Emer Cunningham

An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling

Transmembrane signaling by the phospholipase C-beta (PLC-beta) pathway is known to require at least three components: the receptor, the G protein, and the PLC. Recent studies have indicated that if the cytosol is allowed to leak out of HL60 cells, then G protein-stimulated PLC activity is greatly diminished, indicating an essential role for a cytosolic component(s). We now report the complete purification of one component based on its ability to reconstitute GTP gamma S-mediated PLC activity and identify it as the phosphatidylinositol transfer protein (PI-TP). Based on the in vitro effects of PI-TP, we surmise that it is involved in transporting PI from intracellular compartments for conversion to PI bisphosphate (PIP2) prior to hydrolysis by PLC-beta 2/PLC-beta 3, the endogenous PLC isoforms present in these cells.

ARF and PITP restore GTPγS-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis

BACKGROUND In many cell types, including neutrophils and HL60 cells, there is an absolute requirement for a GTP-dependent step to elicit Ca(2+)-regulated secretion. Neutrophils and HL60 cells secrete lysosomal enzymes from azurophilic granules; this secretion is inhibited by 1% ethanol, indicating that phosphatidate (PA) produced by phospholipase D (PLD) activity may be involved. PLD can use primary alcohols in preference to water during the hydrolytic step, generating the corresponding phosphatidylalcohol instead of PA, its normal product. As ARF (ADP-ribosylation factor) proteins regulate PLD activity and are implicated in constitutive vesicular traffic, we have investigated whether ARF is also required for GTP-dependent secretion in HL60 cells. RESULTS We have used a cell-permeabilization protocol that allows HL60 cells to become refractory to stimulation with GTP gamma S plus 10 microM Ca2+ with regard to secretion and PLD activity. Permeabilization with streptolysin O for 10 minutes permitted the loss of freely diffusable cytosolic proteins, including ARF proteins. Fractions derived from brain cytosol, enriched in ARF proteins, restored secretory function and PLD activity. The major contaminating protein present in these ARF-enriched fractions was identified as phosphatidylinositol transfer protein (PITP). Unexpectedly, PITP was also found to restore GTP gamma S-dependent secretion. Restoration of secretory function was characterized using recombinant proteins, rARF1 and rPITP alpha and rPITP beta. The rARF1 protein restored both secretory function and PLD activity, whereas PITP only restored secretory function. However, both ARF and PITP were capable of stimulating phosphatidylinositol bis phosphate (PIP2) synthesis. CONCLUSIONS ARF and PITP restore secretory function in cytosol-depleted cells when stimulated with GTP gamma S plus Ca2+. We have previously shown that PITP participates in the synthesis of PIP2. In comparison, ARF1 activates PLD, producing PA, which is a known activator of phosphatidylinositol-4-phosphate 5 kinase, the enzyme responsible for PIP2 synthesis. We propose that ARF and PITP both restore exocytosis by a common mechanism-promoting PIP2 synthesis.

ARF1(2–17) does not specifically interact with ARF1-dependent pathways: Inhibition by peptide of phospholipases Cβ, D and exocytosis in HL60 cells

The small GTP‐binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF proteins in regulated exocytosis, we have used the N‐terminal peptide ARF1(2–17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol‐depleted HL60 cells was inhibited by ARF1(2–17). In the presence of endogenous cytosol, ARF1(2–17) also inhibited GTP‐γ‐S‐stimulated PLD activity and exocytosis. Mastoparan Politses jadwagae and mastoparan Vespula lewisii which exhibit similar structural properties to ARF1(2–17) also inhibited GTP‐γ‐S‐stimulated PLD and exocytosis. GTP‐γ‐S‐stimulated phospholipase C‐β (PLC‐β) was also inhibited by ARF(2–17) and mastoparan. In cytosol‐depleted HL60 cells, the ARF(2–17) inhibited the reconstitution of GTP‐γ‐S‐stimulated PLC‐β activity with exogenously‐added PLC‐β1 and phosphatidylinositol transfer protein. We conclude that the widely‐used ARF1(2–17) peptide inhibits both ARF‐independent (i.e. PLC‐β) and ARF‐dependent pathways (i.e. PLD) and therefore cannot be regarded as a specific inhibitor of ARF function.