides (PIP2 and PIP3) since these molecules bind equally well to the GEFs. Importantly, PIP2 and PIP3 increased the potency of PLD activation mediated by the coordinated actions of ARF and its exchange factors indicating the involvement of other pathways in the regulation of PLD in vivo. In particular, it emerged that PIP3 is a more potent activator of PLD than PIP2 in the presence and absence of ARF-GEFs.

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