Inhibition of Phospholipase D by Clathrin Assembly Protein 3 (AP3)

Abstract

15980 -15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P 2 with low affinity. But the PI(4,5)P 2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P 2 . A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.We recently showed that rat brain cytosol contains proteins that inhibit the activity of partially purified brain membrane phospholipase D (PLD) 1 stimulated by ADP-ribosylation factor (ARF) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) (1). Sequential chromatography of the brain cytosolic preparation yielded four inhibitory fractions, which exhibited differential sensitivity to heat. The most heat-labile inhibitor (inhibitor IA) was purified to yield a 150-kDa protein, which, in the accompanying paper (2), we showed to be synaptojanin, a nerve terminal-enriched protein with inositol polyphosphate phosphatase (IPPase) activity, and which inhibits PLD by hydrolyzing PI(4,5)P 2 .We now describe the purification of PLD inhibitor IB, which has an apparent molecular mass of 165 kDa and is less susceptible to heat treatment than synaptojanin. Sequences of tryptic peptides of inhibitor IB suggest that it is identical to clathrin assembly protein 3 (AP3). We further show that AP3 binds to PLD. EXPERIMENTAL PROCEDURESMaterials-The sources of rat brains, bovine brain phosphatidylcholine (PC), phosphatidylethanolamine (PE), GTP␥S, PI(4,5)P 2 , [cholinemethyl-3 H]dipalmitoylphosphatidylcholine ((pam) 2 PC) (50 Ci/mmol), [2-palmitoyl-9,10-3 H](pam) 2 PC (89 Ci/mmol), and n-octyl-␤-D-glucopyranoside were as described previously (2). Phytic acid (inositol hexakisphosphate, or IP 6 ) and monoclonal antibodies to AP3 (clone AP180-I) were obtained from Sigma. PLD, AR...

Phosphatidylinositol 4,5-bisphosphate stimulates vesicle formation from liposomes by brain cytosol

“…PLD is inhibited by endocytic proteins such as synaptojanin, AP180, and amphiphysin. Although AP180 and amphiphysin directly interact with PLD (50,51), the inhibition of PLD by synaptojanin is attributed to its ability to dephosphorylate PtdIns(4,5)P 2 (20,46), which is a cofactor for PLD (51).…”

Inhibition of Phospholipase D by Amphiphysins

“…We have previously shown that synaptojanin and AP180 each inhibit PLD activity (21,22). We have now demonstrated that AmphI and AmphII each inhibit the activities of both PLD1 and PLD2 as a result of direct interaction with these enzymes.…”

“…Two such inhibitory proteins were purified to homogeneity and identified as synaptojanin and clathrin assembly protein 180 (AP180, previously named as clathrin assembly protein 3 or AP3) (21,22). The inhibition of PLD by synaptojanin was attributed to its ability to dephosphorylate PI(4,5)P 2 (21,23), whereas the inhibitory effect of AP180 is a result of its direct interaction with PLD (22).…”

“…Two such inhibitory proteins were purified to homogeneity and identified as synaptojanin and clathrin assembly protein 180 (AP180, previously named as clathrin assembly protein 3 or AP3) (21,22). The inhibition of PLD by synaptojanin was attributed to its ability to dephosphorylate PI(4,5)P 2 (21,23), whereas the inhibitory effect of AP180 is a result of its direct interaction with PLD (22). We have now purified a third type of PLD inhibitor from rat brain and have shown that it comprises amphiphysin I (AmphI) and AmphII, both of which are nerve-terminal proteins that are important for clathrin-mediated endocytosis (24,25).…”

Inhibition of Phospholipase Dα byN-Acylethanolamines

“…Several inhibitors of mammalian PLD have been identified. These inhibitors include fodrin (Lukowski et al, 1996), synaptojanin (Kim et al, 1996), and clathrin assembly protein (Lee et al, 1997), as well as several lipids including ceramide (Venable et al, 1996), alkylphosphate esters (Dittrich et al, 1996), and lysophosphatidyl-Ser (Kawabe et al, 1998). An intriguing observation indicated that oleate-dependent PLD from rat brain was inhibited by several acidic phospholipids, of which polyphosphatidylinositolbisphosphate (PIP 2 ) was the most effective inhibitor (Kanfer et al, 1996).…”