title: Munc-18-1 inhibits phospholipase D activity by direct interaction in an epidermal growth factor-reversible manner.
abstract: Mammalian phospholipase D (PLD) has been reported to be a key enzyme for epidermal growth factor (EGF)-induced cellular signaling, however, the regulatory mechanism of PLD is still unclear. In this report, we found that Munc-18-1 is a potent negative regulator of PLD in the basal state and that its inhibition is abolished by EGF stimulation. We investigated PLD-binding proteins obtained from rat brain extract, and identified a 67-kDa protein as Munc-18-1 by peptide-mass finger-printing. The direct association between PLD and Munc-18-1 was confirmed by in vitro binding analysis using the purified proteins, and their binding sites were identified as the phox homology domain of PLD and multiple sites of Munc-18-1. PLD activity was potently inhibited by Munc-18-1 in vitro (IC50 = 2-5 nm), and the cotransfection of COS-7 cells with Munc-18-1 and PLD inhibited basal PLD activity in vivo. In the basal state, Munc-18-1 coprecipitated with PLD and colocalized with PLD2 at the plasma membrane of COS-7 cells. EGF treatment triggered the dissociation of Munc-18-1 from PLD when PLD was activated by EGF. The dissociation of the endogenous interaction between Munc-18-1 and PLD, and the activation of PLD by EGF were also observed in primary cultured chromaffin cells. These results suggest that Munc-18-1 is a potent negative regulator of basal PLD activity and that EGF stimulation abolishes this interaction.
authors.affiliation: Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea.
authors.forename: Hye Young
authors.initials: HY
authors.lastname: Lee
authors.forename: Jong Bae
authors.initials: JB
authors.lastname: Park
authors.forename: Il Ho
authors.initials: IH
authors.lastname: Jang
authors.forename: Young Chan
authors.initials: YC
authors.lastname: Chae
authors.forename: Jong Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Il Shin
authors.initials: IS
authors.lastname: Kim
authors.forename: Pann-Ghill
authors.initials: PG
authors.lastname: Suh
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Ryu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015536:Down-Regulation; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017384:Sequence Deletion; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C497937:STXBP1 protein, human; C497939:Stxbp1 protein, rat; D033921:Vesicular Transport Proteins; D004815:Epidermal Growth Factor; D010739:Phospholipase D