Munc-18-1 inhibits phospholipase D activity by direct interaction in an epidermal growth factor-reversible manner.
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.
Lee
Hye Young
HY
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, 790-784 Republic of Korea.
Park
Jong Bae
JB
Jang
Il Ho
IH
Chae
Young Chan
YC
Kim
Jong Hyun
JH
Kim
Il Shin
IS
Suh
Pann-Ghill
PG
Ryu
Sung Ho
SH
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*
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