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

doi:10.1074/jbc.272.25.15986

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

From the Laboratory of Cell Signaling, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-0320 and the ^‡Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651

Abstract

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 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)P₂). 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)P₂ with low affinity. But the PI(4,5)P₂ 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)P₂. A bacterially expressed fusion protein, glutathioneS-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.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These authors contributed equally to this work.
↵§ Recipient of a postdoctoral fellowship from the Korean Science and Engineering Foundation. On leave from the Research and Development Center, Miwon Co. Ltd., Seoul, Korea.
↵¶ Present address: Dept. of Fish Pathology, College of Fisheries Science, Pukyong National University, Pusan, Korea.
↵‖ Present address: Dept. of Biochemistry, College of Medicine, Hanyang University, Seoul, Korea.
↵** Recipient of postdoctoral support from the Korean Ministry of Science and Technology (Grant B-02-02-A). On leave from Chungbuk National University, College of Medicine, Chungju, Korea.
↵§§ To whom correspondence should be addressed: National Institutes of Health, Bldg. 3, Rm. 120, 3 Center Dr., MSC 0320, Bethesda, MD 20892-0320. Tel.: 301-496-9646; Fax: 301-480-0357.
↵1 The abbreviations used are: PLD, phospholipase D; ARF, ADP-ribosylation factor; PI(4,5)P₂, phosphatidylinositol 4,5-bisphosphate; IPPase, inositol polyphosphate phosphatase; AP3, clathrin assembly protein 3; PC, phosphatidylcholine; (pam)₂PC, dipalmitoyl PC; PE, phosphatidylethanolamine; IP₆, inositol hexakisphosphate; hPLD1, human PLD1; PLC, phospholipase C; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PEtOH, phosphatidylethanol; PA, phosphatidic acid; GTPγS, guanosine 5′-O-(3′-thiotriphosphate); Mes, 4-morpholineethanesulfonic acid.
- Received September 16, 1996.
- Revision received April 15, 1997.